KR20020059605A - Compositions and methods for tumor-targeted delivery of effector molecules - Google Patents

Abstract

The present disclosure discloses the preparation and use of toxic attenuated tumor-targeted bacterial vectors for the delivery of one or more primary effector molecule (s) to a solid tumor site. The primary effector molecule (s) of the present invention are used in the methods of the invention for the treatment of solid tumors such as carcinoma, melanoma, lymphoma or sarcoma. The present invention relates to the surprising discovery that effector molecules that may be toxic upon systemic administration to a host can be delivered locally to the tumor by a toxic attenuated tumor-target bacteria with reduced toxicity to said host. The present disclosure also discloses the delivery of one or more optional effector molecule (s) (referred to as secondary effector moieties) that can be delivered by the toxicant weakened tumor target bacteria with the primary effector molecule (s).

Description

Technical Field [0001] The present invention relates to a composition for delivering a tumor marker to an effector molecule,

2. BACKGROUND ART

This application is a continuation-in-part of U.S. Patent Application No. 60 / 157,500, 60 / 157,581 and 60 / 157,637, filed October 4, 1999, each of which is incorporated herein by reference.

Neoplasms or tumors are neoplasms that result from abnormal cell growth, which may be benign or malignant. Benign tumors are generally ubiquitous. Malignant tumors generally have the potential to invade neighboring body tissues to destroy the tissue and spread to distant sites leading to death (see, for example, Robins and Angell, 1976, Basic Pathology, 2d Ed., WB Saunders Co., Philadelphia, pp. 68-122). The tumor is said to have metastasized when the tumor spread from one organ or tissue to another.

The main problem in chemotherapy of solid tumors is the delivery of therapeutics, such as drugs, at a concentration sufficient to eradicate tumor cells while minimizing damage to normal cells. Thus, studies in a number of laboratories are directed to the design of biological delivery systems, such as antibodies, cytokines, and viruses, for targeting drugs, prodrug conversion enzymes and / or genes to tumor cells See Crystal, RG, 1995, Science 270: 404-410).

2.1. Cell immunity and cytokines

One strategy for cancer treatment involves the enhancement or activation of cellular immune responses. Successful induction of a cellular immune response against autologous tumors offers several advantages over conventional chemotherapy: 1) immune recognition is only highly specific for tumors; 2) growth at the metastatic site can be suppressed through immunosurveillance; 3) the diversity of immune responses and cognition can compensate for the different resistance mechanisms used by tumor cells; 4) Clonal proliferation of cytotoxic T cells occurs more rapidly than tumor proliferation, which ultimately can produce an antitumor mechanism that overwhelms the tumor; 5) The memory response can inhibit recurrence of the initial condition before physical detection. Clinical studies of patient response have shown that successful immunotherapy is associated with the activation of CD8 + T cells (although the evidence exists for the involvement of CD4 + T cells, macrophages and NK cells) (group I response) The results of the model were verified. 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. 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.

2.2 Tumor necrosis factor (TNF) family of cytokines

The most well-characterized member of the TNF family is TNF-α. TNF-a is known to exert a diversity expression effect on the immune system. TNF- [alpha] is a cytokine that can exert a potent cytotoxic effect directly on tumor cells. TNF-a is generally associated with other mechanisms, such as stimulation of proliferation and differentiation, and the prevention of apoptosis of mononuclear cells (see, e.g., Mangan et al., 1991, J. Immunol. 146: 1541-1546; Ostensen et al., 1987, J. Immunol. 138: 4185-4191), promote tissue factor-type procoagulant activity and inhibit the activity of endothelial cell surface anticoagulants, ultimately forming blood clots in tumors 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] causes a fatal outcome to the host due to diffuse intravascular coagulation.

Other cytokines are also involved in antitumor responses. IL-2 is thought to act as a group I cytokine and also as an antitumor response. For example, spontaneously degenerated melanoma is associated with elevated levels of TNF-a and IL-2 in tumors. See, for example, 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.

Both TNF-α and IL-2 contribute to lymphocyte return, and IL-2 has been shown to induce tumor invasion of natural killer (NK) cells, T-cells and lymphocyte activated killer (LAK) cells 1997, J. Neuropathol. Exp. Neurol. 56: 541 (1986)), as described in the literature (Etter et al., 1998, Cytokine 10: 395-403; Reinhardt et al., 1997, Blood 89: 3837-46; Luscinskas et al., 1996, J. Immunol., 157: 326-35; Kjaergaard et al., 1998, Scand. J. et al., 1996, Vora et al., 1996, Clin. Exp. Immunol., 105: 155-62; Immunol 47, 532-540; Johansson et al., 1996, Nat. Immun. 15: 87-97; and Watanabe et al., 1997, Am. J. Pathol. 150: 1869-80). In the presence of both TNF-a and IL-2, the cytolytic activity of NK and LAK cells is increased even when directed against TNF-non-sensitive cell lines (see, for example, Ostensen et al., 1987, J. Immunol. 138: 4185-4191). However, the therapeutic levels of IL-2 have also been shown to be toxic to the host.

Clearly, dose-limiting toxicity from systemic cytokine administration is a significant impediment to realizing the cytokine potential in cancer therapy. Moreover, systemic cytokine delivery can reduce the return of T cells in the genetic winter, thus contradicting target immunotherapy, other than creating unwanted clinical side effects. 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.

2.3 Transfer of cytokines

Recent animal and clinical studies have attempted to bypass the systemic toxicity of cytokines and administer higher doses via systemic or suboptimal or alternative cytokine delivery methods. In a mouse model, sarcoma-180 tumors were treated with the administration of a fusion liposome-encapsulated TNF-a gene and systemic administration of polyethylene glycol-encapsulated TNF-alpha (which could be localized into tumor vasculature) (Tsutsumi et al. , 1996, Jpn. J. Cancer Res. 87: 1078-1085). Tumorigenesis to TNF-a by endothelial-mononuclear cell-activated polypeptide II has also been reported (Marvin et al., 1999, J. Surg. Res. 63: 248-255; Wu et al., 1996, Cancer Res 59: 205-212).

In a clinical study, complete tumor eradication was observed by administering high doses of TNF-α to patients through isolated limb perfusion with interferon-α or melphalan. However, this technique presents a serious risk to the patient if the cytokine is not completely removed after treatment. Moreover, the treatment alone requires limbic isolation, which is a risk to the patient. Eggermont et al., 1997, Senin. 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].

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. Linear studies by Surgery 32: 301-306 have shown that IL-1β (Carrier) and IL-2 (Saltzman) are directly delivered to the liver and spleen, the site of the original salmonella infection, Reported the use of Salmonella strains with reduced toxicity. Saltzman's study used oral administration of salmonella, which absorbs bacteria by GALT (intestinal lymphatic tissue) and transports them to the liver and spleen. However, such infection is limited to the original site of infection.

2.4. Angiogenesis and tumorigenesis

Another strategy for cancer treatment involves the inhibition of angiogenesis. Angiogenesis is the process of new capillary growth from existing blood vessels. The new capillaries are formed by the process in which endothelial cells of the existing blood vessel decompose and proliferate the nearby basement membrane using a proteolytic enzyme such as a metalloprotease substrate and migrate to the surrounding interstitial tissue to form microtubules. The angiogenic process is very tightly regulated by the interaction between negative and positive factors and is usually restricted to female reproductive cycles and healing of wounds in adults (Malonne et al., 1999, Clin. Exp. Metastasis 17: 1 -14). Abnormal or abnormal regulation of angiogenesis has been implicated in many human diseases including diabetic retinopathy, psoriasis, rheumatoid arthritis, cardiovascular disease and tumorigenesis (Folkman, 1995, Nat. Med. 1: 27-31).

Angiogenesis is an important process for tumor growth and metastasis. Tumor formation is divided into two stages, vascular and vascular. The study demonstrated that angiogenic tumor cells proliferate as soon as cells proliferate from angiogenic tumors. However, since the angiostatic tumor has an equilibrium between cell proliferation and apoptosis, it rarely grows to 2 to 3 ㎣ or more, and this apoptosis is caused by the hypoxic nature of the angiogenic tumor (Folkman, 1995, Nat 1: 27-31). The transition from angiogenesis to vascularization is accompanied by a shift in the balance of angiogenesis regulators from the final balance favoring negative factors toward positive factors such as fibroblast growth factor (FCG) and vascular endothelial growth factor (VEGF) (Cao, 1998, Prog. Mol. Subcell. Biol. 20: 161-176). The shift of the balance between the regulatory factors results in the upregulation of angiogenic factors and the simultaneous downregulation of angiogenic inhibitors (Folkman, 1995, N. Eng. J. Med. 333: 1757-1763).

2.5. Angiogenesis inhibitor

Angiogenic inhibitors are assumed to exist based on several related phenomena that lead to the conclusion that primary tumors often inhibit the growth of its metastasis (Cao, 1998, Prog. Mol. Subcell. Biol. 176). The first of these isolated factors is mouse angiostatin, a 38 kDa proteolytic fragment of plasminogen released into the circulation by primary Lewis lung carcinoma, which prevents the growth of secondary metastasis (O'Reilly et al ., 1994, Cell 79: 315-328). In humans, peptides of 40, 42 and 45 kDa produced by limited proteolysis of plasminogen by metalloelastase have antiangiogenic activity comparable to mouse angiostatin (O'Reilly et al., 1994 , Cell 79: 315-328). Plasminogen itself has no such activity. It is also believed that tumor-associated macrophages contribute to the production of angiostatin because the tumor cells themselves do not have detectable angiostatin mRNA. Macrophage metalloelastase expression is induced by granulocyte colony-stimulating factor (GM-CSF) secreted by tumor cells (Dong et al., 1997, Cell 88: 801-810). In some tumors, the production of angiostatin is facilitated by serine proteases rather than metalloelastases, where serine proteases are produced directly by tumor cells (Gately et al., 1997, Cancer Res. 56: 4887-4890) . Angiostatin was administered to experimental mice with primary tumors at a concentration of 100 mg / kg / day to strongly inhibit tumor growth without toxic side effects. The tumor grows again within two weeks of stopping the angiostatin treatment, indicating that the tumor is reversing to a maintenance state rather than completely killing as a result of the treatment (O'Reilly et al., 1996, Nat. Med. 2: 689 -692).

After the discovery of angiostatin, other angiogenesis inhibitors, including multiple angiostatic peptides, were discovered and isolated. The angiogenesis inhibitor more potent than angiostatin is Kringle 5 (including angiostatin is Kringle domain 1 to 4), which is a peptide containing the fifth Kringle domain of plasminogen. Kringle 5 can be produced by proteolytic digestion of plasminogen and recombinant forms are also active (Cao et al., 1997, J. Biol. Chem. 272: 22924-22928).

Endostatin was isolated analogously to the isolation of angiostatin (O'Reilly et al., 1997, Cell 88: 1-20) and the source is a murine vascular endothelial rather than Lewis lung carcinoma. The peptide has an apparent molecular mass of 20 kDa corresponding to the C-terminus of collagen XVIII (O'Reilly et al., 1997, Cell 88: 1-20), the region of which the sequence is termed NC1 interspersed among the various collagen molecules (Oh et al., 1994, Proc Natl Acad Sci USA 91: 4229-4223, and Rehn et al., 1994, Proc Natl Acad Sci USA 91: 4234-4238). In mice, the growth of Lewis lung carcinoma metastasis is inhibited by the administration of recombinant endostatin 0.3 mg / kg / day, and the primary tumor is reversed to dormant upon administration of the peptide at 20 mg / kg / day. Functional recombinant endostatin can be produced from inclusions in vivo by in vitro or in vivo administration of subcutaneously administered endostatin inclusion complex by denaturation and refolding (O'Reilly et al., 1997, Cell 88: 1- 20). An alternative method of endostatin delivery consisting of intramuscular administration of an endostatin expression plasmid only partially inhibits tumor growth in a mouse model system (Blezinger et al., 1999, Nat. Biotech. 17: 343-348). Similarly, endostatin or angiotensin-encoding plasmids complexed with intramuscularly delivered liposomes partially inhibit tumor growth in a breast cancer nude mouse model (Chen et al., 1999, Cancer Res. 59: 3308-3312).

Recently, a novel antiangiogenic activity has been attributed to C-terminal truncation peptides of serpin (serine protease inhibitor) anti-thrombin (O'Reilly et al., 1999, Science 285: 1926-1928). Full length anti-thrombin has no intrinsic vasoconstriction activity, but upon cleavage of the C-terminal reactive ring of the protein by thrombin, anti-thrombin acquires strong angiogenic activity. The proteolytic fragments are hereinafter referred to as antiangiogenic anti-thrombin.

Other angiogenesis inhibiting peptides known in the art include the 29 kDa N-terminal and 40 kDa C-terminal proteolytic fragments of fibronectin (Homandberg et al., 1985, J. Am. Pathol. 120: 327-332); A 16 kDa proteolytic fragment of prolactin (Clapp et al., 1993, Endocrinology 133: 1292-1299); And a 7.8 kDa proteolytic fragment of platelet factor-4 (Gupta et al., 1995, Proc Natl Acad Sci USA 92: 7799-7803).

In addition to the naturally occurring proteolytic fragments that have been shown to inhibit angiogenesis, a number of synthetic peptides corresponding to known extracellular matrix protein regions have been evaluated for anti-angiogenic activity. Synthetic peptides proven to be functional endothelial inhibitors, i.e., angiogenesis inhibitors, include the 13 amino acid peptides corresponding to fragments of platelet factor-4 (Maione et al., 1990, Cancer Res. 51: 2077-2083); 14 amino acid peptides corresponding to fragments of collagen I (Tolma et al., 1993, J. Cell Biol. 122: 497-511); 19 amino acid peptides corresponding to fragments of thrombospondin I (Tolsma et al., 1993, J. Cell Biol. 122: 497-511); And secreted cysteine-rich extracellular matrix glycoprotein (Ledda et al., 1996, Nature Med. 3: 171 (1991)) in which in vitro cytoprotection is reduced by expression of human melanoma cells and tumorigenesis is reduced in an in vivo nude mouse model -176), a 20 amino acid peptide corresponding to a fragment of SPARC (Sage et al., 1995, J. Cell. Biochem. 57: 1329-1334). Other peptides that inhibit angiogenesis and that are less than 10 amino acids corresponding to fragments of laminin, fibronectin, procollagen and EGF have also been disclosed (Cao, 1998, Prog. Mol. Subcell. Biol. 20: 161-176) .

Small fibronectin peptides that inhibit angiogenesis generally involve RGD synchronization. RGD is a peptide motif (amino acid Arg-Gly-Asp) used by proteins to recognize and bind integrin molecules. Expression of integrin [alpha] _{v [} beta] 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 demonstrated by studies showing that administration of cyclic pentapeptides containing the RGD motif inhibits the activity of the vitronectin receptor type integrin and blocks retinal angiogenesis (Hammes et al., 1996, Nature Medicine 2: 529- 533). Angiogenesis inhibitory effects of integrin blockers such as cyclic pentapeptides and monoclonal antibodies have been shown to promote tumor regression by inducing apoptotic cell death (Brooks et al., 1994, Cell 79: 1157-1164). Peptides containing NGR (amino acid Asn-Gln-Arg), which are RGD motifs and another integrin binding motif, have been shown to significantly enhance tumor suppressor activity.

Another type of cell surface receptor, the inhibition of the activity of the urokinase plasminogen activator (uPA) receptor, also inhibits angiogenesis. The uPA receptor initiates the proteolytic amplification reaction necessary for the basolateral step of angiogenesis upon ligand binding. Inhibition of uPA receptors by receptor antagonists has been implicated in angiogenesis, tumor growth (Min et al., 1996, Cancer Res. 56: 2428-2433) and metastasis (Crowley et al., 1993, Proc. Natl. USA 90: 5021-5025). These antagonists have been identified by the bacteriophage peptide labeling of random peptides (Goodson et al., Proc. Natl. Acad. Sci. USA 91: 7129-7133). The predominant negative forms of the ligand, uPA, of the receptor have also been identified (Min et al., 1996, Cancer Res. 56: 2428-2433).

Although an exciting new approach to cancer therapy has been provided with the discovery of angiostatin, endostatin and other angiostatic inhibitory peptides, the reality of the therapeutic process involving one or more of these peptides is that the vast amount of peptide production (65 kg or 143 lbs (Resulting from the cost and / or labor of producing approximately 1.3 or 6.5 grams of protein per day, depending on the peptide, for the average person), and the duration of treatment (which should be continued if the tumor remains regressed) Do. The two main reasons for administering these peptides in large amounts as described above are first that the majority are degraded in the blood stream, second, only very limited proportions are degraded and the surviving molecules are thought to be tumors. Thus, if an angiogenesis inhibiting protein or peptide can be delivered to the tumor in a more efficient, more cost effective and patient-friendly manner, this would be a great advantage in the field of tumor therapy.

2.6. Bacteriocin series

Colicin E3 (hereinafter referred to as ColE3) is a bacteriocin, a bacterial monoclonal toxin whose selective activity, i.e., its host is immunogenic to the toxin. Bacteriocins are encoded by the host genome or plasmid, may have a broad or narrow range of hosts, may have a simple structure comprising one or two subunits, or may have a plurality of subunit structures (Konisky, 1982, Ann. Rev. Microbiol., 36: 125-144). In addition, the bacteriocin host has immunity to the bacteriocin. The immunity is found in all cells of a given host population, even those that do not express the bacteriocin.

The cytotoxicity of ColE3 is generated 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 common to 30S and 70S ribosomes (Bowman et al., 1971, Proc. Natl. Acad Sci. USA. 68: 964-968) (Meyhack, 1970, Proc. Natl Acad Sci USA). ColE3 activity is unique among RNAse, i. E. It does not cause total degradation of the RNA, but cleaves 49 nucleotides of mRNA molecules from the end to separate the rRNA from mRNA and thereby prevents translation. The ribonuclease activity of ColE3 is in the molecule itself rather than mediated by another protein (Saunders, 1978, Nature 274: 113-114). ColE3 can also penetrate the inner and outer membranes of the target cells.

ColE3 is a 60 kDa protein complex in a naturally occurring form with a 1: 1 ratio of 50 kDa and 10 kDa proteins, with larger subunits having nuclease activity and smaller subunits having the inhibitory action of the 50 kDa subunit Respectively. 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 includes two or more functional domains, an N-terminal region that requires translocation across the target cell membrane, 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 believed to be separated as a result of their interaction with the outer membrane of the target cell upon entry of the toxin (Konisky, 1982, Ann. Rev. Microbiol. 36: 125-144).

The toxicity of the large subunit of ColE3 was used to prevent the spread of the cloned gene in the microorganism. Diaz et al., 1994, Mol. Microbiol. 13: 855-861), the two components of ColE3 were expressed as small (anti-toxic) subunits expressed as chromosomally integrated coding sequences and large subunits expressed from plasmids. Bacteria with a small subunit integrated into the chromosome will be immune to a plasmid expressing the ColE3 large subunit, but will be killed if the plasmid is transduced to another recipient lacking a small subunit.

Coliseum E3 (ColE3) was also tested against mammalian cells (Smarda et al., 1978, Folia Microbiol. 23: 272-277), including leukemia cell model system (Fiska et al., 1979, Experimentia 35: 406-407) And had a large cytotoxic effect. ColE3 activity targets the 40S subunit of the 80S mammalian ribosome (Turnowsky et al., 1973, Biochem. Biophys. Res. Comm. 52: 327-334).

2.7. Bacterial infection and cancer

Early clinical observations have reported cases in which certain cancers have been degenerated in patients with bacterial infections (Nauts et al., 1953, Acta Medica. Scandinavica 145: 1-102, (Suppl.276) JAMA 142: 383-390). Since these observations, Lee et al., 1992, Proc. Natl. Acad. Sci. USA 89: 1847-1851; And Jones et al., 1992, Infect. Immun. 60: 2475-2480) discloses the isolation of mutants of Salmonella typhimurium that are capable of invading HEp-2 (human epidermoid carcinoma) cells in vivo with a considerably large number of wild-type strains. These " invasive " mutations were isolated under aerobic growth conditions of bacteria that normally reverse the ability of wild-type strains to invade HEp-2 animal cells. However, over-invasive Salmonella typhimurium as disclosed in this document involves the risk of pan-invasive infections and can lead to a prevalent bacterial infection in cancer patients.

See Carswell et al., 1975, Proc. Natl. Acad. Sci. USA 72: 3666-3669) showed that mice injected with Calmette-Guerin bacillus (BCG) had increased levels of TNF serum and TNF-positive sera were found in mice with meth A sarcoma and necrosis of other transplanted tumors . ≪ / RTI > As a result of such observations, immunization of cancer patients injected with BCG is currently used in some cancer treatment protocols. BCG therapy is described in 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].

However, among the major gut-associated gobs are TNF-a mediated septic shock, which may have toxic or fatal consequences for the host (Bone, 1992, JAMA 268: 3452-3455; Dinarello et al. , 1993, JAMA 269: 1829-1835). Moreover, dose limiting, systemic toxicity of TNF-a has been a major hurdle to effective clinical use. Modifications that reduce this type of immune response are useful because the level of TNF- [alpha] is not toxic, and more effective concentrations and / or durations of the therapeutic vector can be used.

2.8. Tumor-target bacteria

Genetically engineered Salmonella has been shown to be capable of targeting tumors, has antitumor activity, and is useful for delivering effector genes such as herpes simple thymidine kinase (HSV TK) to fidelity tumors (Pawelek et al., WO 96 / 40238).

2.9 Reduced induction of TNF-α by modified bacterial lipid A

Changes to the lipid composition of tumor-targeted bacteria that alter the immune response as a result of reducing the induction of TNFa production have been proposed by Pawelek et al. (WO 96/40238). Pourec et al. Provided a method for isolating genes from Rhodobacter , which contributes to the production of monophosphoryl lipid A (MLA). MLA acts as an antagonist against septic shock. Also includes a mutant firA encoding the third enzyme UDP-3-O (R-30 hydroxy myristoyl) -glucosamine-acyltransferase in lipid A biosynthesis, (Kelley et al., 1993, J. Biol. Chem. 268: 19866-19874). Faurec et al. Have demonstrated that mutations in the firA gene induce lower levels of TNF [alpha].

In Escherichia coli, the gene msbB (mlt), which contributes to terminal amidification of lipid A, has been identified (Engel, et al., 1992, J. Bacteriol. 174: 6394-6430; Karow and Georgopoulos 1992, J. Somerville et al., 1996, J. Clin. Invest., 97: 359-365; Somerville, WO 97/25061). However, the above references failed to suggest that destruction of the msbB gene in tumor-targeted Salmonella vectors would result in less toxic and more sensitive bacteria to chelating agents.

Problems associated with using bacteria as gene delivery vectors are focused on the general ability of bacteria to directly kill normal mammalian cells as well as their ability to over-stimulate the immune system via TNFa, which may have toxic effects on 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 mimicking the presence of bacteria in the body can be severely impaired (Tschape, 1996, DTW Dtsch Tierarzl Wochenschr 1996 103: 273-7; Ramos et al., 1996, Enferm Infec. Microbiol Clin. 14: 345-51).

Hone and Powell discloses a method for producing Gram-negative bacteria with non-pyrogenic lipid A or LPS in WO 97/18837.

Maskell discloses a mutant strain of Salmonella having a mutation in the msbB gene that induces a low level of TNFa compared to the wild type strain in WO 98/33923.

Bermudes et al. Teach WO 01/13053 to compositions and methods for destroying the msbB gene of Salmonella to produce salmonella with reduced ability to induce TNFα and reduced toxicity compared to the wild type. In some embodiments, some of the above Salmonella strains have increased sensitivity to chelating agents compared to wild-type Salmonella (Low et al., 1999, Nature Biotech. 17: 37-47).

The citation of any reference in Section 2 of this Circle, or in any section, should not be construed as an acknowledgment that such reference is available as prior art to the present invention.

3. Summary of the Invention

The present invention provides a method of delivering one or more primary effector molecule (s) to a solid tumor. In one embodiment, the method delivers one or more primary effector molecules at a high level. In particular, the present invention relates to a method for the treatment of a primary effector molecule (s) that is toxic or undesirable when delivered systemically to a host (e. G., Unwanted immunological effects) Lt; RTI ID = 0.0 > Salmonella < / RTI > with reduced toxicity to the host. The present invention also provides a method of treating a subject suffering from a toxic, attenuated tumor-target bacterium as a vector for delivering one or more primary effector molecule (s) and optionally one or more secondary effector molecule (s) to a suitable site of action, For the manufacture and use of Salmonella. Specifically, the toxic attenuated tumor target bacteria of the present invention are conditioned aerobic bacteria or condition anaerobic bacteria that have been engineered to encode one or more primary effector molecule (s) and optionally one or more secondary effector molecule (s).

The present invention provides a toxin-weakened tumor-targeting bacterium engineered to express a nucleic acid molecule encoding a primary effector molecule at a site of a solid tumor. In certain embodiments, the toxic attenuated tumor-targeted bacteria are engineered to express a nucleic acid molecule encoding a primary effector molecule. In another embodiment, the toxic attenuated tumor target bacteria is engineered to express one or more nucleic acid molecules encoding one or more primary effector molecules. According to this embodiment, a single bacterial strain is engineered to express one or more nucleic acid molecules encoding at least one primary effector molecule at a site of the fidelity tumor. In another embodiment, one or more toxic attenuated tumor-targeting bacterial strains are engineered to express one or more nucleic acid molecules encoding one or more primary effector molecules. In an embodiment of this embodiment, the toxic attenuated tumor target bacterial strain is of the same species. In another embodiment of this embodiment, the toxic attenuated tumor target bacterial strain is of a different species (e. G., Listeria and Salmonella).

The primary effector molecules of the present invention are useful for the treatment of solid tumors such as carcinoma, melanoma, lymphoma or sarcoma. As used herein, the terms " treatment of a solid tumor " or " treating a solid tumor " refer to inhibiting the growth of a tumor or tumor cell, reducing the volume of a tumor, killing a tumor cell, And inhibiting diffusion (transition). In certain embodiments, the primary effector molecule of the invention is a localized immunosuppressive agent that inhibits the growth of tumor or tumor cells at the site of the tumor, kills tumor cells, or prevents the spread of tumor cells to other parts of the body To induce the reaction. Thus, the primary effector molecule provides a therapeutic effect for the treatment of tumors.

The primary effector molecule may be derived from any known organism, such as, but not limited to, animals, plants, bacteria, fungi and protists, or viruses. In a preferred embodiment of one embodiment of the invention said primary effector molecule (s) is derived from a mammal. In a more preferred embodiment of this embodiment, the primary effector molecule (s) is derived from a human. The primary effector molecule of the invention comprises members of the TNF family, angiogenesis inhibitors, cytotoxic polypeptides or peptides, tumor suppressor enzymes and functional fragments thereof.

In certain embodiments, the primary effector molecule of the invention is a member of the TNF family or a functional fragment 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 cytotoxic-inducing ligand (TRAIL), TNF- (LTE), LTX (lymphotoxin beta), OX40L (OX40), and other cytokines such as the activated cytokine (TRANCE), TNF-a-related weak cell apoptosis inducing factor (TWEAK), CD40 ligand Ligand), FasL, CD27L (CD27 ligand), CD30L (CD30 ligand), 4-1BBL, APRIL (proliferation inducing ligand), LIGHT (29 kDa type II transmembrane protein produced by activated T cells) Necrotic factor-type cytokines), TNFSF16, TNFSF17 and AITR-L (ligands of the activation-inducing TNFR family members). In a preferred embodiment, the primary effector molecule of the invention is selected from the group consisting of tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor-beta (TNF-beta), TNF- related activation cytokine (TRANCE), TNF-? -related weak cytotoxic inducer (TWEAK) and CD40 ligand (CD40L), or functional fragments thereof.

In another specific embodiment, the primary effector molecule of the invention is an angiogenesis inhibiting factor or a functional fragment thereof. Examples of angiogenesis inhibitory factors include, but are not limited to endostatin, angiostatin, apomigranin, angiogenesis-inhibiting antithrombin III, a 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragment of fibronectin, a uPA receptor antagonist, A 16 kDa proteolytic fragment, a 7.8 kDa proteolytic fragment of platelet factor-4, an angiogenesis inhibiting 24 amino acid fragment of platelet factor-4, an angiogenesis inhibitor represented by 13.40, an angiogenesis inhibitor of thrombospondin I, There are anti-angiogenic 20 amino acid peptide fragments of peptide fragments, SPARC, RGD and NGR-containing peptides, small angiogenesis inhibitory peptides of laminin, fibronectin, procollagen and EGF, and peptide antagonists of integrin? _V ? ₃ and VEGF receptors . In a preferred embodiment of the present invention, the primary effector molecule of the invention is a functional fragment of endostatin, apomigran or trombospondin I.

In another particular embodiment, the primary effector molecule of the invention is a cytotoxic polypeptide or peptide, or a functional fragment thereof. Examples of cytotoxic polypeptides or peptides include, but are not limited to, members of the bacteriocin family, berotoxin, cytotoxic necrosis factor 1 (CNF1), cytotoxic necrosis factor 2 (CNF2), Pasteurella multiocida toxin PMT), Pseudomonas endotoxin, hemolysin, a strong competitive inhibitor of paranesyltransferase, CAAX tetrapeptide, cyclin inhibitor, Raf kinase inhibitor, CDC kinase inhibitor, caspase, p53, p16 and p21. In a preferred embodiment, the primary effector molecule is a member of the bacteriocin family, but the bacteriocin family member is not a bacteriocin releasing protein (BRP). Examples of members of the bacteriocin family 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, , Festisin A1122, staphylococcine 1580, butyrylcin 7423, pionin R1 or AP41, megasine A-216 and vibriosin. In certain embodiments, the primary effector molecule is cholysin E3.

In another specific embodiment, the primary effector molecule of the invention is a tumor suppressor enzyme or a functional fragment thereof. Examples of tumor suppressor include but are not limited to methionase, asparaginase, lipase, phospholipase, protease, ribonuclease (except for colE3), DNAase and glycosidase. In a preferred embodiment, the primary effector molecule is methionase.

The present invention also provides a method for locally delivering one or more primary effector molecule (s) and one or more secondary effector molecule (s) to a solid tumor by a toxic attenuated tumor-targeted bacterium, such as Salmonella do. In certain embodiments, the toxic attenuated tumor-targeting bacteria are engineered to express a nucleic acid molecule encoding a primary effector molecule and a secondary effector molecule. In another embodiment, a toxic attenuated tumor-targeting bacterium is engineered to express one or more nucleic acid molecules encoding one or more primary effector molecules and one or more secondary effector molecules. According to this embodiment, a single bacterial strain is engineered to express at least one primary effector molecule and at least one nucleic acid molecule encoding at least one secondary effector molecule at the site of the solid tumor. In another embodiment, one or more toxic attenuated tumor-targeting bacterial strains 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 an embodiment of this embodiment, the toxic attenuated tumor target bacterial strain is of the same species. In another embodiment of this embodiment, the toxic attenuated tumor target bacterial strain is of a different species (e. G., Listeria and Salmonella).

The secondary effector molecule (s) of the present invention provides additional antitumor therapeutic activity and can be used to enhance the release of primary effector molecule (s) from toxic attenuated tumor-targeted bacteria and / Improves internalization at sites of sex tumors. The secondary effector molecule (s) of the present invention may further comprise, in addition to the primary effector molecule (s), molecules delivered by the method of the present invention to treat solid tumors such as carcinoma, melanoma, lymphoma or sarcoma Antitumor proteins, including, but not limited to, cytotoxins, enzymes other than bacteriocins, prodrug conversion enzymes, antisense molecules, ribozymes, antigens, etc.).

The secondary effector molecule may be derived from any known organism, such as, but not limited to, an animal, a plant, a bacterium, a fungus and a protist, or a virus. In some embodiments, the secondary effector molecule is derived from a bacterium or a virus. In some preferred embodiments of the invention, the secondary effector molecule is derived from a bacterium (e.g., BRP). In another preferred embodiment of the present invention, said secondary effector molecule (s) are derived from a virus, such as TAT. In yet another preferred embodiment of the present invention, said secondary effector molecule (s) is derived from a mammal. In some preferred embodiments, the secondary effector molecule (s) is derived from a human.

The present invention provides a toxic attenuated tumor-targeting bacterium comprising the plasmid or the effector molecule (s) encoded by the transfected nucleic acid. In a preferred embodiment of the present invention, the toxic weakened tumor target bacterium is Salmonella. When one or more effector molecules (e.g., primary or secondary) are expressed in a toxic attenuated tumor-targeted bacterium, such as salmonella, the effector molecules may be expressed by the same plasmid or nucleic acid, or in one or more plasmids or nucleic acids . The present invention also provides a toxic attenuated tumor-targeting bacterium comprising an effector molecule (s) encoded by a nucleic acid integrated into the bacterial genome. The integrated effector molecule (s) may be endogenous to a toxic attenuated tumor-targeted bacterium, e. G. Salmonella, or the toxic attenuated tumor may be incorporated into the genome of the toxic attenuated tumor- (E. G., By introduction of a nucleic acid that encodes the effector molecule, e. G., A plasmid, a transfection nucleic acid, a transposon, etc.) into the target bacterium. The present invention provides nucleic acid molecules encoding an effector molecule whose nucleic acid is operatively linked to a suitable promoter. The promoter operably linked to the nucleic acid encoding the effector molecule may be homologous (i.e., natural) or heterologous (i.e., the nucleic acid encoding the effector molecule is not native). 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.

The present invention also provides a method for local delivery of one or more fusion proteins comprising a signal sequence and effector molecules by a toxic attenuated tumor-targeted bacterium. In a preferred embodiment, the toxic attenuated tumor target bacteria is treated with an Omp-type protein or a portion thereof (e.g., a signal sequence, a leader sequence, a plasma membrane domain, a transmembrane domain, a plurality of transmembrane domains, Quot; -type protein ", see section 3.1) and a nucleic acid molecule encoding the effector molecule. Although not intending to be limited to the mechanism, the inventors of the present invention have considered that the Omp-type protein acts as a factor for fixing or mooring the effector molecule to the outer membrane, or for arranging the effector molecule on the bacterial outer membrane . In some embodiments, the effector molecule has improved delivery of the bacteria to the outer membrane. In one embodiment, fusion of the effector molecule to the Omp-type protein is used to improve the localization of the effector molecule into the plasma membrane. In some other embodiments, fusion of the effector molecule to the Omp-type protein is used to enhance the release of the effector molecule. Examples of Omp-type proteins include, but are not limited to, at least some of each of the following: OmpA, OmpB, OmpC, OmpD, OmpE, OmpF, OmpT, porin-type protein, PhoA, PhoE, lamB, , Protein A, endoglucanase, peptidoglycan-related lipoprotein (PAL), FepA, FhuA, NmpA, NmpB, NmpC and major extracellular lipoproteins (e.g., LPP). In another embodiment of the invention, the fusion protein of the invention comprises a proteolytic cleavage site. The proteolytic cleavage site may be endogenous to the effector molecule or endogenous to an Omp-type protein, or may be composed of a fusion protein.

The present invention also provides a method for local delivery of a fusion protein comprising a peripeptide and an effector molecule to a solid tumor by a toxic attenuated tumor-targeted bacterium. Peripeptides used in fusion proteins have been shown to promote the delivery of substantially any polypeptide of interest or peptides of interest within the diffusion limit 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). Thus, manipulating tumor-targeted bacteria that are toxic to express a fusion protein comprising a peripeptide and an effector molecule enhances the ability of the effector molecule to be internalized by the tumor cells. In certain embodiments, the toxic attenuated tumor-targeted bacteria are engineered to express a nucleic acid molecule encoding a fusion protein comprising a peripeptide and an effector molecule. In another embodiment, the toxic attenuated tumor target bacteria is engineered to express one or more nucleic acid molecules encoding the one or more fusion proteins comprising the peripeptide and effector molecule. According to these embodiments, the effector molecule may be a primary or secondary effector molecule. Examples of peripeptides include, but are not limited to, peptides derived from HIV TAT protein, Antennapedia homeodomain (pennelaxin), Kaposi fibroblast growth factor (FGF), membrane-translocation sequence (MTS) and herpes simplex virus VP22 .

The present invention also provides a method of topically delivering one or more fusion proteins comprising signal peptides, peripeptides, and effector molecules to a solid tumor by a toxic attenuated tumor-targeted bacterium. In certain embodiments, the toxic attenuated tumor-targeted bacteria are engineered to express one or more nucleic acid molecules that encode one or more fusion proteins comprising signal peptides, peripeptides, and effector molecules. According to this embodiment, the effector molecule may be a primary or secondary effector molecule.

The present invention also provides a method of topically delivering one or more fusion proteins comprising a signal peptide, a proteolytic cleavage site, a peripeptide and an effector molecule to a solid tumor by a toxic attenuated tumor target bacterium. In certain embodiments, the toxic attenuated tumor-targeted bacteria are engineered to express one or more nucleic acid molecules that encode one or more fusion proteins, including signal peptides, proteolytic cleavage sites, peripeptides, and effector molecules. According to this embodiment, the effector molecule may be a primary or secondary effector molecule.

In some embodiments, a single bacterial strain is engineered to express at least one nucleic acid molecule encoding a fusion protein of the invention at the site of the fidelity tumor. In some other embodiments, one or more toxic attenuated tumor-targeted bacterial strains are engineered to express one or more nucleic acid molecules encoding at least one fusion protein of the invention at the site of the solid tumor. In an embodiment of these embodiments, the toxic attenuated tumor target bacterial strain is of the same species. In another embodiment of these embodiments, the toxic attenuated tumor target bacterial strain is of a different species (e. G., Listeria and Salmonella).

The present invention also provides a method of topically delivering one or more fusion proteins of the invention and one or more effector molecules of the invention to a site of a solid tumor with a toxic attenuated tumor target bacteria. Preferably, expression of the fusion protein (s) and effector molecule (s) both in the fidelity tumor site by the toxic attenuated tumor-targeted bacterium expresses only the fusion protein (s) or only the effector molecule (s) Lt; RTI ID = 0.0 > tumor < / RTI > or tumor cell growth.

The present invention also provides for the expression of a bacterial release system in an improved toxicant weakened tumor target bacterium, e. G. Salmonella, and optionally a secondary effector molecule. In a preferred embodiment of the invention, the enhanced release is associated with the expression of a release factor by a toxic attenuated tumor-targeted bacterium. In one embodiment, the release improves the release of the effector molecule from the cytoplasmic membrane or plasma membrane space. The release factor may be endogenous to the toxic attenuated tumor target bacterium, or exogenous (i. E., Encoded by a nucleic acid molecule that is not native to the toxic attenuated tumor target bacterium). The release factor may be encoded by a nucleic acid comprising a plasmid or by a nucleic acid integrated into the genome of a toxic attenuated tumor-targeted bacterium. The release factor may be encoded by the same nucleic acid or plasmid encoding the primary effector molecule, or by a separate nucleic acid or plasmid. The release factor can be encoded by the same nucleic acid or plasmid encoding the secondary effector molecule, or by a separate nucleic acid or plasmid. In a preferred embodiment, the release factor is a bacteriocin releasing protein (BRP). In certain embodiments, the BRP is a chloacin DF13 plasmid, one of the colicin E1-E9 plasmids, or a cholysin A, N or D plasmid. In a preferred embodiment, the BRP is of chloacin DF13 (pCloDF13 BRP). In another embodiment of the present invention, the enhanced release system comprises an overexpression of a protein of interest.

The present invention also provides for the expression of a fusion protein of the invention in an improved toxic attenuated tumor-targeted bacterium, such as Salmonella, for example. In certain embodiments, the release factor is expressed in a cell that also expresses a fusion protein comprising a primary effector molecule fused to an Omp-type protein. In this embodiment, co-expression of the expression factor allows enhanced release of the fusion protein from the plasma membrane space.

In one embodiment, the present invention provides a method of delivering a high level of effector molecule or fusion protein using a modified toxic attenuated tumor-target bacterial strain selectively accumulating in a tumor while expressing an effector molecule or fusion protein . In certain embodiments, the altered toxicant weakened tumor bacterium strain selectively amplifies the effector molecule in the tumor. Although the teachings of the following sections have been specifically discussed with reference to Salmonella for the sake of brevity, the compositions and methods of the present invention are by no means meant to be limited to Salmonella, and include any other bacteria to which the teachings apply. Specifically, the present invention provides a toxic weakly tumor-targeting bacterium which is conditioned aerobic bacteria or conditionally anaerobic bacteria. Examples of toxic attenuated tumor bacteria include, but are not limited to field penetration Escherichia coli, Salmonella, S. Phi, SH Gela S Phi (Shigella spp.), Yersinia Enterobacter nose urticae (Yersinia enterocohtica), L. monocytogenes Jennie S. (Listeria monocytogenies , Mycoplasma hominis , and Streptococcus esophila.

The present invention also provides a pharmaceutical composition comprising a toxin-weakened tumor-targeting bacterium engineered to contain a pharmaceutically acceptable carrier and one or more nucleic acid molecules encoding one or more primary effector molecules. The present invention also provides a pharmaceutical composition comprising a toxicantly attenuated tumor-targeting bacterium engineered to contain a pharmaceutically acceptable carrier and one or more primary effector molecules and one or more nucleic acid molecules encoding one or more secondary effector molecules . The present invention also provides a pharmaceutical composition comprising a toxicant weakly tumor-targeting bacterium engineered to contain a pharmaceutically acceptable carrier and one or more nucleic acid molecules encoding one or more fusion proteins of the invention. Moreover, the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an engineered toxicity attenuating one or more nucleic acid molecules encoding the fusion protein of the invention and one or more effector molecules (i.e., primary and / or secondary molecules) Lt; RTI ID = 0.0 > tumor < / RTI > target bacteria. In a preferred embodiment, the toxic attenuated tumor target bacterium is Salmonella.

The pharmaceutical composition of the present invention is useful for the treatment of solid tumors. Examples of solid tumors include, but are not limited to, sarcomas, carcinomas, lymphomas and other solid tumoral cancers such as, but not limited to, interstitial tumors, central nervous system tumors, breast cancers, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, Thyroid cancer, astrocytoma, glioma, pancreatic cancer, stomach cancer, liver cancer, colon cancer, melanoma, kidney cancer, bladder cancer and mesothelioma.

The present invention relates to a method for the treatment of benign tumor tumor cells, comprising administering to a mammal, preferably a mammal, and most preferably a human, in need of treatment a fulminant oncogenic tumor, a toxic attenuated tumor target bacterium engineered to contain one or more nucleic acid molecules encoding one or more primary effector molecules The method comprising administering to the mammal a therapeutically effective amount of a first effector molecule for the treatment of said solid tumor carcinoma. The present invention also relates to a method of treating a solid tumor cancer, comprising administering to an animal, preferably a mammal, and most preferably a human, in need thereof a therapeutically effective amount of at least one first effector molecule and at least one nucleic acid molecule encoding at least one second effector molecule The method comprising administering to the subject a pharmaceutical composition comprising a reduced toxicity-attenuated tumor-targeted bacterium. The present invention also relates to a method of treating a malignant tumor which has been engineered to contain one or more nucleic acid molecules encoding one or more fusion proteins of the invention in an animal, preferably a mammal, and most preferably a human, There is provided a method of delivering said primary effector molecule for the treatment of said solid tumoral cancer, comprising administering a pharmaceutical composition comprising a bacterium. Furthermore, the present invention provides a method of treating a solid tumor cancer, comprising administering to an animal, preferably a mammal, and most preferably a human, in need of treatment one or more of the fusion proteins of the invention and one or more effector molecules (i. E., Primary and / The method comprising the administration of a pharmaceutical composition comprising a toxic attenuated tumor-targeted bacterium engineered to contain one or more nucleic acid molecules encoding a first effector molecule for the treatment of said solid tumor cells . In a preferred embodiment, the toxic attenuated tumor target bacterium is Salmonella. In certain embodiments, the toxic attenuated tumor target bacteria comprises an enhanced release system.

In some embodiments, toxic attenuated tumor-targeted bacteria that have been engineered to express one or more effector molecules and / or one or more nucleic acid molecules encoding the fusion protein can be used in conjunction with other known cancer therapies. For example, a toxic attenuated tumor-targeted bacterium engineered to express one or more effector molecules and / or one or more nucleic acid molecules encoding a fusion protein can be used with the chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, cisplatin, ipospamide, paclitaxol, taxane, topoisomerase I inhibitors such as CPT-11, topotecan, 9-AC and GG-211, gemcitabine, vinorelbine , Oxaliplatin, 5-fluorouracil (5-FU), leucovorin, vinorelbine, te modal, taxol, cytochalasin B, gramicidin D, emetine, mitomycin, etoposide, But are not limited to, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydro testosterone, melphalan, glucocorticoid, procaine, tetracaine, Lidocaine, propranolol, and puromycin analogs and cytoxic acids. On the one hand, toxic attenuated tumor target bacteria engineered to express one or more effector molecules and / or one or more nucleic acid molecules encoding the fusion protein can be used in conjunction with radiation therapy.

The present invention involves the sequential or simultaneous administration of an anticancer agent and a toxicantly attenuated tumor-targeted bacterium engineered to express one or more effector molecules and / or one or more nucleic acid molecules encoding the fusion protein. The invention encompasses a combination of an anticancer agent with a toxic attenuated tumor-targeted bacterium engineered to express additional or synergistic one or more nucleic acid molecules encoding one or more effector molecules and / or fusion proteins.

The invention also encompasses a combination of an anticancer agent with a toxic attenuated tumor-target bacterium engineered to express one or more effector molecules and / or one or more nucleic acid molecules encoding the fusion protein, having different action sites. Such combinations provide improved therapy based on the double action of the therapeutic agent whether the combination is synergistic or additional. Thus, the novel combination therapies of the present invention provide improved efficacy over any of the drugs used as a single pharmaceutical regimen.

3.1. Definitions and Acronyms

Salmonella used herein includes all Salmonella species, such as Salmonella typhi, Salmonella cholerae, and Salmonella entericidis. The antimicrobial form of Salmonella, for example, a sub-group of Salmonella entericidis, commonly referred to as Salmonella typhimurium, is also included in the present invention.

Homolog: As used herein, the term " homologue " refers to a primary or secondary effector molecule that has similar or identical function but does not necessarily contain similar or identical amino acid sequences or similar or identical structures of primary or secondary effector molecules ≪ / RTI > A polypeptide having at least 30%, at least 35%, at least 40%, at least 45% amino acid sequence identity with the amino acid sequence of the primary or secondary effector molecule disclosed herein, Amino acid sequence which is 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% ; (b) 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 60 contiguous amino acid residues, at least 70 contiguous amino acid 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, A polypeptide encoded by a nucleotide sequence that hybridizes with a nucleotide sequence encoding a primary or secondary effector molecule disclosed herein of contiguous amino acid residues; At least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% , 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identical to the nucleotide sequence. A polypeptide having a structure similar to that of a primary or secondary effector molecule disclosed herein is referred to as a polypeptide having a secondary, tertiary or quaternary structure similar to the primary or secondary effector molecule disclosed herein. The structure of the 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, cyclic dichroism, and crystallographic electron microscopy.

Angiogenesis inhibitory factor: An angiogenesis inhibitory factor is any protein molecule having an anti-angiogenic activity, or a nucleic acid encoding such a protein molecule. In a preferred embodiment, the angiogenesis inhibiting factor is a peptide fragment or cleavage fragment of a larger protein.

Toxic weakness: Toxic weakness is to alter a microorganism or vector so that it is less pathogenic. The end result of the toxic weakening is that the risk of toxicity as well as other side effects are reduced when the microorganism or vector is administered to the patient.

Bacteriocin: Bacteriocin is a bacterial protein toxin that has selective activity, that is, bacteria are immune to toxins. Bacteriocins can be encoded by bacterial host genomes or plasmids, can be toxic to a broad or narrow range of other bacteria, have a simple structure comprising one or two subunits, or they can be multiple subunit structures have. In addition, the host expressing the bacteriocin is immune to the bacteriocin.

Chelating Agent Sensitivity: The chelating agent sensitivity is defined as the effective concentration that affects bacterial growth, or the concentration at which the viability of the bacteria decreases as measured by recoverable colony forming units (c.f.u.).

Derivatives: The term " derivative " in the context of the term " derivative of a polypeptide ", as used herein, refers to a polypeptide, polypeptide, or polypeptide that has been modified by the introduction of amino acid residue substitutions, deletions or additions or by covalent coupling of any type of molecule to the polypeptide Quot; refers to a polypeptide comprising, for example, an amino acid sequence of a primary or secondary effector molecule. The term " derivative " as used herein in connection with " derivatives of primary or secondary effector molecule " refers to, for example, covalently bonding any type of molecule to the primary or secondary effector molecule, Quot; primary " or " secondary " effector molecule. For example, and without limitation, the primary or secondary effector molecule can be altered by, for example, proteolytic cleavage, binding to cellular ligands or other proteins, and the like. Derivatives of primary or secondary effector molecules can be chemically modified using techniques known to those skilled in the art (e.g., by acylation, phosphorylation, carboxylation, glycosylation, selenium modification, and sulfation) . ≪ / RTI > Furthermore, derivatives of primary or secondary effector molecules may contain one or more non-traditional amino acids. Polypeptide derivatives have similar or identical functions to the primary or secondary effector molecules disclosed herein. The term " derivative " in the context of the " derivative of the < RTI ID = 0.0 > msbB < / RTI > toxic attenuated tumor-target Salmonella mutant " refers to a modified msbB Salmonella mutant as defined in international application WO 99/13053 (p. 17) do.

Fragment: As used herein, the term " fragment " refers to a fragment comprising two or more contiguous amino acid residues, five or more contiguous amino acid residues, ten or more contiguous amino acid residues, 15 or more contiguous amino acids residues of the amino acid sequence of a primary or secondary effector molecule At least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 25 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, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues Amino Quot; refers to a peptide or polypeptide comprising an acid sequence.

Functional Fragment: As used herein, the term " functional fragment " refers to a molecule that retains one or more actions of a primary or secondary effector molecule (e.g., enzymatic activity, angiogenesis inhibitory activity or anti- Quot; refers to a fragment of a primary or secondary effector molecule.

Fusion Protein: The term "fusion protein" as used herein refers to an amino acid sequence of a primary or secondary effector molecule, or a functional fragment or derivative thereof, and a heterologous polypeptide (eg, a non-primary or non-secondary ≪ / RTI > the effector molecule).

Omp-type Protein: The Omp-type protein used herein can be any bacterial outer membrane protein or a portion thereof (e.g., a signal sequence, a leader sequence, a plasma membrane region, a transmembrane domain, ). In a particular embodiment, the Omp-type protein is selected from the group consisting of OmpA, OmpB, OmpC, OmpD, OmpE, OmpF, OmpT, , Peptidoglycan-associated lipoprotein (PAL), FepA, FhuA, NmpA, NmpB, NmpC and major extracellular lipoproteins (e.g., LPP).

Purified: The "purified" toxic attenuated tumor-target bacterial strain used herein is substantially free of contaminating proteins or amino acids (eg, debris of dead bacteria) or medium. A toxic attenuated tumor-target bacterial strain substantially free of contaminating proteins or amino acids has less than about 30%, 20%, 10%, or 5% (based on dry weight) of the contaminating protein or amino acid .

Release factors: The release factors used herein include any protein, or functional portion thereof, that enhances the release of bacterial components. In one embodiment, the release factor is a bacteriocin releasing protein. Release factors include, but are not limited to, the bacteriocin release protein (BRP) encoded by the chloacin D13 plasmid, the BRP encoded by the cholinic E1-E9 plasmid, or the BRP encoded by the cholinic A, N or D plasmid .

Septic shock: Septic shock is a condition of internal organ disturbance caused by a complex cytokine cascade initiated by TNF-α. The relative potency of a microorganism or vector that induces TNF-a is used as a measure of its relative ability to induce septic shock.

Tumor Target: A tumor target is defined as the ability to preferentially localize to a cancerous target cell or tissue relative to a non-cancerous corresponding cell or tissue and a replicate. Thus, tumor-target bacteria, such as salmonella, preferentially bind, infect and / or survive in a cancerous target cell or tumor environment.

Toxicity: Toxicity is a relative term that describes the general ability to cause disease, including the ability to kill normal cells or to induce septic shock (see definition).

The strain designation VNP20009 (international applications WO 99/13053), YS1646 and 41.2.9., Which are used in this application, are used interchangeably, each of which refers to a strain deposited with Accession No. 202165 in the American Type Culture Collection. The strain designations YS1456 and 8.7 used herein are used interchangeably, each of which refers to a strain deposited with Accession No. 202164 in the American Type Culture Collection.

BRIEF DESCRIPTION OF THE DRAWINGS The invention may be more fully understood by reference to the following detailed description, an illustrative embodiment of certain embodiments, and the accompanying drawings.

1. Technical Field

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 tumors. More particularly, the present invention relates to the preparation and use of a toxicly attenuated tumor-targeted bacterium, such as salmonella, as a vector for delivering said one or more effector molecule (s) to a site of action, for example a solid tumor, will be. Specifically, the toxic attenuated tumor target bacteria of the present invention are condition aerobic bacteria or condition anaerobic bacteria modified to encode one or more primary effector molecule (s). The primary effector molecule (s) of the present invention include members of the TNF cytokine family, angiogenic inhibitors and cytotoxic polypeptides or peptides. The primary effector molecules of the invention are useful for treating, for example, solid tumors such as carcinoma, melanoma, lymphoma, sarcoma, or metastasis derived from these tumors. The present invention also encompasses a method of delivering to a host a therapeutically effective amount of a first effector molecule (s), such as TNF family members, angiogenesis inhibiting factors and cytotoxic polypeptides or peptides, Lt; RTI ID = 0.0 > immunological < / RTI > complications. The present invention also relates to the delivery of one or more optional effector molecule (s) (referred to as " secondary effector molecules ") that can be delivered by the toxicant weakened tumor target bacteria with the primary effector molecule will be. The secondary effector molecule (s) may provide additional antineoplastic therapeutic activity, enhance the release of the primary effector molecule (s) from the toxic attenuated tumor target bacteria, and / Enhancing the absorption of the primary effector molecule (s) at the site of the fidelity tumor.

4. Brief description of drawing

Figure 1 is the coding sequence for mature human TNF-a. DNA (SEQ ID NO: 3) and protein (SEQ ID NO: 4).

Figure 2 shows the induction of Salmonella VN20009 serC-strain.

Figure 3 shows TNF-a expression from the trc promoter-engineered TNF-a gene integrated chromosomally in Salmonella typhimurium.

Figure 4 shows the coding sequence for the synthetic OmpA signal sequence (nucleotide 1-63) fusion to mature human TNF-a (nucleotides 67-543). DNA (SEQ ID NO: 7) and protein (SEQ ID NO: 8) sequences are shown for the fusion constructs.

FIG. 5 shows plasma membrane localization and processing of OmpA / TNF-a fusion protein in E. coli (strain JM109).

Figure 6 shows the coding sequence for the OmpA signal sequence (nucleotide 1-63) fusion to mature human TRAIL (nucleotides 67-801). DNA (SEQ ID NO: 9) and protein (SEQ ID NO: 10) sequences are shown for the fusion constructs.

Figure 7 shows the expression and processing of the OmpA TRAIL fusion protein in E. coli (strain JM109).

Figure 8 shows the coding sequence for the modified OmpA signal sequence (nucleotide 1-63) fusion to mature (C125A) human IL-2 (nucleotides 64-462). DNA (SEQ ID NO: 11) and protein (SEQ ID NO: 12) sequences are shown for the fusion constructs.

Figure 9 shows plasma membrane localization and processing of mature human IL-2 fused to phoA (8L) or ompA (8L) synthetic signal peptide.

Figure 10 shows the coding sequence for the altered phoA signal sequence (nucleotide 1-63) fusion to mature (C125A) human IL-2 (nucleotides 64-462). DNA (SEQ ID NO: 13) and protein (SEQ ID NO: 14) sequences are shown for the fusion constructs.

Figure 11 shows the in vivo antitumor efficacy of a toxic attenuated strain of Salmonella typhimurium expressing the mature form of human TNF-a.

Figure 12 shows the effect of BRP expression on antitumor efficacy in vivo. This figure shows (1) the PBS control group; (2) VNP20009; And (3) the average tumor size over time in a population of C57BL / 6 mice with B16 melanoma tumors treated with VNP20009 with the pSW1 plasmid containing the BRP gene.

Figure 13 shows the anaerobic induction of β-gal gene expression under control of the pepT promoter in Salmonella. Figure 13A shows the in vitro induction of [beta] -gal expression in response to anaerobic conditions of two Salmonella strains YS1456 and VNP20009. Figure 13B shows in vivo β-gal induction of tumor-versus-host cells of BRP, β-gal, or VNP20009 expressing BRP and β-gal.

Figure 14 shows tetracycline induction of beta -gal gene expression under Tet promoter control in Salmonella. The dose-response shows a linear response to tetracycline up to a concentration of approximately 15 [mu] g / ml, after which the reaction is presumably reduced as a result of the antibiotic action of tetracycline.

Figure 15 shows hexa-histidine-endostatin (HexaHIS-endostatin) expression from the pTrc99a vector. Figure 15A shows the expression of HexaHIS-endostatin from three independent clones transfected into Salmonella (VNP20009). Figure 15B shows the expression of HexaHIS-endostatin from five independent clones transfected into E. coli (DH5a).

Figure 16 shows the expression of HexaHIS-endostatin from plasmid YA3334, and in the asd system (using the trc promoter), HexaHIS-endostatin has the correct size for HexaHIS-endostatin (~25 kD) by Western analysis using anti- (Lanes 1 to 8 correspond to eight independent clones).

Figure 17 shows the efficacy of endostatin expressing VNP20009 cells on C38 mouse colorectal carcinoma. This figure shows (1) the PBS control group; (2) asd ^- carrying the empty YA3334 vector ^- VNP20009; (3) asd ^- expressing hexa histidine-endostatin ^- VNP20009; And (4) graphs the average tumor size over time in a group of mice with established C38 tumors treated with VNP20009 expressing hexahistidine-endostatin and BRP.

Figure 18 shows the efficacy of endostatin expressing VNP20009 cells on DLDl human colon carcinoma. This figure shows (1) the PBS control group; (2) asd ^- carrying the empty YA3334 vector ^- VNP20009; And (3) the average tumor size over time in a population of nude mice with established DLDl tumors treated with VNP20009 expressing hexahistidine-endostatin and BRP.

19 shows the proliferation inhibitory activity of lysates from human endostatin-expressing toxin-attenuated tumor-target Salmonella against endothelial cells. This figure shows inhibition of human intravenous (HUVEC) proliferation of lysates corresponding to bFGF and 8 x 10 ⁸ bacteria. As a control, salmonella containing an empty pTrc vector was used. Each data point is the average of four runs from a typical experiment. Samples were standardized by a number of bacteria.

Figure 20 shows the proliferation inhibitory activity of lysates from platelet factor-4 (amino acids 47-70) and thrombospondin peptide (13.40) expressing toxic attenuated tumor-target Salmonella on endothelial cells. This figure shows inhibition of human intravenous (HUVEC) proliferation of lysates corresponding to bFGF and 3.2 x 10 ⁸ bacteria. As a control, salmonella containing an empty pTrc vector was used. Each data point is the average of four runs from a typical experiment. Samples were standardized by a number of bacteria.

21 shows the structure of the pE3. Shuttle-1 vector.

Figure 22 shows the structure of the ColE3-CA38 vector (GenBank Accession No. AF129270). The nucleotide sequence of the ColE3-CA38 vector is as shown in SEQ ID NO: 1. The ColE3-CA38 vector contains five open reading frames as shown in SEQ ID NOS: 2-5, respectively.

Figure 23 shows the structure of the ColE3-CA38 / BRP-1 vector.

24 is a bar graph showing lethal dose units of collicin E3 produced by each strain.

Figure 25 shows halo analysis for various strains exposed to ultraviolet or x-rays.

Figure 26 shows the efficacy of 41.2.9 / ColE3 on C38 murine colorectal carcinoma.

Figure 27 shows the antitumor activity of 41.2.9 / ColE3 on DLDl human colon carcinoma in NU / Nu mice.

Figure 28 shows the efficacy of 41.2.9 / ColE3 on B16 murine melanoma.

Figure 29 shows the cytotoxicity of Salmonella expressing cloned CNF1 cloned.

Figure 30 shows the expansion and polynuclear formation of HeLa cells (A) exposed to CNFl against normal HeLa cells (B).

Figure 31 shows the msbB portion of the 3 'to 5 ' orientation pCVD442-msbB vector in which the middle of msbB is deleted and contains internal Not1, PacI, SphI, Swal and DraI polylinkers in its place. See FIG.

32 is a restriction map and schematic diagram of the pCVD442-msbB vector for cloning DNA in the DmsbB region and subsequently inserting it on the chromosome. msbBdeI, 5 'to 3' region of DmsbB; mob RP4, a moving element for transferring the plasmid from one strain to another; beta-lactamase gene which confers sensitivity to b-lactam antibiotics such as bla, carbenicillin and ampicillin; sacB, a gene that confers susceptibility to sucrose.

Figure 33 shows the results of 1) pCVD442-Tet-BRP-AB vector, 2) homologous recombination with DmsbB chromosomal replication in Salmonella YS50102, 3) chromosomal integration in Salmonella YS50102 followed by phage transfection into strain VNP20009, Strain 41.2.9-Tet-BRP-AB was generated. oriR6K, plasmid origin of replication; mobRP4, a translocation element for transferring the plasmid from one strain to another; beta-lactamase gene that confers sensitivity to b-lactam antibiotics such as amp, carbenicillin and ampicillin; sacB, a gene that confers susceptibility to sucrose. Ratios are not shown.

Figure 34 shows the cytotoxicity percentages of tetBRPAB clone # 26 and clone # 31 for positive and negative controls (HSC10 and 41.2.9) following 72 h exposure to SKOV3 cells (AveN = 8). Expression of verotoxin was induced by tetracycline (see clones 26 and 31). Tetracycline treatment (+); And tetracycline untreated (-). This coli strain HSC10 was used as a positive control for cytotoxicity percentages.

Figure 35 shows the formation of halo on the blood agar, the absence of tetracycline (2A) and the presence of tetracycline (2B) on the blood agar for toxic attenuated tumor-targeted Salmonella under absence (1A) of tetracycline and presence of tetracycline (1B) (3A) and the presence of tetracycline (3B) toxic weakened tumor target engineered to express tetracycline-inducible SheA Salmonella ≪ / RTI >

36 shows (A) TAT-adapthin fusion protein without hexa histidine labeling, (B) hexahistidine labeled TAT-adapthin fusion protein, and (C) the OmpA-8L signal sequence TAT-apofitin fusion protein is exemplified.

Figure 37 is the coding sequence of the TAT-apofitin fusion protein. DNA (SEQ ID NO: 57) and protein (SEQ ID NO: 58).

Figure 38 is the coding sequence of the hexa histidine-TAT-apofitin fusion protein. DNA (SEQ ID NO: 59) and protein (SEQ ID NO: 60).

Figure 39 shows the efficacy of VNP20009 / cytoxan binding therapy for M27 lung carcinoma growth in C57BL / 6 mice.

Figure 40 shows the efficacy of VNP20009 / mitomycin combination therapy on M27 lung carcinoma growth in C57BL / 6 mice.

Figure 41 shows the efficacy of VNP20009 / cisplatin combination therapy on M27 lung carcinoma growth in C57BL / 6 mice.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention uses a toxicant tumor-targeted bacterial strain to deliver a high level of therapeutic primary effector molecule (s) to the tumor. The present invention provides the advantage of bypassing the potential systemic toxicity of some primary effector molecules (e. G., Septic shock by TNF-a). The present invention delivers one or more primary effector molecule (s) and optionally one or more secondary effector molecule (s) to a solid tumor. More particularly, the present invention relates to a method for the treatment of toxic attenuated tumor-target bacteria (e. G.) As a vector for delivering one or more primary effector molecule (s) and optionally one or more secondary effector , For example the manufacture and use of Salmonella. Specifically, the toxic attenuated tumor target bacteria of the present invention are conditioned aerobic bacteria or conditioned anaerobic bacteria engineered to encode one or more primary effector molecule (s) and optionally one or more secondary effector molecule (s).

A delivery system based on the toxic attenuated tumor target bacteria disclosed herein locally transfers one or more primary effector molecule (s) to a solid tumor. The present invention relates to a method for the treatment or prophylaxis of a primary effector molecule (s) that is toxic or undesirable when delivered systemically to a host (e. G., Unwanted immunological effects) Lt; RTI ID = 0.0 > Salmonella < / RTI > with reduced toxicity to the tumor. The present invention also provides a complex delivery of one or more primary effector molecule (s) delivered by a toxic attenuated tumor-targeted bacterium, e. G. Salmonella, and optionally one or more secondary effector molecule (s). The present invention also provides a complex delivery of different toxic attenuated tumor-targeted bacteria involving one or more different primary effector molecule (s) and / or optionally one or more different secondary effector molecule (s).

The present invention also provides a method for topically delivering said fusion protein by a toxic attenuated tumor-targeted bacterium engineered to express at least one fusion protein comprising an effector molecule at the fidelity tumor (s) site. In one embodiment, the toxic attenuated tumor-targeted bacteria are engineered to express a fusion protein comprising a signal peptide and an effector molecule. In another embodiment, the toxic attenuated tumor-targeted bacteria are engineered to express a fusion protein comprising a peripeptide and an effector molecule. In another embodiment, the toxic attenuated tumor target bacteria is engineered to express a fusion protein comprising a signal peptide, a peripeptide, and an effector molecule. In yet another embodiment, the toxic attenuated tumor target bacteria is engineered to express a fusion protein comprising a signal peptide, a proteolytic cleavage site, a peripeptide, and an effector molecule. Toxic attenuated tumor-targeted bacteria are engineered to express one or more fusion proteins of the invention and one or more effector molecules of the invention.

The present invention also provides a pharmaceutical composition comprising a toxin-weakened tumor-targeting bacterium engineered to contain a pharmaceutically acceptable carrier and one or more nucleic acid molecules encoding one or more primary effector molecules. The present invention also provides a pharmaceutical composition comprising a toxicantly attenuated tumor-targeting bacterium engineered to contain a pharmaceutically acceptable carrier and one or more primary effector molecules and one or more nucleic acid molecules encoding one or more secondary effector molecules . Moreover, the present invention provides a pharmaceutical composition comprising a toxicantly reduced tumor-targeting bacterium engineered to contain a pharmaceutically acceptable carrier and one or more fusion protein and one or more nucleic acid molecules encoding one or more effector molecule.

The present invention provides a method of treating a solid tumor cancer of an animal wherein the method comprises administering to an animal in need of treatment a solid tumor cancer a toxic attenuation engineered to express one or more nucleic acid molecules encoding one or more primary effector molecules Lt; / RTI > bacteria. The present invention also encompasses administering to a mammal in need of such treatment a fungal toxic tumor-negative bacterium engineered to express one or more nucleic acid molecules encoding one or more primary effector molecules and one or more secondary effector molecules The method comprising the steps of: Furthermore, the present invention provides a method of treating a cancer, comprising administering to a mammal in need of such treatment a fungal tumor capable of treating a toxicly attenuated tumor-targeted bacterium engineered to contain one or more fusion proteins and one or more nucleic acid molecules encoding one or more effector molecule Preferably, the animal is a mammal (e.g., a dog, a cat, a horse, a cow, a monkey, or a pig). Examples of solid tumors include, but are not limited to, sarcomas, carcinomas, lymphomas and other solid tumoral cancers such as, but not limited to, interstitial tumors, central nervous system tumors, breast cancers, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, Thyroid cancer, astrocytoma, glioma, pancreatic cancer, stomach cancer, liver cancer, colon cancer, melanoma, kidney cancer, bladder cancer and mesothelioma.

Although not intending to be limited to any one mechanism, the present inventors have found that the delivery of a toxic attenuated tumor target bacterial vector containing the resultant effector molecule (s) of the present invention results in the target agent molecule (s) .

For clarity, the detailed description is divided into the following subsections: Bacterial vector; Primary effector molecules for tumor therapy; A secondary effector molecule for simultaneous expression with a primary effector molecule; Derivatives and homologs thereof; Fusion proteins; An expression vehicle; And delivery methods and compositions.

5.1. Bacterial vector

Any toxic attenuated tumor-targeted bacteria may be used in the methods of the present invention. More specifically, the toxic attenuated tumor target bacteria used in the method of the present invention are condition aerobic bacteria or condition anaerobic bacteria. Conditional aerobic bacteria or conditional anaerobic bacteria that can be used in the method of the present invention include, but are not limited to, intestinal permeable Escherichia coli, Salmonella spp., Shigella esperifie, Yersinia enterocotica, Listeria Monocytogenes, Mycoplasma hominis, and Streptococcus esophila.

Factors contributing to toxic weakening and tumor targeting are disclosed herein and can be used to make or select suitable bacterial strains for use in the methods of the present invention. For example, methods for screening and isolating tumor-targeted bacteria are disclosed in section 6.1 of the international application WO 96/40238, which is incorporated herein by reference, and methods for attenuating the toxicity of the bacteria are described in section 6.2. Examples of toxic attenuated tumor-targeted bacteria are also disclosed in the international application WO 99/13053, which is incorporated herein by reference. In some embodiments of the invention, the bacteria can be modified to become less toxic or very weakened by methods known in the art.

The present invention is directed to a method for the treatment or prophylaxis of a disease or condition comprising administering one or more primary effector molecules (e.g., TNF family members, cytotoxic peptides or polypeptides, tumor suppressor enzymes, or angiogenesis inhibiting factors) Lt; RTI ID = 0.0 > toxic < / RTI > weakened tumor target bacteria. The present invention also provides toxic attenuated tumor-targeting bacteria as a vector for delivering 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 toxic attenuated tumor target bacteria engineered to express one or more nucleic acid molecules encoding an effector molecule and / or a fusion protein is Salmonella.

Although the teachings in the following sections specifically refer to Salmonella, it does not mean that the compositions and methods of the present invention are never limited to Salmonella, and includes any other bacteria to which the teachings apply. Suitable bacterial species include, but are not limited to, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Staphylococcus aureus, Escherichia entericotica, Listeria monocytogenes, ≪ RTI ID = 0.0 > Streptococcus spp., ≪ / RTI >

5.1.1 Salmonella vectors

Any toxic attenuated tumor target bacteria may be used to alter one or more primary effector molecules and optionally one or more secondary effector molecules to encode one or more effector molecules of the invention into a solid tumor using the teachings of the present invention New toxic attenuated tumor-target bacteria can be produced. Moreover, any toxic attenuated tumor-targeting bacteria can be modified to encode one or more fusion proteins, and optionally one or more effector molecules, using the teachings of the present invention to deliver fusion protein and effector molecules of the invention to a solid tumor New toxic attenuated tumor-target bacteria can be produced.

Bacteria such as Salmonella are the causative agent of disease in humans and animals. One such disease that can be caused by Salmonella is sepsis, which is a serious problem due to the high mortality associated with the onset of septic shock (Bone, 1993, Climical Microbiol. Revs. 6: 57-68). Thus, in order to allow the safe use of the Salmonella vector of the present invention, bacterial vectors such as Salmonella attenuate the disease-causing toxicity. In this context, toxic attenuation is not only a traditional definition of altering the microbial vector so that the microbial vector is less pathogenic, but also when administered to a patient with a lower titre of the induced microbial vector to a higher titre of the microbial vector Lt; RTI ID = 0.0 > still < / RTI > compatible with the microbial vector. The end result is to reduce the risk of toxic shock or other side effects of administering the vector to the patient. Such toxic attenuated bacteria are isolated using a number of techniques. For example, toxicity can be attenuated by deletion or destruction of DNA sequences encoding toxic factors that ensure bacterial viability in host cells, particularly macrophages and neutrophils. Such deletion or destruction techniques are well known in the art and include, for example, homologous recombination, chemical mutagenesis, radiation mutagenesis, or transposon mutagenesis. Toxic factors associated with survival in macrophages are usually expressed specifically in the macrophage in response to stimulatory signals, e.g., in response to acidification, or in response to host cell defense mechanisms such as macrophage absorptive action (Fields et al. , 1986, Proc. Natl. Acad. Sci. USA 83: 5189-5193). Table 4 of the international application WO 96/40238 is an exemplary list of Salmonella toxicity factors in which deletion attenuates toxicity.

Another method for attenuating the toxicity of bacterial vectors such as Salmonella is to alter the bacterium's substituents that contribute to bacterial toxicity. For example, lipopolysaccharide (LPS) or endotoxins contribute primarily to the pathological effects of bacterial sepsis. The component of LPS that produces this response is lipid A (" LA "). Elimination or mitigation of the toxic effects of LA produces toxic attenuated bacteria because it reduces the risk of 1) sepsis shock in patients and 2) it can tolerate higher levels of bacterial vectors.

Alteration of the LA content of bacteria such as Salmonella can be accomplished by introducing a mutation in the LPS biosynthetic pathway. Numerous enzymatic steps of LPS biosynthesis and corresponding strains have been identified as well as genes regulating the above in Salmonella (Raetz, 1993, J. Bacteriol. 175: 5745-5733 and references therein). One such exemplary variant is firA, which encodes the enzyme UDP-3-O (R-30 hydroxy myristoyl) -glycosylamine N-acyltransferase, which controls the third step in endotoxin biosynthesis (Kelley et al., 1993, J. Biol. Chem. 268: 19866-19874). The bacterial strain with this type of mutation produces a lipid A that is different from the wild-type lipid A containing the seventh fatty acid hexadecanoic acid (Roy and Coleman, 1994, J. Bacteriol. 176: 1639-1646). Roy and Coleman have demonstrated that in addition to blocking the third step in endotoxin biosynthesis, the firA ^- mutation also reduces the enzymatic activity of lipid A 4 ' kinases that regulate the sixth step of lipid A biosynthesis.

In addition to being toxic, the bacterial vectors of the present invention are tumor-targeted, i.e. the bacteria preferentially bind, infect and / or survive in tumor or tumor cells relative to normal, non-tumor or non-tumor cells. Suitable methods for obtaining toxic attenuated tumor-targeted bacteria are described in Section 6.1 (p. 25-32; tumor-targeting) and Section 6.2.2 (p 43-51; Toxicity reduction). Because the resulting vector is highly specific and super-infectious, the difference in the number of infectious bacteria found in target tumors or tumor cells for noncancerous counterparts is due to the dilution of the microbial culture to allow the use of lower titer microbial vectors as positive results The larger it becomes. Techniques as disclosed in WO 96/40238 can also be used to produce toxic attenuated tumor-targeted salmonella or non-salmonella bacterial vectors.

MsbB disclosed in section 6.1.2, which discloses the features of the strains of ^{Salmonella-Salmonella} illustrative example of a toxic attenuated tumor targeted bacterial strains having the LPS path for reference the international application WO 99/13053, in particular the msbB incorporated by the present invention It is a variant. One characteristic of the msbB ^- Salmonella is the reduced ability to induce a TNF-a response compared to the wild-type bacterial vector. The msbB ^- Salmonella induces the expression of TNF- [alpha] to a level of about 5 to about 40% relative to that induced by wild-type Salmonella.

TNF-alpha responses induced by whole bacteria or isolated or purified LPS can be assessed in vitro or in vivo using commercially available assay systems such as enzyme-linked immunoassay (ELISA). Relative activity is measured using a comparison of TNF- [alpha] production on a colony forming unit (" cfu ") or on the basis of [mu] g / kg. Lower levels of TNF-a per unit baseline indicate reduced TNF-a production. In a preferred embodiment, the msbB ^- salmonella vector is modified to contain one or more primary effector molecule (s) of the invention and optionally one or more secondary effector molecule (s).

The invention also encompasses the use of derivatives of the < RTI ID = 0.0 > msbB ^- toxic < / RTI > attenuated tumor-target Salmonella strains. A derivative of the < RTI ID = 0.0 > msbB ^- toxic < / RTI > attenuated target Salmonella strains was modified to encode one or more primary effector molecule (s) and optionally one or more secondary effector molecule (s) using the teachings of the present invention, New toxic attenuated tumor-target bacteria useful for delivering the molecule (s) to a solid tumor can be produced.

The stability of the toxic attenuated phenotype is important to ensure that the strain is not reverted to a more toxic phenotype during the course of the patient ' s treatment. Such stability can be obtained, for example, by destroying the toxic gene by deletion or other non-return mutation at the chromosomal level rather than at the higher level.

Another way of ensuring the toxic attenuated phenotype is to isolate the bacterium in one or more ways, for example, by mutating a lipid A production pathway, such as the msbB ^- mutant (WO 99/13053) and one or more nutrients or metabolites (Bochner, 1980, J. Bacteriol. 143: 926-933) on nutritional requirements for the product, for example, uracil biosynthesis, purine biosynthesis, arginine biosynthesis. In a preferred embodiment, the toxic attenuated tumor-targeted bacteria encoding or expressing one or more primary effector molecules are toxicized by mutations in AroA, msbB, PurI or SerC. In another embodiment, a toxic attenuated tumor-targeting bacterium encoding one or more primary effector molecules is toxicly attenuated by deletion in AroA, msbB, PurI or SerC.

Thus, any toxic attenuated tumor-target bacteria can be used in the methods of the invention to express one or more primary effector molecule (s) and optionally one or more secondary effector molecule (s) and deliver them to a solid tumor cell have. In a preferred embodiment, the toxic attenuated tumor target bacteria is engineered to express one or more primary effector molecule (s) and optionally one or more secondary effector molecule (s). Moreover, any toxic attenuated tumor-target bacteria can be used in the methods of the invention to express one or more fusion proteins and optionally one or more effector molecules and deliver them to a solid tumor cell. In a preferred embodiment, the toxic attenuated tumor target bacteria is engineered to express one or more fusion proteins and optionally one or more effector molecules.

5.2. Primary effector molecule for tumor therapy

The present invention provides the delivery of primary (and optionally secondary) effector molecule (s) by toxic attenuated tumor-targeted bacteria, e. G. Salmonella. The effector molecule of the invention is a proteinaceous molecule (e.g., a protein, such as, but not limited to, a peptide, a polypeptide, a protein, a post-translationally modified protein, etc.). The present invention also provides a nucleic acid molecule encoding a primary effector molecule of the invention.

The primary effector molecule can be derived from any known organism, such as, but not limited to, animals, plants, bacteria, fungi and protists, or viruses. In a preferred embodiment of the invention, said primary effector molecule (s) is derived from a mammal. In a more preferred embodiment, said primary effector molecule (s) is derived from a human. Primary effector molecules of the invention include members of the TNF family, angiogenic inhibitors, cytotoxic polypeptides or peptides, tumor suppressor enzymes, and functional fragments thereof.

In certain embodiments, the primary effector molecule of the invention is a member of the TNF family or a functional fragment 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 cytotoxic-inducing ligand (TRAIL), TNF- (TWEAK), CD40 ligand (CD40L), LT- [alpha], LT- [beta], OX40L, FasL, CD27L, CD30L, 4-1BBL, APRIL , LIGHT, TL1, TNFSF16, TNFSF17, and AITR-L. In a preferred embodiment, the primary effector molecule of the invention is selected from the group consisting of tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor-beta (TNF-beta), TNF- related activation cytokine (TRANCE), TNF-? -related weak cytotoxic inducer (TWEAK) and CD40 ligand (CD40L), or functional fragments thereof. See, for example, Kwon, B. et al., 1999, Curr. Opin. Immunol. 11: 340-345. In addition, Table 1 below lists the traditional and standardized nomenclature for typical members of the TNF family. In a preferred embodiment of the present invention, the primary effector molecule of the present invention is selected from the group consisting of tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor-beta (TNF-beta), TNF- ), TNF- [alpha] -related activation-induced cytokines (TRANCE), TNF- [alpha] -related weak cytotoxic inducers (TWEAK) or CD40 ligands (CD40L).

TNF family members Traditional nomenclature Standardized nomenclature LT-alpha TNFSF1 TNF-a TNFSF2 LT-beta TNFSF3 OX4OL TNFSF4 CD4OL TNFSF5 F _as L 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

In another specific embodiment, the primary effector molecule of the invention is an angiogenesis inhibiting factor or a functional fragment thereof. Examples of angiogenesis inhibitory factors include, but are not limited to endostatin, angiostatin, apomigranin, angiogenesis-inhibiting antithrombin III, a 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragment of fibronectin, a uPA receptor antagonist, A 16 kDa proteolytic fragment, a 7.8 kDa proteolytic fragment of platelet factor-4, an angiogenesis inhibiting 24 amino acid fragment of platelet factor-4, an angiogenesis inhibitor represented by 13.40, an angiogenesis inhibitor of thrombospondin I, There are anti-angiogenic 20 amino acid peptide fragments of peptide fragments, SPARC, RGD and NGR-containing peptides, small angiogenesis inhibitory peptides of laminin, fibronectin, procollagen and EGF, and peptide antagonists of integrin? _V ? ₃ and VEGF receptors .

In a preferred embodiment of the present invention, the primary effector molecule of the present invention is endostatin. The natural endostatin consists of the C-terminal to 180 amino acids of collagen XVIII (the cDNA encoding the two linked forms of collagen XVIII is GenBank Accession No. AF18081 and AF18082).

In another preferred embodiment of the present invention, the primary effector molecule of the invention is a plasminogen fragment (the coding sequence for plasminogen can be found in GenBank Accession Nos. NM-000301 and A33096). Angiostatin peptides naturally include four Kringle domains of plasminogen, Kringle 1 to Kringle 4. Recombinant Kringle 1, 2 and 3 have the property of inhibiting the angiogenesis of native peptides, whereas Kringle 4 has no such activity (Cao et al., 1996, J. Biol. Chem. 271: 29461-29467). Accordingly, the angiostatin effector molecule of the present invention preferably comprises at least one Kringle domain selected from the group consisting of Kringle 1, Kringle 2 and Kringle 3. In a particular embodiment, the primary effector molecule of the invention is a human angiostatin molecule selected from the group consisting of a 40 kDa isotype, a 42 kDa isotype, a 45 kDa isotype, and combinations thereof. In another embodiment, the 1 effector molecule is the Kringle 5 domain of plasminogen, which is an angiogenesis inhibitor more potent than angiostatin (angiostatin contains Kringle domains 1 to 4).

In another preferred embodiment of the present invention, the primary effector molecule of the present invention is antithrombin III. Antithrombin III (hereinafter referred to as antithrombin) includes a heparin binding domain that binds the protein to the vascular wall and the active site loop that interacts with thrombin. When the antithrombin is bound to heparin, the protein induces a structural change in which the active loop interacts with thrombin to proteolytically cleave the loop by thrombin. (I) the interaction interface between thrombin and antithrombin changes; and (ii) the complex is released from heparin (Carrell, 1999, Science 285 : 1861-1862 and references therein). O'Reilly et al. (O'Reilly et al., 1999, Science 285: 1926-1928) found that truncated antithrombin has potent antiangiogenic activity. Thus, in one embodiment, the angiogenesis inhibiting factor of the present invention is an angiogenesis inhibiting form of antithrombin. In order to deliver the protein to a solid tumor according to the method of the present invention, the bacterial vector is modified to express a full length antithrombin (GendBank Accession Number NM_000488) and a proteolytic enzyme that promotes cleavage of antithrombin, Lt; / RTI > The proteolytic enzyme is selected from the group comprising thrombin, pancreatic elastase, and human neutrophil elastase. In a preferred embodiment, the protease is pancreatic elastase. Methods for recombinant expression of functional pancreatic elastase are described in Shirasu et al., 1987, J. Biochem. 102: 1555-1563.

In another preferred embodiment of the present invention, the primary effector molecule of the invention is a 40 kDa and / or 29 kDa proteolytic fragment of fibronectin. Expression vehicles for these fragments can be prepared by standard procedures using the full length nucleic acid sequence (GenBank Accession No. X02761) encoding the fibronectin precursor protein and the description of the maturation of the encoded protein. In a preferred embodiment, the 40 kDa and / or 29 kDa fragment of the fibronectin is expressed as a cytoplasmic protein by insertion into a plasmid, such as the pTrc99A plasmid, under the control of the trc promoter.

In another preferred embodiment of the present invention, the primary effector molecule of the invention is an urokinase plasminogen activator (uPA) receptor antagonist. In one embodiment of this embodiment, the antagonist is a dominant negative variant of uPA (Crowley et al., 1993, Proc. Natl. Acad. Sci. USA 90: 5021-5025). In another embodiment of this 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 embodiment of this embodiment, the antagonist is a predominantly negative soluble uPA receptor (Min et al., 1996, Cancer Res. 56: 2428-2433).

In another preferred embodiment of the invention, the primary effector molecule of the invention is a 16 kDa N-terminal fragment of prolactin, or a biologically active fragment thereof, comprising approximately 120 amino acids (the coding sequence for prolactin is GenBank accession number NM - 000948 . In certain embodiments, the prolactin fragment has a Cys58-Ser58 variation that bypasses unwanted cross-linking of the protein by disulfide bonds.

In another preferred embodiment of the present invention, the primary effector molecule of the invention is a 7.8 kDa platelet factor-4 fragment. In a particular embodiment, the 7.8 kDa platelet factor-4 fragment is expressed as a fusion protein whose amino terminus comprises the first 35 amino acids of E. coli beta -glucuronidase. In another embodiment, the heparin-binding lysine of platelet factor-4 is mutated into glutamic acid residues, resulting in a variant protein with potent angiogenesis inhibitory activity (Maione et al., 1991, Cancer Res. 51: 2077- 2083). The coding sequence for platelet factor-4 has GenBank accession number NM_002619.

In another preferred embodiment of the present invention, the primary effector molecule of the invention comprises an angiogenesis inhibiting 13 amino acid fragment of platelet factor-4, an angiogenesis inhibitory factor of 13.40, an angiogenesis inhibiting factor 22 of thrombospondin I Amino acid peptide fragment, an angiogenesis inhibiting 20 amino acid peptide fragment of SPARC, laminin, fibronectin, procollagen or a small angiogenic inhibitor of EGF, or a small peptide antagonist of integrin? _V ? ₃ or a small peptide corresponding to a VEGF receptor . In certain embodiments, the small peptides are expressed side by side to increase protein stability. The sequence of the small peptides is described in Cao, 1998, Prog. Mol. Subcell. Biol. 20: 161-176, wherein VEGF receptor antagonists are described in Soker et al., 1993, J. Biol. Chem. 272: 31582-31588. In a very preferred embodiment, said small peptide comprises RGD or NGR motif. In an embodiment of this embodiment, the RGD or NGR containing peptide is provided on the cell surface of the host bacteria by, for example, fusing the nucleic acid encoding the peptide with an in frame with a nucleic acid encoding one or more extracellular loops of OmpA .

In another particular embodiment, the primary effector molecule of the invention is a cytotoxic polypeptide or peptide, or a functional fragment thereof. Cytotoxic polypeptides or peptides are cytotoxic or cell proliferative to cells by inhibiting cell proliferation through, for example, interference of protein synthesis or through interruption of the cell cycle. Such products can act by cleaving rRNA or ribonucleoprotein, inhibiting the elongation factor, cleaving mRNA, or by other mechanisms that reduce protein synthesis to a level at which the cell is not viable.

Examples of cytotoxic polypeptides or peptides include, but are not limited to, members of the bacteriocin family, verotoxins, cytotoxic necrosis factor 1 (CNF1; e. G. Coli CNF 1 and Vibrio fischeri CNFl), cytotoxic necrosis factor (RIP), pseudomonas endotoxin (RIP), and other cytotoxic agents such as, but not limited to, cytotoxic T lymphocytes (CNF2), Pasteurella myrthiocidotoxin (PMT), hemolysin, , DNA, RNA or protein biosynthesis inhibitors, antisense nucleic acids, other metabolic inhibitors (such as DNA or RNA cleaving molecules such as DNase and ribonuclease, proteases, lipases, phospholipases), prodrug conversion enzymes Thymidine kinase from HSV and bacterial cytosine deaminase), photo-activated porphyrin, lysine, lysine A chain, corn RIP, gelonin, cell lethal expansion toxin, Toxin, diphtheria toxin A chain, tricoxanthin, tritin, USP antiviral protein (PAP), mirabilis antiviral protein (MAP), dianthin 32 and 30, avrine, monodrine, Catalytic inhibitors of protein biosynthesis from cucumber seeds (International Application WO 93/24620), Pseudomonas exotoxin, E. coli heat labile toxin, E. coli heat stable toxin, EaggEC stable toxin-1 (EAST), bioactive fragments of cytotoxins , And others known to those skilled in the art. For E. coli and Salmonella toxin, for example, O'Brian and Holmes, Protein Toxins of Escherichia coli and Salmonella in Escherichia and Salmonella, Cellular and Molecular Biology, Neidhardt et al. (Eds. 2788-2802, ASM Press, Washington, DC].

In a preferred embodiment, the primary effector molecule is a member of the bacteriocin family (Konisky, 1982, Ann. Rev. Microbiol. 36: 125-144), although the member of the bacteriocin family is not a bacteriocin releasing protein (BRP). Examples of members of the bacteriocin family 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, (Jayawardene and Farkas-Himsley, 1970, J. Bacteriology vol. 102 pp. 382-388), pestisin A1122, staphylococcine 1580, butyrylcin 7423, piosin R1 or AP41, megasine A- . Most preferably, the primary effector molecule (s) is selected from the group consisting of collins E3 or V, collins A, E1, E2, Ia, Ib, K, L and M (Konisky, 1982, Ann. Rev. Microbiol. : 125-144) is also suitable as the primary effector molecule (s). In another preferred embodiment of this embodiment, the bacteriocin is chloasin and most preferably is chloacin DF13.

In a preferred embodiment, the primary effector molecule (s) are ColEl, ColE2, ColE3, ColE4, ColE5, ColE6, ColE7, ColE8 or ColE. Colicin E3 (ColE3) has a strong cytotoxic effect on mammalian cells including leukemia cell model system (Fiska et al., 1978, Experientia 35: 406-40) (Smarda et al., 1978, Folia Microbial, 23: 272-277). ColE3 cytotoxicity is a quiescent effect of protein synthesis mediated by the 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 native form, ColE3 is a 60 kDa protein complex in which the 50 kDa and 10 kDa proteins are in a 1: 1 ratio, the larger subunits have nuclease activity and the smaller subunits are inhibitors 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. Thus, in one embodiment, when ColE3 acts as a secondary effector molecule, the larger ColE3 subunit or its active fragment is expressed alone or at a higher level than the smaller subunit. In another embodiment of the present invention, the ColE3 50 kDa toxin and the 10 kDa anti-toxin are encoded on a single plasmid in a toxic attenuated tumor-targeted bacterium, e. G. Salmonella. In this embodiment, the toxin / antitoxin serves as a screening system for Salmonella accompanied by the plasmid, so Salmonella that has lost the plasmid is killed by the toxin. In another embodiment, the 10 kDa anti-toxin is chromosomal, apart from the ColE3 toxin on the plasmid, blocking transmission to other bacteria (see section 5.6 above).

In another preferred embodiment, said primary effector molecule (s) is chloacin DF13. Chloacin DF13 acts in a manner similar to ColE3. The protein complex has a molecular weight of 67 kDa. The individual components are 57 kDa and 9 kDa in size. In addition to its ribonuclease activity, DF13 can cause cell potassium leakage.

In another preferred embodiment, the primary effector molecule (s) is collinsin V (Pugsley, AP and Oudega, B. "Methods for Studying Colicins and Their Plasmids" in Plasmids, a Practical Approach 1987, ed. Hardy, Gilson, L. et al., EMBO J. 9: 3875-3884).

In another embodiment, the primary effector molecule (s) is selected from the group consisting of collicin E2 (double subunit collisin which is structurally similar to ColE3 but has endonuclease activity rather than ribonuclease activity); E1, Ia, Ib or K, which forms an ion-permeable channel to disrupt the proton motions of the cell and lead to apoptosis; Collins L that inhibit protein, DNA & RNA synthesis; Colicin M, which causes cellular decay by altering the osmotic environment of the cell; Festisin A1122, which acts in a manner similar to the action of cholinesin B; Staphycocin 1580, a pore-forming bacteriocin; Butyrylsine 7423, which indirectly inhibits RNA, DNA, and protein synthesis through an unknown target; A protein or phoshin P1 that resembles a bacteriophage tail protein that kills cells by releasing respiration from solute transport; Phiosin AP41 with a cholinic E2-acting mode; (See Konisky, 1982, Ann. Rev. Microbiol. 36: 125-144 for bacteriocin), a phospholipase that causes leaks of intracellular material; Colinsin A (Pugsley, A.P. and Oudega, B. "Methods for Studying Colicins and Their Plasmids" in Plasmids, a Practical Approach 1987, ed.

Thus, the primary effector molecule may include any bacteriocin disclosed herein or known in the art, provided that the bacteriocin is not a bacteriocin releasing protein.

In another specific embodiment, the primary effector molecule of the invention is a tumor suppressor enzyme or a functional fragment thereof. Examples of tumor suppressor enzymes include, but are not limited to, methionases, asparaginases, lipases, phospholipases, proteases, ribonucleases, DNAases, and glycosidases. In a preferred embodiment, the primary effector molecule is methionase.

The primary effector molecules of the present invention are useful for the treatment or prevention of, for example, solid tumors such as carcinoma, melanoma, lymphoma or sarcoma.

The present invention provides nucleic acid molecules encoding primary effector molecules. The present invention also provides nucleic acid molecules encoding one or more primary effector molecule (s) and optionally one or more secondary effector molecule (s). The present invention provides a nucleic acid encoding the effector molecule (s) of the invention operatively linked to a suitable promoter. Optionally, the nucleic acid encoding the effector molecule can be operatively linked to other elements involved in transcription, translation, ubiquitination, stability, and the like.

The length of the nucleic acid molecule encoding the primary effector molecule is from about 6 to about 100,000 base pairs. Preferably, the length of the nucleic acid is from about 20 to about 50,000 base pairs. More preferably, the length of the nucleic acid molecule is from about 20 to about 10,000 base pairs. Even more preferably, the length of the nucleic acid molecule is from about 20 to about 4000 basepairs.

5.3. Secondary effector molecule for simultaneous expression with primary effector molecule

In some embodiments of the invention, the primary effector molecule (e.g., a member of the TNF family, a cytotoxic peptide or polypeptide, an angiogenesis inhibitory factor, or a tumor suppressor enzyme) optionally binds another molecule, Lt; / RTI > The secondary effector molecule promotes the release of the contents of the bacterial vector (which expresses one or more primary effector molecules and optionally one or more secondary effector molecules) into the surrounding environment, providing additional therapeutic value and / or promoting release. As used herein, the term " additional therapeutic value " means that the secondary effector molecule is provided by, for example, the primary effector molecule (s), addition or enhancement to the tumor, cell proliferation inhibition or cytotoxic effect Lt; / RTI > Thus, the secondary effector molecule acts as an additional therapeutic and / or release factor. Preferably, the secondary effector molecule is activated or expressed preferentially or specifically at the desired site, i.e., the tumor site, whether the therapeutic agent or the release factor (or both). In some embodiments, the secondary effector molecule is capable of two actions, i.e., promoting the release of bacterial cell contents (e. G., By promoting bacterial cell lysis or similar dissolution) and providing therapeutic values For example by cytotoxicity against tumor cells). In some non-limiting embodiments, the cytotoxicity of the secondary effector molecule may be mediated by the patient ' s immune system; Thus, such secondary effector molecules can act as immunomodulators.

In some embodiments of the invention, the toxic attenuated tumor-target bacterial vector of the invention is used to express one or more secondary effector molecules having anti-tumor activity, i. E., The expression of the secondary effector molecule kills the tumor or tumor cells Manipulate to inhibit its growth.

The secondary effector molecule is a proteinaceous or nucleic acid molecule. The nucleic acid molecule may be double stranded or single stranded DNA or double stranded or single stranded RNA as well as triple nucleic acid molecules. The nucleic acid molecule may function as a ribozyme or an antisense nucleic acid.

Antisense nucleotides are oligonucleotides that bind in a sequence-specific manner to nucleic acids such as mRNA or DNA. Upon binding to an mRNA having a complementary sequence, antisense prevents translation of the mRNA (US Patent Nos. 5,168,053; 5,190,931; 5,135,917; and 5,087,617). Tritone molecules refer to single strands of DNA that bind to and block transcription of double DNA to form a common linear triple molecule (US Patent No. 5,176,996).

Ribozymes are RNA molecules that specifically inhibit or inhibit cell proliferation or expression by cleaving RNA substrates such as mRNA. There are at least five known classes of ribozymes associated with cleavage and / or linkage of the RNA strand. The ribozyme may catalytically cleave the transcription by targeting any RNA transcription (US Patent Nos. 5,272,262; 5,144,019; 5,168,053; 5,180,818; 5,116,742; and 5,093,246).

As described above for the first effector molecule, the nucleic acid encoding or containing the secondary effector molecule is operably linked to a predetermined promoter, and optionally to other transcription, translation, ubiquitination, stability, and / It is operatively associated with the elements. Furthermore, the secondary effector molecule can be expressed using the same promoter and internal ribosome binding site as the primary effector molecule, or using a different promoter than the primary effector molecule.

The length of the nucleic acid molecule encoding the secondary effector molecule is from about 6 base pairs to about 100,000 base pairs. Preferably, the length of the nucleic acid is from about 20 to about 50,000 base pairs. More preferably, the length of the nucleic acid molecule is from about 20 to about 10,000 base pairs. Even more preferably, the length of the nucleic acid molecule is from about 20 to about 4000 basepairs.

The nucleotide sequences of effector molecules encoding the secondary effector molecules disclosed below are well known (see GenBank). (E. G., PCR), probe hybridization of a genomic or cDNA library, expression library < RTI ID = 0.0 > Antibody selection, chemically synthesized, or by methods obtained from commercial sources.

Nucleic acid molecules and nucleotides for use as disclosed herein can be prepared by any method known to those skilled in the art (see, for example, International Application WO 93/01286, U.S. Patents 5,218,088; 5,175,269; and 5,109,124) . ≪ / RTI > Identification of oligonucleotides and ribozymes for use as antisense agents includes methods well known in the art.

5.3.1. Factors that provide additional therapeutic value

In some embodiments of the invention, the toxic attenuated tumor-target bacterial vector of the present invention, which expresses one or more primary effector molecules and is preferably a Salmonella vector, expresses one or more secondary effector molecules with antitumor activity, That is, the expression of the secondary effector molecule increases the cytotoxicity or the cell proliferation inhibitory action of the primary effector molecule by killing the tumor or tumor cell or inhibiting the growth or spreading of the tumor or tumor cell. In one embodiment, the effect of the secondary effector molecule on the tumor is additional to the effect of the primary effector molecule. In a preferred embodiment, the effects are extremely additive or synergistic, i.e. greater than the sum of the effects of the primary and secondary effector molecules administered separately.

In some embodiments, the secondary effector molecule is cytotoxic or cytostatic to the cell by inhibiting the cell through disruption of protein synthesis or disruption of the cell cycle. Such products can act by, for example, cleaving rRNA or ribonucleoproteins, inhibiting prolonging factors, cleaving mRNA, or by other mechanisms that reduce protein synthesis to a level at which the cell is not viable . Examples of such secondary effector molecules include, but are not limited to, saponins, lysines, avrines, and other ribosome inactivating proteins (RIP).

In another embodiment, the secondary effector molecule is an enzyme that modulates the chemical properties of a prodrug converting enzyme or nucleic acid that encodes the above, i. E., A drug that produces a cytotoxic agent. Exemplary examples of prodrug conversion enzymes are listed on page 33 and Table 2 of WO 96/40238 of Powrec, incorporated herein by reference. WO 96/40238 also teaches a method for preparing a secreted fusion protein comprising such a prodrug conversion enzyme. According to the present invention, the prodrug conversion enzyme does not need to be secreted protein when co-expressed with a release factor such as BRP (see section 5.3.2 below). In a particular embodiment, the prodrug transduction enzyme is cytochrome p450 NADPH oxidoreductase, which acts on mitomycin C and formyramycin (Murray et al., 1994, J. Pharmacol. Exp. Therapeut. 270: 645- 649). In another embodiment, the secondary effector molecule (s) are co-expressed with release factors such as BRP and release cofactors (eg, NADH, NADPH, ATP, etc.) that enhance prodrug conversion enzyme activity. In another embodiment of this embodiment, the secondary effector molecule is co-expressed with an release factor such as BRP to release the activated drug (e. G., A drug that is active in the bacterial cytoplasm or plasma membrane and subsequently released from the bacterial vector) .

In yet another embodiment, the secondary effector molecule is an inhibitor of inducible nitric oxide synthase (NOS) or endothelial nitric oxide synthase. Nitrogen oxides (NO) are involved in the regulation of vascular growth and atherosclerosis. NO is formed from L-arginine by nitric oxide synthase (NOS) and regulates immunity, inflammation and cardiovascular responses.

In another embodiment, the secondary effector molecule is a protein associated with cell proliferation, such as a tumorigenesis gene or growth factor, such as bFGF, int-2, hst-1 / K-FGF, FGF- 2 / FGF-6, FGF-8), or by inhibiting the production or activity of a cell receptor or ligand. The inhibition may be at the level of transcription or translation (mediated by a secondary effector molecule that is a ribozyme or triple DNA), or at the level of protein activity (mediated by a secondary effector molecule, an inhibitor of the growth factor pathway such as a dominant negative variant) Can happen.

In yet another embodiment, the secondary effector molecule is a cytokine, a chemokine or an immunomodulatory protein or a nucleic acid encoding the same, such as interleukin-1 (IL-1), interleukin-2 (IL- (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10), interleukin-15 (EMAP2), GM-CSF, IFN- ?, IFN- ?, MIP-3 ?, MIP-3? Or MHC genes such as HLA-B7. The delivery of such immunomodulatory effector molecules increases the latency of host-mediated autoimmunity by modulating the immune system. On the one hand, nucleic acid molecules encoding the ligands for both co-stimulatory molecules, e.g., CD28 and CTLA-4, B7.1 and B7.2, can also be delivered to enhance T cell mediated immunity. Yet another immunomodulator is? -1,3-galactosyltransferase, which is expressed on tumor cells to allow complement-mediated cell killing. Moreover, another immunomodulator is a tumor-associated antigen, that is, a molecule that is expressed by tumor cells specifically and not expressed in non-cancerous corresponding cells, or expressed in tumor cells at a higher level than non-cancerous corresponding cells. Exemplary examples of tumor-associated antigens are described in Kuby, Immunology, W.H. Freeman & Company, New York, NY, 1st Ed. (1992), pp. 515-520. Other examples of tumor-associated antigens are known to those skilled in the art.

In another embodiment, the secondary effector molecule is a Flt-3 ligand or a nucleic acid encoding said molecule. In another embodiment, the secondary molecule is BRP.

In certain embodiments, the secondary effector molecule is not a member of the TNF family when the primary effector molecule is a member of the TNF family. In another particular embodiment, the secondary effector molecule is not an angiogenesis inhibitory factor when the primary effector molecule is an angiogenesis inhibitory factor. In another specific embodiment, the secondary effector molecule is not a cytotoxic peptide or polypeptide when the secondary effector molecule is a cytotoxic peptide or polypeptide. In another particular embodiment, the secondary effector molecule is not a tumor suppressor enzyme when the primary effector molecule is a tumor suppressor enzyme.

5.3.2. Factors promoting the release of the anti-tumor effector into the tumor environment

In some other embodiments of the invention, the toxic attenuated tumor-target bacterial vector of the present invention, which expresses one or more primary effector molecules and is preferably a salmonella vector, infects the bacterial cell membrane (s) Enhancing the delivery of the primary and / or secondary effector molecule (s) by expressing one or more secondary effector molecules that act to enhance the release of intracellular components into the tumor site. These secondary effector molecules that penetrate bacterial cells or enhance their release are referred to as " release factors ". In some embodiments, the release factor also advantageously has anti-tumor activity.

The release factor expressed by the bacterial vector of the present invention may be endogenous or exogenous to the altered toxicant weakened tumor target bacteria (e.g., the toxic weakened tumor target bacterium is encoded by a non-native nucleic acid) . The release factor may be encoded by a nucleic acid comprising a plasmid, or by a nucleic acid integrated into the genome of a toxic attenuated tumor target bacterium. The release factor may be encoded by the same nucleic acid or plasmid encoding the primary effector molecule, or may be encoded by a separate nucleic acid or plasmid. The release factor may be encoded by the same nucleic acid or plasmid encoding the secondary effector molecule, or may be encoded by a separate nucleic acid or plasmid. In one embodiment, the release factor is expressed in a cell that also expresses a fusion protein comprising a primary effector molecule fused to an Omp-type protein. In this embodiment, co-expression of the release factor enhances the release of the fusion protein from the plasma membrane space.

In a preferred embodiment, such factors are either bacteriocin-releasing proteins or BRPs (hereinafter generally referred to as BRPs). The BRPs used in the present invention may originate from any source known in the art, including, but not limited to, the chloacin DF13 plasmid, one of the cholinic E1-E9 plasmids, or the colistin A, N or D plasmid have. In a preferred embodiment, the BRP is of chloacin DF13 (pCloDF13 BRP).

Generally, BRP is a 45 to 52 amino acid peptide that is initially synthesized as a precursor molecule (PreBRP) having a signal sequence that is not cleaved by a signal endopeptidase. BRP activity is believed to be mediated, at least in part, by the detergent resistant outer membrane phospholipase (PldA), and is usually associated with increased degradation of the outer membrane phospholipid (see generally Walp et al., 1995, FEMS Microbiology Review 17: 381-399]. Although not limited to the mechanism, BRP promotes preferential release of plasma membrane components, but release of cytosolic components is also detected to a lesser extent. When properly overexpressed, BRP can induce quasi-lysis and high release of cytoplasmic components by breaking down the bacterial membranes. It is also believed that when the BRP is expressed at an ultra-high level, the protein causes bacterial cell lysis and delivers cell contents by lytic release. In this embodiment, BRP expression may be correlated with BRP activity (e. G., Release of bacterial content). For example, ultra high BRP activity results in bacterial cell lysis of virtually all bacteria. Thus, " ultra-high expression " as used herein is defined as the level of expression of BRP that produces bacterial cell lysis of substantially all bacteria. Suitable BRP activity is associated with partial release or enhancement of bacterial content without unconditional dissolution of the bacterium relative to control bacteria that do not express BRP. Thus, in the above embodiments, moderate overexpression of BRP is defined as an expression level that enhances the release of cellular components substantially without cytolysis of all bacteria. Substantially all of the bacteria used in the present invention are bacteria having 60% or more, preferably 70% or more, more preferably 80%, even more preferably 90% or more, and most preferably 90 to 100%.

In certain embodiments of the present invention, the BRP protein is a pCloDF13 BRP variant that has a dissolution effect unrelated to its protein release action, thereby enhancing protein release without bacterial dissolution (van der Wal et al., 1998, App. Microbiol., 64: 392-398). This embodiment reduces the need for frequent administration of the vector while allowing extended protein release from bacterial vectors. In another specific embodiment, the BRP of the present invention is a shortened C-terminal pCloDF13 BRP, which causes cell lysis in addition to protein release (Luirink et al., 1989, J. Bacteriol. 171: 2673-2679).

In another embodiment of the present invention, the enhanced release system comprises overexpression of a protein of the protein (Sugawara, E. and Nikaido, H., 1992, J. Biol. Chem. 267: 2507-11).

In some embodiments, when the BRP is expressed by the bacterial vectors of the invention, the BRP may be endogenous or exogenous to the altered toxicant weakened tumor target bacteria (e. G., Toxic attenuated tumor target bacteria Which is encoded by a nucleic acid that is not a native. The BRP may be encoded by a nucleic acid containing a plasmid, or by a nucleic acid integrated into the genome of a toxic attenuated tumor-targeted bacterium. The BRP may be encoded by the same nucleic acid or plasmid encoding the primary effector molecule, or may be encoded by a separate nucleic acid or plasmid. The BRP may be encoded by the same nucleic acid or plasmid encoding the secondary effector molecule, or may be encoded by a separate nucleic acid or plasmid. In one embodiment, the BRP-type protein is expressed in a cell that also expresses a fusion protein comprising a primary effector molecule fused to an Omp-type protein. In this embodiment, co-expression of the BRP allows enhanced release of the fusion protein.

In certain preferred embodiments of the invention, the BRP-encoded nucleic acid is encoded by a cholinesin plasmid. In another specific embodiment of the present invention, the BRP encoded nucleic acid comprises a native BRP promoter, wherein the promoter is capable of inhibiting stress in its normal host (BRP, for enterococcus aurea) (e. Which is the SOS promoter that responds to the promoter of the Salmonella, although it is a partial component in Salmonella. In a preferred embodiment, the BRP-encoded 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, for example, Lombardo et al., 1997, J. Bacteriol. -17).

On the other hand, the promoter may be an antibiotic-induced promoter, for example the tet promoter of Tn10 transposon. In a preferred embodiment, the tet promoter is a monomer and the monomer reacts in a totally or other manner in the presence of tetracyclone or its analogs, such as dioxycyclene and anhydrous tetracycline, and genetically stable on- to provide. Such multimers react in a graded manner to the presence of tetracycline and provide a more steerable system for controlling the effector molecule levels. The promoter activity will then be induced by administering an appropriate dose of tetracycline to the patient treated with the toxic attenuated tumor target bacteria of the present invention. The tet inducible expression system was initially introduced into a eukaryotic system, such as Schizosaccaromyces pombe (Faryar and Gatz, 1992, Current Genetics 21: 345-349) and mammalian cells (Lang and Feingold, 1996 , Gene 168: 169-171), recent studies have extended its applicability to bacterial cells. For example, Stieger et al. (1999, Gene 226: 243-252) demonstrated 80-fold induction of firefly luciferase against tet induction upon operative binding to the tet promoter. The advantage of the promoter is that it is induced to a very low level of tetracycline, approximately one-tenth the amount required for antibiotic activity.

5.4. Derivatives and homologs

The present invention further comprises a bacterial vector that has been modified to encode or transfer a derivative, e. G., A non-limiting primary and / or secondary effector molecule, or a fragment, homolog or variant of the nucleic acid encoding it. The derivative, analog or variant is operably active, for example, it may exhibit one or more agonistic activities associated with a full-length wild-type effector molecule. By way of example, such derivatives, homologues or variants as those with the desired therapeutic properties can be used to inhibit tumor growth or tumor cell spread (metastasis). Derivatives or analogs of effector molecules can be tested for activity desired by art-known procedures, e. G., The procedures described herein.

In particular, the variant can be produced by altering the effector molecule coding sequence by substitution, addition (e.g. insertion) or deletion to provide a molecule with the same or increased antitumoral activity relative to the wild type effector molecule. For example, variants of the invention include, but are not limited to, an altered sequence in which the amino acid sequence of the effector molecule (acting as a primary amino acid sequence) produces a silent change using an equivalent amino acid residue in place of the residue in the sequence, Which have at least one conservative substitution).

Any primary or secondary effector encoded nucleic acid of mammalian origin may be altered using methods known in the art using bacterial codon usage. Preferred codon usage is described in Current Protocols in Molecular Biology, Green Publishing Associates, Inc., and John Wiley & New York, and Zhang et al., 1991, Gene 105: 61-72.

In certain embodiments, derivatives, homologues, or variants of the primary or secondary molecule are hybridized to filter-bound DNA at stringent conditions, e. G., At about 45 C in 6X sodium chloride / sodium citrate (SSC) Hybridized to filter-bound DNA at about 45 ° C in stringent conditions, such as 6x SSC, and then hybridized at about 68 ° C in 0.1x SCC / 0.2% SDS, or under other stringent hybridization conditions known to those skilled in the art, / RTI > hybridizes to a nucleotide sequence encoding the primary or secondary molecule under conditions that wash at a temperature of about < RTI ID = 0.0 > , eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, p. 6.3.1-6.3.6 and 2.10.3 Please).

Derivatives or homologs of a primary or secondary effector molecule include, but are not limited to, molecules that are substantially equivalent to the primary or secondary effector molecule or fragment thereof (e.g., in various embodiments, have the same size Greater than or equal to 60% or 70% or 80% or 90% or 95% identical to an aligned sequence that has been subjected to alignment by amino acid sequences or by computer program programs known in the art), or that the encoded nucleic acid has a high stringency , Molecules that contain regions that can hybridize under the appropriate stringency or low stringency conditions to the effector molecule protein effector molecular coding sequence.

To determine the agreement between two amino acid sequences of two nucleic acids, e. G., The sequence of the first effector molecule and other known sequences, the sequences are aligned for optimal comparison purposes (e. G., A secondary amino acid or nucleic acid For optimal alignment with the sequence, a gap can be introduced in the sequence of the primary amino acid or nucleic acid sequence). The amino acid residue or nucleotide is then compared at the corresponding amino acid position or nucleotide position. If the position of the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position of the second sequence, then the molecules match at this position. The match rate between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent match = number of identical positions / total number of positions (e.g., overlapping positions) x100). In one embodiment, the two sequences have the same length.

The measurement of the agreement rate between two sequences can be performed using a mathematical operation. Preferred non-limiting examples of mathematical operations used for comparison of two sequences are described in Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, which is described in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an operation is described in Altschul, et al., 1990, J. Mol. Biol. 215: 403-410. ≪ / RTI > BLAST nucleotide studies can be performed with the NBLAST program (score = 100, wordlength = 12) to obtain nucleotide sequences consistent with the nucleic acid molecules of the present invention. BLAST protein studies can be performed with the XBLAST program (score = 50, wordlength = 3) to obtain amino acid sequences consistent with the protein molecules of the present invention. In order to obtain interstitial alignment for comparison purposes, the interstitial BLAST is described in Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402. On the other hand, it is possible to perform repeated studies using PSI-Blast to detect distant relationships between molecules. If you are using BLAST, Gapped BLAST and PSI-Blast programs, you can use default variables for each program (eg XBLAST and NBLAST). See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical operation used in the comparison of sequences is the computation of Myers and Miller, CABIOS (1989). The above operation is associated with the ALIGN program (Version 2.0), which is part of the GCG sequence alignment software package. If the ALIGN program is used for comparison of amino acid sequences, a PAM120 weight residue table, a gap spacing penalty of 12, and a gap penumbra of 4 can be used. Additional computations for sequencing are known in the art and are described in Torellis and Robotti, 1994, Comput. Appl. Biosci., 10: 3-5; ADVANCE and ADAM; And Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. 85: 2444-8. In FASTA, ktup is a control option that sets sensitivity and research speed. When ktup is 2, the similar regions in the two compared sequences are found by examining pairs of aligned residues; If ktup is 1, a single aligned amino acid is checked. The ktup may be set to 2 or 1 for the protein sequence, or 1 to 6 for the DNA sequence. If ktup is not specified, the default is 2 for protein and 6 for DNA. An additional description of the FASTA variable is given at http://bioweb.pasteur.fr/docs/man/man/fasta.1.htm1#sect2, which is incorporated herein by reference.

On the other hand, protein sequence alignment was performed as described in Higgins et al., 1996, Methods Enzymol. 266: 383-402. ≪ / RTI >

The agreement rate between the two sequences can be measured using techniques similar to those described above without allowing or allowing gaps. In the calculation of the match rate, only exact matches are counted.

Primary effector molecules or secondary effector molecules, or derivatives or analogs thereof, can be prepared by a variety of methods known in the art. The manipulations in this preparation can be performed at the nucleic acid or protein level. For example, the cloned effector molecule coding sequence encoding the effector molecule can be altered by any of a number of strategies known in the art (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). The sequence may be cleaved at a suitable site using a restriction endonuclease (s), followed by further enzymatic modification, isolation, and in vitro coupling. In the manufacture of a modified effector molecule encoding a derivative or homologue of a primary or secondary effector molecule, said altered effector molecule coding sequence is selected from the group consisting of the above-mentioned effector molecule coding sequence regions in which the desired primary or secondary effector molecule activity is encoded, Care must be taken to remain within the same translation reading frame as the native protein not interrupted by the translation stop signal.

It is also contemplated that nucleic acid sequences encoding effector molecules may be used to produce and / or destroy translation, initiation and / or termination sequences, or to generate variants in the coding region and / or to create new restriction endonuclease sites, Can be mutagenized in vivo or ex vivo in order to promote further in vitro modification. In certain preferred embodiments, mutations are generated to produce, for example, more potent variants of the effector molecule encoding nucleic acid sequence. (Hutchinson et al., 1978, J. Biol. Chem. 253: 6551), TAB (TM) linker (Pharmacia), PCR using mutation-containing primers, and the like can be used. In a preferred embodiment, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues of the effector molecule. A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue having a side chain with a pseudo-charge. Sequences of amino acid residues having pseudo-charged side chains are defined in the art. These sequences include, but are not limited to, basic side chains such as lysine, arginine, histidine, acidic side chains such as aspartic acid, glutamic acid, uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine ), Nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, beta-branched side chains such as TM leonin, valine, isoleucine and aromatic side chains such as tyrosine, phenylalanine, Tryptophan, histidine). On the one hand, the mutation can be introduced randomly along all or part of the coding sequence, for example, by a saturation mutation, and the resulting variant can be screened for biological activity to identify variants with activity. Following mutation, the encoded protein can be expressed and the activity of the protein measured.

In another embodiment, the effector molecules or fusion proteins of the invention are made to contain a protease cleavage site.

5.5. Fusion protein

In some embodiments, the invention provides a primary or secondary effector molecule made as a fusion protein (e. G., Covalently linked to a different protein). The present invention provides a nucleic acid encoding such a fusion protein. In some other embodiments of the invention, the nucleic acid encoding the fusion protein of the invention is operably linked to a suitable promoter.

In a specific embodiment, the effector molecule is linked to the effector molecule or fragment thereof (preferably at least the domain or motif of said effector molecule, or the 5 < RTI ID = 0.0 > Or more, 10 or more, 25 or more, 50 or more, 75 or more, or 100 or more amino acids). In certain embodiments, the fusion comprises the fusion of two or more, six or more, at least ten, at least twenty, at least thirty, at least fifty, at least 75, or at least 100 contiguous amino acids of a heterologous polypeptide or fragment thereof . In one embodiment, such a fusion protein or chimeric protein as described above comprises a nucleic acid encoding a primary effector molecule conjugated to the coding sequence of a different protein, such as a TNF-encoding sequence, an angiogenesis inhibiting factor encoding sequence, An enzyme encoding sequence or a cytotoxic polypeptide encoding sequence). Such chimeric products are prepared by linking appropriate nucleic acid sequences encoding the amino acid sequence of interest to a suitable coding frame by methods known in the art and the chimeric product is expressed by methods known in the art Can be expressed in a vehicle. A chimeric nucleic acid comprising a nucleic acid portion encoding an effector molecule fused to any heterologous polypeptide-encoding sequence can be produced. Particular embodiments include fragments of primary or secondary effector molecules of 5 or more, 10 or more, 25 or more, 50 or more, or 100 or more amino acids, or fragments of one or more of the full-length primary or secondary effector molecules Lt; RTI ID = 0.0 > a < / RTI > activity.

In certain embodiments, the fusion protein comprises an affinity label such as a hexahistidine label, or other affinity label that can be used for purification, isolation, identification or expression analysis. In another particular embodiment, the fusion protein comprises a protease cleavage site, such as a metalloprotease or serine cleavage site. In this embodiment, in some cases it is preferred that the protease site corresponding to the protease active at the tumor site is constituted in the fusion protein of the invention. In various embodiments, the effector molecule is an Omp-type protein or fragment thereof (e.g., a signal sequence, a leader sequence, a plasma membrane domain, a transmembrane domain, a plurality of transmembrane domains, &Quot; see section 3.1).

In a preferred embodiment, the effector molecule (primary or secondary) of the invention is expressed as a fusion protein with an outer membrane protein (Omp-type protein). Bacterial outer membrane proteins are the absolutely necessary membrane proteins of bacterial outer membranes, have domains across multiple membranes, and are often bound to one or more lipid short term. The outer membrane proteins are first expressed in a precursor form (pro-Omp) by an amino-terminal signal peptide that, when cleaved by the signal peptidase to produce a mature protein, sends the protein to the outer membrane. In one embodiment, the effector molecule is made as a fusion protein with an Omp-type protein. In this embodiment, the primary effector molecule is improved in delivery of the bacteria to the outer membrane. Although not intending to be limited to the mechanism, the inventors of the present invention have considered that the Omp-type protein acts as a factor for fixing or mooring the effector molecule to the outer membrane, or for arranging the effector molecule on the bacterial outer membrane . In one embodiment, fusion of the effector molecule to the Omp-type protein is used to improve the localization of the effector molecule to the plasma membrane. In yet other embodiments, fusion of the effector molecule to the Omp-type protein is used to enhance the release of the effector molecule. In a particular embodiment, the Omp-type protein is selected from the group consisting of OmpA, OmpB, OmpC, OmpD, OmpE, OmpF, OmpT, , Peptidoglycan-associated lipoprotein (PAL), FepA, FhuA, NmpA, NmpB, NmpC and major extracellular lipoproteins (e.g., LPP). In some embodiments of the invention, the signal sequence is made more hydrophobic (e. G., By insertion of an amino acid in the signal sequence or by substitution with a hydrophobic amino acid, e. G., Leucine). As an illustrative example, see Sections 7.1 to 7.4 below.

In another embodiment of the invention, the fusion protein of the invention comprises a proteolytic cleavage site. The proteolytic cleavage site may be endogenous to the effector molecule or endogenous to an Omp-type protein, or may constitute a fusion protein. In some specific embodiments, the Omp-type protein of the present invention is a hybrid Omp comprising components originating from separate proteins.

In an exemplary embodiment of this embodiment, the Omp-type protein is OmpA; The same principle used to make OmpA-type fusion proteins is applied to other Omp fusion proteins while remembering the structural form of a particular Omp-type protein.

For example, the native Omp protein contains eight semi-parallel transmembrane beta -strands in the 170 amino acid N-terminal domain of the protein. There are extracellular or intracellular loops in the insertion direction of the transmembrane domain between each pair of transmembrane domains. The C-terminal domain consists of 155 amino acids that are placed within the cell and come into contact with a peptidoglycan that presumably occupies the plasma membrane space. An expression vector promoting the production of OmpA fusion protein was generated. For example, Hobb et al. (Hobom et al., 1995, Dev. Biol. Strand. 84: 255-262) inserts into the sequence encoding the third or fourth extracellular loop, RTI ID = 0.0 > open reading frame with < / RTI >

In one embodiment of the invention, a portion of the OmpA fusion protein containing the primary effector molecule has enhanced expression in the plasma membrane. In one embodiment of this embodiment, the fusion protein comprises a signal sequence prior to maturation, or a signal sequence followed by one or more domains of OmpA located at the N-terminus of the primary effector molecule. The signal sequence is cleaved from the mature protein and is not present in the protein. In another embodiment of this embodiment, the primary effector molecule unequally divides the number of domains across the membrane of the OmpA used, so long as the fusion protein is stable at the N-terminus of the OmpA fusion . In yet another embodiment of this embodiment, the primary effector molecule is located between the N- and C-terminal domains of OmpA so that the soluble plasma membrane protein contains the primary effector molecule upon cleavage by the plasma membrane protease in the plasma membrane do. In some embodiments of such embodiments, it is preferred that the bacterial vector expressing the plasma membrane primary effector molecule also co-express BRP to enhance the release of the effector molecule from the bacterial cell.

In another embodiment of the invention, part of the OmpA fusion protein containing the primary effector molecule is on the surface of the extracellular bacteria. In one embodiment of this embodiment, the fusion protein comprises a domain spanning an even or odd membrane of OmpA located at the N-terminus of the primary effector molecule. In another embodiment of this embodiment, the primary effector molecule is located between two extracellular loops of OmpA to be provided to the tumor cells by the bacterial cells. In a particular embodiment, the invention provides an expression plasmid of an effector molecule fusion protein at the bacterial extracellular surface. For example, the plasmid designated Trc (lpp) ompA contains the trc promoter-engineered lipopoly protein (lpp) anchoring sequence fused to the truncated ompA transmembrane sequence. As another example, the plasmid is designated TrcompA and contains the trc promoter-engineered ompA-encoded signal sequence. Such plasmids can be made to include nucleic acids encoding one or more effector molecule (s) of the present invention.

Optionally, the effector molecule is on the front or side of the cleavage sites consistent with the metalloprotease or serine protease enriched in the tumor for release of the molecule into the tumor environment. Whether the primary effector molecule is in front of or at the side of the protease cleavage site varies depending on whether or not the upstream is at the end of the fusion protein or is internal.

Similar fusion proteins can be produced using any of the above Omp type proteins using the strategy described above with respect to OmpA. In the production of such fusion proteins, selection of the Omp-type protein moiety fused to the effector molecule, as will be apparent to those skilled in the art, is preferably performed at a desired location for expression of the effector molecule (e. G., The plasma membrane, Cell membrane boundary, etc.). Fabrication of such fusion proteins for primary effector molecules disclosed herein is also suitable for secondary effector molecules.

In a preferred embodiment, the effector molecule is fused to the peripeptide. Peripeptides used in fusion proteins have been shown to facilitate delivery of polypeptides or peptides of interest to virtually any cell within the diffusion limit for its production or introduction (see, e.g., Bayley, 1999, Nature Biotechnology 17: 8, pp. 1066-1067; Fernandez et al., 1998, Nature Biotechnology 16: 418-420; and Derossi et al., 1998, Trends Cell Biol. 8: 84-87). Thus, manipulating tumor-targeted bacteria that are toxic to express a fusion protein comprising a peripeptide and an effector molecule enhances the ability of the effector molecule to be internalized by the tumor cells. In certain embodiments, the toxic attenuated tumor-targeted bacteria are engineered to express a nucleic acid molecule encoding a fusion protein comprising a peripeptide and an effector molecule. In another embodiment, the toxic attenuated tumor target bacteria is engineered to express one or more nucleic acid molecules encoding the one or more fusion proteins comprising the peripeptide and effector molecule. According to these embodiments, the effector molecule may be a primary or secondary effector molecule. Examples of peripeptides include, but are not limited to, peptides derived from the HIV TAT protein, Antennapedia homeodomain (pennelaxine), Kaposi fibroblast growth factor (FGF), membrane-translocation sequence (MTS), herpes simplex virus VP22, poly 6H), polylysine (e.g., hexaricin; 6K), and polyarginine (e.g., hexaarginine; 6R) (see, for example, Blanke et al., 1996 , Proc Natl Acad Sci USA 93: 8437-8442).

In another preferred embodiment, the fusion protein also comprises signal peptides, peripeptides and effector molecules. In certain embodiments of the above embodiments, the toxic attenuated tumor-targeting bacteria are engineered to express one or more nucleic acid molecules encoding one or more fusion proteins comprising signal peptides, peripeptides, and effector molecules. According to this embodiment, the effector molecule is a primary or secondary effector molecule.

In another preferred embodiment, the fusion protein delivers a signal peptide, proteolytic cleavage site, peripeptide and effector molecule to the solid tumor by a toxic attenuated tumor-targeted bacterium. In certain embodiments, the toxic attenuated tumor-targeted bacteria are engineered to express one or more nucleic acid molecules that encode one or more fusion proteins, including signal peptides, proteolytic cleavage sites, peripeptides, and effector molecules. According to this embodiment, the effector molecule may be a primary or secondary effector molecule.

As a non-limiting example, cholinergic activity can be enhanced by the addition of internalizing peptides derived from HIV TAT, herpes simplex virus VP22, antenna paedias, 6H, 6K and 6R. The fusion may be C-terminal, N-terminal or internal. Internal fusion is particularly preferred when the fusion follows the N-terminal signal sequence cleavage peptide. The fusion protein may further comprise an N-terminal signal sequence, for example an OmpA or C-terminal signal sequence, for example hlyA.

In a preferred embodiment, the effector molecule is fused to the delivery portion of the toxin. A variety of toxins are known to have self-transfer ability, wherein one part of the toxin acts as a transfer factor for the second part of the toxin. See, e.g., Ballard et al., 1996, Proc. Natl. Acad. Sci. USA 93: 12531-12534) also found that anthrax protector (PA), which mediates the inflow of lethal factor (LF) and edema factor into the cytosol compartment of mammalian cells, also has a truncated form of LF (LFn; 255 amino acid residues) To mediate the participation of protein fusion in the cell. Thus, the effector molecules of the present invention may be used in conjunction with the LFn or other toxin systems, such as, but not limited to, the diphtheria toxin A chain moiety 1-193 (Blanke et al., 1996, Proc. Natl. Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, , CNF, cell lethal expansion toxin, alpha-hemolysin, beta-hemolysin and SheA hemolysin (O'Brian and Holmes, 1996, Protein Toxins of Escherichia coli and Salmonella. Cellular and Molecular Biology, Neidhardt et al. eds.), pp. 2788-2802, ASM Press, Washington, DC). In certain embodiments, the primary effector molecule is fused to the transmembrane portion of the toxin. In another particular embodiment, the secondary effector molecule is fused to the transmembrane portion of the toxin.

Methods for making fusion proteins for expression in bacteria are well known in the art, and such methods are within the scope of the present invention (see, for example, Makrides, S., 1996 , Microbiol. Revs 60: 512-538]).

5.6. Expression vehicle

The invention provides engineered toxicant weakened tumor target bacteria encoding one or more primary effector molecules and optionally one or more secondary effector molecules. The present invention provides a toxic attenuated tumor-targeting bacterium comprising the plasmid or the effector molecule (s) encoded by the transfection nucleic acid. In a preferred embodiment of the present invention, the toxic weakened tumor target bacterium is Salmonella. When one or more effector molecules (e.g., primary or secondary) are expressed in a toxic attenuated tumor-targeted bacterium, such as salmonella, the effector molecules may be expressed by the same plasmid or nucleic acid, or in one or more plasmids or nucleic acids . The present invention also provides a toxic attenuated tumor-targeting bacterium comprising an effector molecule (s) encoded by a nucleic acid integrated into the bacterial genome. The integrated effector molecule (s) may be endogenous to a toxic attenuated tumor-targeted bacterium, e. G. Salmonella, or the toxic attenuated tumor may be incorporated into the genome of the toxic attenuated tumor- (E. G., By introduction of a nucleic acid that encodes the effector molecule, e. G., A plasmid, a transfection nucleic acid, a transposon, etc.) into the target bacterium. In a preferred embodiment of the present invention, the toxic weakened tumor target bacterium is Salmonella. The present invention provides nucleic acid molecules encoding an effector molecule whose nucleic acid is operatively linked to a suitable promoter. The promoter operably linked to the nucleic acid encoding the effector molecule may be homologous (i.e., natural) or heterologous (i.e., the nucleic acid encoding the effector molecule is not native).

A nucleotide sequence encoding an effector molecule of the invention or an operative activity homologue or fragment or other derivative thereof may be inserted into a suitable expression vehicle such as a plasmid containing elements necessary for transcription and translation of the inserted protein coding sequence . The necessary transcription and translation signals can be supplied by the effector molecule and / or its side regions. On the one hand, a constitutive DNA sequence encoding a protein of interest in an expression vehicle can be translated into a suitable translation initiation and termination signal by a functional promoter using one of a variety of methods known in the art for the manipulation of DNA And the like. In general, Sambrook et al., 1989, Molecular Biology: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY; Ausubel et al., 1995, Current Protocols in Molecular Biology, Greene Publishing, New York, NY). These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombination (gene recombination). The present invention provides nucleic acid molecules encoding an effector molecule whose nucleic acid is operatively linked to a suitable promoter.

The present invention also provides a toxic attenuated tumor-targeting bacterium that has been modified to encode one or more fusion proteins and optionally one or more effector molecules. The present invention provides a toxic attenuated tumor-targeting bacterium comprising a fusion protein encoded by a plasmid or a transfected nucleic acid. When the one or more fusion protein and / or effector molecules (e.g., primary or secondary) are expressed in a toxic attenuated tumor-targeted bacterium, such as salmonella, the fusion protein and / Or by nucleic acids, or by one or more plasmids or nucleic acids. The present invention also provides a toxic attenuated tumor-targeting bacterium comprising a fusion protein encoded by a nucleic acid integrated into the bacterial genome. The present invention also provides a nucleic acid molecule encoding a fusion protein in which the nucleic acid molecule is operatively linked to a suitable promoter. The nucleotide sequence encoding the fusion protein of the present invention can be inserted into a suitable expression vehicle, for example, a plasmid containing elements necessary for transcription and translation of the inserted protein coding sequence.

In some specific embodiments of the invention, the expression vehicle of the invention is a plasmid. A greater number of suitable plasmids are known to those skilled in the art and are commercially available for the production of the recombinant constructs of the present invention.

Such commercial plasmids include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 " backbone " portions are joined with suitable promoters and structural sequences to be expressed. pBR322 is considered a low copy number plasmid. If higher levels of expression are desired, the plasmid may be a plasmid with a high copy number, for example a pUC backbone. pUC plasmids include, but are not limited to pUC19 (Yanisch-Perron et al., 1985, Gene 33: 103-119) and pBluescript (Stratagene).

The following plasmids are provided as examples and can be used in conjunction with the methods of the present invention. Bacteria: pBs, phagescript, phiX174, pbluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Commercial plasmids with pBR322 " backbone ", such as pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMl (Promega Biotec, Madison, Wis., USA) can also be used. These are combined with suitable promoters and structural sequences to be expressed. pCET, pTS (described in Section 6 of this volume).

In a particular embodiment of the invention, the plasmid encoding the effector molecule is a pTS-TNF-a plasmid, a pTS-BRP plasmid, or a pTS-BRPTNF-a plasmid (disclosed in Section 6 of this disclosure).

In a particular embodiment of the invention, the fusion protein of the invention for secretion into the plasma membrane space comprising the OmpA signal sequence and the primary effector molecule is encoded by the plasmid pIN-III-ompA-Hind, Lt; RTI ID = 0.0 > ompA < / RTI > signal sequence of a linker sequence capable of cloning the coding sequence of the effector molecule. In a preferred specific embodiment, the lac inducible promoter of the pIN-III-ompA-Hind vector is replaced with a pepT or tet promoter (Rentier-Delrue et al. (1988), Nuc. Acids Res. 16: 8726).

The present invention also provides transposon-mediated chromosomal integration of effector molecules. Any transposon plasmid known in the art can be used in the method of the present invention as long as the nucleic acid encoding the effector molecule can be produced from a transposon cassette. For example, the present invention provides a transposon comprising a transposon or a mini transposon, and an MCS.

In some embodiments of the invention, the plasmid of the invention comprises a transposon plasmid, i. E., A transposon inserted with a sequence encoding a molecule of interest. The transposon plasmid contains a transposon cassette in which the cassette is integrated into the bacterial genome. Thus, a nucleic acid encoding the effector molecule or a fusion protein thereof is inserted into the transposon cassette. Thus, transposon insertion integrates the cassette into the bacterial genome. The coding sequence of the effector molecule may be operably linked to the promoter, or may be free of the promoter. In the latter case, expression of the selectable marker is controlled by a promoter at the insertion site of the transposon into the bacterial genome. Colonies of transposon-inserted bacteria are screened for levels of expression of cytokines that are sufficient to promote expression levels that meet the requirements of the invention, e.g., tumor cytotoxicity, stasis, or regression.

In some embodiments, the transposon plasmid comprises, in addition to the transposon, a transposase gene that promotes the insertion of the transposon into the bacterial genome without accompanying the transposon together outside the transcribed repeats of the transposon, Resulting in a bacterial strain in which stable transposon insertion is performed.

The transposons used by the present invention include, but are not limited to, Tn7, Tn9, Tn10, and Tn15. In a preferred embodiment, the transposon plasmid is pNK2883 (ATCC) with an ampicillin resistance gene located outside the Tn10 insertion element, and a nucleic acid encoding one or more effector molecule (s) is inserted between two Tn10 insertion elements Lt; / RTI > cassette). Preferably, the construct is made such that additional sequences encoding other elements are inserted between the two Tn10 insertion elements. In certain embodiments, such elements may optionally include (1) a promoter-free copy that is a selectable marker (eg, SerC, AroA, etc.) for positive selection of bacteria containing the plasmid; (2) a BRP gene, (3) a nucleic acid encoding one or more primary effector molecule (s) (e.g., TNF-a, or a fusion protein thereof, such as OmpA-TNF-a fusion) A promoter for a combined effector molecule (e.g., trc), and (4) a terminator for a nucleic acid that encodes one or more effector molecule (s).

In one embodiment, after the plasmid is appropriately engineered and the ampicillin resistant properties encoded by the plasmid are used to screen for clones with the desired construct, the antibiotic selectivity is removed via plasmid loss, To do this, strains into which the chromosomal transposon is inserted are selected (for example by smearing on a selection medium).

In another specific embodiment, a plasmid pTS comprising a modified target specific transposase gene and a mintransposon containing a promoter-free serC gene and a coding sequence for MCS is used. In another specific embodiment, a plasmid pTS-BRP comprising a modified target specific transposase gene and a mintransposon containing the coding sequence of the promoter-free serC gene and an alkylating agent-induced bacteriocin release factor and MCS is used.

In a preferred embodiment, the transposon plasmid for selection of transposon-mediated chromosomal components comprises

a) a transposable gene for transposon ablation and integration, located outside of the transposon insertion sequence (e.g., outside the transposon cassette);

b) a wild-type coding sequence corresponding to the ribosome binding site and terminator of the wild-type gene (preferably the left TN10 trans < RTI ID = 0.0 > Posson insertion sequence);

c) optionally, a nucleic acid sequence encoding the enhancing nucleic acid (e.g., BRP) between the right and left insertions; And

d) a multiple cloning site (MCS) located between the right and left insert sequences, containing a unique restriction site for binding of the effector molecule in the plasmid, said MCS preferably being immediately behind the release enhancing nucleic acid (if used) TN10 < / RTI > insertion sequence)

.

In another embodiment, gene disruption resulting from random integration of effector molecules onto the host chromosome demonstrates the suitability of the gene location for effector insertion.

In yet another embodiment, the expression vehicle is an extrachromosomal plasmid that is stable, i.e., self-sustaining, without the need for antibiotic screening. In one non-limiting example, the self sustained expression vehicle is a Salmonella toxic plasmid.

For example, in one embodiment of the present invention, the plasmid screening system is maintained by providing an action to defeat bacteria such as Salmonella, and on that basis Salmonella with this deficiency can be screened for those that do not . In one embodiment, the Salmonella of the invention is a nutritional requirement variant and the expression plasmid does not provide a variant or have a biosynthetic enzyme action. Salmonella containing the expression plasmids can be selected by propagating the cells in a growth medium lacking nutrients capable of metabolizing only the desired cells, that is, cells having the expression plasmids. In a highly preferred embodiment of this embodiment, the Salmonella of the invention most preferably has the DAP (meso-diamino pimelic acid) requirement, which is essential by deletion of the asd gene. DAP is a component of a peptidoglycan present in the plasma membrane of gram-negative bacteria necessary for preservation of bacterial outer membrane. The absence of DAP causes bacterial cell lysis resulting from loss of outer membrane integrity. The asd (β-aspartate semialdehyde dehydrogenase) gene encodes a gene in the DAP biosynthetic pathway. Gram negative bacteria lacking asd action can be propagated by feeding DAP to the culture medium. A plasmid carrying an asd gene sequence operatively linked to a homologous or heterologous promoter can be selected, for example, by propagating an expression plasmid of the invention in the absence of DAP by propagating Gram-negative bacteria lacking asd activity See U.S. Patent No. 5,840,483 to Curtiss, III).

Other non-antibiotic screening systems are known in the art and are within the scope of the present invention. For example, a selection system using plasmids containing stable toxins and less stable anti-toxins can be used to screen for bacteria carrying such plasmids.

In another embodiment, the expression vehicle is an extrachromosomal plasmid that is selectable by non-antibiotic means, e. G., A cholinesin plasmid. The colonyplasmic plasmids used in the present invention encode at least the colistin toxin and the anti-colistin (the colistin toxin is more stable than the anti-colistin), so that any bacteria in which the colistin plasmid is lost It is killed as the lasting result of colchicine toxin. In a preferred embodiment, the colistin toxin is a large subunit of ColE3 and the anti-colicin is a small subunit of ColE3.

In another embodiment of the invention, the expression vehicle is a lambda vector, more specifically a luminescent lambda vector. In a preferred embodiment, the bacterial host comprising the lambda vector further comprises a temperature-sensitive λ repressor which is functional at 30 ° C., but not at 37 ° C. Consequently, the bacterial host may be grown and manipulated in vivo at 30 ° C without the expression of primary and / or secondary effector molecules which may be toxic to bacterial cells. Upon introduction of the bacterial strain into a patient, the lambda repressor is inactivated by conventional body temperature and the expression of the primary effector molecule and optionally the secondary effector molecule is activated.

The expression of a nucleic acid sequence encoding an effector molecule or a fusion protein can be regulated by a secondary nucleic acid sequence such that the effector molecule is expressed in a bacterium transformed with a recombinant DNA molecule. For example, expression of effector molecules can be modulated by any promoter / enhancer element known in the art. The promoter / promoter may be homologous (i.e., natural) or heterologous (i.e., non-natural). Promoters that can be used to regulate the expression of effector molecules, such as cytokines or fusion proteins, in bacteria include but are not limited to prokaryotic procoins such as the? -Lactamase promoter (Villa-Kamaroff et al., 1978, Proc Natl Acad Sci USA 75: 3727-3731) or the lac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25; Scientific American, 1980, 242-74 -94). Other promoters that may be included in the present invention include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P _R , lambda P _l , trc, pagC, sulA, pol II (dinA), ruv, recA, uvrA, uvrB, , umuDC, lexA, cea, caa, and recN (Schnarr et al., 1991, Biochimie 73: 423-431). In a preferred embodiment, the promoter is trc (Amann et al., 1988, Gene 69: 301-15).

In a particular embodiment, a toxic attenuated bacterial strain may be treated with x-ray, ultraviolet, alkylating agent, or another DNA damaging agent to increase the expression of said collicin, wherein the primary effector molecule is expressed under the control of an SOS-reactive promoter have. Exemplary SOS-responsive promoters include, but are not limited to ruv, recA, sulA, umuC, dinA, ruv, uvrA, uvrB, uvrD, lexA, cea, caa,

In another preferred embodiment, the promoter has enhanced activity in the tumor environment. For example, the promoter is activated by the anaerobic environment of the tumor, such as the P1 promoter of the pepT gene. Activation of the P1 promoter varies with the FNR transcriptional activator (Strauch et al., 1985, J. Bacteriol. 156: 743-751). In certain embodiments, the P1 promoter is a mutation promoter under anaerobic conditions in which the native P1 promoter, e.g., the activity for anaerobic conditions, is induced at a higher level than the pepT200 promoter that is induced by CRP-cAMP instead of FNR (Lombardo et al. , 1997, J. Bacteriol. 179: 1909-1971). In another embodiment, an anaerobically-derived promoter, such as the potABCD promoter, is used. The potABCD is an operon that is expressed quantitatively from pepT under anaerobic conditions. The promoter of the pepT gene contributing to the expression can be isolated (Lombardo et al., 1997, J. Bacteriol. 179: 1909-1971) and used according to the method of the present invention.

On the other hand, the promoter may be an antibiotic-induced promoter, for example the tet promoter of Tn10 transposon. In a preferred embodiment, the tet promoter is multimerized, e. G., Three times as large. The promoter activity was then induced by administering an appropriate dose of tetracycline to the patient treated with the toxicant weakened tumor target bacteria of the present invention. The tet inducible expression system was initially introduced into eukaryotic systems, such as Fryar and Gatz (1992), Current Genetics 21: 345-349 and mammalian cells (Lang and Feingold, 1996, Gene 168: 169-171), and recent studies have extended its applicability to bacterial cells. For example, Stieger et al. (1999, Gene 226: 243-252), for example, have demonstrated 80-fold induction of firefly luciferase on tet induction upon operative binding to the tet promoter. The advantage of the promoter is that it is induced to a very low level of tetracycline, approximately one-tenth the amount required for antibiotic activity.

Once a plasmid comprising an effector molecule or fusion protein has been made and introduced into the toxic attenuated tumor-targeted bacteria, the effector molecule expression or fusion protein expression can be detected by any of the methods known in the art such as, (Sambrook et al., 1989, Molecular Biology: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY; Ausubel et al., 1995, Current Protocols in Molecular Biology, Greene Publishing, New York, NY).

5.7. Complex therapy

In some embodiments, toxic attenuated tumor-targeted bacteria are used in combination with other known cancer therapies to treat fulminant cancer tumors. In some other embodiments, a fulminant cancer tumor is treated with other known cancer therapies using a toxic attenuated tumor-targeted bacterium engineered to express one or more effector molecules and / or one or more nucleic acid molecules encoding the fusion protein . For example, a toxic attenuated tumor-targeted bacterium engineered to express one or more effector molecules and / or one or more nucleic acid molecules encoding a fusion protein can be used with the chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, cisplatin, ipospamide, paclitaxol, taxane, topoisomerase I inhibitors such as CPT-11, topotecan, 9-AC and GG-211, gemcitabine, vinorelbine , Oxaliplatin, 5-fluorouracil (5-FU), leucovorin, vinorelbine, te modal, taxol, cytochalasin B, gramicidin D, emetine, mitomycin, etoposide, But are not limited to, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydro testosterone, melphalan, glucocorticoid, procaine, tetracaine, Lidocaine, propranolol, and puromycin analogs and cytoxic acids. On the one hand, toxic attenuated tumor-targeted bacteria that have been engineered to express one or more effector molecules and / or one or more nucleic acid molecules that encode a fusion protein can be used with radiation therapy (e.g., gamma or x-ray irradiation) have. Any irradiation therapy protocol can be used depending on the type of cancer being treated. For example, x-ray irradiation may be administered without limitation; In particular, high-energy mega-voltages (above 1 MeV energy) can be used for severe tumors, and electron beam and ortho-voltage x-ray irradiation can be used for skin cancer. Tissue may be exposed to radiation by administering gamma rays that emit radioactive isotopes, such as radioactive isotopes of radium, cobalt, and other elements.

The present invention includes continuous or simultaneous administration of anticancer agents and toxic attenuated tumor target bacteria. The invention includes an additional or synergistic anticancer agent and a combination of toxic attenuated tumor target bacteria.

The present invention also encompasses combinations of one or more anti-cancer agents and a toxic attenuated tumor-targeting bacterium having different action sites. Such combinations provide improved therapy based on the dual action of the therapeutic agent whether the combination is synergistic or additional. Thus, the novel combination therapies of the present invention provide improved efficacy over any of the drugs used as a single pharmaceutical regimen.

The present invention also encompasses the sequential or concomitant administration of an anticancer agent with a toxic attenuated tumor-targeted bacterium engineered to express one or more nucleic acid molecules encoding an effector molecule and / or a fusion protein. The invention encompasses a combination of an anticancer agent with a toxic attenuated tumor-targeted bacterium engineered to express additional or synergistic one or more nucleic acid molecules encoding one or more effector molecules and / or fusion proteins.

The invention also encompasses a combination of an anticancer agent with a toxic attenuated tumor-target bacterium engineered to express one or more effector molecules and / or one or more nucleic acid molecules encoding the fusion protein, having different action sites. Such combinations provide improved therapy based on the double action of the therapeutic agent whether the combination is synergistic or additional. Thus, the novel combination therapies of the present invention provide improved efficacy over any of the drugs used as a single pharmaceutical regimen.

5.8. Delivery method and composition

The present invention provides a method by which one or more primary effector molecules that may be toxic upon systemic delivery to a host can be delivered locally to the tumor by a toxic attenuated tumor-target bacteria having reduced toxicity to said host. In one embodiment, the primary effector molecule is useful for the treatment of sarcoma, lymphoma, carcinoma, or other solid tumors. In some non-limiting embodiments, the effector molecule is useful for inducing a localized immune response at the tumor site.

According to the present invention, a toxic attenuated tumor-target bacterial vector containing a nucleic acid encoding one or more effector molecules and optionally one or more primary effector molecules is used to treat the growth of a tumor in an animal having a solid tumor, Inhibit tumor growth, reduce tumor volume, or prevent the spread of tumor cells.

The present invention provides a pharmaceutical composition comprising a toxic attenuated tumor target bacterium comprising one or more nucleic acid molecules encoding one or more primary effector molecules operatively linked to one or more suitable promoters to an animal in need of treatment of a solid tumor carcinoma The method comprising administering to a subject a therapeutically effective amount of the first effector molecule. The present invention also relates to a method of treating a solid tumor, comprising administering to an animal in need of such treatment a composition comprising one or more primary effector molecules operably linked to one or more suitable promoters and one or more nucleic acid molecules encoding one or more secondary effector molecules, There is provided a method of delivering said primary effector molecule for the treatment of said solid tumor carcinoma, comprising administering a pharmaceutical composition comprising a tumor-targeted bacterium. In one embodiment, the primary effector molecule is a member of the TNF family, a cytotoxic peptide or polypeptide, an angiogenesis inhibiting factor, a tumor suppressor enzyme or a functional fragment thereof.

The invention is directed to a pharmaceutical composition comprising a toxic attenuated tumor target bacterium comprising one or more nucleic acid molecules encoding one or more fusion proteins of the invention operatively linked to one or more suitable promoters to an animal in need of treatment of a solid tumor carcinoma The invention provides a method of delivering one or more fusion proteins of the invention for the treatment of said solid tumors. The invention also relates to a method of treating a solid tumor comprising administering to an animal in need of treatment of a solid tumor tumor one or more of the fusion proteins of the invention operably linked to one or more suitable promoters and one or more nucleic acid molecules encoding one or more effector molecules There is provided a method of delivering one or more fusion proteins of the present invention and one or more effector molecules for the treatment of said solid tumor carcinoma, comprising administering a pharmaceutical composition comprising a target bacterium.

In a preferred embodiment, the toxic attenuated tumor target bacterium is Salmonella. In another embodiment, the toxic attenuated tumor target bacteria comprises an enhanced release system. In a preferred embodiment, the animal is a mammal. In a highly preferred embodiment, the animal is a human.

The present invention also provides a complex delivery of one or more primary effector molecules and optionally one or more secondary effector molecules delivered by a toxic attenuated tumor-targeted bacterium, e. G. Salmonella. The present invention also provides a complex delivery of different toxic attenuated tumor-targeted bacteria involving one or more different primary effector molecules and / or optionally one or more different secondary effector molecules.

The present invention also provides a complex delivery of one or more fusion proteins of the invention delivered by a toxicly attenuated tumor-targeted bacterium, e. G. Salmonella. The present invention also provides a complex delivery of one or more fusion proteins of the invention and optionally one or more effector molecule of the invention delivered by a toxic attenuated tumor-targeted bacterium, for example Salmonella. The present invention also provides a complex delivery of different toxic attenuated tumor-targeted bacteria involving one or more different fusion proteins and / or optionally one or more different effector molecules.

Fidelity tumors include, but are not limited to, sarcomas, carcinomas, lymphomas and other solid tumors such as, but not limited to, interstitium, central nervous system, breast, prostate, , Astrocytoma, glioma, pancreatic cancer, stomach cancer, liver cancer, colon cancer, melanoma, kidney cancer, bladder cancer and mesothelioma. The patient is preferably an animal, such as, but not limited to, a bovine, a pig, a chicken, a dog, a cat, a horse, etc., preferably a mammal, and most preferably a human. Treatment of the solid tumors used herein includes, but is not limited to, inhibiting tumor growth, inhibiting tumor cell proliferation, reducing tumor volume, or inhibiting diffusion (metastasis) of tumor cells to other body parts.

The invention provides a pharmaceutical composition comprising a toxic attenuated tumor target bacteria comprising a pharmaceutically acceptable carrier and one or more nucleic acid molecules encoding one or more primary effector molecules operatively linked to one or more suitable promoters . The present invention provides a method of treating or preventing a toxic, attenuated tumor-targeting bacterium comprising a pharmaceutically acceptable carrier and one or more primary effector molecules operatively linked to one or more suitable promoters and one or more nucleic acid molecules encoding one or more secondary effector molecules ≪ / RTI >

The invention provides a pharmaceutical composition comprising a toxic attenuated tumor target bacterium comprising at least one nucleic acid molecule encoding at least one inventive fusion protein operably linked to a pharmaceutically acceptable carrier and at least one suitable promoter do. The present invention includes a toxic attenuated tumor-targeting bacterium comprising a pharmaceutically acceptable carrier and at least one nucleic acid molecule encoding at least one fusion protein of the invention and at least one effector molecule operatively linked to at least one suitable promoter ≪ / RTI >

The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a toxic attenuated tumor-targeting bacterium. The present invention also provides a pharmaceutical composition comprising a toxic attenuated tumor-targeting bacterium comprising a pharmaceutically acceptable carrier and one or more primary effector molecules and optionally one or more secondary effector molecules. Such compositions comprise a therapeutically effective amount of a toxic attenuated tumor-target salmonella vector comprising a pharmaceutically acceptable carrier and one or more primary effector molecules and optionally one or more secondary effector molecules. The present invention also provides a pharmaceutical composition comprising a toxicant weakened tumor target Salmonella comprising a pharmaceutically acceptable carrier and one or more fusion proteins of the invention and optionally one or more effector molecules. Such compositions comprise a therapeutically effective amount of a toxic attenuated tumor-target salmonella vector comprising a pharmaceutically acceptable carrier and one or more fusion proteins of the invention and optionally one or more effector molecules.

In a particular embodiment, the term " pharmaceutically acceptable " means that it is approved by a federal or state regulatory agency, or that the US pharmacopoeia or animal, and more particularly, a human, do. The term " carrier " refers to a diluent, adjuvant, excipient or vehicle to be administered with a therapeutic agent. Such pharmaceutical carriers may be sterile liquids, such as water and oils, for example oils of animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, olive oil and the like. Salty water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used, especially as a liquid carrier for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk, glycerol, Water, ethanol, and the like. The composition may optionally also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may be taken in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may include standard carriers, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in E.W. Martin, " Remington ' s Pharmaceutical Science ". Such compositions will contain a therapeutically effective amount of a therapeutically effective attenuated tumor-targeted bacterium in purified form together with a suitable amount of carrier to provide a dosage form suitable for the patient. The formulation should be appropriate for the mode of administration.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the intravenous administration composition is a solution in a sterile isotonic aqueous buffer. Optionally, the composition may also include a suspending agent, and a local anesthetic for solving the pain at the injection site, such as lignocaine. In general, the ingredients may be administered in unit dosage form, either separately or together, for example as anhydrous lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the amount of active agent Can supply. When the composition is administered by injection, it may be administered as an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, a sterile injectable water or saline ampoule may be provided so that the ingredients can be mixed prior to administration.

The amount of the pharmaceutical composition of the present invention that is effective for the treatment or prevention of solid tumors will vary depending on the nature of the cancer and can be measured by standard clinical techniques. In addition, in vitro assays may optionally be used to help identify optimal dose ranges. The exact dose used in the formulation will also vary depending on the route of administration and the severity of the cancer and should be determined by the judgment of the practitioner or each patient situation. However, a suitable dosage range will generally be from about 1.0 to about 10 ¹⁰ cfu / kg; Optionally about 1.0 to about 1 x 10 < ⁸ > cfu / kg; Optionally about 1 x 10 ² to about 1 x 10 ⁸ cfu / kg; Optionally about 1 x 10 ⁴ to about 1 x 10 ⁸ cfu / kg; And optionally about 1 x 10 ⁴ to about 1 x 10 ¹⁰ cfu / kg. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

A variety of delivery systems are known and may be used for administration of the pharmaceutical compositions of the present invention. Methods of introduction include, but are not limited to, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intrathecal, intranasal, dermal, and oral routes. The method of introduction may also be within the tumor (e. G., By direct administration to the tumor site).

The composition can be administered by any convenient route, for example by absorption through infusion or bolus injection, epithelial or mucosal skin layers (e.g., oral mucosa, rectum and intestinal mucosa, etc.) It may be administered together with drugs. Administration may be systemic or local. It may also be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, e. G., Intracardiac and intrathecal injection; Intraventricular injection may be facilitated by a catheter attached to an intra-ventricular catheter, for example a reservoir, for example an Ommaya reservoir. Pulmonary administration may be carried out, for example, using inhalers or sprayers and formulations with an aerosol.

In certain embodiments, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; This can be done, for example, by means of local injection, injection, catheter, or graft during surgery, without limitation, wherein the graft is a porous, nonporous or collagenous material, such as a membrane, e.g., a sialastic membrane, Fiber. In one embodiment, administration can be carried out by direct injection into the malignant tumor or site (or entire site) of the neoplastic or prenatal tissue.

Toxic attenuated tumor target bacteria comprising one or more primary effector molecules and optionally one or more secondary effector molecules can be delivered to the controlled release system. Toxic attenuated tumor target bacteria comprising one or more fusion proteins of the invention and optionally one or more effector molecules may also be delivered to the controlled release system. Buchwald et al., 1980, Surgery 88: 507; and Saudek et < RTI ID = 0.0 > et al. al., 1989, N. Engl., J. Med., 321: 574). In another embodiment, polymeric materials can be used (Medical Application of Controlled Release, Langer and Wise (eds.), CRC Press., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, (1983); Levy et al., 1985, Science 228: 190 (1983); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 1989, Ann Neurol. 25: 351, and Howard et al., 1989, J. Neurosung. 71: 105). In yet another embodiment, a controlled release system may be placed in the vicinity of the therapeutic target, i. E. The brain, and thus only a portion of the systemic dose may be required (Goodson, Medical Applications of Controlled Release, vol. 115-138 (1984)).

Other controlled release systems are discussed in Langer, 1990, Science 249: 1527-1533, and include toxic, attenuated (including) one or more primary effector molecule (s) and optionally one or more secondary effector Can be used in connection with the administration of tumor-targeted bacteria.

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. And may be in the form of a form prescribed by a governmental agency regulating the manufacture, use or sale of a pharmaceutical or biological product, optionally in connection with such container (s), and the said post may be manufactured, used or sold for human administration Reflect the approval of the above institution.

The present invention also provides a method of treating such tumors comprising administering a pharmaceutical composition of the invention to an animal in need of treatment of a solid tumor and comprising at least one other known chemotherapy. In certain embodiments, the animal having the fidelity tumor cancer is administered a pharmaceutical composition of the invention and one or more chemotherapeutic agents. Examples of chemotherapeutic agents include, but are not limited to, cisplatin, ipospamide, paclitaxol, taxane, topoisomerase I inhibitors such as CPT-11, topotecan, 9-AC and GG-211, gemcitabine, vinorelbine , Oxaliplatin, 5-fluorouracil (5-FU), leucovorin, vinorelbine, te modal, taxol, cytochalasin B, gramicidin D, emetine, mitomycin, etoposide, But are not limited to, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydro testosterone, melphalan, glucocorticoid, procaine, tetracaine, Lidocaine, propranolol, and puromycin analogs and cytoxic acids.

The present invention includes continuous or simultaneous administration of an anti-cancer agent such as a pharmaceutical composition of the present invention and a chemotherapeutic agent. In certain embodiments, the pharmaceutical compositions of the invention are administered prior to the administration of the anti-cancer agent (e.g., 2 hours, 6 hours, 12 hours, 1 day, 4 days, 6 days, 12 days, 14 days, do. In another specific embodiment, the pharmaceutical compositions of the invention are administered following an anti-cancer agent administration (e.g., 2 hours, 6 hours, 12 hours, 1 day, 4 days, 6 days, 12 days, 14 days, . In certain embodiments, the pharmaceutical compositions of the invention are administered concurrently with an anti-cancer agent. The invention encompasses a combination of an anticancer agent with a toxic attenuated tumor-targeted bacterium engineered to express additional or synergistic one or more nucleic acid molecules encoding one or more effector molecules and / or fusion proteins.

The invention also encompasses a combination of an anticancer agent with a toxic attenuated tumor-target bacterium engineered to express one or more effector molecules and / or one or more nucleic acid molecules encoding the fusion protein, having different action sites. Such combinations provide improved therapy based on the double action of the therapeutic agent whether the combination is synergistic or additional. Thus, the novel combination therapies of the present invention provide improved efficacy over any of the drugs used as a single pharmaceutical regimen.

In one embodiment, an animal with a solid tumor carcinoma is administered a pharmaceutical composition of the present invention and treated with radiation therapy (e.g., gamma or x-ray irradiation). In certain embodiments, the present invention provides a method of treating or preventing cancer that has proved inconvenient to irradiation therapy. The pharmaceutical composition may be administered at the same time as the irradiation therapy. Alternatively, the irradiation regimen may be followed by administration of the pharmaceutical composition of the invention, preferably at least 1 hour, 5 hours, 12 hours, 1 day, 1 week, 1 month, more preferably several months (for example up to 3 months) You can do it later.

Irradiation therapies performed subsequent to, concurrently with, or subsequent to administration of the pharmaceutical compositions of the present invention may be performed by any method known in the art. Any irradiation therapy protocol can be used depending on the type of cancer being treated. For example, x-ray irradiation may be administered without limitation; In particular, high-energy mega-voltages (over 1 MeV energy) can be used for severe tumors, and electron beam and ortho-voltage x-ray irradiation can be used for skin cancer. Tissue may be exposed to radiation by administering gamma rays that emit radioactive isotopes, such as radioactive isotopes of radium, cobalt, and other elements.

The present invention also relates to a method of treating cancer with a composition that is weak as an alternative to the radiation therapy, wherein irradiation therapy is proven or proven to be too toxic to the treating patient, i. E. Producing an unacceptable or unbearable side effect ≪ / RTI >

5.9. Evidence of therapeutic or prophylactic utility of the pharmaceutical compositions of the present invention

The pharmaceutical compositions of the invention are preferably tested in vitro and subsequently in vivo for the desired therapeutic or prophylactic activity prior to use in humans. For example, an in vitro assay, which can be used to determine whether administration of a particular pharmaceutical composition is indicated, can be accomplished by exposing the patient tissue sample to a culture fluid and exposing the pharmaceutical composition to a pharmaceutical composition or otherwise administering the composition, And an analysis of an in vitro cell culture fluid for observing the effect of the composition as described above.

The pharmaceutical composition of the present invention can be administered orally or parenterally by contacting the immune cells with the test pharmaceutical composition or a control and measuring the ability of the test pharmaceutical composition to modulate (e.g., increase) the biological activity of the immune cell You can test for the ability to grow. The ability of a test composition to modulate the biological activity of an immune cell can be assessed by detecting the expression of a cytokine or antigen, detecting the proliferation of the immune cell, detecting activation of a signaling molecule, , Or by detecting the differentiation of the immune cells. Techniques known to those skilled in the art can be used to measure these activities. For example, cell proliferation can be analyzed by ³ H-thymidine binding assay and trypan blue cell count. Cytokine and antigen expression can be determined by, for example, immunoassay, including but not limited to Western blot, immunohistochemistry, chemiluminescent immunoassay, ELISA (enzyme linked immunosorbent assay), sandwich immunoassay, Analysis can be performed by immobilization, immobilization analysis, immunofluorescence analysis, fluorescence immunoassay, protein A immunoassay, and FACS analysis. Activation of the signal sequence can be analyzed, for example, by kinase analysis and ElectroMobility Change Analysis (EMSA). The effector action of T-cells can be measured, for example, by 51 Cr-release assay (Palladino et al., 1987, Cancer Res. 47: 5074-5079 and Blachere et al., 1993, J. Immunotherapy 14: 352 -356).

The pharmaceutical compositions of the invention can be tested for their ability to reduce tumor formation in animals suffering from cancer. The pharmaceutical compositions of the invention may also be tested for their ability to alleviate one or more symptoms associated with fidelity tumor cancer. Moreover, the pharmaceutical composition of the present invention can be tested for its ability to increase the survival time of patients suffering from fulminant tumor cancers. Techniques known to those skilled in the art can be used to analyze the action of the pharmaceutical compositions of the invention in an animal.

In various specific embodiments, in vitro assays are performed using typical cells of the cell type as described above in relation to fidelity tumor cells, to determine whether the pharmaceutical composition of the invention has a desired effect depending on the cell type .

The pharmaceutical compositions of the present invention for use in therapy may be tested in suitable animal model systems, including but not limited to rats, mice, chickens, cows, monkeys, pigs, dogs, rabbits and the like, prior to testing in humans. For in vivo testing, any animal model system known in the art may be used prior to administration to humans.

The following series of examples are provided as examples of the present invention, and they do not limit the scope of the present invention.

6. EXAMPLES: Expression of TNF-α by Toxic Weakened Tumor Target Salmonella

The following examples demonstrate that toxic attenuated tumor-targeted bacteria containing nucleic acid molecules encoding members of the TNF family, such as Salmonella, can express members of the TNF family.

6.1 Production of TNF-α plasmid

The plasmids disclosed herein exemplify certain embodiments of the invention. As is known to one of ordinary skill in the art, the promoter and / or effector molecule encoding nucleic acid, such as the trc promoter and / or TNF- [alpha] encoding nucleic acid, may be introduced into other suitable promoter or effector molecules by methods known in the art Can be replaced.

The plasmid Trc99A (commercially available from Pharmacia) or TrcHisB (commercially available from InVitrogen) was used for plasmid-based bacterial expression of the effector molecule encoding nucleic acid using the trc promoter. Both plasmids use the Nco I site as the initiation codon followed by the multiple cloning site.

6.1.1. pCET plasmid

For expression of the plasmid-based bacteria of the effector molecule encoding nucleic acid using the dual λP _L or λP _R promoter, pCET plasmids were prepared as follows. Plasmid pCE33 (Elvin et al., 1990, Gene 87: 123-126) was cleaved by restriction enzyme Cla I and terminal inactivated by bombinucleases and then serially cleaved by restriction enzyme BamHI. The resulting 1.4 kb fragment was then ligated to the 2.1 kb Ssp I / Bam HI fragment of pUC19 (commercially available from GIBCO) to generate plasmid pCI. Plasmid pCI was digested with restriction enzyme BamHI and terminated with a murine nuclease and then digested with restriction enzyme AflII. The resulting 3.1 kb band was isolated. The plasmid TrcHisB was partially digested with restriction enzyme ClaI, terminated with T4 DNA polymerase and then digested with AflII. The resulting 0.6 kb band containing the minisicron and terminator was then ligated to the 3.1 kb pCI fragment to generate the plasmid pCET. As in the case of Trc99A or TrcHisB, pCET uses the TrcHisB multicloning site following the NcoI site as the initiation codon. The propagation of any plasmid containing bacteria containing the lambda P _L or lambda P _R promoter was carried out at 30 ° C.

6.1.2. pTS plasmid

Transposon-mediated chromosome integration and effector molecule coding The plasmid pTS was constructed as follows using serine primed nutrient screening of nucleic acids. Plasmid pNK2883 (commercially available from ATCC) was digested with restriction enzyme BamHI and the 4.8 kb band was isolated. The Salmonella typhimurium serC encoding nucleic acid was isolated from Escherichia coli strain 14028 by PCR using the forward primer of the sequence GAAGATCTTCCGGAGGAGGGGAAATG (SEQ ID NO: 1) and the reverse primer of the sequence CGGGATCCGAGCTCGAGGGCCCGGGAAAGGATCTAAGAAGATCC (SEQ ID NO: 2). The PCR reaction mixture was digested with restriction enzymes BglII and BamHI, and a 1.1 kb PCR product was isolated and ligated to 4.8 pNK2883 fragment to produce plasmid pTS. A cloning site adjacent to 3 ' of the serC encoded nucleic acid was obtained for insertion of the effector molecule encoding nucleic acid.

6.1.3. pTS-TNF-a plasmid

A plasmid (pTS-TNF-α) was prepared as follows for the integration of the pTS-mediated chromosome of the trc promoter-engineered human TNF-α encoding nucleic acid. The plasmid PYA3332 is the ASD plasmid PYA272 (U.S. Patent No. 5,840,483 to Curtis III) whose replication origin has been replaced with that of the colE1 plasmid (Bazaral and Helsinki, 1970, Biochem 9: 399-406). Plasmid PYA3332 was digested with restriction enzyme Nco I and terminated with a murine nuclease. The end-inactivated fragment was then digested with restriction enzyme HindIII and a 3.3 kb DNA fragment was isolated. The following E. coli-optimized human TNF-alpha encoding nucleic acid (Pennica et al., 1984 Nature 312: 724-729 and Salztman, et al., 1996, Cancer Biotherapy 11: 145-153) I, terminated with T4 DNA polymerase, and then digested with restriction enzyme HindIII. The resulting 0.5 kb fragment was ligated to the 3.3 kb PYA3332 fragment to obtain the plasmid Asd34TNF-a. Asd34TNF-? Was then digested with restriction enzyme BglII, and a 1.1 kb fragment encoding the trc promoter-engineered TNF-alpha encoding nucleic acid was ligated to the BamHI site of pTS to obtain the plasmid pTS-TNF- ?.

6.1.4. pTS-BRP plasmid

The plasmid pTS-BRP was prepared as follows using the transposon-mediated chromosome integration of the BRP-encoding nucleic acid and the serine raw nutrient screening of the effector molecule-encoding nucleic acid. BRP encoded nucleic acid was isolated from the plasmid pSWI (commercially available from Bio101, Vista, CA) by PCR using the forward primer of the sequence CCGACGCGTTGACACCTGAAAACTGGAG (SEQ ID NO: 5) and the sequence CCGACGCGTGAAAGGATCTCAAGAAGATC (SEQ ID NO: 6) And cloned into a TOPO-TA cloning plasmid (commercially available from InVitrogen, Carlsbad, Calif.) To give pBRP # 5. Plasmid pBRP # 5 was digested with restriction enzymes ApaI and BamHI, and the resulting 0.6 kb band containing the BRP-encoded nucleic acid was ligated to the 5.9 kb ApaI / BamHI proto-pts fragment to obtain plasmid pTS-BRP. The cloning site of both 5 'and 3' of the BRP-encoded nucleic acid was provided for insertion of the effector molecule encoding nucleic acid.

6.1.5. pTS-BRPTNF-a plasmid

A plasmid (pTS-BRPTNF-α) was constructed as follows to integrate pTS-mediated chromosomes of BRP and trc promoter-engineered TNF-α encoding nucleic acid. For the production of pTS-TNF- [alpha], the plasmid Asd34TNF- [alpha] described above was digested with restriction enzyme BglII and a 1.1 kb fragment encoding the trc promoter-engineered TNF-alpha encoding nucleic acid was ligated to the BamHI site of pTS- BRPTNF-a was obtained.

6.2. Integration of effector molecule encoding nucleic acid into Salmonella host chromosome

The system disclosed herein uses A. sero-Salmonella strains that are auxotrophic for serine or glycine, and plasmids that restore serine / glycine primacy during chromosome integration into actively transcribed regions. However, it is well known in the art that other selectable markers can also be used in the selection of chromosomal components, and such markers are within the scope of the present invention (Kleckner et al., 1991, Meth. Enzymol. -180).

The pTS or pTS-BRP plasmid containing the effector molecule encoding nucleic acid can be introduced into the serC-Salmonella strain by a number of means well known in the art, for example by chemical transformation and electroporation. Following introduction of the effector molecule-encoding nucleic acid, Salmonella was propagated in an ampicillin-containing growth medium for a minimum of 2 hours, more preferably 6 hours or more. Bacteria were then placed in a medium capable of screening for serine, for example, M56 medium (Atlas, RM "Handbook of Microbiological Media." LC Parks, ed. CRC Press, Boca Raton, Florida, 1993) . Bacteria with chromosomal integration of effector molecule encoding nucleic acids can be grown in the above selection medium. The effector molecule encoding nucleic acid expression is then measured as exemplified below. The effector molecule encoding nucleic acid expression can be measured by any of a number of methods known to those skilled in the art, for example by enzymatic activity, biological activity, northern blot analysis or Southern blot analysis.

6.2.1. Delivery and expression of Salmonella-expressing TNF-α

The trc promoter-engineered TNF-α-encoding nucleic acid was inserted into the BamHI site of pTS-BRP as described above to obtain the pTS-BRPTNF-α plasmid. The plasmid pTS-BRPTNF-α was isolated from the toxin-weakened strain VNP20009 (produced by serC- in the genotype of ΔmsbB, ΔpurI, ΔserC (FIG. 2) by standard methods known in the art WO 99/13053). Although not intending to be limited by the mechanism, integration of the plasmid into the bacterial genome allows activation of the serC encoded nucleic acid and produces the serC ⁺ phenotype. Thus, bacteria with chromosomal integration of the TNF-alpha encoding nucleic acid were selected by plating the electroporated bacteria on adenine supplemented M56 agar plates. Bacteria were further characterized by loss of resistance to ampicillin, indicating plasmid loss and simultaneous loss of plasmid-based TNF-a expression.

To examine and quantify TNF-a expression by the tumor-targeted bacteria of the present invention, Salmonella with the chromosomal integration of the TNF-alpha encoding nucleic acid was propagated overnight and samples of the culture were used for Western blot analysis. Specifically, the expression of TNF-α from a typical serC ⁺ , an ampicillin-sensitive clone represented by pTS-BRPTNF-α clone 2, is shown in FIG. Western blot analysis showed that the bacterial protein corresponding to 3.9 × 10 ⁷ cfu of pTS-BRPTNF-α clone 2 bacteria expressed more than 50 ng of TNF-α (lane 5), indicating that TNF-α expression was 10 ng / Bacteria at a level of 10 ⁷ or higher. Thus, human TNF-a was successfully expressed from chromosome-integrated and trc-promoter-engineered TNF-a coding nucleic acid in Salmonella.

7. Example: Toxigenic weakened tumor expressing OMPA fusion protein target bacteria

Plasma membrane fragmentation by fusion to various signal peptides varies depending on both the signal peptide and the protein. For example, a protein may be localized in the plasma membrane compartment of bacteria by fusion of the signal peptide to the amino terminus of the protein. Without limitation, it is believed that the plasma membrane ubiquitin promotes the release of the components by allowing only a single membrane to pass through, in order to allow germ components (e.g., proteins) to be released into the environment. In contrast, cytoplasmic ubiquitin requires that it pass through both the inner and outer membranes of bacteria in order for the components to be released into the environment. Moreover, the plasma membrane ubiquitination of some proteins may also aid biological activity.

A variety of methods known in the art may be used to target the effector molecule of the invention to the plasma membrane. This example demonstrates that the fusion of a signal peptide to the amino terminus of an effector molecule such as TNF- [alpha], TRAIL (a TNF- [alpha] -dependent cell death-inducing ligand) and interleukin-2 (IL-2) And that subsequent machining takes place.

7.1. Processing of OMPA-TNF-a fusion protein

TNF- [alpha] expression in four different clones expressing the plasmid-based trc promoter-engineered ompA-TNF- [alpha] fusion protein in JM109 bacteria was examined by Western blot analysis of whole cell lysates. A precursor fusion protein of mature TNF-α was cleaved by a signal peptidase, an enzyme located in the plasma membrane, to prove the plasma membrane localization. In all four clones, overexpression of TNF-a upon induction by IPTG resulted in TNF-a as a double line migrating at approximately 20 kd (Fig. 5, lane 4-7) Corresponds to all processed forms. For comparison, Salmonella strains with chromosomal integrated TNF-alpha encoding nucleic acid expressing the mature (processed) form of TNF- alpha were used as positive controls (Fig. 5, lane 3). TNF-a expression was not detected in bacteria lacking the TNF-alpha encoding nucleic acid (Fig. 5, lane 2).

These results demonstrate that fusion of mature human TNF-a protein to E. coli ompA signal peptide (shown in Fig. 4) is plasmocalized and processed upon expression in E. coli. Furthermore, it is not known whether overexpression of secreted proteins would be toxic to bacterial hosts as a result of overwhelming normal secretion machinery. This demonstration of the expression of engineered ompA-TNF-a fusion protein indicated that the normal secretion device could modulate the expression of high levels of secretory protein.

7.2. Processing of OMPA-TRAIL fusion protein

The ability of the ompA signal peptide to protoplastically localize members of the TNF family was extended to another member of the TNF family, TRAIL (a TNF-alpha-related extracellular extracellular ligand). For this experiment, the trc promoter-engineered TRAIL-encoding nucleic acid encoding the mature form of human TRAIL (hTRAIL) was fused to the coding sequence of the ompA signal peptide (shown in Figure 6). Encoding two different ompA / TRAIL junctions, the NcoI site, and encoding the NdeI site (see Figure 6 for NdeI containing sequences) were examined. Western analysis for the two types of clones is shown in Fig. Using an anti-hTRAIL antibody, Western blot analysis showed that the bacterium overexpressing the NcoI-conjugated ompA-TRAIL was in the processed (28.2 kd) and untreated (30.2 kd) forms of hTRAIL (Figure 7, lanes 2-4) , While bacteria expressing NdeI-conjugated ompA-TRAIL expressed only the processed form (Fig. 7, lane 4 to 7), indicating that the NdeI junction provides more efficient processing.

The results demonstrate that fusion of the mature human TRAIL protein to the E. coli ompA signal peptide produces plasma membrane localization and processing. Furthermore, it is not known whether overexpression of secreted proteins would be toxic to bacterial hosts as a result of overwhelming normal secretion machinery. This demonstration of the expression of the engineered ompA-TRAIL fusion protein indicated that the normal secretion device can modulate the expression of high levels of secretory protein.

7.3. Processing of OMPA (8L) -IL-2 fusion protein

The secondary effector molecule (IL-2) was expressed as a fusion protein. The fusion of mature (C125A) hIL-2 to the wild-type OmpA signal sequence used above for TNF-a and TRAIL did not allow for the processing of IL-2. Mature human (C125A) IL-2 was fused to a modified ompA signal peptide (Figure 8), designated ompA (8L), to examine plasma membrane localization and processing of human IL-2 cytokines. The modified ompA signal peptide was altered by replacing the amino acids 6-17 of the ompA signal with those shown in Fig. Expression and processing are shown in Fig. 9 (lanes 6 and 7). Each lane represents a single clone. Western blot analysis showed that substantially complete processing was observed in the ompA (8L) signal peptide (Figure 9, lanes 6 and 7).

7.4. Processing of PHOA (8L) -IL-2 fusion protein

The second fusion protein was examined for plasma membrane 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 (shown in Figure 10) fused to the modified phoA signal peptide (shown in phoA (8L)) was examined. Figure 9 shows the expression and processing. Partial processing of the synthetic phoA (8L) signal peptide (Figure 9, lanes 4 and 5) was observed while more complete processing was observed for the ompA (8L) signal peptide (Figure 9, lanes 6 and 7).

The results indicate that the localization and processing of IL-2 is provided by different signal peptides. The results also demonstrate that the plasmalicular ubiquitination of proteins by fusion to various signal peptides varies with both signal peptide and protein.

The results of this fusion protein study indicate that the secondary effector molecule of the invention, such as IL-2, can be expressed and localized into bacterial plasma membranes by fusion with protein signal peptides such as OmpA or PhoA. As will be apparent to those skilled in the art, other signal sequences may be used to cause the plasmal membrane localization of the effector molecule. As will also be apparent to those skilled in the art, other effector molecules of the present invention may be substituted with the effector molecules disclosed in the present Examples.

8. Example: Antitumor efficacy of salmonella (DELTA MSBB, DELTA PURI) expressing the mature form of TNF-a

The following experiments demonstrate that a toxic attenuated tumor-targeted bacterium, such as Salmonella, containing nucleic acids encoding a primary effector molecule (e.g. a member of the TNF family), for example Salmonella, delivers the primary effector to a mammalian tumor, To the extent possible.

The ability of Salmonella typhimurium to enhance the antitumor efficacy of TNF-a was evaluated in a rats colon 38 carcinoma model. For this experiment, 1 ㎣ tumor fragments derived from colon 38 tumors were transplanted into C57BL / 6 mice and the tumors were grown at an average size of approximately 0.3 g, wherein the animals were divided into the following treatment groups (n = 10) Randomly divided: 1) untreated group; 2) Salmonella typhimurium (ΔmsbB, ΔpurI, serC); And 3) pTS-BRPTNF-alpha (clone 2 described above). Each group of mice was either untreated or injected intravenously once with a suitable bacterial strain of 1 x 10 ⁶ cfu. Tumor size was measured weekly starting with bacterial inoculation.

In the group treated with toxic attenuated tumor-target Salmonella expressing TNF-a, tumor regression occurred by two weeks of treatment and complete regression was observed in six of the animals within four weeks of treatment (FIG. 11). Tumors of the untreated group grew gradually in size whereas tumors of the group treated with the strain Salmonella typhimurium ([Delta] msbB, [Delta] purI, serC) showed partial regression between 3 and 4 weeks of treatment, The tumor size gradually increased (Fig. 11).

The results demonstrate that toxic attenuated tumor-target Salmonella can express effector molecules such as members of the TNF family and deliver them to tumors. Salmonella as described above is useful for the treatment of tumors and provides improved tumor regression results as compared to the Salmonella strains that do not express the TNF family members.

The demonstration of complete tumor regression by salmonella expressing TNF-a from chromosomal integrated nucleic acids indicates that biologically effective expression can be generated from chromosome-integrated effector molecule-encoding nucleic acids.

9. Example: Enhanced delivery of nucleic acid molecules by BRP-expressing bacteria

In order to demonstrate that BRP activity can enhance the release of plasmid from tumor-target toxicity-attenuated bacteria, e. G. Salmonella, a second plasmid (pTrc99a with AMP marker) used as a release marker as well as BRP on the plasmid Which is a tumor-target toxicity-weakened Salmonella strain. To analyze the activity of BRP, Salmonella with or without BRP was grown in culture medium by standard methods. The remaining supernatant was then removed from the resulting supernatant by centrifugation and filtration, and then the bacterial-removed supernatant was added to competent cells and transformed. These " accepting " cells were then plated onto LB amp to search for absorption of the AMP marker plasmid. An increase in the number of AMP resistant colonies with BRP will indicate that more plasmid has been released into the medium from strains expressing BRP. The results are summarized in Table 2 below:

Plasmid Amp Colony Average # / Transformation pTrc99a alone 125 pTrc99a + BRP (pSW1) 383

The results demonstrate that the presence of BRP increased the amount of amp plasmid secreted into the medium. Thus, transformation into " recipient cells " by supernatants from BRP expressing cells provided a larger number of colonies. The results demonstrate that BRP enhanced the release of secondary effector molecules, including nucleic acid plasmids. Thus, the results indicate that BRP is useful for plasmid release or DNA delivery. In addition, the Salmonella strains were able to express BRP and transfer DNA, and maintained the replication ability as a group.

10. Example: BRP expression which does not impair tumor targeting or tumor suppression ability of toxic weakened tumor target Salmonella

The following examples demonstrate that toxic attenuated tumor-targeted bacteria can be engineered to express BRP with one or more effector molecules to enhance delivery of effector molecules to the tumor without inhibiting the ability of the bacteria to target the tumor do.

Fidelity tumor models were obtained by subcutaneously injecting B16 melanoma cells into the right posterior abdominal region of C57BL / 6 mice. For the tumor transplantation, the cells by trypsinization Remove from the flank, washed, and suspended in phosphate buffered saline with 2.5 x 10 ⁶ cells / ㎖ on. 0.2 ml of the cell suspension fraction was injected on day 0 for a total of 5 x 10 ⁵ cells / mouse. At approximately 10 days post-transplantation, when the tumor volume reached 150-200 ㎣, the mice were randomly divided into three groups of ten and given different treatments. 0.2 ml of PBS was administered to the control group (curve # 1 in FIG. 12). Another group received 0.2 ml of Salmonella VNP20009 containing 2 x 10 < ⁶ > cfu / mouse of a toxic attenuated tumor target strain (curve # 2 in Figure 12). The third group was administered 0.2 ml containing 2 x 10 ⁶ cfu / mouse of the toxic attenuated tumor-bearing strain of Salmonella containing the plasmid pSWl containing the BRP gene under the control of the intrinsic promoter (curve # 3 of Figure 12 ). Although the BRP gene is capable of inducing SOS in E. coli, the present inventors have found that the present invention is not limited to the mechanism, but is a low level or a low level, which is a partial constituent of the Salmonella and thus further enhanced by the SOS properties of the tumor environment. It is believed to produce normal levels of BRP protein. BRP Expression VNP20009 Salmonella-injected mice exhibited almost the same antitumor response as that of non-BRP-expressing VNP20009, suggesting that the survival or tumor targeting ability of these Salmonella strains was not altered by BRP expression, The ability is not changed. The results of BRP expression on toxic attenuated tumor-targeted Salmonella show the effect of expression of secreted HSV-thymidine kinase (HSV-TK), which results in loss of tumor suppressor ability of VNP20009 as a result of expression (Pawelek et al., 1997, Cancer Res 57: 4537-4544). Thus, the BRP system can be used without further modification to enhance the delivery of primary and / or secondary effector molecules to the tumor.

11. Example: expression of pepT promoter vehicle

This example demonstrates in vitro and in vivo expression of a nucleic acid molecule encoding an informative gene such as beta-gal under the control of a pepT promoter in a toxic attenuated tumor-targeted bacterium, e. G. Salmonella.

11.1 Preparation of pepT-BRP-βGAL Expression Plasmid

The pepT promoter was cloned by PCR amplification of the region from the isolated colony of wild-type Salmonella typhimurium (ATCC 14028) using the following primers:

Forward: 5'-AGT CTA GAC AAT CAG GCG AAG AAC GG-3 '(SEQ ID NO: 15)

Inverse: 5'-AGC CAT GGA GTC ACC CTC ACT TTT C-3 '(SEQ ID NO: 16)

The PCR conditions were: 1 cycle of 95 ° C for 5 minutes; 95 ° C for 1 minute, 65 ° C for 1 minute, 72 ° C for 2 minutes, and 72 ° C for 10 minutes. The PCR product was cloned into a PCR 2.1 cloning vector (Invitrogen, Carlsbad, Calif.) And referred to as PepT / PCR 2.1.

The PepT / PCR 2.1 vector was digested with NcoI and XbaI. The pepT fragment was gel-isolated and ligated into? -Gal Zterm digested with the same enzyme. Zterm (Temporary Genbank Bank No. 296495) is a promoter-free β-gal plasmid produced by cloning the βGal open reading frame into pUC19. The resulting plasmid was referred to as pepT-beta GAL.

11.2. In vitro expression of pepT-βGAL and measurement of pepT-βGAL activity

(CC13 in Fig. 13A; see WO 96/40238 for the gene structure of the strain) or VNP20009 (CC16 in Fig. 13A) with pepT-? GAL to an OD ₆₀₀ of ~0.5-0.8 under anaerobic or aerobic conditions . β-gal activity was determined by the method of Birge and Low, 1974, J. Mol. Biol. 83: 447-457. The results are shown in Fig. 13A, which demonstrates that the b-gal activity is induced approximately 14 to 24 fold during bacterial growth under anaerobic conditions.

11.3. In vivo expression of pepT-βGAL and measurement of pepT-βGAL activity

cells of Salmonella strain YS1456 harboring both the pepT-? GAL expression plasmid, BRP expression plasmid (pSW1 (BIO101, Vista, California) containing the pCloDF13 BRP coding sequence under the control of the intrinsic promoter) Intravenous injection. At day 5 post-injection, the tumor and liver were homogenized and the bacteria isolated to show that the presence of pepT-β-gal and / or BRP plasmid did not interfere with the tumor targeting ability of the bacterium. Βgal activity was also measured to determine whether active βgal could be measured in vivo using the tumor and hepatic homogenate and whether the pepT promoter was induced in an anaerobic tumor environment. The results shown in Figure 13B indicate that the pepT promoter activity is very high in the tumor environment. There was no significant increase in liver expression of βgal relative to background levels, which is believed to be due to low activity of the pepT promoter in the aerobic liver environment and / or low targeting of the bacterial vector in the liver.

12. Example: Tetracycline Induced Expression System

This example demonstrates the expression of a nucleic acid molecule encoding an informative gene such as beta-gal under the control of a tet-promoter in a toxic attenuated tumor-targeted bacterium, e. G. Salmonella.

The tet promoter was cloned from mini-TN10 transposon by PCR amplification using the following primers:

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).

The PCR conditions were: 1 cycle of 95 ° C for 5 minutes; 1 minute at 95 ° C, 1 minute at 60 ° C, 2 minutes at 72 ° C for 35 cycles, and 10 minutes at 72 ° C for 1 cycle.

The ~ 400 bp PCR fragment was gel isolated and cloned into the PCR 2.1 vector (Invitrogen). The PCR 2.1 / tet promoter vector was digested with NcoI and BamHI. The -400 bp tet promoter fragment was gel-isolated and ligated into the promoter-free [beta] -gal vector Zterm cut with the same two enzymes. The ligation mixture was transformed and the transformed bacteria were plated on tetracycline / X-gal plates. Positive colonies were isolated based on their blue color. Extracts from a number of positive clones were prepared and compared to β-gal activity in the presence of tetracycline [Birge and Low, 1974, J. Mol. Biol. 83: 447-457. One clone was isolated and analyzed for beta-gal activity over a range of tetracycline concentrations. The above analysis (Fig. 14) demonstrates that beta-gal activity by tetracycline is induced in a dose-dependent manner.

13. Example: Inhibition of tumor growth of toxic weakened tumor target Salmonella expressing endostatin

The following examples demonstrate the in vivo efficacy of endostatin-expressing toxic-attenuated tumor-target salmonella production and tumor treatment by Salmonella as described above.

13.1 Construction of Endostatin Expression Plasmid

Endostatin was PCR amplified from human placenta cDNA library using the following primers:

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).

The resulting PCR product was cloned into a PCR 2.1 vector (Invitrogen). Hexahistidine-endostatin was PCR amplified using the prepared plasmids with the following primers as templates:

(SEQ ID NO: 21): 5'-GTG TCC ATG GCT CGG CGG GCA AGT GTC GGG ACT GAG CAT CAT CAT CAT CAT CAT CAT CAC AGC CAC CGC GAC TTC-3 '

5'-GTG CGG ATC CCT ACT TGG AGG CAG TCA TGA AGC TG-3 '(SEQ ID NO: 22).

The PCR conditions were 95 ° C for 1 minute, 95 ° C for 1 minute, 55 ° C for 1 minute, and 30 ° C at 72 ° C for 2 minutes. And one cycle at 72 캜 for 10 minutes.

The product was a DNA fragment with NcoI (5 ') and BamHI (3') restriction sites encoding human endostatin with the peptide sequence MARRASVGTDHHHHHH (SEQ ID NO: 23) at the amino terminus.

The PCR product was digested with NcoI and BamHI and the 550 bp product was gel-isolated and ligated into the pTrc99A vector pre-digested with the same enzymes. The ligation reaction product was transformed into E. coli DH5a and toxic attenuated tumor target Salmonella VNP20009.

The hexa histidine-endostatin coding sequence was also cloned into the expression vector YA3334 as an NcoI / BamHI fragment. YA3334 is the asd plasmid PYA272 (Curtiss III, U.S. Patent No. 5,840,483), whose origin of replication has been replaced by ColE1 (Bazaral and Helsinki, 1970, Biochem 9: 399-406). Plasmid DNA prepared from positive clones was isolated and transformed into Salmonella strain 8324 (VNP20009 with asd mutation). The strain was generated according to the method disclosed in U.S. Patent No. 5,840,483 to Curtis III.

13.2. In Vitro Expression of Endostatin by Toxic Weakened Tumor Target Salmonella

pTrc99A- hexa-histidine-endostatin and proliferation by different strains of E. coli DH5α containing the plasmid and Salmonella VNP20009 middle log phase (OD ₆₀₀ ~0.6-0.8), to each culture at this point, 0.1 mM for the trc promoter activity induced It was divided into two with IPTG and the other with no IPTG. After an additional 3 hours of propagation, the bacterial extract was prepared and the expression of hexa histidine-endostatin was confirmed by Western blot analysis using an anti-histidine antibody (Clontech, Palo Alto, Calif.). 15A and 15B demonstrate pTrc99A hexa histidine-endostatin (HexHIS-endostatin) expression in E. coli DH5a and Salmonella VNP20009, respectively. While the trc promoter does not show activity in E. coli in the absence of IPTG, the same promoter is constitutively active in Salmonella. The hexa histidine-endostatin is expressed in a single band of approximately 25 kD, the molecular weight predicted for the fusion protein.

The hexahistidine-endostatin fusion protein was similarly expressed from YA3334 using the trc promoter in expression control. As shown in Fig. 16, a protein having a predicted mass of 25 kDa was detected. In Figure 16, all bacterial cultures from which the sample was derived were induced with 0.1 mM IPTG for 3 hours.

13.3. C38 endostatin expression toxicity to mouse colorectal carcinoma weakened tumor target Salmonella efficacy

Colon 38 tumor sections of 2 x 2 x 2 cm in volume were subcutaneously transplanted into 9 week old female C57BL / 6 mice. When the tumor volume reached 1000,, the above was removed and cut into 2x2x2 단 fragments. The fragments were passed serially for additional periods and the 2x2x2 fragments generated were subcutaneously implanted in the right flank of female C57BL / 6 mice. When the tumor volume reached 150-200 대략 at approximately 24 days after transplantation, the mice were randomly divided into 10 groups of 6 and provided different treatments for each of the groups. One control was given 0.2 ml of PBS. Another control group was given 0.2 ml containing a control asd plasmid, i.e., 1 x 10 ⁶ cfu of a toxic attenuated tumor target strain of Salmonella VNP20009 with an asd plasmid without insert, as described in Section 5.6 above. The first experimental group was provided with 0.2 ml containing 1x10 ⁶ cfu of VNP20009 expressing hexa histidine-endostatin in asd plasmid. The second experimental group provided VNP20009 with the same expression construct and the additional expressed BRP as the first group.

Figure 17 shows the results of the above experiment demonstrating tumor suppressive efficacy by VNP20009 expressing hexahistein-endostatin. After 60 days of treatment, the median tumor size in VNP20009 salmonella expressing endostatin was approximately 13% of the median tumor size of the control animals and was 30% or less smaller than the median tumor size of animals treated with VNP20009 Salmonella with empty vectors. Of the living animals, a majority showed stopped tumor growth as indicated by a small change in overall tumor size, and one showed strong tumor regression. The incomplete penetration or efficacy of the treatment best reflects the incomplete delivery system of endostatin, according to the discovery of endostatin accumulating inclusion bodies [O'Reilly et al., 1997, Cell 88: 277-285] . The endostatin delivery system is enhanced by the expression of BRP. BRP expression is usually regulated by its intrinsic promoter, which exhibits an SOS response in bacteria. BRP expression was shown to reduce the mean tumor volume by approximately 6% of the mean tumor volume of the control group. Furthermore, in the case of mice treated with hexa histidine-endostatin and BRP, a large number of said mice showed a significant reduction in tumor volume over time, wherein said tumor volume regressed to less than about 10% of the initial tumor volume . The effect of the BRP appears to be a dual effect: first, the BRP itself may have antitumor activity; second, the BRP promotes the release of the plasma membrane content and promotes the release of cytoplasmic content, including endostatin, Prevent proteins from accumulating in inclusion bodies.

13.4. Endodastatin Expression Toxicity to DLD Human Colon Carcinoma Weakened Tumor Target Salmonella Efficacy

Cultures of logarithmic growthed DLD1 cells were trypsinized, washed with PBS, and the cells were resuspended in a suspension of 5 x ¹⁰⁶ cells / ml in PBS. 0.1 ml of a single cell suspension containing 5 x ¹⁰⁶ cells each was injected subcutaneously into the right side abdomen of a 9-week-old nude female mouse (Charles River, Nu / Nu-CD1). Animals were randomly divided into three groups of 10 animals each at 10 to 15 days after the injection of the mice or when the tumor volume reached 200 to 400..

The first group of mice was the control group, and 0.3 ml of PBS was injected into each animal. The second group received 0.3 ml containing 1 x 10 < ⁶ > cfu of the toxic attenuated tumor target strain of Salmonella VNP2009 with a control asd plasmid. The third group was provided with 0.3 ml containing 1 x 10 ⁶ cfu of a toxic attenuated tumor target strain of Salmonella VNP20009 with a hexa histidine-endostatin fusion protein and an asd plasmid expressing BRP. The tumors were monitored and measured twice a week. FIG. 18 is a graph showing the tumor volume after providing the three treatments, demonstrating the inhibitory effect of hexa histidine-endostatin expression toxicity attenuated tumor-target Salmonella on growth of DLDl human colon carcinoma.

VNP20009 with empty vector PYA3332 was not able to significantly inhibit tumor growth. However, VNP20009 expressing endostatin and BPR could inhibit tumor growth. The results demonstrate that the combination of endostatin + BRP elevates the antitumor effect of any one VNP20009 (strain 8324) with the PYA3332 vector.

14. Example: Expression of Angiogenic Inhibitors by Toxic Weakened Tumor Target Salmonella

The following examples illustrate the methods used to engineer tumorigenic bacteria, such as Salmonella, which are toxic to express angiogenesis inhibiting agents, thrombospondin AHR, platelet factor-4 and apomigranin.

14.1 Production of plasmid containing nucleic acid sequence encoding thrombospondin AHR

(SEQ ID NO: 24) (see, for example, patent application No. C07K-14/78), which corresponds to the angiogenesis inhibitory homologous region (AHR) of thrombospondin, Optimized for expression in Salmonella DNA sequence: GCG TAC CGC TGG CGC CTG TCC CAT CGC CCG AAA ACC GGC TTT ATC CGC GTG GTG ATG TAC GAA GGA (SEQ ID NO: 25) was generated. Complementary oligonucleotides (oligo 13: 40-1 and oligo 13: 40-2) were prepared to synthesize the peptides. The 5 'end sequence encoding the processing region of OMPA and the SpeI restriction site was added. At the 3 'end, the stop codon was added by the BamHI restriction site. The two oligos were annealed to generate double stranded DNA fragments. The DNA fragment was digested with SpeI / BamHI and ligated to the SpeI / BamHI cleavage vector pTrc801IL2 to produce a plasmid pTrc801-13.40 containing the full-length modified OmpA leading sequence. Upon processing, the sequence produces a full length 13.40 thrombospondin peptide.

13.40-1:

5'gtgt actagt gtggcgcaggcg GCGTACCGCTGGCGCCTGTCCCATCGCCCGAAAACCGGCTTTATCCGCGTGGTGATGTACGAAGGCTAA ggatccgcgc 3 '(SEQ ID NO: 26)

13.40-2:

5 'gcgc ggatcc TTAGCCTTCGTACATCACCACGCGGATAAAGCCGGTTTTCGGGCGATGGGACAGGCGCCAGCGGTACGC cgcctgcgccac actagt acac3' (SEQ ID NO: 27)

(Restriction area is italicized and OmpA machining recognition area is underlined).

14.2. Production of plasmid containing nucleic acid sequence encoding platelet factor-4 peptide (47-70)

Consisting of the C-terminal amino acid residues 47-70 of platelet factor-4 (see PF-4; Mainoe et al., 1990, Science 247: 77-79 and Jouan et al., 1999, Blood 94: 984-993) The peptides were codon-optimized for expression in Salmonella. The peptides shown below include DLQ-motifs that contribute to the inhibitory activity of CFU-GM pre-cells and clusters of basic amino acids that are the major heparin-binding domain.

Platelet factor-4:

MSSAAGFCASRPGLLFLGLLLLPLVVAFASA EAEEDG DLQ CLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLK NGRKICL DLQ APLY KK II KK LLES (SEQ ID NO: 28)

Signal peptide = underline & bold line

Lys 61, 62, 65, 66 = major heparin binding domain (bold line)

DLQ (7-9, 54-56) = inhibitory activity on CFU-GM senescent cells (bold line)

Complementary oligonucleotides (oligo PF4-1 and oligo PF4-2) were prepared to synthesize the peptides. A 5 'end sequence encoding the OmpA processing region and SpeI restriction site was added. At the 3 'end, the stop codon was added by the BamHI restriction site. The two oligos were annealed to generate double stranded DNA fragments. The fragment was restriction cleaved and ligated to the SpeI / BamHI restriction vector pTrc801 to produce the plasmid pTrc801-PF4. In processing, the sequence produces a full length PF-4 (47-70) peptide.

Oligo PF4-1

5'cttcacta gt gtggcgcaggcg AACGGCCGCAAAATCTGCCTGGACCTGCAGGCGCCGCTGTACAAAAAAATCATCAAAAAACTGCTGGAAAGCTAA ggatcc gcg3 '(SEQ ID NO: 29)

Oligo PF4-2

5'cgc ggatcc TTAGCTTTCCAGCAGTTTTTTTGATGATTTTTTTTTAGCGGCGCCTGCAGGTCCAGGCAGATTTTGCGGCCGTT cgcctgcgccac actagt gaag3 '(SEQ ID NO: 30)

(Restriction area is italicized and OmpA machining recognition area is underlined).

14.3. Production of a plasmid containing a nucleic acid sequence encoding apomigran

The angiogenesis inhibiting peptide apomigran (IYSFDGRDIMTDPSWPQKVIWHGSSPHGVRLVDNYCEAWRTADTAVTGLASPLSTGKILDQKAYSCANRLIVLCIENSFMTDARK (SEQ ID NO: 31: see international application WO 99/29856) corresponds to the C-terminus of the proteolytic fragment of collagen XV. Oligonucleotides (oligo Apom5F and oligo Apom6F) Was designed to amplify the DNA fragment from human cDNA. At the 5 'end, sequences coding for the OmpA processing region and the SpeI restriction site were added, at the 3' end, the stop codon was added by the BamHI restriction site:

Oligo Apom5F: 5'-ggcttc actagt gtggcgcaggcg ATATACTCCTTTGATGGTCG-3 '(SEQ ID NO: 32)

Oligo Apom6R: 5'- cgc ggatcc TTACTTCCTAGCGTCTGTCATGAAACTG-3 '(SEQ ID NO: 33)

(Restriction area is italicized and OmpA machining recognition area is underlined).

Fragments of exact size were obtained by PCR using placental cDNA as template. The PCR product was digested with SpeI / BamHI and ligated to a SpeI / BamHI restricted vector pTrc801 containing the modified ompA signal sequence to produce the plasmid pTrc801-Apom. Upon processing, the sequence produces an ampholgene peptide.

14.4. Angiogenesis inhibitory peptides produced by Salmonella inhibiting endothelial cell proliferation

pTrcOmpA-endostatin, pTrc801-PF4, and pTrc801-13.40 plasmids were electroporated with the tumor-negative Salmonella VNP20009 strain, which has been attenuated. Salmonella strains expressing pTrcOmpA-endostatin, pTrc801-PF4, and pTrc801-13.40 were purchased from Feldman et al., 2000, Cancer Res. 60: 1503-1506 and Blezinger et al., 1999, Nature Biotech. 17: 343-348. ≪ / RTI > 5 ml of the culture of individual colonies was grown for 4 hours. The cell pellet was resuspended in 1/20 volume of HUVEC medium containing 100 mg / ml of gentamicin and three consecutive freezing / thaw cycles were performed to produce a cell lysate. The lysate was clarified by centrifugation and sterilized by filtration using a 0.2 mm syringe filter. 10, 25 or 50 ml of the lysate was added to human intravenous endothelial cells (HUVEC) in 96 well plates containing 100 ml of 2% FCS + 10 ng / ml FGF containing basal medium. As a control, salmonella containing an empty pTrc vector was used. Plates were incubated for 72 hours and proliferation was measured by MST assay (Mosman et al., 1983, J. Immunol. Methods 65: 55-63).

Preliminary results in Figures 19 and 20 show that platelet factor-4 peptide (PF4-2), thrombospondin peptide 13.40 (13.40-3) and endostatin produced by Salmonella have a proliferation inhibitory activity of 40-60%.

15. Example: Expression of Bacteriocin Family Members by Toxic Weakened Tumor Target Salmonella

This example demonstrates that a toxic attenuated target bacterium containing a nucleic acid encoding a bacteriocin family member, such as Salmonella, can express the bacteriocin family member.

15.1. Production of COLE3 plasmid

Plasmids disclosed herein serve to illustrate examples of certain embodiments of the invention. As will be apparent to those skilled in the art, promoters such as trc promoter and / or bacteriocin encoding nucleic acid and / or effector molecule encoding nucleic acids can be replaced by other suitable promoter or effector molecules by methods known in the art.

15.1.1. pE3. Shuttle-1 intermediate vector plasmid

pE3. Shuttle-1 is a multiple cloning site for cloning / selection into the plasmid vector ColE3-CA38 (SEQ ID NO: 34) and an intermediate vector used in the production of cassettes containing lacZ fragments. To facilitate cloning of BRP into E3, BRP was first cloned onto an intermediate shuttle vector (Figure 21). The vector contains a lacZ fragment that can be used to screen for clones on lactose in bacterial strains mutated on chromosome lacZ. The BRP fragment was then cloned into the E3 plasmid SmaI site (Figure 22) as a cassette containing the lacZ alpha complementary fragment. The lacZ fragment makes it possible to screen the insert at this stage (i.e., Lac +). The natural E3 plasmid does not have an antibiotic screening marker (Figure 23), but selection for the presence of the plasmid is possible by halo analysis (Pugsley, AP and Oudega, B. "Methods for Studying Colicins and Their Plasmids" in Plasmids , a Practical Approach 1987, edited by KG Hardy, Gilson, L. et al., EMBO J. 9: 3875-3884). The shuttle vector will facilitate the cloning of BRP onto the E3 plasmid, as well as the cloning of any DNA that can bind E3 or E3 / BRP. The new E3 / BRP plasmid was then transformed to 41.2.9 and tested for activity. A preliminary halo formation assay has demonstrated that the presence of BRP on the plasmid does not interfere with the ability of the strain to produce E3. 41.2.9. To determine whether E3 / BRP had enhanced activity compared to 41.2.9.E3, the lethal dose of E3 produced by each strain was determined (Figure 24). 41.2.9 E3 / BRP produces 100% more lethal units than 41.2.9 E3 alone, demonstrating that the strain has improved activity compared to 41.2.9 E3 alone.

15.1.2. Backlight "puncture" analysis for E3 activity

The susceptibility test strain (SK522) was grown to an OD ₆₀₀ of 0.8. 100 [mu] l of the test strain was added to 3 ml of warm (~ 55 [deg.] C) LB soft agar (for 100x15 mm dish) and quickly poured onto LB agar plates. The plate was shaken gently to spread evenly over the plate, and the agar was allowed to solidify for 10-15 minutes. Colonies of this E. coli or Salmonella that desired E3 activity assay were isolated with a sterile toothpick and "punctured" on the agar. The agar plate was then inverted and incubated overnight at 37 ° C. As the next day secreted collissin E3 kills the susceptible strain, a halo or transparent band appears around the E3 puncture. The colonies can be further treated with any of a variety of SOS-inducing agents, such as alkylating agents (e.g. mitomycin), ultraviolet or X-rays to further increase E3 production or secretion.

The results of one of these backlight analyzes are shown in Fig. When the bacterial strain secretes colchicine in the presence of a susceptible strain grown on bacterial cotton on the petri dish, the secreted colicin is diffused to kill bacterial cells contained in the bacterial cotton and dissolve them to produce a transparent band or halo . The size of the halo corresponds to the amount of secreted collins. The results shown in Figure 25 represent a number of strains. No luminescence is observed around the strain containing no colE3-CA38 plasmid. In the absence of induction, colicin is produced by Salmonella strains. It is also clear that all of these backlights increase in size in a dose-dependent manner by various types of derivations (e.g., alkylating agents, UV light, X-rays).

15.1.3. Overlay analysis on selectivity E3 clones

The transformants were plated on LB at various dilution ratios (1: 10,000) and grown at 37 ° C for 2 hours. The susceptibility test strains were then prepared as described above in the halo analysis and poured onto soft agar and poured. After solidifying for 10 minutes, the plate was turned upside down and incubated overnight at 37 ° C. Small transparent bands (resembling bacteriophage plaques) with small colonies (or colonies) appear in the middle of the next day.

15.1.4. &Quot; Plaque " or backlight tablet analysis

The small colonies in the center of the transparent band in the above-mentioned nested agar are then isolated using a sterile Pasteur pipette. If no colonies are seen in one halo or there are multiple colonies, the entire halo is picked up with a sterile Pasteur pipette. Transfer the colony or halo to 500 μl of LB. Dilute (up to 1: 10,000), resramble on LB agar and grow at 37 ° C for 2 hours. The overlay was then poured into the susceptible test strain as outlined above. The next day, all or most of these colonies have a halo around them.

16. Example: In vivo E3 injection, and measurement of retention of plasmid in salmonella

The following examples demonstrate retention of the colE3-CA38 plasmid in Salmonella in vivo.

Tumor and liver homogenates from 2 mice on the 30th day after injection of either 41.29 (or 41.2.9E3-CA38) were used in the study. In the following description, L is liver and T is tumor. All four homogenates were plated for CFU and the colonies were harvested for analysis by msbB PCR and for colchicine production. Almost ordered cultures of colonies similar to 41.2.9 were obtained from all homogenates. Five colonies were picked from each for colchicine and PCR analysis. An additional 30 colonies were picked from the 41.2.9 E3 T and L plates for further analysis when they appeared to be a mixture of collissin producers and non-producers in the 41.2.9E3 liver homogenate. Based on the results, an additional 100 colonies from 41.2.9E3 tumors and liver were picked and tested for colchicine production and msbB PCR. The distribution and plasmid retention were calculated from the combined data.

Table 3 shows the results of measurement of retention of plasmids in Salmonella by injection of E3 in vivo.

group CFU / ml Tissue weight CFU / g Colicin + number msb PCR + The retained plasmid% 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

In order for the colE3 plasmid to have an effect in vivo and to carry other genes to the in vivo tumor site, the colE3 plasmid must remain viable in vivo. The results obtained in these experiments were surprising and advantageous because the targets of the effector were tumors and thus would have little effect on the liver itself.

17. EXAMPLES: Variations in the M27 lung tumor model 41.2.9. Tumor targeting of the strain

The following experiments demonstrate the ability of the Salinella strains 41.2.9 colE3 and 41.2.9 colE3 BRP and 41.2.9 colE3 BRP-m (modified BRP) to target tumors.

The Salmonella strains listed in Table 4 below were injected into the animals with M27 vP tumor and the animals were killed on day 7. For the calculation of cfu / g, the organ weight was analyzed the following day. Tumors and liver were homogenized and plated on msbB to determine colony forming units (cfu). In Groups 1, 2, 4, and 6, the strains all accumulated in the liver in the range of 6x10 ⁴ to 4x10 ⁶ cfu / g in the liver and accumulate approximately 4x10 ⁸ cfu / g Rkwl in the tumors. Table 4 summarizes the data for all of the above groups and shows them as average cfu / g. All strains showed good tumor accumulation (10 ⁸ cfu / g or more) and provided positive tumor-to-liver ratios. BRP colE3 had the best rate, but not necessarily better than all other available strains. E3 and E3BRP strains accumulate at very high levels in tumors with a tumor-to-liver ratio of 100 to 200: 1.

group Strain Tumor (T) Liver (L) cfu / organization g Ratio (tumor: liver) One 41.2.9 / E3 T 5.1 ± 1.1 × ^{10 8} 131: 1 One 41.2.9 / E3 L 3.9 ± 3.6 × 10 ⁶ 2 41.2.9 / E3BRP T 4.6 ± 2.7 × ^{10 8} 209: 1 2 41.2.9 / E3BRP L 2.2 ± 1.3 × 10 ⁶ 6 ¹ 41.2.9 / E3BRP _m T 3.5 ± 0.15 × ^{10 8} 90: 1 6 ¹ 41.2.9 / E3BRP _m L 3.9 ± 3.6 × 10 ^{6 1} BRP _m has been modified to contain a point mutation at position 96 (G to A resulting in glycine causing an amino acid change to arginine) and position 114 (T to A resulting serine to change the amino acid to threonine) BRP. The variant BRP _m no longer causes similar degradation but can still secrete proteins from the bacteria (van der Wal, F., Koningstein, G., Ten Hagen, CM, Oudega, B. and Luirink, J. et al. (1998) Optimization of Bacteriocin Release Protein (BRP) -Medated Protein Release by Escherichia coli: Random Mutagenesis of the pCloDF13-Derived BRP Gene to Microbiology vol.64 pp 392-398).

18. Example: Effect of 41.2.9 / COLE3 on C38 mouse colorectal carcinoma

The following example demonstrates the ability of 41.2.9 / colE3 to inhibit the growth of C38 mouse colorectal carcinoma.

Colon 38 tumor fragments (2x2x2 ㎣) were subcutaneously transplanted into C57BL / 6 mice (female, 9 weeks old). When the tumor volume reached 1,000,, the tumor was removed from the mice under sterile conditions and cut into small pieces (about 2 x 2 x 2 ㎣ / fragment) and the process repeated for 5 cycles. The fragments were subcutaneously implanted in the right side abdomen of the mice using a tumor transplantation needle on the 0th day of tumor transplantation.

Animals were randomized on Day 0 of Salmonella administration when the tumor volume reached 150-200 ㎣. Freeze stock solutions of 41.2.9 and 41.2.9 / ColE3 were thawed at room temperature and diluted with PBS to a final concentration of 7.5 x 10 ⁶ cfu / ml, respectively. A 0.2 ml aliquot of bacterial suspension (1.5 x 10 ⁶ CFU / mouse) was subcutaneously administered to mice as a group expressed on day 0. The bacterial suspension was diluted to 1 x 10 ³ CFU, plated on an msb plate, and incubated overnight to determine the number of administered cfu. The tumors were measured twice a week until the end of the experiment. Three tumors from each group (ColE3) were excised and processed to measure retention of cfu and plasmid.

group: mouse One Untreated control group 8 2 41.2.9 (1.5x10 < ⁶ > / mouse) 8 3 41.2.9 / ColE3 (1.5x10 < ⁶ > / mouse) 8

The results for the efficacy of 41.29 / ColE3 on C38 mouse colon carcinoma are shown in Fig. The data demonstrate that mice treated by intravenous infusion of VNP20009 (41.2.9) can significantly inhibit the growth of C38 mouse colorectal carcinoma. In addition, when mice were treated with VNP20009 containing the ColE3 plasmid, tumor regression was achieved (i.e., tumors were smaller at the end of the experiment than at the beginning).

19. Example: Antitumor activity of VNP20009 / COLE3 on DLD1 human colon carcinoma in nude mice

The following example demonstrates the enhanced ability of the Salmonella variant VNP20009 / ColE3 (41.2.9 / ColE3) to inhibit the growth of DLDl human colon carcinoma compared to Salmonella variant 41.2.9.

The logarithmic growth of DLD1 cells was trypsinized, washed with PBS and resuspended in a suspension of 5 x ¹⁰⁶ cells / ml in PBS. A single cell suspension (0.1 ml) was injected subcutaneously into the right lateral abdomen of a 9-week-old nude female mouse (Charles River, Nu / Nu-CD1) at day 0. Ten to fifteen days after the tumor transplantation or when the tumor volume reached 300 to 400,, 10 animals were used in each group and randomized. CFUs of Salmonella strains 41.2.9 and 41.2.9 / ColE3 were counted day by day. Bacteria (41.2.9 and 41.2.9 / ColE3) were diluted to 1 x 10 ⁷ CFU / ml. A 0.2 ml aliquot of bacterial suspension (2 x 10 ⁶ CUF / mouse) was injected subcutaneously into mice on the indicated day. The bacterial suspension was diluted to 1 x 10 ³ CFU, 100 μl of each solution was plated on an msbB plate, and the plate was incubated overnight. The next day, bacterial colonies were counted. Tumors were measured twice a week.

group: mouse One Untreated control (PBS) 10 2 41.2.9 (2x10 < ⁶ > / mouse) 10 3 41.2.9 / ColE3 (2x10 < ⁶ > / mouse) 10

The antitumor activity results of 41.2.9 / ColE3 on DLDl human colon carcinoma in nude mice are shown in Fig. Strain 41.2.9 containing colicin E3 shows improved activity compared to strain 41.2.9 alone.

Example: Efficacy of 41.2.9 / COLE3 on B16 mouse melanoma in C57BL / 6 mice

The following example demonstrates the ability of Salmonella strain 41.2.9 / ColE3 to inhibit the growth of B16-F10 melanoma.

Removed by a B16-F10 cell proliferation group number trypsinization, washed with PBS, which was a composition material 5 x 10 ⁶ cells / ㎖ in PBS. A single cell suspension (0.1 ml) was injected subcutaneously into the right flank of 9 week old C57BL / 6 mice (female) at day 0. Ten animals were used in each group and were randomized at day 9 when the tumor volume reached 150-200 ㎣. Frozen stock solutions of Salmonella clone 41.2.9 and 41.2.9 / ColE3 were thawed at room temperature and diluted to a final concentration of 7.5 x 10 ⁶ cfu / ml respectively. A 0.2 ml aliquot of bacterial suspension (1.5 x 10 ⁶ CFU / mouse) was injected subcutaneously into mice as the group indicated on day 9. The bacterial suspension was diluted to 1 x 10 ³ CFU, plated on an msbB plate, and the plates were incubated overnight to determine the number of administered cfu of bacteria. Tumors were measured twice weekly until the end of the experiment.

group: mouse One Untreated control group 10 3 41.2.9 (1.5x10 < ⁶ > / mouse) 10 5 41.2.9 / ColE3 (1.5x10 < ⁶ > / mouse) 10

The efficacy of 41.2.9 / ColE3 on B16 murine melanoma in C57BL / 6 mice is shown in Fig. The data demonstrate that 41.2.9 (41.2.9) subcutaneously injected mice can significantly inhibit the growth of B16 mouse melanoma. In addition, mice treated with 41.2.9 / ColE3 showed significantly reduced tumor size at the initial time (up to 37 days) compared with 41.2.9 alone. This finding is very important because smaller tumor sizes can be more easily accepted for other therapies (eg, chemotherapeutic agents and x-rays).

21. Example: Antitumor efficacy of 41.2.9 / E3 combined with BRP

The following example demonstrates that the simultaneous expression of BRP and E3 in Salmonella variant 41.2.9 increases the antitumor efficacy of the variant.

Simultaneous expression of BRP and E3 in salmonella strain 41.2.9 increases the amount of E3 isolated from bacteria in vitro. If BRP increases the amount of E3 secreted from Salmonella in vivo, it can be assumed that this additional extracellular E3 will be readily available to tumor cells and thus cytotoxicity of these cells will be increased. In this experiment, four groups of animals (10 animals per group) were tested:

Group number process One Control (untreated) 2 41.2.9 3 41.2.9 / E3 4 41.2.9 / E3 / BRP

The model used in the experiment was human lung cancer strain HTB177. The cells were transplanted subcutaneously into the lateral abdomen of the mice on the first day. When tumors reached approximately 500,, animals were injected at day 14 with 1 x 10 ⁶ cfu of the strain described in the above table, or in the case of group 1 by intravenous injection. The tumor volume was measured weekly until 24 days. The results in Table 5 show that 41.2.9 itself can inhibit tumor growth (40% inhibition) while combination with E3 can increase antitumor efficacy (63%). However, when the strains having both E3 and BRP were used in this model, the antitumor efficacy was further enhanced (67% inhibition compared to the untreated control) and the enhanced inhibition was very marked at an earlier time 5).

The tumor inhibition rate for untreated controls Strain 17th 20 days 24th 41.2.9 50 38 40 41.2.9 / E3 63 58 63 41.2.9 / E3 / BRP 97 82 67

In conclusion, treatment with Salmonella with both cytotoxic colicin E3 and enhanced secretion system BRP increases antitumor efficacy compared to untreated control and 41.2.9 / E3 alone.

22. EXAMPLES: Combination of colicin E3-containing cell monolayer and x-ray treatment

The following example demonstrates that the combination of 41.2.9 with two doses of x-rays increases the survival time of the mice, which appears for x-ray alone.

The schedule was as follows: On day 0, the tumors were transplanted by subcutaneous administration of B16F10 melanoma (5 x 10 ⁵ cells / mouse) to the right side of the middle trunk of 100C57B6 female mice (5-7 weeks old). On day 8, Salmonella 41.2.9 containing choline E3 was injected and on days 12 and 26 x-rays were administered.

The results of the combination of salmonella containing colicin E3 and x-ray treatment are shown in Table 6.

category n = () Days up to 1g Average T / C A Mock 15Gy (6) 12, 12, 18, 18, 21 17 1.0 J 15Gy x-ray 12dpt, 26dpt (9) 14, 14, 18, 21, 25, 35, 35, 67, 67 33 1.9 K 41.2.9 + 15Gy x-ray 12dpt, 26dpt Regressive # 1, 2 (9) 21, 28, 35, 35, 56, 60, 60, 60, 67 47 2.8 L 41.2.9 / E3 + 15Gy x-ray 12dpt, 26dpt Regression d32 (9) 28, 39, 53, 56, 56, 60, 67, 74, 78 57 3.3

The data demonstrate that the combination of 41.2.9 with two doses of x-rays significantly increases the survival time of the mice exhibited by x-ray alone. E3 further increased the survival time of the mice that appeared for 41.2.9 + x-rays.

23. Example: Expression of Cytotoxic Necrosis Factor by Tumor Target Bacteria

The following example demonstrates the expression of this coli cytotoxic necrosis factor (CNF1) by tumor-targeted bacteria.

Cytotoxic necrosis factors include, for example, coli cytotoxic necrosis factor 1 (CNF1; Falbo et al., 1993, Infect. Immun. 61: 4904-4914), Vibrio fiserian CNF1 (Lin et al., 1998, Biochem. Biophys. Res. Comm., 250: 462-465) and E. coli cytotoxic necrosis factor 2 (CNF2, Sugai et al., 1999, Infect. Immun. 67: 6550-6557). The CNF family also includes Pasteurella mithiothida singlet (PMT), which shares an 80% conserved residue with a 27% match residue with the n-terminal part of CNF2 (Oswald et al., 1994, Proc. Acad, Sci USA 91: 3814-3818).

(SEQ ID NO: 35) and 5'-CACAGAGCTCGCGCTAACAAAACAGCACAAGGGAG-3 '(SEQ ID NO: 36) and primer (front) 5'-GTGTCATGAAAATGGGTAACCAATGGCAAC-3' (SEQ ID NO: 35) from E. coli J96 (ATCC 700336) ≪ / RTI > Approximately 3100 pb product was obtained and cloned into pTrc99adml NcoI and SacI sites for DNA sequencing as well as protein expression using this colony as DNA cloning host. DNA sequencing was performed by standard methods at Yale University's Keck Biotechnology Laboratory. The DNA sequencing has proved that the cloned PCR product is CNF1 presenting only 6 small sequence changes out of 3065 base pairs.

The CNF1 plasmid was electroporated into E. coli DNA cloning host DH5a and Salmonella strain YS1646 (International Application WO 99/13053). The expression of CNF1 was measured in E. coli DNA cloning host and Salmonella strain YS1649 using standard LDH analysis (Promega, Madison, Wis., Cytotox 96 (R)). Figure 29 shows that the presence of the CNF-containing plasmids improves cytotoxicity. Using subsequent analysis, Salmonella with the CNF-containing plasmid also showed other known properties of CNFl such as polynuclear formation (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 Figure 30 clearly show that the presence of CNFl in Salmonella results in expected polynucleation and cell enlargement.

24. Example: Expression of Verotoxin by Tumor-Mediated Bacteria

The following example demonstrates the cytotoxicity of verotoxin AB produced by tumor-targeted bacteria engineered to express verotoxin AB.

Berotoxin (synthetic HSC10 toxin, Shiga toxin, shiga toxin, Shigella toxin). The toxin was isolated from the colchicine-producing E. coli strain HSC10, which was originally believed to be a cholycin (Farkas-Himsley et al., 1995, Proc. Natl. Acad Sci 92 (15): 6996-7000). While this has a long history of antitumor activity, particularly antitumor activity against ovarian cancer and brain tumors, the antitumor activity is associated with purified formulations that are not associated with complete live bacteria.

Berotoxin was cloned from E. coli HSC10 (ATCC 55227) using primers based on the published sequence for berotoxin I and was confirmed by DNA-sequencing in the Yalekak biotechnology center using standard DNA sequencing techniques . Expression of verotoxin was achieved using the BRP gene under the control of tetracycline-inducible promoter poly- sistronic with the verotoxin A and B subunits. The tetracycline-inducible BRP verotoxin AB was cloned into a vector for chromosome integration using the msbB gene.

24.1. Production of vectors

24.1.1. AB amplification and cloning

Verotoxin AB (AB) was generated by PCR using the following primers:

H19B-7:

Forward: 5'-GTGTCCATGGCTAAAACATTATTAATAGCTGCATCGC-3 '(SEQ ID NO: 37); And

QSTX-R1:

Reverse: 5'-GTGTCTGCAGAACTGACTGAATTGAGATG-3 '(SEQ ID NO: 38).

These primers also contain external NcoI (5 ') and PstI (3') restriction endonuclease sites for cloning into the NcoI and PstI sites of ptrc99A.

24.1.2. Amplification and Cloning of TETBRP

TetBRP-AB was constructed in the intermediate vector pSP72-F6 / R6. TetBRP was generated by PCR using the following primers:

Tet-5 ':

Forward 5'-GTGTAGATCTTTAAGACCCACTTTCACATTTAAGTTG-3 '(SEQ ID NO: 39) and

BRP-TET-3 ':

Inverse 5'-CACAGGATCCTTACTGAACCGCGATCCCCG-3 '(SEQ ID NO: 40).

These primers contain BglII (5 ') and BamHI (3') restriction endonuclease sites for cloning into the BglII and BamHI sites of the pSP72-F6 / R6 vector.

24.1.3. Subcloning of AB into pSP72-F6 / R6-TETBRP

ptrc99A-AB was cleaved with BamHI and AvaI restriction endonuclease and AB was also removed to insert into pSP72F6 / R6-TetBRP truncated with BamHI and AvaI restriction endonuclease. The pSP72F6 / R6 vector contains a plurality of restriction endonuclease sites to clone lacZ-alpha trans-complement in addition to a portion of the-gal gene. The vector (pSP72F6 / R6-TetBRP) and the AB insert were both dissolved on 0.8% 1XTAE agarose gel and purified using a 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. Cells were plated on LB plates containing 100 [mu] g / ml Amp and 40 [mu] g / ml X-gal. Positive colonies were screened based on their resistance to ampicillin and the presence of a functional beta-gal gene (positive colonies were blue).

24.1.4. Subcloning of TETBRP-AB into pCDV442

cleaved with NotI and SfiI restriction endonuclease to subclone pSP72F6 / R6-TetBRP-AB into pCVD442 vector (also truncated with NotI and SfiI restriction endonuclease).

24.1.5. msbB chromosome vector

A vector capable of homologous recombination with DmsbB in the chromosome of strain VNP20009 (a.k.a. YS1646, international application WO 99/13053) was constructed with suicide vector pCVD442 (Donnenberg and Kaper, 1991, Infection and Immunity 59: 4310-4317). 5 'and 3' sections of msbB deletion (msbB-5 ': forward 5'-GTG TGA GCT CGA TCA ACC AGA 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' GTG TGC ATG CGG GGT TAA TTA AGG GGG CGG CCG CGT GGT ATT GGT TGA ACC GAC GGT CAT GAC ATG GC-3 '(SEQ ID NO: 43) and inverted 5'-GTG TCT CGA GGA TAT CAT TCT GGC CTC TGA CGT TGT G-3 '(SEQ ID NO: 44)). The primers also bind to the 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 through the common SphI site PacI, SphI, SfiI, SwaI and DraI to facilitate cloning of DNA fragments into DmsbB for stable chromosome integration without antibiotic resistance. This vector is referred to as pCVD442-msbB (see FIGS. 32 and 33).

To clone the Tet-BRP-AB into pCVD442-msbB, the Tet-BRP-AB plasmid DNA was restriction cleaved and the appropriate DNA was purified and the ligation reaction containing these two components was performed using T4 ligase . The coupling reaction was then converted to DH5 1 pir and colonies were screened for the presence and orientation of the Tet-BRP-AB. The Tet-BRP-AB clone was transformed with SM101 pir (Donnenberg and Kaper, 1991) and the plasmid was designated pCVD442-Tet-BRP-AB. SM10 1 pir colonies were screened for the Tet-BRP-AB gene by PCR and the Sm10 1 pirclone pCVD442-Tet-BRP-AB was selected for use as a hybridization donor for Salmonella strains. SM10 1 pir containing pCVD442-Tet-BRP-AB was transformed into Salmonella by the standard method (Davis, RW ,; Botstein, D., and Roth, JR 1980. Advanced Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor) Hybridization was carried out with strain YS50101 (a spontaneous derivative of the tetracycline resistant strain YS82 (Above, Low et al., 1999) resistant to Difco MacConkey agar) and incubated with 50 ug / ml carbenicillin and 300 ug / 0.0 > streptomycin < / RTI > (strep). The resulting YS50102-pCVD442-Tet-BRP-AB clone was tested for pCVD442-Tet-BRP-AB gene by PCR.

24.2 Strain 41.2.9 Transfer of pCVD442-Tet-BRP-AB Chromosomally integrated into 41.2.9 (YS1646) to generate Tet-BRP-AB

Using bacteriophage P22 (strain HT105 / 1 int-201; Davis et al., 1980) 41.2.9 was transfected with carbenicillin resistance using the YS50102-Tet-BRP-AB strain as donor. The presence of the bla and sac genes from pCVD442 was confirmed by the presence of carb ^r (or 41.2.9-pCVD-Tet-BRP-AB-1) containing both DmsbB and DmsbB-Tet-BRP-AB genes amp ^r ) suc ^s . Strain 41.2.9-Tet-BRP-AB-1 was plated on LB sucrose to select suc ^r carb ^s derivatives and LB-sucrose agar plates were prepared without NaCl and the plates were cultured at 30 ° C And the DmsbB gene was deleted and the DmsbB-Tet-BRP-AB gene was left according to the method of Donnenberg and Kaper, 1991 (FIG. 33, # 4). After proliferation of the colonies on the plate, they were transferred to a grid on an msbB plate to detect the presence of clones lacking both carbenicillin and sucrase markers and cloned into either the antibiotic and sucrose containing plates . The resulting clones were examined for the presence of the Tet-BRP-AB gene by PCR. One such derivative containing the chromosomal integrated Tet-BRP-AB gene and lacking sucrose sensitivity and carbenicillin resistance was designated as 41.2.9-Tet-BRP-verotoxin AB.

41.2.9-Tet-BRP-verotoxin AB was tested for cytotoxicity in vitro using standard LDH cytotoxicity assay (Cytotox ⁹⁶ (TM); Promega, Madison, Wis.). The results shown in Figure 34 demonstrate the toxic nature of verotoxin-expressing clones 26 and 31. Clones 26 and 31 had significantly higher cytotoxic percentages when treated with tetracycline than when not treated with tetracycline.

25. Example: Expression of hemolysin by tumor-target bacteria

The following examples demonstrate that tumor-targeting bacteria can be engineered to express hemolytic proteins such as hemolysin under structural or inducible regulation.

Hemolysin is a well-known cytotoxic protein with the ability to dissolve red blood cells (Beutin, 1991, Med. Microbiol. Immunol 180: 167-182). SheA (Genbank Number ECO238954) is a silent hemolysin that is found in E. coli with most wild-type not normally expressed (Fernandez et al., 1998, FEMS Microbiol Lett 168: 85-90). SheA (aka hlyE (Genbank Number U57430) was cloned from the wild type E. coli (strain 2507, E. coli Genetic Stock Center, Yale University) under the standard PCR conditions with the following primers: 5'-TTTTTTCCAT GGCTATTATG ACTGAAATCG TTGCAGATAA AACGG-3 (SEQ ID NO: 45) and (reverse) 5'-TTTTTTAAGC TTCCCGGGTC AGACTTCAGG TACCTCAAAG AGTGTC-3 '(SEQ ID NO: 46). The correct size of the PCR product was cloned into the NcoI and HindIII sites of ptrc99a (Pharmacia) in order to place it under the trc promoter, a partial constituent. The PCR product was also cloned into a tet-bgal-Z-term vector (described above) that had been cut with NcoI and EcoRV. The E. coli DH5a (Gibco) was subsequently transformed with the plasmid and plated on blood agar (tripik soy agar with 5% blood volume) without and with the addition of 0.2 ug / ml tetracycline. Positive colonies containing a clear halo around the colonies representing hemolysis were picked. Positive colonies were transformed with Salmonella YS501 by standard plasmid purification and rescreened for halo.

The formation of constitutive halo for the trc99a structure is shown in Figure 35 (2A and 2B), wherein the halo is observed without the addition or addition of tetracycline. Tetracycline-dependent halo formation of tetracycline-promoter-engineered SheA is shown in Figure 35 (3A and 3B), wherein the halo is not observed without the addition of tetracycline. These results demonstrate that tumor-target bacteria can express hemolytic proteins under constitutive or inducible regulation.

26. Example: Expression of Methionase by Tumor Target Bacteria

The following examples demonstrate that toxic attenuated tumor-targeted bacteria, such as Salmonella, can be engineered to express methionine.

Methionase is an enzyme that degrades methionine, an essential amino acid necessary for tumor growth. Methods of inhibiting tumor growth by administering purified methionase or methods of administering DNA or viral vectors that encode methionases have been disclosed (International Patent Application WO 00/29589, Number (Xu) and Tan (Tan) . Su and Tan did not disclose the use of tumor-specific bacterial vectors for the delivery of methionases and required large amounts of methionine to obtain efficacy by purified proteins. A novel method of delivering methionase directly to a tumor is to use the tumor-targeted bacteria to express the enzyme.

The following primers were used in Genbank No. 1. L43133 was prepared to produce methionase from Pseudomonas putida: < RTI ID = 0.0 >

Front: METH-XHOI

5'-CCGCTCGAGATGCACGGCTCCAACAAGCTCCCA-3 '(SEQ ID NO: 47); And

Station: METH-BAM

5'-CGCGGATCCTTAGGCACTCGCCTTGAGTGCCTG-3 '(SEQ ID NO: 48)

Using the primers listed above (4 mM) and the isolated colony of Pseudomonas putida as a template, the sequence of methionase was amplified by PCR under the following conditions: 1 cycle of 94 ° C for 5 minutes followed by 94 ° C for 1 minute , 35 cycles of 60 캜 for 1 minute and 72 캜 for 2 minutes. The final amplification step at 72 ° C for 10 minutes was included as the final step of the PCR reaction. The PCR product was solubilized on 0.8% 1X TAE agarose gel and a PCR product with an expected size (~ 1196 bp) for methionase was identified. The band was excised from the gel and purified using a quiagen gel extraction kit.

Both the obtained pSP72 vector and the isolated gel purified methionine gene were digested with restriction enzymes XhoI and BamHI. The cleaved vector and methionase were dissolved in 0.8% 1X TAE agarose gel. The linearized vector and the cleavage product corresponding to the cleaved methionine gene were excised from the gel and purified using a quiagen gel extraction kit. The linearized vector and insert (methionine) were ligated together using T4 ligase. Dh5a was transformed into E. coli cells by heat shock method. After recovery, the cells were plated on LB medium containing 100 mg / ml ampicillin (Amp) to select cells containing the complete pSP72 vector. Amp resistant colonies and the presence of the pSP72 vector containing the methionine gene was confirmed by plasmid production using restriction digest with the quiagen mini-prep kit and the enzymes EcoRI and PspHI. Clone # 9 was sent to Yale 's Yale sequencing facility for sequencing. Sequencing was performed using both SP6 (forward) and T7 (reverse) sequencing primers. The results demonstrate that the TGA stop codon is 100% identical to the published methionine sequence except that it was changed to TAA by PCR.

Methionase activity can be measured using the methionine assay described in Hori et al., 1996, Cancer Research 56: 2116-2122.

27. Example: Expression of apopentin protein as TAT fusion in toxic attenuated tumor-targeted bacteria

The following examples demonstrate that toxic attenuated tumor-targeted bacteria can be engineered to express and secrete fusion proteins including effector molecules and peripeptides such as TAT, Antennapedia, VP22 and Kaposi FGF MTS.

27.1. Production of TAT-Apophedin Vectors

The canary virus (CAV) protein, apophedin, is known to induce apoptosis in neoplastic cells upon delivery by adenoviral vectors (Noteborn et al., 1999, Gene Therapy 6: 882-892).

In order to produce a protein that can be transcribed in the cytoplasm of Salmonella and carried to the nucleus of the tumor cell and still have the ability to cause cell death, the apoptin protein is conjugated to a peptide derived from human immunodeficiency virus (HIV) TAT protein (Schwartze et al., 1999, Science 285: 1596-1572). Since TAT protein fusion has also been shown to be functional upon fusion to poly-histidine (hexa-histidine) amino acids that increase both positive charges and promote protein purification (Schwartze et al., 1999), the TAT- Tin fusion occurred in the presence and absence of the hexa-histidine (Figs. 36A and B). Furthermore, the TAT-adapthin fusion can be generated in the presence or absence of the OmpA-8L signal sequence (Figures 36A and C).

The above-mentioned apopentin and hexa-histadine apophedin were collected using a superimposed oligonucleotide. The apophedrin-encoded nucleic acid sequence was generated by PCR using the following oligonucleotides:

TAP1: 5'-GATCCCATGG CTTATGGCAG AAAAAAACGC CGTCAGCGCC GTCGCATGAA CGCGCTGCAG GAAGATACCC CGCCGGGCCC GTCCACCGTG TTTCGCCCGC CG-3 '(SEQ ID NO: 49)

TAP2: 5'-GGGACAGGGT GATGGTGATG CCCGCGATGC CGATGCGGAT TTCGCGGCAA TGCGGGGTTT CCAGCGGGCG GGAGGAGGTC GGCGGGCGAA ACACGGTGGA CGG-3 '(SEQ ID NO: 50)

TAP3: 5'-GGCATCGCGG GCATCACCAT CACCCTGTCC CTGTGCGGCT GCGCGAACGCGCGCGCGCCG ACCCTGCGCT CCGCGACCGC GGATAACTCC GAAAACACCG GC-3 '(SEQ ID NO: 51)

TAP4: 5'-GCGATATTCG GACGGATCGC AGGAGCGTTT TTTGGACGGC GGTTTCGGCT GATCGGTGCG CAGATCCGGG ACGTTTTTAA AGCCGGTGTT TTCGGAGTTA TCCGCGGTCG C-3 '(SEQ ID NO: 52)

TAP5: 5'-CCTGCGATCC GTCCGAATAT CGCGTCTCCG AACTGAAAGA ATCCCTGATC ACCACCACCC CGTCCCGCCC GCGCACCGCC CGCCGCTGCA TCCGCCTCTG AAACGTTCAT G-3 '(SEQ ID NO: 53)

TAP6: 5'-CATGAAGCTT TCAGAGGCGG ATGCAGCGGC GGGCGGTGCG-3 '(SEQ ID NO: 54).

The nucleic acid sequence encoding the hexa-stadin-containing version of the TAT-adhofine fusion protein is referred to as the TAP 2-TAT 6 oligonucleotide and the TAT 6H1 oligonucleotide (5'-GATCCCATGG CTCATCACCA TCACCACCAT TATGGCCGCA AAAAACGCCG TCAGCGCCGT CGCATGAACG CGCTGCAGGA AGATACCCCG CCGGGCCC- ; SEQ ID NO: 55). (5'-GATCCCATGG CTAAAAAGAC GTCTGCTCT TGCTGTTAGC GCTGACTAGT GTAGCGCAGG CCTATGGCCG CAAAAAACGC CGTCAGCGCC-3 '; SEQ ID NO: 56), which encodes the OmpA8L-containing version of the TAT-adapthin fusion protein, Lt; RTI ID = 0.0 > TAP1-TAP6 < / RTI > oligonucleotides using PCR.

Each oligonucleotide is formulated in a stock solution at a concentration of 4 [mu] M. Using pre-mixed PCR reaction beads (Pharmacia, ready-to-use beads), 2 [mu] l of each oligonucleotide was used. The PCR reaction was carried out for 5 minutes at 95 ° C for 1 cycle; 35 cycles at 95 占 폚 for 1 minute, 60 占 폚 for 1 minute, and 72 占 폚 for 1 minute; And one cycle at 72 캜 for 10 minutes. The PCR reaction was then extracted with phenol / chloroform, precipitated with ethanol, resuspended in water, and restriction cleaved with NcoI and HindIII. The restricted PCR products were digested by gel electrophoresis and the correct size products (approximately 420 and 450 bp for TAT-adapthin and hexahistidine-TAT-adipoffin, respectively) were excised from the gel and standard molecules Biological techniques were used to isolate. The product was ligated to Nco I and Hind III truncated ptrc99a (Pharmacia) to generate the ptrc99a-TAT-adapthin construct. An accurate DNA sequence was obtained for both the TAT-Apophedin (Figure 37) and the hexa-histidine TAT-Apophedin (Figure 38).

27.2 Demonstration of secretion and uptake of TAT-

The toxic attenuated tumor-targeted bacteria were transformed into ptrc99a-TAT-adhphtinin constructs by standard techniques known in the art (e. G., Heat shock or electroporation) and cultured in media. The supernatant of the bacterial culture is tested for the presence of TAT-adhoptin using techniques known to those skilled in the art (e.g. Western blot analysis or ELISA). Once the presence of TAT-adhopitin is confirmed in the supernatant of the bacterial culture, the bacterial culture supernatant is incubated with mammalian cells (e.g., NIH3T3, CHO293 and 293T cells) and the TAT- Is confirmed by means of an apoptin assay known to those skilled in the art.

27.3 Demonstration of TAT-Apoptin Absorption in the Tumor

Toxic attenuated tumor-targeted bacteria engineered to express TAT-apoptin or apophedin are administered intravenously to the B16 tumor model. The mice are killed several days after the administration of the bacteria and the organ weight is measured. Tumors may be treated with TAT-afrofutin or with apoptin using a cytotoxic assay known to those skilled in the art, such as DNA staging and fluorescent in situ cell killing assay kit (Boehringer Mannheim, Mannheim, Germany) Analyze for presence and paralysis. Further, the size of the tumor is analyzed to determine the antitumor activity of the TAT-apophedin. Tumors are also homogenized and streaked to determine colony forming units (c.f.u.).

28. EXAMPLES: Efficacy of VNP20009 and chemotherapeutic combinations on growth of M27 lung carcinoma in mice

The following examples demonstrate that administration of a toxicly attenuated tumor-targeting bacterium with a chemotherapeutic agent can synergistically or additionally inhibit the growth of a solid tumor, such as a lung cancer species.

28.1. The efficacy of combination of VNP20009 and cytoxan or VNP20009 and mitomycin for the growth of M27 lung carcinoma in mice

M27 mouse lung carcinoma cells (1 x 10 ⁶ / ml x 1 ml) stored in liquid nitrogen were recovered by rapid thawing at 37 ° C and DMEM culture medium containing 10% fetal calf serum (FCS) at 5% CO ₂ at 37 ° C Ml < / RTI > After passage of the cells for 2 generations, large-scale M27 cells were harvested by trypsinization, washed with 1 x PBS and resuspended to 2.5 x 10 ⁶ cells / ml in 1 x PBS for tumor implantation. M27 cell suspension was subcutaneously implanted into the right lateral abdomen with 100 C57BL / 6 mice (female, 8 weeks, 20 g; 5 x 10 ⁵ cells / mouse) at day 0. The mice were randomly divided into 10 groups, each group consisting of 10 mice.

Salmonella strain VNP20009 was diluted to 5 x 10 ⁶ CFU / ml with 1 x PBS according to the standard dilution procedure of the present invention. Each mouse was administered intravenously with 0.2 ml of salmonella (1 x 10 ⁶ CFU / mouse) diluted on day 12 according to Table 6 below. To measure the actual number of infused bacteria, the bacterial suspension of 5 x 10 ⁶ CFU / ml was further diluted to 1 x 10 ³ CFU / ml and plated onto nutrient agar (MsbB plates; international application WO 99/13053) Respectively. The number of colonies formed the following day was counted.

Mitomycin C (Sigma) and cytoxic (Sigma) were administered to mice according to Table 7 below. The second dose of mitomycin C was administered to the combination group on day 22 except for those treated with only mitomycin C due to the large tumor size. 200 mpk was administered to each mouse treated with VNP20009 alone or with VNP20009 + chemotherapy, because severe toxic reactions were observed in the group treated with VNP20009 + cytoxan (Cipro, Bayer Inc., West Haven, CT) It is because. Tumor volume was measured twice a week until the end of the experiment. The behavior, appearance and mortality of the animals were monitored daily. The mice were kept in a clean room. The bedding was changed twice a week and the mice were provided with sufficient food and drinking water.

group Number of mice Untreated control group 10 3 mpk mitomycin C, intravenous, 15 days 10 5 mpk mitomycin C, intravenous, 15 days 10 150 mpk Cytoxin, intraperitoneal, 15 days 10 200 mpk Cytoxin, intraperitoneal, 15 days 10 VNP20009, 1x10 ⁶ / mouse, iv, 12 10 VNP20009, 1x10 ⁶ / mouse, i.v., 12 +3 mpk mitomycin C, intravenous, 15 and 22 days 10 VNP20009, 1x10 ⁶ / mouse, i.v., 12 +5 mpk mitomycin C, intravenous, 15 and 22 days 10 VNP20009, 1x10 ⁶ / mouse, iv, 12 +150 mpk between toksan, intraperitoneal, 15 days 10 VNP20009, 1x10 ⁶ / mouse, iv, 12 +200 mpk between toksan, intraperitoneal, 15 days 10

As shown in Fig. 39, the combined treatment with VNP20009 + cytoxane inhibited the growth of M27 lung cancer species more than VNP20009 alone treatment or cytokine treatment alone. As shown in Fig. 40, the combination of VNP20009 + mitomycin C inhibited the growth of the M27 lung cancer species more than mitomycin C alone. However, the combination of VNP20009 + mitomycin C did not inhibit the growth of M27 lung cancer species more than VNP20009 alone (Fig. 40). These results suggest that toxic attenuated tumor-targeted bacteria may be co-administered with chemotherapeutic agents to synergistically or additionally inhibit the growth of solid tumors such as lung cancer.

28.2. The efficacy of VNP20009 and cisplatin combination on the growth of M27 lung carcinoma in mice

(1 x 10 ⁶ / ml x 1 ml) stored in liquid nitrogen was recovered by rapid thawing at 37 ° C and DMEM culture medium 25 containing 10% fetal bovine serum (FCS) at 37 ° C and 5% CO ₂ Ml < / RTI > After passage of the cells for 2 generations, large-scale M27 cells (about 90-95% saturation) were recovered by trypsin treatment, washed with 1 x PBS and resuspended in 2.5 x 10 < ⁶ > Cells / ml. M27 cell suspension (0.2 ml) was subcutaneously implanted into the right lateral abdomen with 36 C57BL / 6 mice (female, 8 weeks, 20 g; 5 x 10 ⁵ cells / mouse) at day 0. The mice were randomly divided into several groups, each group consisting of 9 mice.

Salmonella strain VNP20009 was diluted to 5 x 10 ⁶ CFU / ml with 1 x PBS according to the standard dilution procedure of the present invention. Each mouse was dosed with 0.2 ml of Salmonella (1 x ¹⁰⁶ CFU / mouse) diluted on day 12 according to the following Table 8 on the tail. To measure the actual number of infused bacteria, the 5 x 10 ⁶ CFU / ml bacterial suspension was further diluted to 1 x 10 ³ CFU / ml and plated on MsBb plates. The number of colonies formed the following day was counted.

Cisplatin was administered to mice on day 14 and bacteria were injected on day 2 (Table 8 below). The cisplatin was diluted with ordinary saline to 0.5 mg / ml and then administered. Tumor volume was measured twice a week until the end of the experiment. The behavior, appearance and mortality of the animals were monitored daily. The mice were kept in a clean room. The bedding was changed twice a week and the mice were provided with sufficient food and drinking water.

group Number of mice Untreated control group 9 VNP20009, 1 x 10 ⁶ / mouse, intravenously, day 12 9 5 mpk cisplatin, intraperitoneal, q.w x 2, 14 days, 19 days 9 VNP20009, 1x10 ⁶ / mouse, i.v., day 12 +5 mpk cisplatin, intraperitoneal, qw x 2, 14 days, 19 days, 33 days 9

As shown in FIG. 41, the combined treatment with VNP20009 + cisplatin inhibited the growth of M27 lung cancer species more than VNP20009 alone treatment or cisplatin alone treatment. These results suggest that toxic attenuated tumor-target bacteria can be co-administered with chemotherapeutic agents such as cisplatin to synergistically or additionally inhibit the growth of solid tumors such as lung cancer.

The present invention is not intended to be limited to the specific embodiments disclosed 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 drawings. Such modifications are intended to fall within the scope of the appended claims.

The contents of the various references cited herein are incorporated herein by reference in their entirety.

Claims (99)

A conditionally aerobic bacterium or conditionally anaerobic bacterium which comprises at least one nucleic acid molecule encoding at least one primary effector molecule operatively linked to one or more promoters. At least one primary effector molecule operatively linked to at least one promoter, and at least one nucleic acid molecule encoding at least one secondary effector molecule, wherein the aerobic bacterium is a condition aerobic bacterium or a condition anaerobic bacterium. The toxicantly attenuated tumor-target bacterium of claim 1 or 2, wherein at least one of the primary effector molecules is a member of the TNF family. 4. The method of claim 3, wherein the TNF family member is selected from the group consisting of tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor-alpha (TNF- alpha), TNF- CD40L, LT- [beta], OX40L, CD40L, FasL, CD27L, CD30L, and CD30L, associated kinase (TRANCE), TNF- [alpha] -related weak cytotoxic inducer (TWEAK) 1BBL, APRIL, LIGHT, TL1, TNFSF16, TNFSF17 or AITR-L. 3. The method of claim 1 or 2, wherein at least one of the primary effector molecules is an angiogenesis inhibiting factor. 6. The method of claim 5, wherein the angiogenesis inhibitory factor is endostatin, angiostatin, angiogenesis inhibiting antithrombin III, 29 kDa N-terminal and 40 kDa C-terminal proteolytic fragments of fibronectin, uPA receptor antagonist, 16 kDa protein of prolactin A 7.8 kDa proteolytic fragment of platelet factor-4, an angiogenesis-inhibiting 24 amino acid fragment of platelet factor-4, an angiogenesis inhibitor represented by 13.40, an angiogenesis inhibiting 22 amino acid peptide fragment of thrombospondin I, An angiogenesis inhibiting 20 amino acid peptide fragment of SPARC, RGD and NGR containing peptides, a small angiogenesis inhibiting peptide of laminin, fibronectin, procollagen and EGF, and a peptide antagonist of integrin? _V ? ₃ , or a toxic attenuated Tumor target bacteria. 3. The method of claim 1 or 2, wherein at least one of the primary effector molecules is a bacteriocin family member, but the bacteriocin is not a BRP. 8. The method of claim 7, wherein the bacteriocin family members are selected from the group consisting of ColE1, ColE1a, ColE1b, ColE2, ColE3, ColE4, ColE5, ColE6, ColE7, ColE8, ColE9, colicin A, colicin K, colicin L, colicin M, Toxic attenuated tumor-target bacteria, DF13, festisin A1122, staphylococcine 1580, butyrylcin 7423, pionin R1 or AP41, megasine A-216, vibriosin or microsin M15. 4. The toxicantly attenuated tumor-targeting bacterium of claim 1 or 2, wherein the primary effector molecule is a tumor suppressor enzyme. 10. The method of claim 9, wherein the tumor suppressor enzyme is methionase, asparaginase, lipase, phospholipase, protease, DNAase or glycosidase. The toxic attenuated tumor-targeting bacterium of claim 1 or 2, wherein the primary effector molecule is a hemolysin, berotoxin, CNF1, CNF2 or PMT. The toxic attenuated tumor-targeted bacterium of claim 1 or 2, wherein the primary effector molecule is derived from an animal, plant, bacterium or virus. 3. The method of claim 2, wherein the secondary effector molecule is an immunomodulator, an anti-tumor protein, a prodrug conversion enzyme, an antisense molecule, a ribozyme, or an antigen. The toxin-weakened tumor-targeting bacterium according to claim 1 or 2, which is Salmonella. The toxic attenuated tumor target bacterium of claim 1, further comprising an enhanced release system. 3. The method of claim 2, wherein the secondary effector molecule is a bacteriocin release factor (BRP). A toxic attenuated tumor target bacterium comprising at least one nucleic acid molecule encoding at least one fusion protein operatively linked to one or more promoters, wherein the toxic attenuated tumor target bacterium is a condition aerobic bacterium or condition anaerobic bacterium and the fusion protein Said toxin-weakened tumor-targeting bacterium comprising said signal sequence and effector molecule. A toxic attenuated tumor target bacterium comprising at least one nucleic acid molecule encoding at least one fusion protein operatively linked to one or more promoters, wherein the toxic attenuated tumor target bacterium is a condition aerobic bacterium or condition anaerobic bacterium and the fusion protein Said toxin-weakened tumor-targeting bacterium comprising said peripeptide and effector molecule. 19. The toxicant attenuated tumor-targeting bacterium of claim 18, wherein the fusion protein also comprises a signal sequence. The toxicantly attenuated tumor-targeting bacterium of claim 17 or 19 wherein the signal sequence is an OmpA-type protein. 20. The method of claim 18 or 19, wherein the peripeptide is selected from the group consisting of HIV TAT protein, Antennapedia homeodomain (pennelaxin), Kaposi fibroblast growth factor (FGF), membrane-translocation sequence (MTS) Toxic attenuated tumor-target bacteria derived from hexa histidine, hexa lysine or hexaarginine. 20. A toxicant attenuated tumor-target bacterium according to any one of claims 17 to 19, wherein the effector molecule is a primary or secondary effector molecule. 20. A toxin-weakened tumor-targeting bacterium according to any one of claims 17 to 19, further comprising at least one nucleic acid molecule encoding at least one effector molecule operatively linked to at least one promoter. 24. The method of claim 23, wherein the effector molecule is a primary or secondary effector molecule. A condition comprising at least one nucleic acid molecule encoding at least one primary effector molecule operatively linked to one or more promoters; a aerobic bacterium or condition; an anaerobic germ; a toxic attenuated tumor; and a pharmaceutical composition comprising a bacterium and a pharmaceutically acceptable carrier . Conditions comprising one or more primary effector molecules operatively linked to one or more promoters and one or more nucleic acid molecules encoding one or more secondary effector molecules aerobic bacterium or condition anaerobic gut toxin weakened tumor target bacterium and pharmaceutically acceptable Lt; / RTI > acceptable carrier. 26. The pharmaceutical composition according to claim 25 or 26, wherein at least one of the primary effector molecules is a member of the TNF family. 28. The method of claim 27, wherein the TNF family member is selected from the group consisting of tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor- alpha (TNF- alpha), TNF- CD40L, LT- [beta], OX40L, CD40L, FasL, CD27L, CD30L, and CD30L, associated kinase (TRANCE), TNF- [alpha] -related weak cytotoxic inducer (TWEAK) 1BBL, APRIL, LIGHT, TL1, TNFSF16, TNFSF17 or AITR-L. 26. The pharmaceutical composition according to claim 25 or 26, wherein at least one of the primary effector molecules is an angiogenesis inhibiting factor. 30. The method of claim 29, wherein the angiogenesis inhibitory factor is selected from the group consisting of endostatin, angiostatin, angiogenesis inhibiting antithrombin III, a 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragment of fibronectin, a uPA receptor antagonist, a 16 kDa protein of prolactin A 7.8 kDa proteolytic fragment of platelet factor-4, an angiogenesis-inhibiting 24 amino acid fragment of platelet factor-4, an angiogenesis inhibitor represented by 13.40, an angiogenesis inhibiting 22 amino acid peptide fragment of thrombospondin I, A pharmaceutical composition comprising an angiogenic inhibitory 20 amino acid peptide fragment of SPARC, RGD and NGR containing peptides, a small angiogenesis inhibitory peptide of laminin, fibronectin, procollagen and EGF, and a peptide antagonist of integrin? _V ? ₃ , or a VEGF receptor. 26. The pharmaceutical composition according to claim 25 or 26, wherein at least one of the primary effector molecules is a bacteriocin family member, provided that the bacteriocin is not a BRP. 31. The method of claim 31, wherein the bacteriocin family member is selected from the group consisting of ColE1, ColE1a, ColE1b, ColE2, ColE3, ColE4, ColE5, ColE6, ColE7, ColE8, ColE9, colicin A, colicin K, colicin L, colicin M, DF13, festisin A1122, staphylococcine 1580, butyrylcin 7423, pionin R1 or AP41, megasine A-216, vibriosin or microsine M15. 26. The pharmaceutical composition according to claim 25 or 26, wherein at least one of the primary effector molecules is a tumor suppressor enzyme. 34. The pharmaceutical composition according to claim 33, wherein the tumor suppressor enzyme is methionase, asparaginase, lipase, phospholipase, protease, DNAase or glycosidase. 26. The pharmaceutical composition according to claim 25 or 26, wherein at least one of the primary effector molecules is hemolysin, berotoxin, CNF1, CNF2 or PMT. 26. The pharmaceutical composition according to claim 25 or 26, wherein the primary effector molecule is derived from an animal, plant, bacterium or virus. 27. The pharmaceutical composition according to claim 26, wherein the second effector molecule is an immunomodulator, an anti-tumor protein, a prodrug conversion enzyme, an antisense molecule, a ribozyme or an antigen. 27. The pharmaceutical composition according to claim 25 or 26, wherein the toxic attenuated tumor target bacterium is salmonella. 26. The pharmaceutical composition of claim 25, wherein the toxic attenuated tumor target bacteria also comprises an enhanced release system. 27. The pharmaceutical composition according to claim 26, wherein the secondary effector molecule is a bacteriocin releasing factor. One or more nucleic acid molecules encoding one or more fusion proteins operatively linked to one or more promoters, wherein the fusion protein comprises a signal sequence and an effector molecule, wherein the fusion protein is a condition aerobic bacterium or a condition anaerobic bacterium, A pharmaceutical composition comprising a pharmaceutically acceptable carrier. One or more nucleic acid molecules that encode one or more fusion proteins operatively linked to one or more promoters, wherein the fusion protein comprises a peri-peptide and an effector molecule, wherein the fusion protein is a condition aerobic bacterium or a condition anaerobic bacterium, A pharmaceutical composition comprising a pharmaceutically acceptable carrier. 43. The pharmaceutical composition of claim 42, wherein the fusion protein further comprises a signal sequence. 44. The pharmaceutical composition according to claim 41 or 43, wherein the signal sequence is an OmpA-type protein. 44. The method according to claim 42 or 43, wherein the peripeptide is selected from the group consisting of HIV TAT protein, Antennapedia homeodomain (pennelaxin), Kaposi fibroblast growth factor (FGF), membrane-translocation sequence (MTS) Hexa-histidine, hexa-lysine or hexaarginine. 44. The pharmaceutical composition according to any one of claims 41 to 43, wherein the effector molecule is a primary or secondary effector molecule. 44. The pharmaceutical composition according to any one of claims 41 to 43, further comprising at least one nucleic acid molecule encoding the at least one effector molecule in which the toxic attenuated tumor target bacteria is operatively linked to one or more promoters. A condition comprising one or more nucleic acid molecules encoding one or more primary effector molecules operably linked to one or more promoters for a patient in need of treatment for a solid tumor cancer, wherein the aerobic bacterium is a condition aerobic bacterium or a condition anaerobic bacterium; A method of delivering the at least one primary effector molecule to a patient for treatment of the cancer, comprising administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier. Comprising administering to a patient in need of treatment of a solid tumor cancer one or more primary effector molecules operably linked to one or more promoters and one or more nucleic acid molecules encoding one or more secondary effector molecules, Delivering said at least one primary effector molecule and at least one secondary effector molecule to said patient for the treatment of said cancer, comprising administering to said patient a pharmaceutical composition comprising a toxicantly attenuated tumor-targeted bacterium and a pharmaceutically acceptable carrier. How to. 49. The method of claim 48 or 49, wherein at least one of the primary effector molecules is a member of the TNF family. 52. The method of claim 50, wherein the TNF family member is selected from the group consisting of tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor-alpha (TNF- alpha), TNF- CD40L, LT- [beta], OX40L, CD40L, FasL, CD27L, CD30L, and CD30L, associated kinase (TRANCE), TNF- [alpha] -related weak cytotoxic inducer (TWEAK) 1BBL, APRIL, LIGHT, TL1, TNFSF16, TNFSF17 or AITR-L. 49. The method of claim 48 or 49, wherein at least one of the primary effector molecules is an angiogenesis inhibitory factor. 53. The method of claim 52, wherein the angiogenesis inhibitory factor is selected from the group consisting of endostatin, angiostatin, angiogenesis inhibiting antithrombin III, a 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragment of fibronectin, a uPA receptor antagonist, a 16 kDa protein of prolactin A 7.8 kDa proteolytic fragment of platelet factor-4, an angiogenesis-inhibiting 24 amino acid fragment of platelet factor-4, an angiogenesis inhibitor represented by 13.40, an angiogenesis inhibiting 22 amino acid peptide fragment of thrombospondin I, A small angiogenesis inhibiting peptide of laminin, fibronectin, procollagen and EGF, and a peptide antagonist of integrin [alpha] _{v [} beta] ₃ , or a VEGF receptor, wherein the angiogenesis inhibiting 20 amino acid peptide fragment of SPARC, RGD and NGR containing peptides. 49. The method of claim 48 or 49 wherein at least one of the primary effector molecules is a bacteriocin family member, provided that the bacteriocin is not a BRP. 55. The method of claim 54, wherein the bacteriocin family member is selected from the group consisting of ColE1, ColE1a, ColElb, ColE2, ColE3, ColE4, ColE5, ColE6, ColE7, ColE8, ColE9, Colicin A, Colicin K, Colicin L, Colicin M, DF13, festisin A1122, staphylococcine 1580, butyrylline 7423, pionin R1 or AP41, megasine A-216, vibriosin or microsine M15. 49. The method of claim 48 or 49 wherein at least one of the primary effector molecules is a tumor suppressor enzyme. 57. The method of claim 56, wherein the tumor suppressor enzyme is methionase, asparaginase, lipase, phospholipase, protease, DNAase or glycosidase. 49. The method of claim 48 or 49 wherein at least one of the primary effector molecules is hemolysin, verotoxin, CNF1, CNF2 or PMT. 49. The method of claim 48 or 49 wherein at least one of the primary effector molecules is derived from an animal, plant, bacterium, or virus. 50. The method of claim 49, wherein at least one of the secondary effector molecules is an anti-tumor protein, a prodrug conversion enzyme, an antisense molecule, a ribozyme, or an antigen. 49. The method of claim 48 or 49, wherein the toxic attenuated tumor target bacterium is Salmonella. 49. The method of claim 48, wherein the toxic attenuated tumor target bacteria also comprises an enhanced release system. 50. The method of claim 49, wherein the second effector molecule is a bacteriocin release factor. A method of treating a patient suffering from a solid tumor, comprising administering to a patient in need of such treatment one or more nucleic acid molecules encoding one or more fusion proteins operably linked to one or more promoters, said conditioned aerobic bacteria or condition anaerobic bacteria, A method of delivering one or more fusion proteins to a patient for treatment of said cancer, said method comprising administering to said patient a pharmaceutical composition comprising a toxic attenuated tumor target bacteria comprising a molecule and a pharmaceutically acceptable carrier. A method of treating a patient suffering from fulminant tumor cancer comprising administering to a patient in need of such treatment one or more nucleic acid molecules encoding one or more fusion proteins operably linked to one or more promoters and expressing said fusion protein in a fermentative or effector A method of delivering said at least one fusion protein to said patient for treatment of said cancer, comprising administering to said patient a pharmaceutical composition comprising a toxic attenuated tumor target bacteria comprising a molecule and a pharmaceutically acceptable carrier. 65. The method of claim 65, wherein the fusion protein also comprises a signal sequence. 67. The method according to claim 64 or 66, wherein the signal sequence is an OmpA-type protein. 66. The method of claim 65 or 66 wherein the peripeptide is selected from the group consisting of HIV TAT protein, Antennapedia homeodomain (pennelaxin), Kaposi fibroblast growth factor (FGF), membrane-translocation sequence (MTS) Hexa-histidine, hexa- lysine or hexaarginine. 66. The method according to any one of claims 64 to 66, wherein the effector molecule is a primary or secondary effector molecule. 66. The method of any one of claims 64 to 66, further comprising one or more nucleic acid molecules that encode one or more effector molecules in which the toxic attenuated tumor-target bacteria is operatively linked to one or more promoters. One or more chemotherapeutic agents, and one or more nucleic acid molecules encoding one or more primary effector molecules operably linked to one or more promoters. Aerobic bacterium or condition anaerobic germ. Toxic weakened tumor. Target bacterium and pharmaceutically acceptable A method of treating a solid tumor cancer in an animal, comprising administering a pharmaceutical composition comprising a possible carrier. One or more chemotherapeutic agents, and one or more primary effector molecules operatively linked to one or more promoters and one or more nucleic acid molecules encoding one or more secondary effector molecules. Aerobic bacterium or condition. A method of treating a solid tumor carcinoma in an animal, comprising administering a pharmaceutical composition comprising a target bacterium and a pharmaceutically acceptable carrier. 72. The method of claim 71 or 72 wherein at least one of the primary effector molecules is a member of the TNF family. 73. The method of claim 73, wherein the TNF family member is selected from the group consisting of tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor- alpha (TNF- alpha), TNF- CD40L, LT- [beta], OX40L, CD40L, FasL, CD27L, CD30L, and CD30L, associated kinase (TRANCE), TNF- [alpha] -related weak cytotoxic inducer (TWEAK) 1BBL, APRIL, LIGHT, TL1, TNFSF16, TNFSF17 or AITR-L. 72. The method of claim 71 or 72 wherein at least one of the primary effector molecules is an angiogenesis inhibitory factor. Wherein the angiogenesis inhibitory factor is selected from the group consisting of endostatin, angiostatin, angiogenesis inhibiting antithrombin III, a 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragment of fibronectin, a uPA receptor antagonist, a 16 kDa protein of prolactin A 7.8 kDa proteolytic fragment of platelet factor-4, an angiogenesis-inhibiting 24 amino acid fragment of platelet factor-4, an angiogenesis inhibitor represented by 13.40, an angiogenesis inhibiting 22 amino acid peptide fragment of thrombospondin I, A small angiogenesis inhibiting peptide of laminin, fibronectin, procollagen and EGF, and a peptide antagonist of integrin [alpha] _{v [} beta] ₃ , or a VEGF receptor, wherein the angiogenesis inhibiting 20 amino acid peptide fragment of SPARC, RGD and NGR containing peptides. 72. The method of claim 71 or 72 wherein at least one of the primary effector molecules is a bacteriocin family member, provided that the bacteriocin is not a BRP. 78. The method of claim 77, wherein the bacteriocin family members are selected from the group consisting of ColE1, ColE1a, ColElb, ColE2, ColE3, ColE4, ColE5, ColE6, ColE7, ColE8, ColE9, colicin A, colicin K, colicin L, colicin M, DF13, festisin A1122, staphylococcine 1580, butyrylline 7423, pionin R1 or AP41, megasine A-216, vibriosin or microsine M15. 72. The method of claim 71 or 72 wherein at least one of the primary effector molecules is a tumor suppressor enzyme. 79. The method of claim 79, wherein the tumor suppressor enzyme is methionase, asparaginase, lipase, phospholipase, protease, DNAase or glycosidase. 72. The method of claim 71 or 72 wherein at least one of the first effector molecules is hemolysin, verotoxin, CNF1, CNF2 or PMT. 72. The method of claim 71 or 72 wherein the primary effector molecule is derived from an animal, plant, bacterium or virus. 83. The method of claim 82, wherein the second effector molecule is an immunomodulator, an anti-tumor protein, a prodrug conversion enzyme, an antisense molecule, a ribozyme, or an antigen. 73. The method of claim 71 or 72, wherein the toxic attenuated tumor target bacterium is salmonella. 74. The method of claim 71, wherein the toxic attenuated tumor target bacteria also comprises an enhanced release system. 73. The method of claim 72, wherein the second effector molecule is a bacteriocin release factor. One or more chemotherapeutic agents, and one or more nucleic acid molecules encoding one or more fusion proteins operatively linked to one or more promoters, wherein the conditioned aerobic bacteria or conditioned anaerobic bacteria comprise the signal sequence and the effector molecule A method of treating a solid tumor carcinoma in an animal, comprising administering a toxic attenuated tumor-targeting bacterium and a pharmaceutically acceptable carrier. One or more chemotherapeutic agents, and one or more nucleic acid molecules that encode one or more fusion proteins operatively linked to one or more promoters, wherein the conditioned aerobic bacteria or conditioned anaerobic bacteria comprise the ferripeptide and the effector molecule A method of treating a solid tumor carcinoma in an animal, comprising administering a pharmaceutical composition comprising a toxic attenuated tumor-targeted bacterium and a pharmaceutically acceptable carrier. 90. The method of claim 88, wherein the fusion protein also comprises a signal sequence. 87. The method of claim 87 or 89, wherein the signal sequence is an OmpA-type protein. 88. The method of claim 88 or 89 wherein the peripeptide is selected from the group consisting of an HIV TAT protein, an Antennapedia homeodomain (pennelaxin), a Kaposi fibroblast growth factor (FGF), a membrane-translocation sequence (MTS) Hexa-histidine, hexa-lysine or hexaarginine. 90. The method according to any one of claims 87 to 89, wherein the effector molecule is a primary or secondary effector molecule. 89. The method of any one of claims 87 to 89, further comprising at least one nucleic acid molecule encoding a one or more effector molecules wherein the toxic attenuated tumor target bacteria is operatively linked to one or more promoters. A method of treating a solid tumor cancer in an animal, comprising administering to the animal a pharmaceutical composition comprising at least one chemotherapeutic agent, and a pharmaceutically acceptable carrier and a toxic attenuated tumor target bacteria. A fusion protein comprising an OmpA-type protein and an effector molecule. A fusion protein comprising a signal sequence, a peripeptide and an effector molecule. 96. The fusion protein of claim 96, wherein the signal sequence is an OmpA-type protein. 96. The method of claim 96, wherein the peripeptide is selected from the group consisting of an HIV TAT protein, an Antennapedia homeodomain (pennelaxin), a Kaposi fibroblast growth factor (FGF), a membrane-translocation sequence (MTS), a herpes simplex virus VP22, , Hexa lysine or hexaarginine. 96. The fusion protein of claim 95 or 96, wherein the effector molecule is a primary or secondary effector molecule.