U.S. patent application number 10/738423 was filed with the patent office on 2004-11-18 for compositions and methods for tumor-targeted delivery of effector molecules.
This patent application is currently assigned to Vion Pharmaceuticals, Inc.. Invention is credited to King, Ivan C..
Application Number | 20040229338 10/738423 |
Document ID | / |
Family ID | 33425499 |
Filed Date | 2004-11-18 |
United States Patent
Application |
20040229338 |
Kind Code |
A1 |
King, Ivan C. |
November 18, 2004 |
Compositions and methods for tumor-targeted delivery of effector
molecules
Abstract
The present application discloses the preparation and use of
attenuated tumor-targeted bacteria vectors for the delivery of one
or more primary effector molecule(s) to the site of a solid tumor.
The primary effector molecule(s) of the invention is used in the
methods of the invention to treat a solid tumor cancer such as a
carcinoma, melanoma, lymphoma, or sarcoma. The invention relates to
the surprising discovery that effector molecules, which may be
toxic when administered systemically to a host, can be delivered
locally to tumors by attenuated tumor-targeted bacteria with
reduced toxicity to the host. The application also discloses to the
delivery of one or more optional effector molecule(s) (termed
secondary effector molecules) which may be delivered by the
attenuated tumor-targeted bacteria in conjunction with the primary
effector molecule(s).
Inventors: |
King, Ivan C.; (New Haven,
CT) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
Vion Pharmaceuticals, Inc.
|
Family ID: |
33425499 |
Appl. No.: |
10/738423 |
Filed: |
December 16, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10738423 |
Dec 16, 2003 |
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09645415 |
Aug 24, 2000 |
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60157500 |
Oct 4, 1999 |
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60157581 |
Oct 4, 1999 |
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60157637 |
Oct 4, 1999 |
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Current U.S.
Class: |
435/252.3 ;
424/200.1; 424/93.2 |
Current CPC
Class: |
A61K 31/675 20130101;
A61K 31/704 20130101; A61K 31/7048 20130101; A61K 48/00 20130101;
A61K 31/4745 20130101; A61K 33/243 20190101; A61K 31/513 20130101;
Y02A 50/30 20180101; C12N 15/74 20130101; A61K 35/74 20130101; A61K
31/337 20130101; A61K 31/7068 20130101; A61K 45/06 20130101; A61K
38/00 20130101; A61K 31/337 20130101; A61K 2300/00 20130101; A61K
31/675 20130101; A61K 2300/00 20130101; A61K 31/7068 20130101; A61K
2300/00 20130101; A61K 31/704 20130101; A61K 2300/00 20130101; A61K
31/4745 20130101; A61K 2300/00 20130101; A61K 31/7048 20130101;
A61K 2300/00 20130101; A61K 31/513 20130101; A61K 2300/00 20130101;
A61K 33/24 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
435/252.3 ;
424/200.1; 424/093.2 |
International
Class: |
A61K 048/00; C12N
001/21; A61K 039/02 |
Claims
1.-99. (Canceled)
100. A method of inhibiting the growth or reducing the volume of a
solid tumor cancer, comprising administering to a subject having a
solid tumor cancer an effective amount of one or more
chemotherapeutic agents and an effective amount of a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and an
attenuated tumor-targeted bacteria, wherein said attenuated
tumor-targeted bacteria is a facultative aerobe or facultative
anaerobe.
101. A method of inhibiting the growth or reducing the volume of a
solid tumor cancer, comprising administering to a subject having a
solid tumor cancer an effective amount of one or more
chemotherapeutic agents and an effective amount of 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 primary effector molecules
operably linked to one or more promoters, wherein said attenuated
tumor-targeted bacteria is a facultative aerobe or facultative
anaerobe.
102. A method of inhibiting the growth or reducing the volume of a
solid tumor cancer, comprising administering to a subject having a
solid tumor cancer an effective amount of one or more
chemotherapeutic agents and an effective amount of 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 primary effector molecules and
one or more secondary effector molecule operably linked to one or
more promoters, wherein said attenuated tumor-targeted bacteria is
a facultative aerobe or facultative anaerobe.
103. A method of inhibiting the growth or reducing the volume of a
solid tumor cancer, comprising administering to a subject having a
solid tumor cancer an effective amount of mitomycin C, cytoxan or
cisplatin and an effective amount of a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and an attenuated
tumor-targeted bacteria, wherein said attenuated tumor-targeted
bacteria is Salmonella.
104. A method of inhibiting the growth or reducing the volume of a
solid tumor cancer, comprising administering to a subject having a
solid tumor cancer an effective amount of mitomycin C, cytoxan or
cisplatin and an effective amount of 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 primary effector molecules operably
linked to one or more promoters, wherein said attenuated
tumor-targeted bacteria is Salmonella.
105. A method of inhibiting the growth or reducing the volume of a
solid tumor cancer, comprising administering to a subject having a
solid tumor cancer an effective amount of mitomycin C, cytoxan or
cisplatin and an effective amount of 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 primary effector molecules and one
or more secondary effector molecule operably linked to one or more
promoters, wherein said attenuated tumor-targeted bacteria is
Salmonella.
106. The method of claim 100, 101 or 102, wherein at least one of
the chemotherapeutic agents is cisplatin, ifosfamide, a taxane, a
topoisomerase I inhibitor, 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, a glucocorticoid, a puromycin
homolog or cytoxan.
107. The method of claim 100, 101 or 102, wherein the attenuated
tumor-targeted bacteria is Escherichia coli, Shigella, Yersinia
enterocohtica, Listeria monocytogenies, Mycoplasma hominis or
Streptococcus.
108. The method of claim 103, 104 or 105, wherein the Salmonella is
an msbB.sup.- Salmonella mutant.
109. The method of claim 101, 102, 104 or 105, wherein at least one
of the primary effector molecules is a TNF family member,
anti-angiogenic factor, a tumor inhibitory enzyme, hemolysin,
verotoxin, CNF1, CNF2, PMT, or a bacteriocin family member with the
proviso said bacteriocin is not BRP.
110. The method of claim 102 or 105, wherein at least one of the
secondary effector molecules is an immunomodulating agent, an
anti-tumor protein, a pro-drug converting enzyme, an antisense
molecule, a ribozyme, an antigen or a bacteriocin release
factor.
111. The method of claim 100, 101, 102, 103, 104 or 105, wherein
the solid tumor is a tumor 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 or
mesothelioma.
112. The method of claim 100, 101, 102, 103, 104 or 105, wherein
the subject is a human.
Description
[0001] This application claims priority to U.S. provisional patent
applications Nos. 60/157,500, 60/157,581, and 60/157,637, filed on
Oct. 4, 1999, the contents of each of which is incorporated herein
by reference its entirety.
1. FIELD OF THE INVENTION
[0002] The present invention relates to the delivery of one or more
primary effector molecule(s) 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 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 is a
facultative aerobe or facultative anaerobe which is modified to
encode one or more primary effector molecule(s). The primary
effector molecule(s) 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 solid 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 molecule(s) 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 molecule(s)
(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 molecule(s)
provide additional anti-tumor therapeutic activity, enhance release
of the primary effector molecule(s) from the attenuated
tumor-targeted bacteria, and/or enhance uptake of the primary
effector molecule(s) at the appropriate site of action, e.g., at
the site of a solid tumor.
2. BACKGROUND OF THE INVENTION
[0003] 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 tumors 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 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.
[0004] 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).
[0005] 2.1. Cellular Immunity and Cytokines
[0006] 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 al., 1999, Eur. J. Immunol. 29:284-290.
[0007] 2.2. Tumor Necrosis Factor (TNF) Family of Cytokines
[0008] The best characterized member of the TNF family is
TNF-.alpha.. TNF-.alpha. is known to exert pleiotropic effects on
the immune system. TNF-.alpha. is a cytokine which can exert potent
cytotoxic effects directly on tumor cells. TNF-.alpha. 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 al., 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., 1992, Ann. Rev. Immunol. 10:411-452).
However, as a result of these properties, systemic administration
of TNF-.alpha. results in lethal consequences in the host due to
disseminated intravascular coagulation.
[0009] 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-.alpha. and IL-2. See, e.g., Beutler and Cerami, 1989, Annu.
Rev. Immunol. 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.
[0010] Both TNF-.alpha. and IL-2 aid in lymphocyte homing, and IL-2
has been shown to induce tumor infiltration of natural killer (NK)
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-.alpha. 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.
[0011] 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 Schirrmacher et al., 1998, Clin. Cancer Res.
4:2635-2645.
[0012] 2.3. Delivery of Cytokines
[0013] 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-.alpha. gene, and systemic administration
of polyethylene glycol-encapsulated TNF-.alpha., which could
localize to the tumor vasculature (see Tsutsumi et al., 1996, Jpn.
J. Cancer Res. 87:1078-1085). Sensitization of tumors to
TNF-.alpha. 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).
[0014] In clinical studies, complete tumor eradication has been
observed following high-dose TNF-.alpha. administration to patients
via isolated limb perfusion, in combination with interferon-.alpha.
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 al., 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 al., 1996, Cancer
Res. 59, 205-212.
[0015] Previous studies by Carrier et al, 1992, J. Immunol.
148:1176-81, Saltzman et al., 1997, Cancer Biother. Radiopharm.
12:37-45, Saltzman et al., 1997, J. Pediat. Surgery 32:301-306 have
reported the use of attenuated Salmonella strains to deliver
IL-1.beta. (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 are 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.
[0016] 2.4. Angiogenesis and Tumorigenesis
[0017] 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).
[0018] 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 mm.sup.3 because of the 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, 1995, N. Eng. J. Med.
333:1757-1763).
[0019] 2.5. Anti-Angiogenic Factors
[0020] 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 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 45 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 produced directly by
the tumor cells (Gately et al., 1997, Cancer Res. 56:4887-4890).
Administration of angiostatin at a concentration of 100 mg/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).
[0021] 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).
[0022] 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 NC1 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).
[0023] Recently, a novel anti-angiogenic activity has been
attributed to a C-terminal truncation peptide of the Serpin (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.
[0024] 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).
[0025] 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 14 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 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).
[0026] 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
.alpha..sub.v.beta..sub.3 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-angiogenic effect of integrin blockers 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
[0027] 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).
[0028] While 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 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.
[0029] 2.6. Bacteriocin Family
[0030] 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.
[0031] 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.
[0032] In its naturally occurring form, ColE3 is a 60 kDa protein
complex consisting of a 50 kDa and a 10 kDa protein in a 1:1 ratio,
the larger subunit having the nuclease activity and the smaller
subunit having inhibitory function of the 50 kDa subunit. Thus, the
50 kDa protein acts as a cytotoxic protein (or toxin), and the 10
kDa 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).
[0033] 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
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.
[0034] 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 40S subunit of the 80S mammalian ribosome (Turnowsky et
al., 1973, Biochem. Biophys. Res. Comm. 52:327-334).
[0035] 2.7. Bacterial Infections and Cancer
[0036] Early clinical observations reported cases in which certain
cancers were reported to regress in patients with bacterial
infections, See Nauts et al., 1953, Acta Medica. Scandinavica
145:1-102, (Suppl. 276); 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
typhimurium that were able to invade HEp-2 (human epidermoid
carcinoma) cells in vitro in significantly greater numbers than the
wild-type strain. The "hyperinvasive" mutants were isolated under
conditions of aerobic growth of the bacteria that normally repress
the ability of wild-type strains to invade HEp-2 animal cells.
However, such hyperinvasive Salmonella typhimurium as described by
Lee et al. and Jones et al. carry the risk of pan-invasive
infection and could lead to wide-spread bacterial infection in the
cancer patient.
[0037] 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. 20:695-701; Barth and Morton, 1995, Cancer 75 (Suppl.
2):726-734; Friberg, 1993, Med. Oncol. Tumor. Pharmacother.
10:31-36 for reviews of BCG therapy.
[0038] However, TNF-.alpha.-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-.alpha. has been the major
barrier to effective clinical use. Modifications which reduce this
form of an immune response would be useful because TNF-.alpha.
levels would not be toxic, and a more effective concentration
and/or duration of the therapeutic vector could be used.
[0039] 2.8. Tumor-Targeted Bacteria
[0040] 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).
[0041] 2.9. Decreased Induction of TNF-.alpha. By Modified
Bacterial Lipid A
[0042] Modifications to the lipid composition of tumor-targeted
bacteria which alter the immune response as a result of decreased
induction of TNF.alpha. 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. 268:19866-19874).
Pawelek et al. showed that mutations in the firA gene induce lower
levels of TNF.alpha..
[0043] In Escherichia coli, the gene msbB (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
TNF.alpha. 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.
[0044] 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 TNF.alpha. 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, Enferm Infec. Microbiol. Clin. 14: 345-51).
[0045] Hone and Powell, WO97/18837 ("Hone and Powell"), disclose
methods to produce gram-negative bacteria having non-pyrogenic
Lipid A or LPS.
[0046] Maskell, WO98/33923, describes a mutant strain of Salmonella
having a mutation in the msbB gene which induces TNF.alpha. at a
lower level as compared to a wild type strain.
[0047] 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
TNF.alpha. 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.
[0048] 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 INVENTION
[0049] The present invention provides methods for delivering one or
more primary effector molecule(s) 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 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 molecule(s) 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 molecule(s)
and optionally, one or more secondary effector molecule(s).
[0050] 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).
[0051] 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.
[0052] 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 molecule(s) is
derived from a mammal. In a more preferred mode of this embodiment,
the primary effector molecule(s) is derived from a human. The
primary effector molecules of the invention include members of the
TNF family, anti-angiogenic factors, cytotoxic polypeptides or
peptides, tumor inhibitory enzymes, and functional fragments
thereof.
[0053] 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-.alpha. (TNF-.alpha.), tumor
necrosis factor-.beta. (TNF-.beta.), TNF-.alpha.-related
apoptosis-inducing ligand (TRAIL), TNF-.alpha.-related
activation-induced cytokine (TRANCE), TNF-.alpha.-related weak
inducer of apoptosis (TWEAK), CD40 ligand (CD40L), LT-.alpha.
(lymphotoxin alpha), LT-.beta. (lymphotoxin beta), OX40L (OX40
ligand), FasL, CD27L (CD27 ligand), CD30L (CD30 ligand), 4-1BBL,
APRIL (a proliferation-inducing ligand), LIGHT (a 29 kDa type II
transmembrane protein produced by activated T cells), TL1 (a tumor
necrosis factor-like cytokine), TNFSF16, TNFSF17, 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-.alpha. (TNF-.alpha.), tumor necrosis
factor-.beta. (TNF-.beta.), TNF-.alpha.-related apoptosis-inducing
ligand (TRAIL), TNF-.alpha.-related activation-induced cytokine
(TRANCE), TNF-.alpha.-related weak inducer of apoptosis (TWEAK),
and CD40 ligand (CD40L), or a functional fragment thereof.
[0054] 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 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 .alpha..sub.v.beta..sub.3 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.
[0055] 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, ColE1, ColE1a, ColE1b ColE2, ColE3, ColE4,
ColE5, ColE6, ColE7, ColE8, ColE9, Colicins A, Colicin K, Colicin
L, Colicin M, cloacin DF13, pesticin A1122, staphylococcin 1580,
butyricin 7423, pyocin R1 or AP41, megacin A-216, and vibriocin. In
a specific embodiment, the primary effector molecule is colicin
E3.
[0056] 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.
[0057] The present invention also provides methods for local,
combinatorial delivery of one or more primary effector molecule(s)
and one or more secondary effector molecule(s) 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 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).
[0058] The secondary effector molecule(s) of the invention provide
additional anti-tumor therapeutic activity, enhance release of the
primary effector molecule(s) 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
molecule(s) 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 molecule(s) by the
methods of the invention to treat a solid tumor cancer such as a
carcinoma, melanoma, lymphoma, or sarcoma.
[0059] 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 molecule(s) is derived from a bacterium (e.g.
BRP). In other preferred embodiments of the invention, the
secondary effector molecule(s) is derived from a virus (e.g., TAT).
In yet other preferred embodiments of the invention, the secondary
effector molecule(s) is derived from a mammal. In certain preferred
embodiments, the secondary effector molecule(s) is derived from a
human.
[0060] The invention provides attenuated tumor-targeted bacteria
comprising effector molecule(s) 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. The invention also provides attenuated tumor-targeted
bacteria comprising effector molecule(s) which are encoded by a
nucleic acid which is integrated into the bacterial genome.
Integrated effector molecule(s) 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). 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, lexA, cea, caa, recN and
pagC.
[0061] 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, lamB, .beta.-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.
[0062] 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 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.
[0063] 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.
[0064] 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.
[0065] 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).
[0066] 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 protein(s) and effector molecule(s)
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 protein(s) alone or the effector
molecule(s) alone is expressed.
[0067] 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 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 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 a preferred embodiment, the
release factor is a Bacteriocin Release Protein (BRP). In a
specific embodiment, the BRP is that of the cloacin DF13 plasmid,
one of colicin E1-E9 plasmids, or the colicin A, N or D plasmids.
In a preferred embodiment, the BRP is of cloacin DF13 (pCloDF13
BRP). In another embodiment of the invention, the enhanced release
system comprises overexpression of a porin protein.
[0068] 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 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.
[0069] 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.
[0070] 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. 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.
[0071] 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,
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.
[0072] 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 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.
[0073] 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, 5-fluorouracil
(5-FU), leucovorin, vinorelbine, temodal, taxol, cytochalasin B,
gramicidin D, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, 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.
[0074] 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.
[0075] 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.
[0076] 3.1. Definitions and Abbreviations
[0077] As used herein, Salmonella encompasses all Salmonella
species, including: Salmonella typhi, Salmonella choleraesuis, and
Salmonella enteritidis. Serotypes of Salmonella are also
encompassed herein, for example, typhimirium, a subgroup of
Salmonella enteritidis, commonly referred to as Salmonella
typhimurium.
[0078] 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 55%,
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 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 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
55%, 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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).
[0084] 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.sup.-
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.
[0085] 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 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.
[0086] 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 function of the primary or
secondary effector molecule (e.g., enzymatic activity,
anti-angiogenic activity, or anti-tumor activity of the effector
molecule).
[0087] 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
polypeptide (e.g., a non-primary or non-secondary effector
molecule).
[0088] Omp-like protein: As used herein, an Omp-like protein
includes any bacterial outer membrane protein, or portion thereof
(e.g., signal sequence, leader sequence, periplasmic region,
transmembrane domain, multiple transmembrane domains, or
combinations thereof). In specific embodiments, the Omp-like
protein is at least a portion of OmpA, OmpB, OmpC, OmpD, OmpE,
OmpF, OmpT, a porin-like protein, PhoA, PhoE, lamB,
.beta.-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.
[0089] 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.
[0090] 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.
[0091] Septic shock: Septic shock is a state of internal organ
failure due to a complex cytokine cascade, initiated by
TNF-.alpha.. The relative ability of a microorganism or vector to
elicit TNF-.alpha. is used as one measure to indicate its relative
ability to induce septic shock.
[0092] 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.
[0093] 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).
[0094] As used herein, the strain designations VNP20009
(International Publication No. WO 99/13053), YS1646 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.
[0095] 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
[0096] FIG. 1. Coding sequence for the mature human TNF-.alpha..
Both DNA (SEQ ID NO:3) and protein (SEQ ID NO:4) sequences are
indicated.
[0097] FIG. 2. Derivation of the Salmonella VNP20009
serC-strain.
[0098] FIG. 3. TNF-.alpha., expression from a
chromosomally-integrated trc promoter driven TNF-.alpha., gene in
Salmonella typhimurium.
[0099] FIG. 4. Coding sequence for the synthetic OmpA signal
sequence (nucleotides 1-63) fusion to the mature human TNF-.alpha.
(nucleotides 67-543). Both DNA (SEQ ID NO:7) and protein (SEQ ID
NO:8) sequences are indicated for the fusion construct.
[0100] FIG. 5. Periplasmic localization and processing of an
OmpA/TNF-.alpha., fusion protein in E-coli (JM109 strain).
[0101] FIG. 6. Coding sequence for the OmpA signal sequence
(nucleotides 1-63) fusion to the mature human TRAIL (nucleotides
67-801). Both DNA (SEQ ID NO:9) and protein (SEQ ID NO:10)
sequences are indicated for the fusion construct.
[0102] FIG. 7. Expression and processing of an OmpA TRAIL fusion
protein in E-coli (JM109 strain).
[0103] 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
NO:12) sequences are indicated for the fusion construct.
[0104] FIG. 9. Expression and processing of mature human IL-2 fused
to the phoA(8L) or ompA (8L) synthetic signal peptides.
[0105] FIG. 10. Coding sequence for the modified phoA signal
sequence (nucleotides 1-63) fusion to the mature (C125A) human IL-2
(nucleotides 64-462). Both DNA (SEQ ID NO:13) and protein (SEQ ID
NO:14) sequences are indicated for the fusion construct.
[0106] FIG. 11. In vivo anti-tumor efficacy of an attenuated strain
of Salmonella typhimurium expressing the mature form of human
TNF-.alpha..
[0107] FIG. 12. Effect of BRP expression on anti-tumor efficacy in
vivo. The figure shows a graphic representation of mean tumor size
over time of a C57BL/6 mouse population with B16 melanoma tumors
treated with (1) a PBS control; (2) VNP20009; and (3) VNP20009
harboring the pSW1 plasmid, which comprises the BRP gene.
[0108] FIG. 13. Anaerobic induction of .beta.-gal gene expression
under the control of the pepT promoter in Salmonella. FIG. 13A
demonstrates the in vitro induction of .beta.-gal expression in
response to anaerobic conditions of two strains of Salmonella, YS
1456 and VNP20009. FIG. 13B demonstrates the in vivo induction of
.beta.-gal in tumor v. liver cells of VNP20009 Salmonella
expressing BRP, .beta.-gal, or BRP and .beta.-gal.
[0109] FIG. 14. Tetracycline induction of .beta.-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 .mu.g/ml, after which there
response declines, presumably as a result of the antibiotic
function of Tetracycline.
[0110] 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 (DH5.alpha.). Even numbered lanes indicate extracts from
uninduced cultures, whereas odd numbered lanes indicate the
corresponding IPTG-induced cultures.
[0111] FIG. 16. Expression of HexaHIS-endostatin from the plasmid
YA3334: HexaHIS-endostatin in the asd system (utilizing the trc
promoter) is able to express a band of the correct size for
HexaHIS-endostatin (.about.25 kD) by Western analysis with a
anti-histidine antibody (lanes 1-8 correspond to eight independent
clones).
[0112] 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.sup.- VNP20009 carrying an empty YA3334 vector; (3) asd
.sup.-VNP20009 which expresses hexahistidine-endostatin; (4) and
VNP20009 which expresses hexahistidine-endostatin and BRP.
[0113] FIG. 18. Efficacy of VNP20009 cells expressing endostatin on
DLD1 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.sup.- VNP20009 carrying an empty YA3334 vector;
and (3) VNP20009 which expresses hexahistidine-endostatin and
BRP.
[0114] 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
lysates corresponding to 8.times.10.sup.8 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.
[0115] FIG. 20. Anti-proliferative activity of lysates from
attenuated tumor-targeted 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.times.10.sup.8
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.
[0116] FIG. 21. Construction of the pE3.shuttle-1 Vector.
[0117] FIG. 22. Construction of the Col E3-CA38 Vector (GenBank
Accession Number 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.
[0118] FIG. 23. Construction of the Col E3-CA38/BRP-1 vector.
[0119] FIG. 24. Bar Graph showing the amount of lethal units of
colicin E3 produced by each strain.
[0120] FIG. 25. Halo assay for various strains exposed to
ultraviolet light or x-rays.
[0121] FIG. 26. Efficacy of 41.2.9/Col E3 on C38 murine colon
carcinoma.
[0122] FIG. 27. Anti-tumor activity of 41.2.9/Col/E3 on DLD1 human
colon carcinoma in NU/Nu mice.
[0123] FIG. 28. Efficacy of 41.2.9/Col E3 on B16 murine
melanoma.
[0124] FIG. 29. Cytotoxicity of Salmonella expressing cloned E.
coli CNF 1.
[0125] FIG. 30. Hela cells exposed to CNF1 (A) show enlargement and
multinucleation relative to normal Hela cells (B).
[0126] 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 Not1, PacI, SphI,
SfiI, SwaI and DraI polylinker in its place (SEQ ID NO:61). See
FIG. 32.
[0127] 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.
[0128] 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-lactam
antibiotic such as carbenicillin and ampicillin. sacB, the gene
which confers sensitivity to sucrose. Note: not drawn to scale.
[0129] FIG. 34. Percent cytotoxicity of tetBRPAB clone #26 and
clone #31 compared to positive and negative controls (HSC 10 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 HSC 10 was used as a positive control for
percent cytotoxicity.
[0130] 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).
[0131] 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.
[0132] FIG. 37. Coding sequence for TAT-apoptin fusion protein.
Both DNA (SEQ ID NO:57) and protein (SEQ ID NO:58) sequences are
indicated.
[0133] FIG. 38. Coding sequence for hexahistidine-TAT-apoptin
fusion protein. Both DNA (SEQ ID NO:59) and protein (SEQ ID NO:60)
sequences are indicated.
[0134] FIG. 39. Efficacy of VNP20009/cytoxan combination therapy on
M27 lung carcinoma growth in C57BL/6 mice.
[0135] FIG. 40. Efficacy of VNP20009/mitomycin combination therapy
on M27 lung carcinoma growth in C57BL/6 mice.
[0136] FIG. 41. Efficacy of VNP20009/cisplatin combination therapy
on M27 lung carcinoma growth in C57BL/6 mice.
5. DETAILED DESCRIPTION OF THE INVENTION
[0137] The present invention utilizes attenuated tumor-targeted
strains of bacteria to deliver high levels of therapeutic primary
effector molecule(s) 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-.alpha.). The present invention provides delivery of one or
more primary effector molecule(s) and optionally, one or more
secondary effector molecule(s) 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 molecule(s) 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 molecule(s)
and optionally, one or more secondary effector molecule(s).
[0138] The attenuated tumor-targeted bacterial-based delivery
system presently described provides local delivery of one or more
effector molecule(s) 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 molecule(s) and
optionally, one or more secondary effector molecule(s) 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 molecule(s) and/or optionally, one or
more different secondary effector molecule(s).
[0139] 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
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.
[0140] 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 carrier 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.
[0141] 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.
[0142] 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 molecule(s) at the site of a
tumor by delivery of the attenuated tumor-targeted bacterial vector
containing the effector molecule(s).
[0143] 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.
[0144] 5.1. Bacterial Vectors
[0145] 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 tumor-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.
[0146] Factors contributing to attenuation and tumor-targeting are
described herein and 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 WO96/40238, which are incorporated herein by reference.
Examples of attenuated tumor-targeted bacteria are also described
in International Application WO99/13053, 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.
[0147] 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 cytotoxic 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.
[0148] 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
monocytogenies, Mycoplasma hominis, Streptococcus spp., wherein the
bacterium is a facultative aerobe or facultative anaerobe.
[0149] 5.1.1 Salmonella Vectors
[0150] 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.
[0151] 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, 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 83:5189-5193). Table 4 of International Publication WO 96/40238
is an illustrative list of Salmonella virulence factors whose
deletion results in attenuation.
[0152] Yet 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 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.
[0153] 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.sup.-
mutation also decreases enzymatic activity of lipid A 4' kinase
that regulates the sixth step of lipid A biosynthesis.
[0154] 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 43-51; 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.
[0155] An illustrative example of an attenuated tumor-targeted
bacterium having an LPS pathway mutant is the msbB.sup.- Salmonella
mutant described in International Publication WO99/13053, which is
incorporated herein by reference in its entirety; see especially
Section 6.1.2 which describes the characteristic of the msbB.sup.-
Salmonella mutant. One characteristic of the msbB.sup.- Salmonella
is decreased ability to induce a TNF-.alpha. response compared to
the wild-type bacterial vector. The msbB.sup.- Salmonella induce
TNF-.alpha. expression at levels of about 5 percent to about 40
percent compared to the levels induced by wild-type Salmonella.
[0156] The TNF-.alpha. 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-.alpha. production on a per
colony forming unit ("c.f.u.") or on a .mu.g/kg basis, is used to
determine relative activity. Lower TNF-.alpha. levels on a per unit
basis indicate decreased induction of TNF-.alpha. production. In a
preferred embodiment, the msbB.sup.- Salmonella vector is modified
to contain one or more primary effector molecule(s) and optionally,
one or more secondary effector molecule(s) of the invention.
[0157] The present invention also encompasses the use of
derivatives of msbB.sup.- attenuated tumor-targeted Salmonella
mutants. Derivatives of msbB.sup.- attenuated tumor-targeted
Salmonella mutants can be modified using the teaching of the
invention to encode one or more primary effector molecule(s) and
optionally, one or more secondary effector molecule(s) to produce a
novel attenuated tumor-targeted bacteria useful for the delivery of
one or more effector molecule(s) of the invention to a solid
tumor.
[0158] 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.
[0159] 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.sup.- 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.sup.- 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, PurI or
SerC. 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, PurI or SerC.
[0160] Accordingly, any attenuated tumor-targeted bacteria may be
used in the methods of the invention to express and deliver one or
more primary effector molecule(s) and optionally, one or more
secondary effector molecule(s) to a solid tumor cancer. In
preferred embodiments, the attenuated tumor-targeted bacteria are
constructed to express one or more primary effector molecule(s) 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 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.
[0161] 5.2. Primary Effector Molecules for Tumor Therapy
[0162] The invention provides for delivery of primary (and
optionally secondary) effector molecule(s) 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.
[0163] 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 molecule(s) is derived from a
mammal. In a more preferred embodiment, the primary effector
molecule(s) 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.
[0164] 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-.alpha. (TNF-.alpha.), tumor
necrosis factor-.beta. (TNF-.beta.), TNF-.alpha.-related
apoptosis-inducing ligand (TRAIL), TNF-.alpha.-related
activation-induced cytokine (TRANCE), TNF-.alpha.-related weak
inducer of apoptosis (TWEAK), CD40 ligand (CD40L), LT-.alpha.,
LT-.beta., OX4OL, CD4OL, FasL, CD27L, CD30L, 4-1BBL, APRIL, LIGHT,
TL1, TNFSF16, TNFSF17, and AITR-L. In a preferred embodiment, a
primary effector molecule of the invention is tumor necrosis
factor-.alpha. (TNF-.alpha.), tumor necrosis factor-.beta.
(TNF-.beta.), TNF-.alpha.-related apoptosis-inducing ligand
(TRAIL), TNF-.alpha.-related activation-induced cytokine (TRANCE),
TNF-.alpha.-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-.alpha. (TNF-.alpha.), tumor necrosis factor-.beta.
(TNF-.beta.), TNF-.alpha.-related apoptosis-inducing ligand
(TRAIL), TNF-.alpha.-related activation-induced cytokine (TRANCE),
TNF-.alpha.-related weak inducer of apoptosis (TWEAK), or CD40
ligand (CD40L).
1TABLE 1 TNF FAMILY MEMBERS Classic Nomencalture Standardized
Nomenclature LT-.alpha. TNFSF1 TNF-.alpha. TNFSF2 LT-.beta. TNFSF3
OX4OL TNFSF4 CD4OL TNFSF5 F.sub.asL TNFSF6 CD27L TNFSF7 CD30L
TNFSF8 4-1BBL TNFSF9 TRAIL TNFSF10 TRANCE TNFSF11 TWEAK TNFSF12
APRIL TNFSF13 LIGHT TNFSF14 TL1 TNFSF15 -- TNFSF16 -- TNFSF17
AITR-L TNFSF18
[0165] 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 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 .alpha..sub.v.beta..sub.3 and the VEGF receptor.
[0166] In a preferred embodiment of the invention, a primary
effector molecule of the invention is endostatin. Naturally
occurring endostatin consists of the C-terminal .about.180 amino
acids of collagen XVIII (cDNAs encoding two splice forms of
collagen XVIII have Genbank Accession No. AF 18081 and AF
18082).
[0167] 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.sub.--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 isoform, 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).
[0168] In another preferred embodiment 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.sub.--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).
[0169] 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.
[0170] 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).
[0171] 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.sub.--000948).
In a specific embodiment, said prolactin fragment has a Cys58-Ser58
mutation to circumvent undesired cross-linking of the protein by
disulfide bonds.
[0172] 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
.beta.-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.sub.--002619.
[0173] 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, fibronectin, procollagen, or
EGF, or small peptide antagonists of integrin
.alpha..sub.v.beta..sub.3 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. 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.
[0174] 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.
[0175] 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. coli 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 inactiviting 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. coli heat-labile toxin, E. coli
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 coli and Salmonella in Escherichia and Salmonella,
Cellular and Molecular Biology, Neidhardt et al. (eds.), pp.
2788-2802, ASM Press, Washington, D.C. for a review of E. coli and
Salmonella toxins.
[0176] 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). Examples
of bacteriocin family members, include, but are not limited to,
ColE1, ColE1a, ColE1b ColE2, ColE3, ColE4, ColE5, ColE6, ColE7,
ColE8, ColE9, Colicin A, Colicin K, Colicin L, Colicin M, cloacin
DF13, pesticin A1122, staphylococcin 1580, butyricin 7423, 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 molecule(s) 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.
[0177] In a preferred embodiment, the primary effector molecule(s)
is ColE1, ColE2, ColE3, ColE4, ColE5, ColE6, ColE7, ColE8, 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 80S
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 60 kDa protein complex consisting of a 50 kDa and
a 10 kDa protein in a 1:1 ratio, the larger subunit having the
nuclease activity and the smaller subunit having inhibitory
function of the 50 kDa subunit. Thus, the 50 kDa protein acts as a
cytotoxic protein (or toxin), and the 10 kDa 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
50 kDa toxin and 10 kDa 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).
[0178] In another preferred embodiment, the primary effector
molecule(s) is cloacin DF13. Cloacin DF13 functions in an analogous
manner to ColE3. The protein complex is of 67 KDa molecular weight.
The individual components are 57 kDa and 9 kDa in size. In addition
to its ribonuclease activity, DF13 can cause the leakage of
cellular potassium.
[0179] In another preferred embodiment, the primary effector
molecule(s) 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).
[0180] In another embodiment, the primary effector molecule(s) is
colicin E2 (a dual subunit colicin similar to ColE3 in structure
but with endonuclease rather than ribonuclease activity); colicins
A, E1, Ia, Ib, or K, which form ion-permeable channels, causing a
collapse of the proton motive force of the cell and leading to cell
death; colicin L which inhibits protein, DNA & RNA synthesis;
colicin M which causes cell sepsis by altering the osmotic
environment of the cell; pesticin 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).
[0181] 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.
[0182] 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, methionase, asparaginase, lipase,
phospholipase, protease, ribonuclease, DNAase, and glycosidase. In
a preferred embodiment, the primary effector molecule is
methionase.
[0183] 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.
[0184] The invention provides nucleic acid molecules encoding a
primary effector molecule. The invention also provides nucleic acid
molecules encoding one or more primary effector molecule(s) and
optionally, one or more secondary effector molecule(s). The
invention provides nucleic acids encoding effector molecule(s) of
the invention which is operably linked to an appropriate promoter.
Optionally, the nucleic acids encoding an effector molecule(s) may
be operably linked to other elements that participate in
transcription, translation, localization, stability and the
like.
[0185] 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.
[0186] 5.3. Secondary Effector Molecules for Co-Expression with
Primary Effector Molecules
[0187] 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 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., in 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.
[0188] 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.
[0189] 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.
[0190] Antisense nucleotides are oligonucleotides that bind in a
sequence-specific manner to nucleic acids, such as mRNA or DNA.
When bound to mRNA that has complementary sequences, antisense
prevents translation of the mRNA (see, e.g., U.S. Pat. Nos.
5,168,053; 5,190,931; 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.
Pat. No. 5,176,996).
[0191] 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. Pat. No.
5,272,262; U.S. Pat. No. 5,144,019; and U.S. Pat. Nos. 5,168,053,
5,180,818, 5,116,742 and 5,093,246).
[0192] 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.
[0193] 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.
[0194] 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.
[0195] 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. Pat. 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.
[0196] 5.3.1. Factors Providing Additional Therapeutic Value
[0197] 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.
[0198] 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).
[0199] 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 molecule(s) 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).
[0200] 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.
[0201] 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).
[0202] 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 (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-.gamma., IFN-.alpha., MIP-3a, SLC, MIP-3p, 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,
.alpha.-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 tumor cell and not in
the non-cancerous counterpart cell, or is expressed in the tumor
cell at a higher level than in the non-cancerous counterpart cell.
Illustrative examples of tumor-associated antigens are described in
Kuby, Immunology, W.H. Freeman and Company, New York, N.Y.,
1.sup.st 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.
[0203] 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.
[0204] 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.
[0205] 5.3.2. Factors that Promote the Release of Anti-Tumor
Effector Molecules into the Tumor Environment
[0206] 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
membrane(s) or enhance the release of intracellular components into
the extracellular environment, e.g. at the tumor site, thereby
enhancing the delivery of the 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.
[0207] 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.
[0208] 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 DF13 (pCloDF13 BRP).
[0209] 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 (PldA) 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 BRP
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, preferably more than 70%, more preferably 80%, still more
preferably more than 90% and most preferably 90-100% of
bacteria.
[0210] In a specific embodiment of the invention, the BRP protein
is a pCloDF13 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 bacterial vector, while reducing the need
for frequent administration of the vector. In another specific
embodiment, the BRP of the invention is a pCloDF13 BRP with a
shortened C-terminus, which in addition to protein release causes
cell lysis (Luirink et al., 1989, J. Bacteriol. 171:2673-2679).
[0211] 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.
[0212] 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.
[0213] 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).
[0214] Alternatively, the promoter can be an antibiotic-induced
promoter, such as the tet promoter of the Tn10 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 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 {fraction
(1/10)}th of the dosage required for antibiotic activity.
[0215] 5.4. Derivatives and Analogs
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C. followed by
one or more washes in 0.2.times.SSC/0.1% SDS at about 50-65.degree.
C., under highly stringent conditions, e.g., hybridization to
filter-bound nucleic acid in 6.times.SSC at about 45.degree. C.
followed by one or more washes in 0.1.times.SSC/0.2% SDS at about
68.degree. 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).
[0220] 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.
[0221] 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).times.100). In one embodiment, the two
sequences are the same length.
[0222] 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. 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 of a 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 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.1.html#sect2, the
contents of which are incorporated herein by reference.
[0223] 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.
[0224] 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.
[0225] 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 Press, Cold Spring Harbor, N.Y.). 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.
[0226] 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.RTM. 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.
[0227] In other embodiments, the effector molecules or fusion
proteins of the invention are constructed to contain a protease
cleavage site.
[0228] 5.5. Fusion Proteins
[0229] 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 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.
[0230] 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 TNF-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 fused 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.
[0231] 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").
[0232] 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 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.
[0233] 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, lamB, .beta.-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.
[0234] 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.
[0235] 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.
[0236] For example, the native OmpA protein contains eight
anti-parallel transmembrane .beta.-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 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.
[0237] 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.
[0238] 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 molecule(s) of the invention.
[0239] 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.
[0240] Similar fusion proteins may be constructed with any of the
Omp-like proteins using the strategies described above in terms of
OmpA. In the construction of such fusion proteins, as will be
apparent to one of ordinary skill in the art, the selection of the
portion of the Omp-like protein to be fused to 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.
[0241] 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).
[0242] 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.
[0243] 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.
[0244] 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, 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.
[0245] 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 (LFn; 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-hemolysins,
enterotoxins, cytotoxins, EAggEC stable toxin 1 (EAST), CNFs,
cytolethal distending toxin, .alpha.-hemolysins, .beta.-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 molecule 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.
[0246] 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).
[0247] 5.6. Expression Vehicles
[0248] 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 molecule(s) 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 molecule(s) which are
encoded by a nucleic acid molecule which is integrated into the
bacterial genome. Integrated effector molecule(s) 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 molecule encoding the
effector molecule becomes integrated into the genome of the
attenuated tumor-targeted bacteria. 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).
[0249] 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,
N.Y.; Ausubel et al., 1995, Current Protocols in Molecular Biology,
Greene Publishing, New York, N.Y. 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.
[0250] 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 appropriate expression
vehicle, e.g., a plasmid which contains the necessary elements for
the transcription and translation of the inserted protein-coding
sequence.
[0251] 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.
[0252] Such commercial plasmids include, for example, pKK223-3
(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega
Biotec, Madison, Wis., 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 pUC19 (see
e.g., Yanisch-Perron et al. 1985, Gene 33:103-119) and pBluescript
(Stratagene).
[0253] 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,
pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3,
pKK233-3, pDR540, pRIT5 (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, Wis., USA). These are combined with an appropriate
promoter and the structural sequence to be expressed. pCET, pTS (as
described in Section 6 herein).
[0254] In specific embodiments of the invention, a plasmid encoding
an effector molecule is the pTS-TNF-.alpha. plasmid, the pTS-BRP
plasmid, or the pTS-BRPTNF-.alpha. plasmid as described in Section
6 herein.
[0255] 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).
[0256] 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.
[0257] 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.
[0258] 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.
[0259] Transposons to be utilized by the present invention include
but are not limited to Tn7, Tn9, Tn10 and Tn5. In a preferred
embodiment, the transposon plasmid is pNK2883 (ATCC) having an
ampicillin resistance gene located outside the Tn10 insertion
elements and the nucleic acids encoding one or more effector
molecule(s) is inserted between the two Tn10 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 Tn10 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 molecule(s) (such as TNF-.alpha., or a fusion
protein thereof, e.g., an OmpA-TNF-.alpha. fusion), (4) a
terminator for the nucleic acid encoding the one or more effector
molecule(s).
[0260] 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).
[0261] 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 serC gene and an MCS. In another specific embodiment,
the plasmid pTS-BRP is used which comprises an altered target
specificity transposase gene and a minitransposon, containing the
coding sequences for a promoterless serC gene, and alkylating
agent-inducible bacteriocin release factor, and an MCS.
[0262] In a preferred embodiment, a transposon plasmid for
selection of transposon-mediated chromosomal integrants,
comprises:
[0263] a) a transposase gene, for transposon excision and
integration, located outside of the transposon insertion sequence
(e.g., outside of the transposon cassette);
[0264] 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;
[0265] c) optionally, between the right and left insertion
sequences is a nucleic acid sequence encoding a release enhancing
nucleic acid (e.g., BRP); and
[0266] 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.
[0267] 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.
[0268] 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.
[0269] 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 (.beta.-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. Pat. No. 5,840,483
to Curtiss, III).
[0270] 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.
[0271] 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.
[0272] In other embodiments of the invention, the expression
vehicle is a .lambda. vector, more specifically a lysogenic
.lambda. vector. In a preferred embodiment, the bacterial host
comprising the .lambda. vector further comprises a
temperature-sensitive .lambda. repressor which is functional at
30.degree. C. but not 37.degree. C. Consequently, the bacterial
host can be grown and manipulated in vivo at 30.degree. 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 .lambda. repressor is
inactivated by normal body temperature and expression of the
primary effector molecule, and optionally a secondary effector
molecule, is activated.
[0273] 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 .beta.-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 PL 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 73:423-431). In a
preferred embodiment, the promoter is trc, (see, e.g., Amann et
al., 1988, Gene 69:301-15).
[0274] 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.
[0275] 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 P 1
promoter of the pepT gene. Activation of the P 1 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 pepTunder
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.
[0276] Alternatively, the promoter can be an antibiotic-induced
promoter, such as the tet promoter of the Tn10 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 low levels of
tetracycline, approximately {fraction (1/10)}th of the dosage
required for antibiotic activity.
[0277] 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 Spring
Harbor Press, Cold Spring Harbor, N.Y.; Ausubel et al., 1995,
Current Protocols in Molecular Biology, Greene Publishing, New
York, N.Y.).
[0278] 5.7. Combination Therapy
[0279] 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 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 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.
[0280] 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.
[0281] 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.
[0282] 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.
[0283] 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.
[0284] 5.8. Methods and Compositions for Delivery
[0285] 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.
[0286] 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.
[0287] 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 primary effector molecules and one or more secondary
effector molecules operably linked to one or more appropriate
promoters. In one embodiment, the primary effector molecule is a
TNF family member, a cytotoxic peptide or polypeptide, an
anti-angiogenic factor, a tumor inhibitory enzyme, or a functional
fragment thereof.
[0288] 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.
[0289] 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.
[0290] 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.
[0291] 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.
[0292] 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 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).
[0293] 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 primary 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
primary effector molecules and one or more secondary effector
molecules operably linked to one or more appropriate promoters.
[0294] 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 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 and
one or more effector molecules operably linked to one or more
appropriate promoters.
[0295] The present invention also provides a pharmaceutical
composition comprising a pharmaceutically acceptable carrier 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 carrier. 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.
[0296] 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 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 carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. 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.
[0297] 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.
[0298] 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 generally from about 1.0
c.f.u./kg to about 1.times.10.sup.10 c.f.u./kg; optionally from
about 1.0 c.f.u./kg to about 1.times.10.sup.8 c.f.u./kg; optionally
from about 1.times.10.sup.2 c.f.u./kg to about 1.times.10.sup.8
c.f.u./kg; optionally from about 1.times.10.sup.4 c.f.u./kg to
about 1.times.10.sup.8 c.f.u./kg; and optionally from about
1.times.10.sup.4 c.f.u./kg to about 1.times.10.sup.10 c.f.u./kg.
Effective doses may be extrapolated from dose-response curves
derived from in vitro or animal model test systems.
[0299] 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).
[0300] 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.
[0301] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment; this may be achieved by, for example,
and not by way of limitation, local infusion during surgery, by
injection, by means of a catheter, or by means of an implant, said
implant being of a porous, non-porous, or gelatinous material,
including membranes, such as sialastic membranes, or fibers. In one
embodiment, administration can be by direct injection at the site
(or former site) of a malignant tumor or neoplastic or
pre-neoplastic tissue.
[0302] The attenuated tumor-targeted bacteria comprising one or
more primary effector molecules and optionally, one or more
secondary effector molecules may be delivered in a controlled
release system. The attenuated tumor-targeted bacteria comprising
one or more fusion 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, Fla. (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 (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)).
[0303] 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 molecule(s) and
optionally, one or more secondary effector molecule(s).
[0304] 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 container(s) 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.
[0305] 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-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.
[0306] 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 nucleic acid molecules encoding one or more effector molecules
and/or fusion proteins that are additive or synergistic.
[0307] 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.
[0308] 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.
[0309] 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.
[0310] 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.
[0311] 5.9. Demonstration of Therapeutic or Prophylactic Utility of
Pharmaceutical Compositions of the Invention
[0312] The pharmaceutical compositions of the invention are
preferably tested in vitro, and then in vivo 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 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.
[0313] 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 .sup.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 51Cr-release assay (see,
e.g., Palladino et al., 1987, Cancer Res. 47:5074--5079 and
Blachere et al., 1993, J. Immunotherapy 14:352-356).
[0314] 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.
[0315] In various specific embodiments, in vitro assays can be
carried 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.
[0316] 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, mice,
chicken, cows, monkeys, pigs, dogs, rabbits, etc. For in vivo
testing, prior to administration to humans, any animal model system
known in the art may be used.
[0317] 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-.alpha. by Attenuated Tumor-Targeted
Salmonella
[0318] 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.
[0319] 6.1. Construction of TNF-.alpha. Plasmids
[0320] 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-.alpha. encoding nucleic acids may be replaced with other
appropriate promoter or effector molecules by methods known in the
art.
[0321] 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.
[0322] 6.1.1. The pCET Plasmid
[0323] For plasmid-based bacterial expression of effector molecule
encoding nucleic acids using a dual .lambda.P.sub.L, or
.lambda.P.sub.R 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 I/Bam HI fragment of pUC19 (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 Cla1, 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
bacteria harboring any plasmid containing the .lambda.P.sub.L, or
.lambda.P.sub.R promoter, was performed at 30.degree. C.
[0324] 6.1.2. The pTS Plasmid
[0325] 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 serC 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 (SEQ ID
NO: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 serC encoding nucleic
acid was present for the insertion of effector molecule-encoding
nucleic acids.
[0326] 6.1.3. The pTS-TNF-.alpha. Plasmid
[0327] A plasmid (pTS-TNF-.alpha.), for the pTS-mediated
chromosomal integration of a trc promoter-driven human TNF-.alpha.
encoding nucleic acid, was constructed as follows. Plasmid PYA3332
is the ASD plasmid PYA272 (see, e.g., U.S. Pat. No. 5,840,483 to
Curtiss, III) with the origin of replication replaced by that of
the colE1 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-.alpha.
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-.alpha.. Asd34TNF-.alpha. was then cleaved
with restriction enzyme Bgl II, and the 1.1 kb fragment, encoding
the trc promoter-driven TNF-.alpha. encoding nucleic acid, and
ligated into the Bam HI site of pTS to give plasmid
pTS-TNF-.alpha..
[0328] 6.1.4. The pTS-BRP plasmid
[0329] 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 acid was
isolated from plasmid pSWI (commercially available from Bio101,
Vista, Calif.) by PCR using a forward primer, of sequence
CCGACGCGTTGACACCTGAAAACTGGAG (SEQ ID NO:5), and a reverse primer,
of sequence CCGACGCGTGAAAGGATCTCAAGAAGATC (SEQ ID NO:6), and cloned
into a TOPO-TA cloning plasmid (commercially available from
InVitrogen, Carlsbad, Calif.) 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 I/Bam 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.
[0330] 6.1.5. The pTS-BRPTNF-.alpha. Plasmid
[0331] A plasmid (pTS-BRPTNF-.alpha.), for the pTS-mediated
chromosomal integration of the BRP and trc promoter-driven
TNF-.alpha. encoding nucleic acids, was constructed as follows.
Plasmid Asd34TNF-.alpha., described above for the construction of
pTS-TNF-.alpha., was cleaved with restriction enzyme Bgl II, and
the 1.1 kb fragment, encoding the trc promoter-driven TNF-.alpha.
encoding nucleic acid, was ligated into the Bam HI site of pTS-BRP
to give plasmid pTS-BRPTNF-.alpha..
[0332] 6.2. Integration of Effector Molecule-Encoding Nucleic Acid
into the Salmonella Host Chromosome
[0333] The system described here employs .DELTA.serC-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.
[0334] pTS or pTS-BRP plasmids containing effector
molecule-encoding nucleic acids may be introduced into
serC-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,
Fla., 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. 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.
[0335] 6.2.1. Delivery and Expression of Salmonella-Expressed
TNF-.alpha.
[0336] A trc promoter-driven TNF-.alpha. encoding nucleic acid was
inserted into the Bam HI site of pTS-BRP to give a plasmid, denoted
pTS-BRPTNF-.alpha., as described above. Plasmid pTS-BRPTNF-.alpha.
was electroporated into an attenuated strain of S. typhimurium,
strain VNP20009, (see International Publication WO 99/13053)
constructed to be serC- such that the genotype was .DELTA.msbB,
.DELTA.purI, .DELTA.serC (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 serC
encoding nucleic acid and leads to a serC.sup.+ phenotype.
Accordingly, bacteria harboring a chromosomal integration of the
TNF-.alpha. 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-.alpha. expression.
[0337] In order to examine and quantify TNF-.alpha. expression by
the tumor-targeted bacteria of the invention, Salmonella harboring
a chromosomal integration of the TNF-.alpha. encoding nucleic acids
were grown overnight, and a measured sample of the culture was used
in Western blot analysis. Specifically, TNF-.alpha. expression from
a representative serC.sup.+, ampicillin-sensitive clone, denoted
pTS-BRPTNF-.alpha. Clone 2, is shown in FIG. 3. Western blot
analysis revealed that bacterial protein, corresponding to
3.9.times.10.sup.7 cfu of pTS-BRPTNF-.alpha. Clone 2 bacteria (Lane
1), expressed more than 50 ng TNF-.alpha. (Lane 5), indicating
expression of TNF-.alpha. at levels greater than 10 ng/10.sup.7
bacteria. Therefore, the human TNF-.alpha. was successfully
expressed from a chromosomally-integrated, trc promoter-driven,
TNF-.alpha. encoding nucleic acid in Salmonella.
7. EXAMPLE
Attenuated Tumor-Targeted Bacteria Expressing ompA Fusion
Proteins
[0338] 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 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.
[0339] 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-.alpha.,
TRAIL (TNF-.alpha.-related apoptosis-inducing ligand), and
interleukin-2 (IL-2) results in the periplasmic localization and
subsequent processing of proteins.
[0340] 7.1. Processing of an ompA-TNF-.alpha. Fusion Protein
[0341] TNF-.alpha. expression in four different clones, expressing
a plasmid-based trc promoter-driven ompA-TNF-.alpha. 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-.alpha. by signal
peptidase, an enzyme located in the periplasm. In all four clones,
following induction with IPTG, overexpression of TNF-.alpha.
resulted in the appearance of TNF-.alpha. as a doublet migrating at
approximately 20 kd (FIG. 5, lanes 4-7), corresponding to both
unprocessed and processed forms. For comparison, a Salmonella
strain harboring a chromosomally-integrated TNF-.alpha. encoding
nucleic acids, expressing the mature (processed) form of
TNF-.alpha., was used as a positive control (FIG. 5, lane 3).
TNF-.alpha. expression was not detected in bacteria lacking the
TNF-.alpha. encoding nucleic acids (FIG. 5, lane 2).
[0342] These results demonstrated that fusion of the mature human
TNF-.alpha. 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
ompA-TNF-.alpha. fusion protein indicated that the normal secretory
apparatus was capable of accommodating the high-level expression of
secreted proteins.
[0343] 7.2. Processing of an ompA-Trail Fusion Protein
[0344] The ability of the ompA signal peptide to periplasmically
localize TNF family members was extended to TRAIL
(TNF-.alpha.-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 ompA/TRAIL junctions
were examined, one encoding an Nco 1 site and one encoding an Nde1
site (See FIG. 6 for Nde1 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 processed (28.2 kd) and unprocessed (30.2 kd) forms of hTRAIL
(FIG. 7, lanes 2-4), whereas bacteria over-expressing 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.
[0345] 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.
[0346] 7.3. Processing of an ompA(8L)-IL-2 Fusion Protein
[0347] 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-.alpha. and TRAIL, 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 FIG. 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).
[0348] 7.4. Processing of an phoA(8L)-IL-2 Fusion Protein
[0349] 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 (C125A) 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).
[0350] 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.
[0351] 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 periplasm by 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 (.DELTA.msbB, .DELTA.purI)
Expressing the Mature Form of TNF-.alpha.
[0352] 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.
[0353] The ability of TNF-.alpha. expression to increase the
anti-tumor efficacy of Salmonella typhimurium was evaluated in a
staged murine Colon 38 carcinoma model. For these experiments, 1
mm.sup.3 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 typhimurium (.DELTA.msbB, .DELTA.purI,
serC.sup.-) (parental strain); and 3) pTS-BRPTNF-.alpha. (Clone 2
described above). Mice in each group either remained untreated or
received a single intravenous injection of 1.times.10.sup.6 cfu of
the appropriate bacterial strain. Tumor size was measured weekly,
beginning at the time of bacteria inoculation.
[0354] In the group receiving attenuated tumor-targeted Salmonella
expressing TNF-.alpha., 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
(.DELTA.msbB, .DELTA.purI, .DELTA.serC) strain displayed partial
regression between weeks 3-4 following treatment, after which
tumors progressively increased in size (FIG. 11).
[0355] 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.
[0356] The demonstration of complete tumor regression, by
Salmonella expressing TNF-.alpha. from chromosomally-integrated
nucleic acid, indicates that biologically effective expression can
result from chromosomally integrated-effector molecule encoding
nucleic acids.
9. EXAMPLE
Enhanced Delivery of Nucleic Acid Molecules by BRP Expressing
Bacteria
[0357] 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:
2 TABLE 2 Average # of Amp Plasmid Colonies/Transformation pTrc99a
alone 125 pTrc99a + BRP (pSW1) 383
[0358] 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
[0359] 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.
[0360] 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 detached from the flask by
trypsinization, washed, and suspended in phosphate buffered saline
at 2.5.times.10.sup.6 cells/ml. An aliquot of 0.2 ml of the cell
suspension, for a total of 5.times.10.sup.5 cells/mouse, was
injected on Day 0. When tumor volumes reached 150-200 mm.sup.3,
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 0.2 mls
of PBS. Another group received 0.2 ml containing 2.times.10.sup.6
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 2.times.10.sup.6 c.f.u./mouse of the attenuated
tumor-targeted strain of Salmonella comprising pSW1, 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 (Pawelek 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
pepT Promoter Expression Vehicles
[0361] This example demonstrates the in vitro and in vivo
expression of a nucleic acid molecule encoding reporter such as
.beta.-gal under the control of the pepT promoter in an attenuated
tumor-targeted bacteria such as Salmonella.
[0362] 11.1. Construction of pepT-BRP-.beta.GAL Expression
Plasmids
[0363] 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:
[0364] Forward: 5'-AGT CTA GAC AAT CAG GCG AAG AAC GG-3' (SEQ ID
NO:15)
[0365] Reverse: 5'AGC CAT GGA GTC ACC CTC ACT TTT C-3' (SEQ ID
NO:16).
[0366] The PCR conditions consisted of 1 cycle of 95.degree. C. for
5 minutes, 35 cycles of 95.degree. C. for 1 minute, 65.degree. C.
for 1 minute, 72.degree. C. for 2 minutes and 1 cycle of 72.degree.
C. for 10 minutes. The PCR product was cloned into the PCR 2.1
cloning vector (Invitrogen, Carlsbad, Calif.), and is referred to
as PepT/PCR 2.1.
[0367] The PepT/PCR 2.1 vector was digested with NcoI and XbaI. The
pepT fragment was gel isolated and ligated into the .beta.-gal
Zterm vector digested with the same enzymes. Zterm (Temporary
Genbank Bankit No. 296495) is a promoterless .beta.-gal plasmid
generated by cloning the .beta.Gal open reading frame into pUC19.
The resultant plasmid was called pepT-.beta.GAL.
[0368] 11.2. In Vitro Expression of pepT-.beta.GAL and Measurement
of pepT-.beta.GAL Activity
[0369] Salmonella strains YS1456 (CC14 in FIG. 13A; for the genetic
make up of the strain, see WO 96/40238) or VNP20009 (CC 16 in FIG.
13A) harboring pepT-.beta.GAL were grown under either anaerobic or
aerobic conditions to an OD.sub.600 of .about.0.5-0.8. .beta.-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 .beta.-gal
activity upon growth of the bacteria under anaerobic
conditions.
[0370] 11.3. In Vivo Expression of pepT-.beta.GAL and Measurement
of pepT-.beta.GAL Activity
[0371] Cells of the Salmonella strain YS 1456 harboring the
pepT-.beta.gal expression plasmids, a BRP expression plasmid (pSWI
from BIO101 (Vista, California), which comprises the pCloDF13 BRP
coding sequence under the control of its native promoter) or both
expression plasmids were injected intravenously into tumor bearing
mice. Five days post injection, tumors and livers were homogenized
and bacteria were isolated to show that the presence of plasmids
for pepT-.beta.gal 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 .beta.gal activity to determine
whether active .beta.gal 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 .beta.gal 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
[0372] This example demonstrates the expression of a nucleic acid
molecule encoding a reporter gene such as .beta.-gal under the
control of the tet promoter in an attenuated tumor-targeted
bacteria such as Salmonella.
[0373] The tet promoter was cloned from a mini-TN10 transposon by
PCR amplification using the following primers:
3 Forward: 5'-GGA TCC TTA AGA CCC ACT TTC ACA TTT AAG T-3' (SEQ ID
NO:17) Reverse: 5'-GGT TCC ATG GTT CAC TTT TCT CTA TCA C-3'. (SEQ
ID NO:18)
[0374] The PCR conditions were as follows: one cycle of 95.degree.
C. for 5 minutes; 35 cycles of 95.degree. C. for 1 minute,
60.degree. C. for 1 minute, 72.degree. C. for 2 minutes; and one
cycle of 72.degree. C. for 10 minutes.
[0375] The .about.400 bp 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 .about.400 bp tet
promoter fragment was gel isolated and ligated into the
promoterless .beta.-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
.beta.-gal activity in the presence of tetracycline. One clone was
isolated and assayed for .beta.-gal expression over a range of
tetracycline concentrations. The results of the assay, which
demonstrate the induction of .beta.-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
[0376] The following example demonstrates the generation of
endostatin-expressing attenuated tumor-targeted Salmonella, and the
in vivo efficacy of tumor treatment by such Salmonella.
[0377] 13.1 Construction of Endostatin Expression Plasmids
[0378] Endostatin was PCR amplified from a human placental cDNA
library using the following primers:
4 Forward: 5'-GTG TCC ATG GCT CGG CGG GCA AGT GTC GGG ACT GAC CAT
(SEQ ID NO:21) CAT CAT CAT CAT CAT CAC AGC CAC CGC GAC TTC-3'
Reverse: 5'-GTG CGG ATC CCT ACT TGG AGG CAG TCA TGA AGC TG-3'. (SEQ
ID NO:22)
[0379] 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:
5 Forward: 5'-GTG TCC ATG GGG CAC AGC CAC CGC GAC TTC CAG-3' (SEQ
ID NO:19) Reverse: 5'-ACA CGA GCT CCT ACT TGG AGG CAG TCA TGA AGC
T-3'. (SEQ ID NO:20)
[0380] The conditions for the PCR amplification consisted of 1
cycle of 95.degree. C. 5 min; 30 cycles of 95.degree. C. for 1 min,
55.degree. C. for 1 minute, and 72.degree. C. for 2 minutes; and 1
cycle of 72.degree. C. for 10 minutes.
[0381] The resulting product was a DNA fragment with NcoI (5') and
BamHI (3') restriction sites encoding human endostatin having the
peptide sequence MARRASVGTDHHHHHH (SEQ ID NO:23) at its amino
terminus.
[0382] The PCR product was digested with NcoI and BamHI and the 550
bp 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 DH5.alpha. and the
attenuated tumor-targeted Salmonella strain VNP20009.
[0383] 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. Pat. No. 5,840,483)
with the origin of replication replaced by that of the ColE1
(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. Pat. No. 5,840,483).
[0384] 13.2. In Vitro Expression of Endostatin by Attenuated
Tumor-Targeted Salmonella
[0385] Different strains of Salmonella VNP20009 and E. coli
DH5.alpha. strains containing the pTrc99A-hexahistidine-endostatin
plasmid were grown to mid-log phase (O.D..sub.600.about.0.6-0.8),
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, Calif.).
FIGS. 15A and 15B show the results of the Western blots which
demonstrate pTrc99A hexahistidine-endostatin (HexHIS-endostatin)
expression in E. coli DH5.alpha. 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 25 kD, which is the predicted molecular weight for
the fusion protein.
[0386] 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.
[0387] 13.3. Efficacy of Attenuated Tumor-Targeted Salmonella
Expressing Endostatin on C38 Murine Colon Carcinoma
[0388] Colon 38 tumor fragments of 2.times.2.times.2 mm.sup.3
volume were implanted subcutaneously in 9 week old female C57BL/6
mice. When the tumor volumes reached 1000 mm.sup.3, they were
removed, cut into fragments of 2.times.2.times.2 mm.sup.3. The
fragments were serially passaged for further cycles and the
resulting 2.times.2.times.2 mm.sup.3 fragments were implanted
subcutaneously at the right flanks of female C57BL/6 mice. When
tumor volumes reached 150-200 mm.sup.3, 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 0.2 mls of PBS. Another control group received 0.2 ml
containing 1.times.10.sup.6 c.f.u. of the 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
1.times.10.sup.6 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.
[0389] 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.
[0390] 13.4. Efficacy of Attenuated Tumor-Targeted Salmonella
Expressing Endostatin on DLD Human Colon Carcinoma
[0391] Cultures of DLD1 cells grown in log phase were trypsinized,
washed with PBS and the cells reconstituted to a suspension of
5.times.10.sup.7 cells/ml in PBS, 0.1 ml aliquots of single cell
suspensions, each containing 5.times.10.sup.6 cells, were injected
subcutaneously into the right flanks of 9-week old nude female mice
(Nu/Nu-CD1 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.sup.3.
[0392] The first group of mice was the control group, and each
received an 0.3 ml injection of PBS. The second group of mice
received 0.3 ml containing 1.times.10.sup.6 c.f.u. of the
attenuated tumor-targeted strain of Salmonella VNP20009 carrying a
control asd plasmid. The third group of mice received 0.3 ml
containing 1.times.10.sup.6 c.f.u. 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 treatments, demonstrating the inhibitory effect of the
hexahistidine-endostatin expressing attenuated tumor targeted
Salmonella on the growth of DLD1 human colon carcinoma.
[0393] 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
[0394] 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.
[0395] 14.1. Construction of a Plasmid Containing the Nucleic Acid
Sequence Encoding Thrombospondin AHR
[0396] The peptide sequence, TiP 13.40: AYRWRLSHRPKTGFIRVVMYEG (SEQ
ID NO: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 NO:25). Complementary 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 BamHI restriction site. The two oligos
were annealed to generate the double stranded DNA fragment. The DNA
fragment was cut with SpeI/BamHI and ligated to the SpeI/BamHI 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.
[0397] Oligo 13.40-1:
6 5'gtgtactagtgtggcgcaggcgGCGTACCGCTGGCGCCTGTCCCATCGCCCGAAAACC (SEQ
ID NO:26) GGCTTTATCCGCGTGGTGATGTACGAAGGCTAAggatccgcgc 3'
[0398] Oligo 13.40-2:
7 5'gcgcggatccTTAGCCTTCGTACATCACCACGCGGATAAAGCCGGTTTTCGGGC (SEQ ID
NO:27) GATGGGACAGGCGCCAGCGGTACGCcgcctgcgccacactagtacac 3'
[0399] (Restriction sites are italicized and the OmpA processing
recognition site is underlined.)
[0400] 14.2. Construction of a Plasmid Containing the Nucleic Acid
Sequence Encoding Platelet Factor-4 Peptide (47-70)
[0401] 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.
[0402] Platelet Factor-4:
[0403] MSSAAGFCASRPGLLFLGLLLLPLVVAFASAEAEEDGDLQCLCVKTTSQV
RPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLOAPLYKKIIKKLLES (SEQ ID
NO:28)
[0404] Signal peptide=underlined & in bold
[0405] Lys 61,62, 65,66=major heparin binding domain (in bold)
[0406] DLQ (7-9, 54-56)=inhibitory activity on CFU-GM progenitor
cells (in bold)
[0407] Complementary oligonucleotides (oligo PF4-1 and oligo PF4-2)
were produced to 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 SpeI/BamHI restricted vector pTrc801 to
produce the plasmid pTrc801-PF4. When processed, the sequence
produces the full length PF-4 (47-70) peptide.
[0408] Oligo PF4-1
8 5'cttcactagtgtggcgcaggcgAACGGCCGCAAAATCTGCCTGGACCTGCAGGCGCCGCT
(SEQ ID NO:29) GTACAAAAAAATCATCAAAAAACTGCTGGAAAGCTAA ggatcc
gcg3'
[0409] Oligo PF4-2
9 5'cgcggatccTTAGCTTTCCAGCAGTTTTTTGATGATTTTTTTGTACAGCGGCGCCTG (SEQ
ID NO:30) CAGGTCCAGGCAGATTTTGCGGCCGTTcgcctgcgccacactagtg- aag3'
[0410] (Restriction sites are italicized and the ompA processing
recognition site is underlined.)
[0411] 14.3. Construction of a Plasmid Containing the Nucleic Acid
Sequence Encoding Apomigren
[0412] The anti-angiogenic peptide apomigren
(IYSFDGRDIMTDPSWPQKVIWHGSSPHG- VRLVDNYCEA
WRTADTAVTGLASPLSTGKILDQKAYSCANRLIVLCIENSFMTDARK (SEQ ID NO:31; see,
e.g., International Publication No. WO99/29856) corresponds to the
C-terminus of restin, which is a proteolytic fragment of collagen
XV. Oligonucleotides (oligo Apom5F 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:
10 Oligo Apom5F: 5'- ggcttc actagt gtggcgcaggcg
ATATACTCCTTTGATGGTCG -3' (SEQ ID NO:32) Oligo Apom6R: 5'- cgc
ggatcc TTACTTCCTAGCGTCTGTCATGAAACTG -3' (SEQ ID NO:33)
[0413] (Restriction sites are italicized and the OmpA processing
recognition site is underlined.) A fragment of the correct size was
obtained by PCR using placental cDNA as template. The PCR product
was cut with SpeI/BamHI and ligated to the SpeI/BamHI restricted
vector pTrc801 containing the modified ompA signal sequnce to
produce the plasmid pTrc801-Apom. When processed, the sequence
produces the Apomigren peptide.
[0414] 14.4. Anti-Angiogenic Peptides Produced by Salmonella
Inhibiting Endothelial Cell Proliferation
[0415] 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 by Feldman et al.,
2000, Cancer Res. 60:1503-1506 and Blezinger et al., 1999, Nature
Biotech. 17:343-348. Five-ml cultures of individual colonies were
grown for 4 hours. Cell lysates were produced by resuspending the
cell pellet in {fraction (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-five or fifty ml of the
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).
[0416] 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 between 40-60%.
15. EXAMPLE
Expression of a Bacteriocin Family Member by Attenuated
Tumor-Targeted Salmonella
[0417] 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.
[0418] 15.1. Construction of ColE3 Plasmids
[0419] 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 molecules by methods known in the
art.
[0420] 15.1.1. The pE3.Shuttle-1 Intermediate Vector Plasmid
[0421] 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 NO: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 mutation(s) in chromosomal
lacZ. The BRP fragment was then cloned into the E3 plasmid SmaI
site (FIG. 22) as a cassette containing the lacZ alpha
complementation fragment. The lacZ fragment makes insert selection
possible (i.e. Lac+) at this step. Although the naturally occurring
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 E3/BRP. The
new E3/BRP 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 E3/BRP 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 E3/BRP
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.
[0422] 15.1.2. Halo "Stab" Assay for E3 Activity
[0423] The sensitive tester strain (SK522) is grown to an
OD.sub.600 of 0.8. One hundred .mu.l of tester strain is added to 3
ml of warm (.about.55.degree. C.) LB soft agar (for a 100.times.15
mm 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.degree. 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.
[0424] 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 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.
[0425] 15.1.3. Overlay Assay for Selective E3 Clones
[0426] Transformants are plated with various dilutions (up to
1:10,000) onto LB and grown for 2 hours at 37.degree. C. The
sensitive tester strain is then prepared as above in the halo assay
and an overlay poured with soft agar. After allowing to solidify
for 10 minutes, the plate is then inverted and incubated overnight
at 37.degree. 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.
[0427] 15.1.4. "Plaque" or Halo Purification Assay
[0428] 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 500 .mu.L of LB. Dilutions (up to 1:10,000) are made and
replated on LB agar and allowed to grow for 2 hours at 37.degree.
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
[0429] The following example demonstrates the retention of the
colE3-CA38 plasmid in Salmonella in vivo.
[0430] 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 and msbB PCR. Distribution and plasmid
retention were calculated from the combined date.
[0431] The results of the E3 Injection in vivo, Determination of
the Percent retention of plasmid in Salmonella are shown below in
Table 3.
11TABLE 3 number % positive Tissue positive in msbB % plasmid
Tissue CFU/ml weight CFU/gm for colicin PCR retention 41.29L
1.07E+03 1.33 4.02E+03 0/5 100% n/a 41.29T 1.26e+07 0.26 2.42E+08
0/5 100% n/a 41.29E3L 1.15E+04 2.34 2.46E+04 87/135 100% 64.44
41.29E3T 1.09e+06 0.35 1.56E+07 134/135 100% 99.26
[0432] 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
[0433] 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.
[0434] 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
4.times.10.sup.8 cfu/g with varying accumulation in the livers
ranging from 6.times.10.sup.4 to 4.times.10.sup.6 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 108 c.f.u./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.
12TABLE 4 Tumor (T) Ratio Group Strain Liver (L) cfu/g tissue
(Tumor:Liver) 1 41.2.9/E3 T 5.1 .+-. 1.1 .times. 10.sup.8 131:1 1
41.2.9/E3 L 3.9 .+-. 3.6 .times. 10.sup.6 2 41.2.9/E3BRP T 4.6 .+-.
2.7 .times. 10.sup.8 209:1 2 41.2.9/E3BRP L 2.2 .+-. 1.3 .times.
10.sup.6 .sup. 6.sup.1 41.2.9/E3BRP.sub.m T 3.5 .+-. 0.15 .times.
10.sup.8 90:1 .sup. 6.sup.1 41.2.9/E3BRP.sub.m L 3.9 .+-. 3.6
.times. 10.sup.6 .sup.1BRP.sub.m 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., Oudega, B. and Luirink, J.
(1998) Optimization of Bacteriocin Release Protein (BRP)-Mediated #
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
[0435] The following example demonstrates the ability of
41.2.9/ColE3 to inhibit the growth of C38 murine colon
carcinoma.
[0436] Colon 38 tumor fragment (2.times.2.times.2 mm.sup.3) was
implanted in C57BL/6 mice (female, Age: 9 weeks) subcutaneously.
After tumor volume reached to 1,000 mm.sup.3, the tumors were
removed from the mice under sterile condition and cut into small
fragments (about 2.times.2.times.2 mm.sup.3 mm.sup.3/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.
[0437] Animals were randomized on Day 0 of Salmonella
administration when tumor volume reached 150-200 mm.sup.3. 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.times.10.sup.6
cfu/ml, respectively. Aliquots of 0.2 ml bacterial suspension
(1.5.times.10.sup.6 CFU/mouse) were administered intravenously into
mice as group indicated on Day 0. The bacteria suspension were
diluted to 1.times.10.sup.3 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 (ColE3) were
dissected and processed for determining cfu and retention of
plasmid.
[0438] Groups:
13 Mice 1. Untreated control 8 2. 41.2.9 (1.5 .times.
10.sup.6/mouse) 8 3. 41.2.9/ColE3 (1.5 .times. 10.sup.6/mouse)
8
[0439] The results for the efficacy of 41.2.9/ColE3 on C38 murine
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 murine 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
[0440] The following example demonstrates the enhanced ability of
Salmonella mutant VNP20009/ColE3 (41.2.9/ColE3) to inhibit the
growth of DLD1 human colon carcinoma relative to Salmonella mutant
41.2.9.
[0441] DLD1 cells grown in log phase were removed by
trypsinization, washed with PBS, and reconstituted to
5.times.10.sup.7 cell/ml PBS. Single cell suspensions (0.1 ml) were
injected into Nude mice (Nu/Nu-CD1 female, Age: 9 weeks; from
Charles River) subcutaneously on Day 0 (5.times.10.sup.6
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 300-400 mm.sup.3. 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 1.times.10.sup.7 CFU/ml.
Aliquots of 0.2 ml bacterial suspensions (2.times.10.sup.6
CFU/mouse) were injected intraveneously into mice on days
indicated. The bacteria suspension was diluted to 1.times.10.sup.3
CFU, plated each solutions 100 ul on msbB plates and the plates
incubated overnight. The bacteria colonies were counted next day.
The tumors were measured twice per week.
[0442] Groups:
14 Mice 1. Untreated control (PBS) 10 2. 41.2.9 (2 .times.
10.sup.6/mouse) 10 3. 41.2.9/ColE3 (2 .times. 10.sup.6/mouse)
10
[0443] 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 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/6 Mice
[0444] The following example demonstrates the ability of Salmonella
mutant 41.2.9/ColE3 to inhibit the growth B16-F10 melanoma.
[0445] B16-F10 cells grown in log phase were removed by
trypsinization, washed with PBS, and reconstituted to
5.times.10.sup.6 cell/ml PBS. Single cell suspensions (0.1 ml) were
injected into C57BL/6 mice (female, Age: 9 weeks) subcutaneously on
Day 0 (5.times.10.sup.5 cells/mouse) at right flank. Ten animals
were used in each group, and randomized at day 9, when tumor volume
reached 150-200 mm. 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.times.10.sup.6 cfu/ml, respectively.
Aliquots of 0.2 ml bacterial suspension (1.5.times.10.sup.6
CFU/mouse) were administered intravenously into mice as group
indicated on Day 9. The bacteria suspension were diluted to
1.times.10.sup.3 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.
[0446] Groups:
15 Mice 1. Untreated control 10 3. 41.2.9 (1.5 .times.
10.sup.6/mouse) 10 5. 41.2.9/ColE3 (1.5 .times. 10.sup.6/mouse)
10
[0447] The results of the efficacy of 41.2.9/ColE3 on B16 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
[0448] The following example demonstrates that the coexpression of
BRP and E3 in Salmonella mutant 41.2.9 increases the anti-tumor
efficacy of mutant.
[0449] The coexpression of BRP 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
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:
16 Group number Treatment 1 Control (no treatment) 2 41.2.9 3
41.2.9/E3 4 41.2.9/E3/BRP
[0450] The model used in this experiment was the human lung
carcinoma line HTB177. The cells were implanted into the flank of
mice subcutaneously on day 1. When the tumors reached to
approximately 500 mm.sup.3, on day 14 the animals were injected by
intravenous injection with 1.times.10.sup.6 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 BRP 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).
17TABLE 5 Percent Tumor Growth Inhibition Compared to Untreated
Control Strain Day 17 Day 20 Day 24 41.2.9 50 38 40 41.2.9/E3 63 58
63 41.2.9/E3/BRP 97 82 67
[0451] In conclusion, treatment with Salmonella carrying both the
cytotoxic colicin E3 and the enhanced secretion system BRP 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
[0452] 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.
[0453] The schedule was as follows: At day 0, tumors were implanted
by the administration of B16F10 melanoma (5.times.10.sup.5
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.
[0454] The results of the combination of colicin E3-containing
Salmonella with x-ray treatment are shown in Table 6.
18TABLE 6 Category n = ( ) Days to 1 g mean T/C A sham 15Gy (6) 12,
12, 18, 18, 18, 21 17 1.0 J 15Gy x-rays (9) 14, 14, 18, 21, 25, 35,
35, 33 1.9 12dpt, 26dpt 67, 67 K 41.2.9 + 15Gy (9) 21, 28, 35, 35,
56, 60, 60, 47 2.8 x-rays12dpt, 26dpt 60, 67 regression #1, 2 L
41.2.9/E3 + 15Gy (9) 28, 39, 53, 56, 56, 60, 67, 57 3.3 x-rays
12dpt, 26dpt 74, 78 regression d32
[0455] 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
[0456] The following example demonstrates that the expression of E.
coli cytotoxic necrotic factor 1 (CNF 1) by tumor-targeted
bacteria.
[0457] 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 CNF1 (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).
[0458] CNF1 was cloned from E. coli J96 (ATCC 700336) by PCR using
the primers (forward) 5'-GTGTCATGAAAATGGGTAACCAATGGCAAC-3' (SEQ ID
NO:35) and (reverse) 5'-CACAGAGCTCGCGCTAACAAAACAGCACAAGGGAG-3' (SEQ
ID NO:36) using standard PCR. An approximately 3100 bp 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 sequencing confirmed that the cloned PCR product was CNF 1 with
only minor sequence variation of 6 of 3065 base pairs.
[0459] The CNF1 plasmid was electroporated into an E. coli DNA
cloning host DH05.alpha. and Salmonella strain YS1646
(International Publication No. WO 99/13053). The expression of CNF1
was determined in the E. coli DNA cloning host and Salmonella
strain YS1649 using a standard LDH assay (Promega, Madison, Wis.,
Cytotox 96.RTM.). 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 CNF1
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
[0460] The following example demonstrates the cytotoxicity of
verotoxin AB produced by tumor-targeted bacteria engineered to
express verotoxin AB.
[0461] 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 tumors, however, the
antitumor activity is associated with purified preparations, not
with whole live bacteria.
[0462] 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.
[0463] 24.1. Construction of Vectors
[0464] 24.1.1. Amplification and Cloning of AB
[0465] Verotoxin AB (AB) was generated by PCR using the following
primers:
19 H19B-7: forward: 5'-GTGTCCATGGCTAAAACATTATTAATAGCTGCATCGC-3';
(SEQ ID NO:37) and QSTX-R1: reverse
5'-GTGTCTGCAGAACTGACTGAATTGAGATG-3'. (SEQ ID NO: 38)
[0466] These primers also contain outer NcoI(5') and PstI(3')
restriction endonuclease sites for cloning into the NcoI and PstI
sites of ptrc99A.
[0467] 24.1.2. Amplification and Cloning of TetBRP
[0468] 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 NO:39) and
BRP-TET-3': reverse 5'-CACAGGATCCTTACTGAACCGCGATCCCCG-3' (SEQ ID
NO:40). These primers contain BglII(5') and BamHI(3') restriction
endonuclease sites for cloning into BglII and BamHI sites of
pSP72--F6/R6 vector.
[0469] 24.1.3. Subcloning of AB into pSP72--F6/R6-TetBRP
[0470] 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 .beta.-gal gene for
lacZ-alpha complementation in trans. Both the vector
(pSP72F6/R6-TetBRP) and the AB insert were resolved on a 0.8%
1.times.TAE agarose gel and purified using the Qiagen gel
extraction kit. The vector and insert were ligated using T4 ligase
and transformed into DH5a E. coli cells using the heat shock
method. The cells were plated to LB plates containing 100 .mu.g/ml
Amp and 40 .mu.g/ml X-gal. Positive colonies were selected based on
ampicillin resistance and the presence of a functional .beta.-gal
gene (positive colonies were blue).
[0471] 24.1.4. Subcloning of TetBRP-AB into pCDV442
[0472] 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.
[0473] 24.1.5. msbB Chromosomal Vector
[0474] 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 CCG TTA ACC CTC TGA C-3'
(SEQ ID NO: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
NO: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 NO:43) and reverse 5'-GTG TCT CGA GGA TAT CAT TCT GGC CTC TGA
CGT TGT G-3' (SEQ ID NO:44). These primers also contain outer SacI
(5') and AvaI (3') restriction endonuclease sites to facilitate
cloning into the SacI and SalI sites of pCVD442 when these two
fragments are joined via a common SphI site and generate internal
NotI, 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).
[0475] 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 DH5 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 SM10 1 pir (Donnenberg and Kaper,
1991, supra) and the plasmid designated pCVD442-Tet-BRP-AB.
Colonies of SM10 1 pir were screened for Tet-BRP-AB gene by PCR,
and a SM10 1 pir clone pCVD442-Tet-BRP-AB was chosen for use as a
mating donor to Salmonella strains. SM1 1 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 50 .mu.g/mL carbenicillin (carb) and 300 .mu.g/mL
streptomycin (strep). The resulting YS50102-pCVD442-Tet-BRP-AB
clones were checked for pCVD442-Tet-BRP-AB gene by PCR.
[0476] 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
[0477] 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 carb.sup.r (or
amp.sup.r) suc.sup.s 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
suc.sup.r carb.sup.s derivative to remove the DmsbB gene and leave
the DmsbB-Tet-BRP-AB gene according to the methods of Donnenberg
and Kaper, 1991, supra (FIG. 33, #4) except that the LB-sucrose
agar plates were made without NaCl, and the plates were incubated
at 30.degree. 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.
[0478] 41.2.9-Tet-BRP-verotoxin AB was tested for cytotoxicity in
vitro using a standard LDH cytotoxicity assay (Cytotox.sup.96.RTM.;
Promega, Madison, Wis.). 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 Hemolysin by Tumor-Targeted Bacteria
[0479] The following example demonstrates that tumor-targeted
bacteria can be engineered to express hemolytic proteins such as
hemolysin constitutively or under inducible control.
[0480] 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 ECO238954) 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) 5'-TTTTTTCCAT GGCTATTATG
ACTGAAATCG TTGCAGATAA AACGG-3' (SEQ ID NO:45) and (reverse)
5'-TTTTTTAAGC TTCCCGGGTC AGACTTCAGG TACCTCAAAG AGTGTC-3' (SEQ ID
NO: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 DH5a (Gibco) were then transformed with the
plasmids and plated to blood agar (trypic soy agar with 5% sheep
blood; BioMerieux, Lombard, Ill.) 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.
[0481] 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-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
[0482] The following example demonstrates that attenuated
tumor-targeted bacteria such as Salmonella can be engineered to
express methionase.
[0483] 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. WO00/29589 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.
[0484] The following primers were generated for methionase from
Pneudomas putida based upon Genbank No. L43133:
20 Forward: METH-XHOI 5'-CCGCTCGAGATGCACGGCTCCAACAAGC (SEQ ID
NO:47) TCCCA-3'; and Reverse: METH-BAM
5'-CGCGGATCCTTAGGCACTCGCCTTGAGT (SEQ ID NO:48) GCCTG-3'
[0485] 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:
[0486] one cycle of 94.degree. C. for 5 minutes, followed by 35
cycles of: 94.degree. C. for 1 minute, 60.degree. C. for 1 minute
and 72.degree. C. for 2 minutes. A final amplification step of
72.degree. C. for 10 minutes was included as the last step of the
PCR reaction. PCR products were resolved on 0.8% 1.times.TAE
agarose gel and a PCR product of the expected size for methionase
(.about.1196 bp) was identified. The band was excised from the gel
and purified using the Qiagen gel extraction kit.
[0487] 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% 1.times.TAE agarose gel. The products of the
digestion 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 Dh5a 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.
[0488] 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
Tumor-Targeted Bacteria
[0489] 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.
[0490] 27.1. Construction of Tat-Apoptin Vectors
[0491] 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).
[0492] 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 (FIGS. 36A and B). Further, the TAT-apoptin fusion
can be generated with and without an OmpA-8L signal sequence (FIGS.
36A and C).
[0493] The apoptin and hexahistadine apoptin are assembled using
overlapping oligonucleotides. The nucleic acid sequence encoding
apoptin was generated by PCR using the following
oligonucleotides:
21 TAP1: 5'-GATCCCATGG CTTATGGCAG AAAAAAACGC CGTCAGCGCC (SEQ ID
NO:49) GTCGCATGAA CGCGCTGCAG GAAGATACCC CGCCGGGCCC GTCCACCGTG
TTTCGCCCGC CG-3' TAP2: 5'-GGGACAGGGT GATGGTGATG CCCGCGATGC
CGATGCGGAT (SEQ ID NO:50) TTCGCGGCAA TGCGGGGTTT CCAGCGGGCG
GGAGGAGGTC GGCGGGCGAA ACACGGTGGA CGG-3' TAP3: 5'-GGCATCGCGG
GCATCACCAT CACCCTGTCC CTGTGCGGCT (SEQ ID NO:51) GCGCGAACGC
GCGCGCGCCG ACCCTGCGCT CCGCGACCGC GGATAACTCC GAAAACACCG GC-3' TAP4:
5'-GCGATATTCG GACGGATCGC AGGAGCGTTT TTTGGACGGC (SEQ ID NO:52)
GGTTTCGGCT GATCGGTGCG CAGATCCGGG ACGTTTTTAA AGCCGGTGTT TTCGGAGTTA
TCCGCGGTCG C-3' TAP5: 5'-CCTGCGATCC GTCCGAATAT CGCGTCTCCG
AACTGAAAGA (SEQ ID NO:53) ATCCCTGATC ACCACCACCC CGTCCCGCCC
GCGCACCGCC CGCCGCTGCA TCCGCCTCTG AAAGCTTCAT G-3' TAP6:
5'-CATGAAGCTT TCAGAGGCGG ATGCAGCGGC GGGCGGTGCG C-3' (SEQ ID
NO:54)
[0494] 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 NO:55). The nucleic acid sequence encoding the
OmpA8L-containing version of the TAT-apoptin fusion protein is
generated from the PCR product of TAP1-TAP6 oligonucleotides by PCR
using TAP6 oligonucleotide and omp8LF1 oligonucleotide
(5'-GATCCCATGG CTAAAAAGAC GGCTCTGGCG CTTCTGCTCT TGCTGTTAGC
GCTGACTAGT GTAGCGCAGG CCTATGGCCG CAAAAAACGC CGTCAGCGCC-3'; SEQ ID
NO:56).
[0495] Each oligonucleotide is formulated into a stock solution
which is 4 .mu.M in concentration. Using premixed PCR reaction
beads (Pharmacia, Ready-to-go beads), 2 .mu.l of each
oligonucleotide was used. The PCR reaction consisted of one cycle
at 95.degree. for 5 minutes; thirty-five cycles at 95.degree. C.
for 1 minute, 60.degree. C. for 1 minute, 72.degree. C. for 1
minute; and one cycle at 72.degree. C. for 10 minutes. The PCR
reaction was then extracted with 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 bp 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).
[0496] 27.2. Demonstration of Secretion and Uptake of
Tat-Apoptin
[0497] 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.
[0498] 27.3. Demonstration the Uptake of Tat-Apoptin
Intratumorally
[0499] 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.f.u.).
28. EXAMPLE
Efficacy of the Combination of VNP20009 and Chemotherapeutic Agents
on the Growth of M27 Lung Carcinoma in Mice
[0500] 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.
[0501] 28.1. Efficacy of the Combination of VNP20009 and Cytoxan or
VNP20009 and Mitomycin C on the Growth of M27 Lung Carcinoma in
Mice
[0502] Liquid nitrogen stored M27 murine lung carcinoma cells
(1.times.10.sup.6/ml.times.1 ml) were recovered by rapidly thawing
the cells at 37.degree. C. and cultured with 10 ml of DMEM culture
medium containing 10% fetal calf serum (FCS) at 37.degree. C., 5%
CO.sub.2. After passing the cells for two generations, M27 cells in
log phase were removed by trypsinization, washed with 1.times.PBS,
and reconstituted to 2.5.times.10.sup.6 cells/ml with 1.times.PBS
for tumor implantation. An M27 cell suspension was implanted into
100 C57BL/6 mice (female, aged 8 weeks, 20 g; 5.times.10.sup.5
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.
[0503] Salmonella strain VNP20009 was diluted to 5.times.10.sup.6
CFU/ml with 1.times.PBS with our standard dilution procedures. Each
mouse was intravenously administered 0.2 ml of diluted Salmonella
(1.times.10.sup.6 CFU/mouse) on day 12 according to Table 6; infra.
In order to determine the actual number of injected bacteria, the
5.times.10.sup.6 CFU/ml bacterial suspensions were further diluted
to 1.times.10.sup.3 CFU/ml and plated on nutrient agar (MsbB
plates; International Publication No. WO 99/13053). The colonies
formed were counted the next day.
[0504] 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 tumor. 200 mpk of Cipro (Bayer Inc., West Haven, Conn.) 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.
22TABLE 7 Group Number of Mice No treatment control 10 3 mpk
mitomycin C, i.v., day 15 10 5 mpk, mitomycin C, i.v., day 15 10
150 mpk cytoxan, i.p., day 15 10 200 mpk cytoxan, i.p., day 15 10
VNP20009, 1 .times. 10.sup.6/mouse i.v., day 12 10 VNP20009, 1
.times. 10.sup.6/mouse i.v., day 12 + 3 mpk 10 mitomycin C, i.v.,
days 15 & 22 VNP20009, 1 .times. 10.sup.6/mouse i.v., day 12 +
5 mpk 10 mitomycin C, i.v., days 15 & 22 VNP20009, 1 .times.
10.sup.6/mouse i.v., day 12 + 150 mpk 10 cytoxan, i.p., day 15
VNP20009, 1 .times. 10.sup.6/mouse i.v., day 12 + 200 mpk 10
cytoxan, i.p., day 15
[0505] 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
chemotherapeutic agent may act synergistically or additively to
inhibit the growth of solid tumors such as lung carcinoma.
[0506] 28.2. Efficacy of the Combination of VNP20009 and Cisplatin
on the Growth of M27 Lung Carcinoma in Mice
[0507] Liquid nitrogen stored M27 murine lung carcinoma cells
(1.times.10.sup.6/ml.times.1 ml) were recovered by rapidly thawing
the cells at 37.degree. C. and cultured with 25 ml of DMEM culture
medium containing 10% fetal calf serum (FCS) at 37.degree. C., 5%
CO.sub.2. After passing the cells for two generations, M27 cells in
log phase (about 90-95% saturation) were removed by trypsinization,
washed with 1.times.PBS, and reconstituted to 2.5.times.10.sup.6
cells/ml with 1.times.PBS for tumor implantation. An M27 cell
suspension (0.2 ml) was implanted into 36 C57BL/6 mice (female,
aged 8 weeks, 20 g; 5.times.10.sup.5 cells/mouse) subcutaneously at
the right flank on day 0. The mice were randomly divided into
groups with each group consisting of 9 mice.
[0508] Salmonella strains VNP20009 was diluted to 5.times.10.sup.6
CFU/ml with 1.times.PBS with our standard dilution procedures. Each
mouse was administered via the tail vein 0.2 ml of Salmonella
(1.times.10.sup.6 CFU/mouse) on day 12 according to Table 8, infra.
In order to determine the actual number of injected bacteria, the
5.times.10.sup.6 CFU/ml bacterial suspensions were further diluted
to 1.times.10.sup.3 CFU/ml and plated on MsBb plates. The colonies
formed were counted the next day.
[0509] 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.
23TABLE 8 Group Number of Mice Control (no treatment) 9 VNP20009, 1
.times. 10.sup.6/mouse i.v., on day 12 9 5 mpk cisplatin, i.p. qw
.times. 2, on day 14, 19 9 VNP20009, 1 .times. 10.sup.6/mouse i.v.,
on day 12 + 5 mpk 9 cisplatin, i.p. qw .times. 2, on day 14, 19,
33
[0510] 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.
[0511] 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.
[0512] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
61 1 26 DNA Artificial Sequence Forward primer 1 gaagatcttc
cggaggaggg gaaatg 26 2 44 DNA Artificial Sequence Reverse primer 2
cgggatccga gctcgagggc ccgggaaagg atctaagaag atcc 44 3 477 DNA Homo
sapiens CDS (1)...(474) 3 atg gta cgt agc tcc tct cgc act ccg tcc
gat aag ccg gtt gct cat 48 Met Val Arg Ser Ser Ser Arg Thr Pro Ser
Asp Lys Pro Val Ala His 1 5 10 15 gta gtt gct aac cct cag gca gaa
ggt cag ctg cag tgg ctg aac cgt 96 Val Val Ala Asn Pro Gln Ala Glu
Gly Gln Leu Gln Trp Leu Asn Arg 20 25 30 cgc gct aac gcc ctg ctg
gca aac ggc gtt gag ctc cgt gat aac cag 144 Arg Ala Asn Ala Leu Leu
Ala Asn Gly Val Glu Leu Arg Asp Asn Gln 35 40 45 ctc gtg gta cct
tct gaa ggt ctg tac ctg atc tat tct caa gta ctg 192 Leu Val Val Pro
Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu 50 55 60 ttc aag
ggt cag ggc tgc ccg tcg act cat gtt ctg ctg act cac acc 240 Phe Lys
Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr 65 70 75 80
atc agc cgt att gct gta tct tac cag acc aaa gtt aac ctg ctg agc 288
Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser 85
90 95 gct atc aag tct ccg tgc cag cgt gaa act ccc gag ggt gca gaa
gcg 336 Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu
Ala 100 105 110 aaa cca tgg tat gaa ccg atc tac ctg ggt ggc gta ttt
caa ctg gag 384 Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe
Gln Leu Glu 115 120 125 aaa ggt gac cgt ctg tcc gca gaa atc aac cgt
cct gac tat cta gat 432 Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg
Pro Asp Tyr Leu Asp 130 135 140 ttc gct gaa tct ggc cag gtg tac ttc
ggt att atc gca ctg 474 Phe Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile
Ile Ala Leu 145 150 155 taa 477 4 158 PRT Homo sapiens 4 Met Val
Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His 1 5 10 15
Val Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg 20
25 30 Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn
Gln 35 40 45 Leu Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser
Gln Val Leu 50 55 60 Phe Lys Gly Gln Gly Cys Pro Ser Thr His Val
Leu Leu Thr His Thr 65 70 75 80 Ile Ser Arg Ile Ala Val Ser Tyr Gln
Thr Lys Val Asn Leu Leu Ser 85 90 95 Ala Ile Lys Ser Pro Cys Gln
Arg Glu Thr Pro Glu Gly Ala Glu Ala 100 105 110 Lys Pro Trp Tyr Glu
Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu 115 120 125 Lys Gly Asp
Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp 130 135 140 Phe
Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 5
28 DNA Artificial Sequence Forward primer 5 ccgacgcgtt gacacctgaa
aactggag 28 6 29 DNA Artificial Sequence Reverse primer 6
ccgacgcgtg aaaggatctc aagaagatc 29 7 543 DNA Artificial Sequence
Fusion construct 7 atg aaa aag aca gct atc gcg att gca gtg gca ctg
gct ggt ttc gct 48 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu
Ala Gly Phe Ala 1 5 10 15 acc gta gcg cag gcc cat atg gta cgt agc
tcc tct cgc act ccg tcc 96 Thr Val Ala Gln Ala His Met Val Arg Ser
Ser Ser Arg Thr Pro Ser 20 25 30 gat aag ccg gtt gct cat gta gtt
gct aac cct cag gca gaa ggt cag 144 Asp Lys Pro Val Ala His Val Val
Ala Asn Pro Gln Ala Glu Gly Gln 35 40 45 ctg cag tgg ctg aac cgt
cgc gct aac gcc ctg ctg gca aac ggc gtt 192 Leu Gln Trp Leu Asn Arg
Arg Ala Asn Ala Leu Leu Ala Asn Gly Val 50 55 60 gag ctc cgt gat
aac cag ctc gtg gta cct tct gaa ggt ctg tac ctg 240 Glu Leu Arg Asp
Asn Gln Leu Val Val Pro Ser Glu Gly Leu Tyr Leu 65 70 75 80 atc tat
tct caa gta ctg ttc aag ggt cag ggc tgc ccg tcg act cat 288 Ile Tyr
Ser Gln Val Leu Phe Lys Gly Gln Gly Cys Pro Ser Thr His 85 90 95
gtt ctg ctg act cac acc atc agc cgt att gct gta tct tac cag acc 336
Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr 100
105 110 aaa gtt aac ctg ctg agc gct atc aag tct ccg tgc cag cgt gaa
act 384 Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu
Thr 115 120 125 ccc gag ggt gca gaa gcg aaa cca tgg tat gaa ccg atc
tac ctg ggt 432 Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile
Tyr Leu Gly 130 135 140 ggc gta ttt caa ctg gag aaa ggt gac cgt ctg
tcc gca gaa atc aac 480 Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu
Ser Ala Glu Ile Asn 145 150 155 160 cgt cct gac tat cta gat ttc gct
gaa tct ggc cag gtg tac ttc ggt 528 Arg Pro Asp Tyr Leu Asp Phe Ala
Glu Ser Gly Gln Val Tyr Phe Gly 165 170 175 att atc gca ctg taa 543
Ile Ile Ala Leu 180 8 180 PRT Artificial Sequence Fusion construct
8 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1
5 10 15 Thr Val Ala Gln Ala His Met Val Arg Ser Ser Ser Arg Thr Pro
Ser 20 25 30 Asp Lys Pro Val Ala His Val Val Ala Asn Pro Gln Ala
Glu Gly Gln 35 40 45 Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu
Leu Ala Asn Gly Val 50 55 60 Glu Leu Arg Asp Asn Gln Leu Val Val
Pro Ser Glu Gly Leu Tyr Leu 65 70 75 80 Ile Tyr Ser Gln Val Leu Phe
Lys Gly Gln Gly Cys Pro Ser Thr His 85 90 95 Val Leu Leu Thr His
Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr 100 105 110 Lys Val Asn
Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr 115 120 125 Pro
Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly 130 135
140 Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn
145 150 155 160 Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gln Val
Tyr Phe Gly 165 170 175 Ile Ile Ala Leu 180 9 801 DNA Artificial
Sequence Fusion construct 9 atg aaa aag aca gct atc gcg att gca gtg
gca ctg gct ggt ttc gct 48 Met Lys Lys Thr Ala Ile Ala Ile Ala Val
Ala Leu Ala Gly Phe Ala 1 5 10 15 acc gta gcg cag gcc cat atg gct
aac gag ctg aag cag atg cag gac 96 Thr Val Ala Gln Ala His Met Ala
Asn Glu Leu Lys Gln Met Gln Asp 20 25 30 aag tac tcc aaa agt ggc
att gct tgt ttc tta aaa gaa gat gac agt 144 Lys Tyr Ser Lys Ser Gly
Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser 35 40 45 tat tgg gac ccc
aat gac gaa gag agt atg aac agc ccc tgc tgg caa 192 Tyr Trp Asp Pro
Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln 50 55 60 gtc aag
tgg caa ctc cgt cag ctc gtt aga aag atg att ttg aga acc 240 Val Lys
Trp Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr 65 70 75 80
tct gag gaa acc att tct aca gtt caa gaa aag caa caa aat att tct 288
Ser Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser 85
90 95 ccc cta gtg aga gaa aga ggt cct cag aga gta gca gct cac ata
act 336 Pro Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile
Thr 100 105 110 ggg acc aga gga aga agc aac aca ttg tct tct cca aac
tcc aag aat 384 Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn
Ser Lys Asn 115 120 125 gaa aag gct ctg ggc cgc aaa ata aac tcc tgg
gaa tca tca agg agt 432 Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp
Glu Ser Ser Arg Ser 130 135 140 ggg cat tca ttc ctg agc aac ttg cac
ttg agg aat ggt gaa ctg gtc 480 Gly His Ser Phe Leu Ser Asn Leu His
Leu Arg Asn Gly Glu Leu Val 145 150 155 160 atc cat gaa aaa ggg ttt
tac tac atc tat tcc caa aca tac ttt cga 528 Ile His Glu Lys Gly Phe
Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg 165 170 175 ttt cag gag gaa
ata aaa gaa aac aca aag aac gac aaa caa atg gtc 576 Phe Gln Glu Glu
Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val 180 185 190 caa tat
att tac aaa tac aca agt tat cct gac cct ata ttg ttg atg 624 Gln Tyr
Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met 195 200 205
aaa agt gct aga aat agt tgt tgg tct aaa gat gca gaa tat gga ctc 672
Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu 210
215 220 tat tcc atc tat caa ggg gga ata ttt gag ctt aag gaa aat gac
aga 720 Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp
Arg 225 230 235 240 att ttt gtt tct gta aca aat gag cac ttg ata gac
atg gac cat gaa 768 Ile Phe Val Ser Val Thr Asn Glu His Leu Ile Asp
Met Asp His Glu 245 250 255 gcc agt ttt ttc ggg gcc ttt tta gtt ggc
taa 801 Ala Ser Phe Phe Gly Ala Phe Leu Val Gly 260 265 10 266 PRT
Artificial Sequence Fusion construct 10 Met Lys Lys Thr Ala Ile Ala
Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala
His Met Ala Asn Glu Leu Lys Gln Met Gln Asp 20 25 30 Lys Tyr Ser
Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser 35 40 45 Tyr
Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln 50 55
60 Val Lys Trp Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr
65 70 75 80 Ser Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn
Ile Ser 85 90 95 Pro Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala
Ala His Ile Thr 100 105 110 Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser
Ser Pro Asn Ser Lys Asn 115 120 125 Glu Lys Ala Leu Gly Arg Lys Ile
Asn Ser Trp Glu Ser Ser Arg Ser 130 135 140 Gly His Ser Phe Leu Ser
Asn Leu His Leu Arg Asn Gly Glu Leu Val 145 150 155 160 Ile His Glu
Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg 165 170 175 Phe
Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val 180 185
190 Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met
195 200 205 Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr
Gly Leu 210 215 220 Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys
Glu Asn Asp Arg 225 230 235 240 Ile Phe Val Ser Val Thr Asn Glu His
Leu Ile Asp Met Asp His Glu 245 250 255 Ala Ser Phe Phe Gly Ala Phe
Leu Val Gly 260 265 11 465 DNA Artificial Sequence Fusion construct
11 atg aaa aag acg gct ctg gcg ctt ctg ctc ttg ctg tta gcg ctg act
48 Met Lys Lys Thr Ala Leu Ala Leu Leu Leu Leu Leu Leu Ala Leu Thr
1 5 10 15 agt gta gcg cag gcc gct cct act agc tcg agc act aag aaa
act caa 96 Ser Val Ala Gln Ala Ala Pro Thr Ser Ser Ser Thr Lys Lys
Thr Gln 20 25 30 ctg caa ttg gag cat ctg ctg ctg gat ctg cag atg
att ctg aat ggc 144 Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met
Ile Leu Asn Gly 35 40 45 atc aat aac tac aag aac cct aag ctg act
cgc atg ctg act ttc aaa 192 Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr
Arg Met Leu Thr Phe Lys 50 55 60 ttc tac atg ccg aaa aag gct acc
gag ctc aaa cat ctc cag tgc ctg 240 Phe Tyr Met Pro Lys Lys Ala Thr
Glu Leu Lys His Leu Gln Cys Leu 65 70 75 80 gaa gag gaa ctg aag ccg
ctg gag gaa gta ctt aac ctg gca cag tct 288 Glu Glu Glu Leu Lys Pro
Leu Glu Glu Val Leu Asn Leu Ala Gln Ser 85 90 95 aag aac ttc cac
ctg cgt ccg cgt gac ctg atc tcc aac atc aat gta 336 Lys Asn Phe His
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val 100 105 110 atc gtt
ctt gag ctg aag gga tcc gaa acc acc ttc atg tgc gaa tac 384 Ile Val
Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr 115 120 125
gct gac gaa acc gcc acc att gtg gag ttc ctg aac cgt tgg atc acc 432
Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr 130
135 140 ttt gcc caa tcg atc att agc acg tta act taa 465 Phe Ala Gln
Ser Ile Ile Ser Thr Leu Thr 145 150 12 154 PRT Artificial Sequence
Fusion construct 12 Met Lys Lys Thr Ala Leu Ala Leu Leu Leu Leu Leu
Leu Ala Leu Thr 1 5 10 15 Ser Val Ala Gln Ala Ala Pro Thr Ser Ser
Ser Thr Lys Lys Thr Gln 20 25 30 Leu Gln Leu Glu His Leu Leu Leu
Asp Leu Gln Met Ile Leu Asn Gly 35 40 45 Ile Asn Asn Tyr Lys Asn
Pro Lys Leu Thr Arg Met Leu Thr Phe Lys 50 55 60 Phe Tyr Met Pro
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu 65 70 75 80 Glu Glu
Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser 85 90 95
Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val 100
105 110 Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu
Tyr 115 120 125 Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg
Trp Ile Thr 130 135 140 Phe Ala Gln Ser Ile Ile Ser Thr Leu Thr 145
150 13 465 DNA Artificial Sequence Fusion construct 13 atg aaa cag
tcg act ctg gcg ctt ctg ctc ttg ctg tta gcg ctg act 48 Met Lys Gln
Ser Thr Leu Ala Leu Leu Leu Leu Leu Leu Ala Leu Thr 1 5 10 15 agt
gtg gcc aaa gcg gct cct act agc tcg agc act aag aaa act caa 96 Ser
Val Ala Lys Ala Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln 20 25
30 ctg caa ttg gag cat ctg ctg ctg gat ctg cag atg att ctg aat ggc
144 Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly
35 40 45 atc aat aac tac aag aac cct aag ctg act cgc atg ctg act
ttc aaa 192 Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr
Phe Lys 50 55 60 ttc tac atg ccg aaa aag gct acc gag ctc aaa cat
ctc cag tgc ctg 240 Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His
Leu Gln Cys Leu 65 70 75 80 gaa gag gaa ctg aag ccg ctg gag gaa gta
ctt aac ctg gca cag tct 288 Glu Glu Glu Leu Lys Pro Leu Glu Glu Val
Leu Asn Leu Ala Gln Ser 85 90 95 aag aac ttc cac ctg cgt ccg cgt
gac ctg atc tcc aac atc aat gta 336 Lys Asn Phe His Leu Arg Pro Arg
Asp Leu Ile Ser Asn Ile Asn Val 100 105 110 atc gtt ctt gag ctg aag
gga tcc gaa acc acc ttc atg tgc gaa tac 384 Ile Val Leu Glu Leu Lys
Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr 115 120 125 gct gac gaa acc
gcc acc att gtg gag ttc ctg aac cgt tgg atc acc 432 Ala Asp Glu Thr
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr 130 135 140 ttt gcc
caa tcg atc att agc acg tta act taa 465 Phe Ala Gln Ser Ile Ile Ser
Thr Leu Thr 145 150 14 154 PRT Artificial Sequence Fusion construct
14 Met Lys Gln Ser Thr Leu Ala Leu Leu Leu Leu Leu Leu Ala Leu Thr
1 5 10 15 Ser Val Ala Lys Ala Ala Pro Thr Ser Ser Ser Thr Lys Lys
Thr Gln 20 25 30 Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met
Ile Leu Asn Gly 35 40 45 Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr
Arg Met Leu Thr Phe Lys 50 55 60 Phe Tyr Met Pro Lys Lys Ala Thr
Glu Leu Lys His Leu Gln Cys Leu 65 70
75 80 Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln
Ser 85 90 95 Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn
Ile Asn Val 100 105 110 Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr
Phe Met Cys Glu Tyr 115 120 125 Ala Asp Glu Thr Ala Thr Ile Val Glu
Phe Leu Asn Arg Trp Ile Thr 130 135 140 Phe Ala Gln Ser Ile Ile Ser
Thr Leu Thr 145 150 15 26 DNA Artificial Sequence Forward primer 15
agtctagaca atcaggcgaa gaacgg 26 16 25 DNA Artificial Sequence
Reverse primer 16 agccatggag tcaccctcac ttttc 25 17 31 DNA
Artificial Sequence Forward primer 17 ggatccttaa gacccacttt
cacatttaag t 31 18 28 DNA Artificial Sequence Reverse primer 18
ggttccatgg ttcacttttc tctatcac 28 19 33 DNA Artificial Sequence
Forward primer 19 gtgtccatgg ggcacagcca ccgcgacttc cag 33 20 34 DNA
Artificial Sequence Reverse primer 20 acacgagctc ctacttggag
gcagtcatga agct 34 21 72 DNA Artificial Sequence Forward primer 21
gtgtccatgg ctcggcgggc aagtgtcggg actgaccatc atcatcatca tcatcacagc
60 caccgcgact tc 72 22 35 DNA Artificial Sequence Reverse primer 22
gtgcggatcc ctacttggag gcagtcatga agctg 35 23 16 PRT Homo sapiens 23
Met Ala Arg Arg Ala Ser Val Gly Thr Asp His His His His His His 1 5
10 15 24 22 PRT Artificial Sequence Peptide sequence TiP 13.40 24
Ala Tyr Arg Trp Arg Leu Ser His Arg Pro Lys Thr Gly Phe Ile Arg 1 5
10 15 Val Val Met Tyr Glu Gly 20 25 66 DNA Artificial Sequence
Nucleotide sequence encoding TiP13.40 25 gcgtaccgct ggcgcctgtc
ccatcgcccg aaaaccggct ttatccgcgt ggtgatgtac 60 gaaggc 66 26 101 DNA
Artificial Sequence Oligonucleotide 26 gtgtactagt gtggcgcagg
cggcgtaccg ctggcgcctg tcccatcgcc cgaaaaccgg 60 ctttatccgc
gtggtgatgt acgaaggcta aggatccgcg c 101 27 101 DNA Artificial
Sequence Oligonucleotide 27 gcgcggatcc ttagccttcg tacatcacca
cgcggataaa gccggttttc gggcgatggg 60 acaggcgcca gcggtacgcc
gcctgcgcca cactagtaca c 101 28 101 PRT Homo sapiens 28 Met Ser Ser
Ala Ala Gly Phe Cys Ala Ser Arg Pro Gly Leu Leu Phe 1 5 10 15 Leu
Gly Leu Leu Leu Leu Pro Leu Val Val Ala Phe Ala Ser Ala Glu 20 25
30 Ala Glu Glu Asp Gly Asp Leu Gln Cys Leu Cys Val Lys Thr Thr Ser
35 40 45 Gln Val Arg Pro Arg His Ile Thr Ser Leu Glu Val Ile Lys
Ala Gly 50 55 60 Pro His Cys Pro Thr Ala Gln Leu Ile Ala Thr Leu
Lys Asn Gly Arg 65 70 75 80 Lys Ile Cys Leu Asp Leu Gln Ala Pro Leu
Tyr Lys Lys Ile Ile Lys 85 90 95 Lys Leu Leu Glu Ser 100 29 106 DNA
Artificial Sequence Oligonucleotide 29 cttcactagt gtggcgcagg
cgaacggccg caaaatctgc ctggacctgc aggcgccgct 60 gtacaaaaaa
atcatcaaaa aactgctgga aagctaagga tccgcg 106 30 106 DNA Artificial
Sequence Oligonucleotide 30 cgcggatcct tagctttcca gcagtttttt
gatgattttt ttgtacagcg gcgcctgcag 60 gtccaggcag attttgcggc
cgttcgcctg cgccacacta gtgaag 106 31 85 PRT Homo sapiens 31 Ile Tyr
Ser Phe Asp Gly Arg Asp Ile Met Thr Asp Pro Ser Trp Pro 1 5 10 15
Gln Lys Val Ile Trp His Gly Ser Ser Pro His Gly Val Arg Leu Val 20
25 30 Asp Asn Tyr Cys Glu Ala Trp Arg Thr Ala Asp Thr Ala Val Thr
Gly 35 40 45 Leu Ala Ser Pro Leu Ser Thr Gly Lys Ile Leu Asp Gln
Lys Ala Tyr 50 55 60 Ser Cys Ala Asn Arg Leu Ile Val Leu Cys Ile
Glu Asn Ser Phe Met 65 70 75 80 Thr Asp Ala Arg Lys 85 32 44 DNA
Artificial Sequence Oligonucleotide 32 ggcttcacta gtgtggcgca
ggcgatatac tcctttgatg gtcg 44 33 37 DNA Artificial Sequence
Oligonucleotide 33 cgcggatcct tacttcctag cgtctgtcat gaaactg 37 34
7117 DNA E. coli 34 cccgggcact tccggggcat gagtatgtga tatccggggc
tgcaccccgg accccgccaa 60 cacatcacgg gccacaaaat tttttgtggc
ccgctctgcg ttttctaagt gttatccctc 120 ctgatttcta aaaaattttc
cacctgaact tgacagaaaa aacgatgacg agtacttttt 180 gatctgtaca
taaacccagt ggttttatgt acagtattaa tcgtgtaatc aattgtttta 240
acgcttaaaa gagggaattt ttatgagcgg tggcgatgga cgcggccata acacgggcgc
300 gcatagcaca agtggtaaca ttaatggtgg cccgaccggg cttggtgtag
gtggtggtgc 360 ttctgatggc tccggatgga gttcggaaaa taacccgtgg
ggtggtggtt ccggtagcgg 420 cattcactgg ggtggtggtt ccggtcatgg
taatggcggg gggaatggta attccggtgg 480 tggttcggga acaggcggta
atctgtcagc agtagctgcg ccagtggcat ttggttttcc 540 ggcactttcc
actccaggag ctggcggtct ggcggtcagt atttcagcgg gagcattatc 600
ggcagctatt gctgatatta tggctgccct gaaaggaccg tttaaatttg gtctttgggg
660 ggtggcttta tatggtgtat tgccatcaca aatagcgaaa gatgacccca
atatgatgtc 720 aaagattgtg acgtcattac ccgcagatga tattactgaa
tcacctgtca gttcattacc 780 tctcgataag gcaacagtaa acgtaaatgt
tcgtgttgtt gatgatgtaa aagacgagcg 840 acagaatatt tcggttgttt
caggtgttcc gatgagtgtt ccggtggttg atgcaaaacc 900 taccgaacgt
ccgggtgttt ttacggcatc aattccaggt gcacctgttc tgaatatttc 960
agttaataac agtacgccag cagtacagac attaagccca ggtgttacaa ataatactga
1020 taaggatgtt cgcccggcag gatttactca gggtggtaat accagggatg
cagttattcg 1080 attcccgaag gacagcggtc ataatgccgt atatgtttca
gtgagtgatg ttcttagccc 1140 tgaccaggta aaacaacgtc aagatgaaga
aaatcgccgt cagcaggaat gggatgctac 1200 gcatccggtt gaagcggctg
agcgaaatta tgaacgcgcg cgtgcagagc tgaatcaggc 1260 aaatgaagat
gttgccagaa atcaggagcg acaggctaaa gctgttcagg tttataattc 1320
gcgtaaaagc gaacttgatg cagcgaataa aactcttgct gatgcaatag ctgaaataaa
1380 acaatttaat cgatttgccc atgacccaat ggctggcggt cacagaatgt
ggcaaatggc 1440 cgggcttaaa gcccagcggg cgcagacgga tgtaaataat
aagcaggctg catttgatgc 1500 tgctgcaaaa gagaagtcag atgctgatgc
tgcattgagt tctgctatgg aaagcaggaa 1560 gaagaaagaa gataagaaaa
ggagtgctga aaataattta aacgatgaaa agaataagcc 1620 cagaaaaggt
tttaaagatt acgggcatga ttatcatcca gctccgaaaa ctgagaatat 1680
taaagggctt ggtgatctta agcctgggat accaaaaaca ccaaagcaga atggtggtgg
1740 aaaacgcaag cgctggactg gagataaagg gcgtaagatt tatgagtggg
attctcagca 1800 tggtgagctt gaggggtatc gtgccagtga tggtcagcat
cttggctcat ttgaccctaa 1860 aacaggcaat cagttgaaag gtccagatcc
gaaacgaaat atcaagaaat atctttgaga 1920 ggaagttatg ggacttaaat
tggatttaac ttggtttgat aaaagtacag aagattttaa 1980 gggtgaggag
tattcaaaag attttggaga tgacggttca gttatggaaa gtctaggtgt 2040
gccttttaag gataatgtta ataacggttg ctttgatgtt atagctgaat gggtaccttt
2100 gctacaacca tactttaatc atcaaattga tatttccgat aatgagtatt
ttgtttcgtt 2160 tgattatcgt gatggtgatt ggtgatcaaa tattatcagg
gatgagttga tatacgggct 2220 tctagtgttc atggatgaac gctggagcct
ccaaatgtag aaatgttata ttttttattg 2280 agttcttggt tataattgct
ccgcaatgat ttaaataagc attatttaaa acattctcag 2340 gagaggtgaa
ggtggagcta aaaaaaagta ttggtgatta cactgaaacc gaattcaaaa 2400
aatttattga agacatcatc aattgtgaag gtgatgaaaa aaaacaggat gataacctcg
2460 agtattttat aaatgttact gagcatccta gtggttctga tctgatttat
tacccagaag 2520 gtaataatga tggtagccct gaaggtgtta ttaaagagat
taaagaatgg cgagccgcta 2580 acggtaagtc aggatttaaa cagggctgaa
atatgaatgc cggttgttta tggatgaatg 2640 gctggcattc tttcacaaca
aggagtcgtt atgaaaaaaa taacagggat tattttattg 2700 cttcttgcag
tcattattct gtctgcatgt caggcaaact atatccggga tgttcagggc 2760
gggaccgtat ctccgtcatc aacagctgaa gtgaccggat tagcaacgca gtaacccgaa
2820 atcctctttg acaaaaacaa agcgtgtcag gctgattctg atgcgctttt
tttttgaaat 2880 gtcacaaaaa ttccatgtgg gagatgggat ctaaaatcct
cgtgcagaac tttccatcca 2940 gggggagaaa acttgtcgtt ttgagccgtt
cggtgttcag aacgcacgaa accgatcgcg 3000 cgcatcgctt tcgtgaatag
ttatgcaggc ccctgaaaac gattctgacg cgttttttcg 3060 gttttgcctg
gtgttttcct gtctttttgc gttttttgcg tcagaacgcg tctgagggcg 3120
ttttaagggg tgcgtacaac gggagttatg gtaaatggat cggtttttcg ggaaggatcg
3180 acaggatttg ccgttgggtg tagtgtaagc gactgaaaaa caaacgcccc
gtaaatcgtg 3240 ctctcaccgc caagattgat cacgaaatta cagggcgccg
ggttccgcgt ttcccgatgg 3300 gaaagcgcgg ttagttaaac tgtgtaccga
gagaaatcgt atcacatgag cgccgtactt 3360 caacgcttca gggaaaaatt
accgcacaaa ccgtactgta cgaacgattt cgcgtacggc 3420 gttcgcattc
tgccgaaaaa cattgccatt cttgcccgtt tcatccagca gaaccagcca 3480
catgcactgt actggcttcc ctttgacgtg gaccggacgg gggcatcaat cgactggagc
3540 gaccggaatt gtccggcccc gaacatcacc gtaaaaaatc cccgtaacgg
gcacgcgcat 3600 ctgctctacg cgctcgccct tcctgtgaga actgcgccgg
atgcatcggc ttcggcgctc 3660 agatacgctg ccgctattga gcgtgcgttg
tgtgaaaaac tgggcgcgga tgtgaattac 3720 agcggcctga tctgcaaaaa
tccgtgccac cctgaatggc aggaagtgga atggcgcgag 3780 gaaccctaca
ctctcgacga actggctgat tatctcgatt tgagcgcctc agcgcgccgt 3840
agcgtcgata aaaattacgg gctggggcga aactgctatc tgttcgaaaa gggccgtaaa
3900 tgggcttacc gggctattcg tcagggctgg ccggcattct cacaatggct
tgatgcggtg 3960 attcagcgtg tcgaaatgta caacgcatcg ctccccgttc
cgctttcacc tcctgaatgt 4020 cgggctattg gcaagagtat tgcgaaatac
acgcacagga acttcacgcc ggaaactttc 4080 gcacagtatg tggctgatac
gcacacgcca gaaattcagg ctacacgcgg tcgcaagggc 4140 ggttctaagt
ctaagcgcgg cacagtagct acatcagcac gcacgctgaa accttgggag 4200
aaattaggga tcagtcgcgc ctggtattac caactgaaaa aacgaggtct cgtagagtag
4260 accaaataag cctatatcag ataacagcgc ctttttggcg cctttttgag
cagcttggtt 4320 tgttgctatt tccctcgttg aatcccgcaa tggcgcggct
ttccgcatga ttgaggtggt 4380 agcgctcgcc gcagtctcat gaccgagcgt
agcgagcgaa tgagcgagga agcgcaaagg 4440 cgtccggtgg tgcatgtggc
acttacgcgc cggggcttag tggttctgcg gtttcgccgg 4500 tggtctgggt
agcttctcca gctcgttaat cagcggttgt agtcggttca catccacctg 4560
tcttgtgact tcctttcgca gaaactggag caggaacgca cgcagttgcg cttcttccgg
4620 cctccgtacc cttgccagca tggctgcccc cacaatgact ttttgcgccg
tgtccaggct 4680 tcggctcttc gccttcaggc gctgtaatct ggcctcagct
tcggcaatct tctgttcgag 4740 tgttctgctc atttcgtgac tccgtgcgcg
gtgaaaaatc gcattttagc gcgtcactgg 4800 tagtttaaaa actaaactgg
cataatgcac ggcacatcac gaagtgcgca cttatacaat 4860 ctccacttcg
tttcgattgt gtgcggtctg cgacgctaaa agaaaacggc aaaaaggcat 4920
tacggcagaa atggcgattt atcatctcag catgaaaatc atttcgcgaa aaaacggcta
4980 cagtgctgtt gcttctgctg cctaccgttc cggctctgtc atacccgatg
accgtaccgg 5040 attaatccac gattacaccc gtaaacgcgg cgttgatgat
gcggtcattc tcacccctgc 5100 gaatgcaccg tcctggtgtg ttgaccgttc
cgttctttgg aatgcggtcg agaaagccga 5160 acagcgccgg aactcccagc
tggcaaggga ggttgaactc gccattcccc gtgagatttc 5220 ccgcgaggcc
gcacgggaga ccgttctcgc tttgtccggg aaaactttgt cagtcggggc 5280
atgattgccg atgtggcgtt ccatcacatg gaccggacca atccccatgc gcacatcatg
5340 ctgaccacga gagctgtcgg ggaaacggga ttcgcaggaa aggtcaggga
tggaacgacc 5400 gggcactcgc cgagacgtgg cgcgcatcat gggctgacca
tgcgaacaga gcgcttgcga 5460 acgccggcta ccaggaagag atagaccatc
gttcatacga gcgtcaggga ctggagaaag 5520 cgccggcctt cacctcggaa
aggctgcctg tgcgatggaa aaacggggaa tggaaacaga 5580 acgcggtgag
cagaaccgtc tgattaacag ccttaacctg gaaatacagg tttcccgcac 5640
gcagcttgct ctcaggacgg ttcaggaaac gcagcgtaag cgggaactca gcgatgctgc
5700 acgtcgtgca gtggaagccc ttaacctgac cattcccgct gcgaatgcct
cagcggatac 5760 cctgcgggaa ttcattgcca cgctgccgca ggaatgcggg
aacgcgtggg agatgacccc 5820 ggagttcctg gcgatgagcg ggaaggtgaa
cgacatcgaa cgtgagggga atgcgctgct 5880 gaaagagcag gccattctcg
aaaaggagat gaccggactc aaaaaagcac gccctgtcgc 5940 gtcccttctg
tcagagattc ccctgatgac atgggctgaa ccggaatacc gcaaaagaca 6000
actccgtttc ttggaaactc gggaaacaga ttgaatctct tcgccgcacc tacagggccg
6060 tgaaagaacg ggacattccc gcccgtcgtc aggcctttga aacgcagtgg
aatacgtgga 6120 ttgcgccgga atggcagagc tgaaagaaaa actgtcagca
cgggaagcgg agcggcgcag 6180 ggaggagccc gaagcggaag cgcgccggaa
ggaacaggag catgaggcgc ggctgaaacg 6240 tcatgataac caccgtctga
gccgtgaaac ggcattagtc ggggttatta cggagctggg 6300 gcgtgccaga
gagccgggaa cgggcaggat aacccgctac atgatgttga gtaacagagc 6360
cggagaattc acggtatggg gtgatgagct ggcgcattac ccccagagtg ttcatgaccc
6420 ggtgaatgtt tacctgtcgc caggcggggc tgtgatggtc tcggatatac
gtgagggaat 6480 gccagaatct catgagacga tggcgcggcc tgagcgtgtg
agaatgtatt ccggtgcgac 6540 ggtccggcat gtactggaac agatgcgcca
ggggtggccc tcttacggtt ttccggcgct 6600 gccgcatcac tggccggata
atttttattt cagcgacgac cgcaggcccg tagcctctcc 6660 gctgccgtct
gcgcaccggg tggacgtcac cgcttatgcg gcaccggagc aactcatgcc 6720
cgttgtattt tcgacagagc gaaacagcag gacgctgaat ctgctgttgt gcaaagggcc
6780 ggaggaagtg cttgtcggat ttgtgcgcca ggaggacggg ctgcgtcccg
ttcttgcgct 6840 tccgtcgccg gattacagtc atctgatggt cagcaccatc
acggagaacg gggtatgcct 6900 ggcaggttac ggagaagcta taaaccatga
tgcggatact ccgtacccac cggaaccgca 6960 cctgatgcag ttccggctca
aaggccatca tgacaggctt ttggctgctg tccacaaacc 7020 ggaagagatg
ccggattatc tcttccgtca actcggtttt aatcagacct ggcatgagtg 7080
gaagcgggac gaacagcaca ggcaacaaca acgccgc 7117 35 30 DNA Artificial
Sequence Forward primer 35 gtgtcatgaa aatgggtaac caatggcaac 30 36
35 DNA Artificial Sequence Reverse primer 36 cacagagctc gcgctaacaa
aacagcacaa gggag 35 37 37 DNA Artificial Sequence Forward primer 37
gtgtccatgg ctaaaacatt attaatagct gcatcgc 37 38 29 DNA Artificial
Sequence Reverse primer 38 gtgtctgcag aactgactga attgagatg 29 39 37
DNA Artificial Sequence Forward primer 39 gtgtagatct ttaagaccca
ctttcacatt taagttg 37 40 30 DNA Artificial Sequence Reverse primer
40 cacaggatcc ttactgaacc gcgatccccg 30 41 40 DNA Artificial
Sequence Forward primer 41 gtgtgagctc gatcaaccag caagccgtta
accctctgac 40 42 67 DNA Artificial Sequence Reverse primer 42
gtgtgcatgc ggggggccat ataggccggg gatttaaatg caaacgtccg ccgaaacgcc
60 gacgcac 67 43 71 DNA Artificial Sequence Forward primer 43
gtgtgcatgc ggggttaatt aagggggcgg ccgcgtggta ttggttgaac cgacggtgct
60 catgacatcg c 71 44 37 DNA Artificial Sequence Reverse primer 44
gtgtctcgag gatatcattc tggcctctga cgttgtg 37 45 45 DNA Artificial
Sequence Forward primer 45 ttttttccat ggctattatg actgaaatcg
ttgcagataa aacgg 45 46 46 DNA Artificial Sequence Reverse primer 46
ttttttaagc ttcccgggtc agacttcagg tacctcaaag agtgtc 46 47 33 DNA
Artificial Sequence Forward primer 47 ccgctcgaga tgcacggctc
caacaagctc cca 33 48 33 DNA Artificial Sequence Reverse primer 48
cgcggatcct taggcactcg ccttgagtgc ctg 33 49 102 DNA Artificial
Sequence Oligonucleotide 49 gatcccatgg cttatggcag aaaaaaacgc
cgtcagcgcc gtcgcatgaa cgcgctgcag 60 gaagataccc cgccgggccc
gtccaccgtg tttcgcccgc cg 102 50 103 DNA Artificial Sequence
Oligonucleotide 50 gggacagggt gatggtgatg cccgcgatgc cgatgcggat
ttcgcggcaa tgcggggttt 60 ccagcgggcg ggaggaggtc ggcgggcgaa
acacggtgga cgg 103 51 102 DNA Artificial Sequence Oligonucleotide
51 ggcatcgcgg gcatcaccat caccctgtcc ctgtgcggct gcgcgaacgc
gcgcgcgccg 60 accctgcgct ccgcgaccgc ggataactcc gaaaacaccg gc 102 52
111 DNA Artificial Sequence Oligonucleotide 52 gcgatattcg
gacggatcgc aggagcgttt tttggacggc ggtttcggct gatcggtgcg 60
cagatccggg acgtttttaa agccggtgtt ttcggagtta tccgcggtcg c 111 53 111
DNA Artificial Sequence Oligonucleotide 53 cctgcgatcc gtccgaatat
cgcgtctccg aactgaaaga atccctgatc accaccaccc 60 cgtcccgccc
gcgcaccgcc cgccgctgca tccgcctctg aaagcttcat g 111 54 41 DNA
Artificial Sequence Oligonucleotide 54 catgaagctt tcagaggcgg
atgcagcggc gggcggtgcg c 41 55 98 DNA Artificial Sequence
Oligonucleotide 55 gatcccatgg ctcatcacca tcaccaccat tatggccgca
aaaaacgccg tcagcgccgt 60 cgcatgaacg cgctgcagga agataccccg ccgggccc
98 56 100 DNA Artificial Sequence Oligonucleotide 56 gatcccatgg
ctaaaaagac ggctctggcg cttctgctct tgctgttagc gctgactagt 60
gtagcgcagg cctatggccg caaaaaacgc cgtcagcgcc 100 57 551 DNA
Bacteriophage CDS (7)...(408) modified_base (1)...(1) n=a, c, g, or
t 57 nagacc atg gct tat ggc aga aaa aaa aga aga cag aga aga aga atg
48 Met Ala Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met 1 5 10
aac gcg ctg cag gaa gat acc ccg ccg ggc ccg tcc acc gtg ttt cgc 96
Asn Ala Leu Gln Glu Asp Thr Pro Pro Gly Pro Ser Thr Val Phe Arg 15
20 25 30 ccg ccg acc tcc tcc cgc ccg ctg gaa acc ccg cat tgc cgc
gaa atc 144 Pro Pro Thr Ser Ser Arg Pro Leu Glu Thr Pro His Cys Arg
Glu Ile 35 40 45 cgc atc ggc atc gcg ggc atc acc atc acc ctg tcc
ctg tgc ggc tgc 192 Arg Ile Gly Ile Ala Gly Ile Thr Ile Thr Leu Ser
Leu Cys Gly Cys 50 55 60 gcg aac gcg cgc gcg ccg acc ctg cgc tcc
gcg acc gcg gat aac tcc 240 Ala Asn Ala Arg Ala Pro Thr Leu Arg Ser
Ala Thr Ala Asp Asn Ser 65 70 75 gaa aac acc ggc ttt aaa aac gtc
ccg gat ctg cgc acc gat cag ccg 288 Glu Asn Thr Gly Phe Lys Asn Val
Pro Asp Leu Arg Thr Asp Gln Pro 80 85 90 aaa ccg ccg tcc aaa aaa
cgc tcc tgc gat ccg tcc gaa tat cgc gtc 336 Lys Pro Pro Ser Lys Lys
Arg Ser Cys Asp Pro Ser Glu Tyr Arg Val 95 100
105 110 tcc gaa ctg aaa gaa tcc ctg atc acc acc acc ccg tcc cgc ccg
cgc 384 Ser Glu Leu Lys Glu Ser Leu Ile Thr Thr Thr Pro Ser Arg Pro
Arg 115 120 125 acc gcc cgc cgc tgc atc cgc ctc tgaaagcttg
gctgttttgg cggatgagag 438 Thr Ala Arg Arg Cys Ile Arg Leu 130
aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat
498 ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc aga 551
58 134 PRT Bacteriophage 58 Met Ala Tyr Gly Arg Lys Lys Arg Arg Gln
Arg Arg Arg Met Asn Ala 1 5 10 15 Leu Gln Glu Asp Thr Pro Pro Gly
Pro Ser Thr Val Phe Arg Pro Pro 20 25 30 Thr Ser Ser Arg Pro Leu
Glu Thr Pro His Cys Arg Glu Ile Arg Ile 35 40 45 Gly Ile Ala Gly
Ile Thr Ile Thr Leu Ser Leu Cys Gly Cys Ala Asn 50 55 60 Ala Arg
Ala Pro Thr Leu Arg Ser Ala Thr Ala Asp Asn Ser Glu Asn 65 70 75 80
Thr Gly Phe Lys Asn Val Pro Asp Leu Arg Thr Asp Gln Pro Lys Pro 85
90 95 Pro Ser Lys Lys Arg Ser Cys Asp Pro Ser Glu Tyr Arg Val Ser
Glu 100 105 110 Leu Lys Glu Ser Leu Ile Thr Thr Thr Pro Ser Arg Pro
Arg Thr Ala 115 120 125 Arg Arg Cys Ile Arg Leu 130 59 444 DNA
Bacteriophage modified_base (1)...(1) n=a, c, g, or t 59 nagacc atg
gct cat cac cat cac cac cat tat ggc cgc aaa aaa cgc 48 Met Ala His
His His His His His Tyr Gly Arg Lys Lys Arg 1 5 10 cgt cag cgc cgt
cgc atg aac gcg ctg cag gaa gat acc ccg ccg ggc 96 Arg Gln Arg Arg
Arg Met Asn Ala Leu Gln Glu Asp Thr Pro Pro Gly 15 20 25 30 ccg tcc
acc gtg ttt cgc ccg ccg acc tcc tcc cgc ccg ctg gaa acc 144 Pro Ser
Thr Val Phe Arg Pro Pro Thr Ser Ser Arg Pro Leu Glu Thr 35 40 45
ccg cat tgc cgc gaa atc cgc atc ggc atc gcg ggc atc acc atc acc 192
Pro His Cys Arg Glu Ile Arg Ile Gly Ile Ala Gly Ile Thr Ile Thr 50
55 60 ctg tcc ctg tgc ggc tgc gcg aac gcg cgc gcg ccg acc ctg cgc
tcc 240 Leu Ser Leu Cys Gly Cys Ala Asn Ala Arg Ala Pro Thr Leu Arg
Ser 65 70 75 gcg acc gcg gat aac tcc gaa aac acc ggc ttt aaa aac
gtc ccg gat 288 Ala Thr Ala Asp Asn Ser Glu Asn Thr Gly Phe Lys Asn
Val Pro Asp 80 85 90 ctg cgc acc gat cag ccg aaa ccg ccg tcc aaa
aaa cgc tcc tgc gat 336 Leu Arg Thr Asp Gln Pro Lys Pro Pro Ser Lys
Lys Arg Ser Cys Asp 95 100 105 110 ccg tcc gaa tat cgc gtc tcc gaa
ctg aaa gaa tcc ctg atc acc acc 384 Pro Ser Glu Tyr Arg Val Ser Glu
Leu Lys Glu Ser Leu Ile Thr Thr 115 120 125 acc ccg tcc cgc ccg cgc
acc gcc cgc cgc tgc atc cgc ctc t 427 Thr Pro Ser Arg Pro Arg Thr
Ala Arg Arg Cys Ile Arg Leu 130 135 140 gaaagcttgg ctgtttt 444 60
140 PRT Bacteriophage 60 Met Ala His His His His His His Tyr Gly
Arg Lys Lys Arg Arg Gln 1 5 10 15 Arg Arg Arg Met Asn Ala Leu Gln
Glu Asp Thr Pro Pro Gly Pro Ser 20 25 30 Thr Val Phe Arg Pro Pro
Thr Ser Ser Arg Pro Leu Glu Thr Pro His 35 40 45 Cys Arg Glu Ile
Arg Ile Gly Ile Ala Gly Ile Thr Ile Thr Leu Ser 50 55 60 Leu Cys
Gly Cys Ala Asn Ala Arg Ala Pro Thr Leu Arg Ser Ala Thr 65 70 75 80
Ala Asp Asn Ser Glu Asn Thr Gly Phe Lys Asn Val Pro Asp Leu Arg 85
90 95 Thr Asp Gln Pro Lys Pro Pro Ser Lys Lys Arg Ser Cys Asp Pro
Ser 100 105 110 Glu Tyr Arg Val Ser Glu Leu Lys Glu Ser Leu Ile Thr
Thr Thr Pro 115 120 125 Ser Arg Pro Arg Thr Ala Arg Arg Cys Ile Arg
Leu 130 135 140 61 1565 DNA Salmonella 61 gatatcattc tggcctctga
cgttgtgatg gtcgcacgtg gcgatctggg cgttgaaatc 60 ggcgatccgg
agctggttgg tatccagaaa gcgctgattc gccgtgcgcg tcagctaaac 120
cgcgcagtca tcaccgcaac gcaaatgatg gagtcgatga tcaccaaccc gatgccgacc
180 cgtgcggaag tgatggacgt ggcgaacgcc gtcctggatg gcacggatgc
ggttatgctg 240 tctgccgaaa ccgcagccgg tcagtatcct tctgaaaccg
ttgccgcaat ggcgcgcgtc 300 tgcctgggcg cagaaaaaat ccccagcatc
aatgtgtcta aacaccgtct cgacgtgcag 360 ttcgacaacg ttgaagaagc
cattgccatg tctgcgatgt atgcggcaaa ccatctgaaa 420 ggcgttaccg
cgatcatcac catgacggaa tccggtcgta ccgcgctaat gacttcccgt 480
atcagctccg gcctgccgat tttcgccatg tcgcgccatg aacgcacgct gaacctgacc
540 gcgctctatc gcggagtaac gccggtgcat tttgatagcg cggctgatgg
cgttgtcgcg 600 gcacatgaag ctgttaatct gctgcgcgat aaagggtatc
tggtttccgg cgacctggtt 660 atcgtgaccc agggcgatgt catgagcacc
gtcggttcaa ccaataccac gcggccgccc 720 ccttaattaa ccccgcatgc
ggggggccat ataggccggg gatttaaatg caaacgtccg 780 ccgaaacgcc
gacgcactgt gttccagata tagtcaaaaa ccggattacc ctgattatga 840
aacatcgccg ccattttttg cccctgagag gccatcagca tggctggaat gtcgacgccc
900 cagccatgcg gtacgagaaa aatgactttt tcgtcgttac gacgcatctc
ctcgataatc 960 tccagacctt cccagtcaac acgctgttga atttttttcg
gaccgcgcat cgccaactca 1020 gccatcatcg ccattgcctg tggcgcggtg
gcgaacatct catcgacaat cgcttcgcgc 1080 tcagcttcgc tacgctgcgg
aaagcacaac gacagattaa ttagcgcccg gcgacgagaa 1140 ctcttcccca
gccgtccggc aaaacgcccc agcgtcgcca gcaaagggtc gcggaatgat 1200
gccggtgtta atgcgatccc cgccattgcc gccgcgccca accaggcgcc ccaatactgt
1260 ggatagcgaa aggatttttc gaattcaggg atatactcac tattattttt
tttggtttcc 1320 atgcttttcc agggtctgct gacgcgaaaa ggaattgtga
atagtgtagc gacgtctgcg 1380 tctcacacaa aacaaaaaag ccggcacaca
tcgcgtaccg gctctgtcag cgcatttgtt 1440 aatcgaagcg cagttgcggc
agaacctctt tcacctgtgc caggtattca cgacgatctg 1500 accccgtcag
accttccgtg cgcggcaatt ttgctgtcag agggttaacg gcttgctggt 1560 tgatc
1565
* * * * *
References