Protease inhibitor: protease sensitive expression system composition and methods improving the therapeutic activity and specificity of proteins delivered by bacteria

Description

1. FIELD OF THE INVENTION

This invention is generally in the field of therapeutic delivery systems, systems and methods for providing co-expression of protease inhibitors with genetically engineered protease sensitive therapeutic constructs, and chimeric proteins.

2. BACKGROUND OF THE INVENTION

Citation or identification of any reference herein, or any section of this application shall not be construed as an admission that such reference is available as prior art to the present application. The disclosures of each of these publications and patents are hereby incorporated by reference in their entirety in this application, and shall be treated as if the entirety thereof forms a part of this application.

Tumor-targeted bacteria offer tremendous potential advantages for the treatment of solid tumors, including the targeting from a distant inoculation site and the ability to express therapeutic agents directly within the tumor (Pawelek et al., 1997, Tumor-targeted Salmonella as a novel anticancer agent, Cancer Research 57: 4537-4544; Low et al., 1999, Lipid A mutant salmonella with suppressed virulence and TNF-alpha induction retain tumor-targeting in vivo, Nature Biotechnol. 17: 37-41). However, the primary shortcoming of tumor-targeted bacteria investigated in the human clinical trials (Salmonella strain VNP20009 and its derivative TAPET-CD; Toso et al., 2002, Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma, J. Clin, Oncol. 20: 142-152; Meir et al., 2001, Phase 1 trial of a live, attenuated Salmonella Typhimurium (VNP20009) administered by direct Intra-tumoral (IT) injection, Proc Am Soc Clin Oncol 20: abstr 1043); Nemunaitis et al., 2003, Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients, Cancer Gene Therapy 10: 737-744) is that no significant antitumor activity has been observed, even in patients where the bacteria was documented to target the tumor. One method of increasing the ability of the bacteria to kill tumor cells is to engineer the bacteria to express conventional bacterial toxins (e.g., WO 2009/126189, WO 03/014380, WO/2005/018332, WO/2008/073148, US 2003/0059400 U.S. Pat. Nos. 7,452,531, 7,354,592, 6,962,696, 6,923,972, 6,863,894, 6,685,935, 6,475,482, 6,447,784, 6,190,657 and 6,080,849, each of which is expressly incorporated herein by reference), but these approaches pose risks of systemic toxicity.

Use of protein toxins for treatment of various disorders including inflammation, autoimmunity, neurological disorders and cancer has long-suffered from off-target toxicity. Enhancing toxin specificity, which offers the potential to eliminate side effect, has been achieved by several different means, such as attachment of a specific antibodies or peptide ligand (e.g., Pseudomonas endotoxin A (PE-ToxA) antibody conjugate, known as an immunotoxin). Based upon the binding specificity of the attached antibody moiety for a specific target, enhanced specificity of the target is achieved. Other toxins have been engineered to achieve specificity based upon their site of activation. For example, proaerolysin requires proteolytic activation to become the cytotoxic protein aerolysin. Substitution of the natural protease cleavage site for a tumor-specific protease cleavage site (e.g., that of the prostate specific antigen (PSA) protease or urokinase) results in a toxin selectively activated within tumors (Denmeade et al. WO 03/018611 and Denmeade et al. U.S. Pat. No. 7,635,682). Another similar activation system has utilized ubiquitin fusion, coupled with a hydrolysable tumor protease (e.g., PSA) sequence and a toxin (e.g., saporin), as described by Tschrniuk et al. 2005 (Construction of tumor-specific toxins using ubiquitin fusion technique, Molecular Therapy 11: 196-204). However, while some specificity is engendered and thus these activated protein types are useful in the present invention as modified herein, in these types of engineered toxins, off-target toxicity can occur. In the case of the Pseudomonas immunotoxin, several dose-limiting toxicities have been identified. Vascular leakage syndrome (VLS) is associated with hypoalbuminemia, edema, weight gain, hypotension and occasional dyspnea, which is suggested to occur by immunotoxin-mediated endothelial cell injury (Baluna et al., 2000, Exp. Cell Res. 258: 417-424), resulting in a dose-limiting toxicity. Renal injury has occurred in some patients treated with immunotoxins, which may be due to micro-aggregates of the immunotoxin (Frankel et al., 2001, Blood 98: 722a). Liver damage from immunotoxins is a frequent occurrence that is believed to be multifactorial (Frankel, 2002, Clinical Cancer Research 8: 942-944). To date, antibodies linked to proteinaceous toxins have limited success clinically. One explanation for the off target toxicity is that although a specific agent is targeted to the tumor and/or specifically activated there, the agent is also toxic if it diffuses out of the tumor, which is likely to occur due to the high osmotic pressure that occurs within tumors (Jain, R. K., 1994, Barriers to drug delivery in solid tumors, Scientific American 271 (11): 58-65). Once activated inside the tumor and having diffused back outside, toxins such as aerolysin remain active and are able to contribute to non-target toxicity. Never-the-less, delivery of targeted pro-toxins is of interest by targeted bacteria if systemic toxicity can be overcome and the toxin remains active only at the target site.

Seed et al., WO/2009/014650 have suggested the fusion of proteases with Vibrio cholerae exotoxins. These authors suggest that protease (proteinase) inhibitors may hamper the activity of the fusions. They teach ways to maintain fusion protein activity and conclude for example: “Thus, it is possible to keep granzyme fusion proteins active in plasma through formulations using chondroitin sulfates”. In the context of delivery by a tumor-localized vector, such activity would be expected to contribute to toxic side effects since the toxin would remain active in the blood and reach other organs of the body.

Use of secreted proteins in live bacterial vectors has been demonstrated by several authors. Holland et al. (U.S. Pat. No. 5,143,830, expressly incorporated in its entirety herein by reference) have illustrated the use of fusions with the C-terminal portion of the hemolysin A (hlyA) gene, a member of the type I secretion system. When co-expressed in the presence of the hemolysin protein secretion channel (hlyBD) and a functional TolC, heterologous fusions are readily secreted from the bacteria. The type I secretion system that has been utilized most widely, and although it is currently considered the best system available, is thought to have limitations for delivery by attenuated bacteria (Hahn and Specht, 2003, FEMS Immunology and Medical Microbiology, 37: 87-98). Those limitations include the amount of protein secreted and the ability of the protein fused to it to interfere with secretion. Improvements of the type I secretion system have been demonstrated by Sugamata and Shiba (2005 Applied and Environmental Micobiology 71: 656-662) using a modified hlyB, and by Gupta and Lee (2008 Biotechnology and Bioengineering, 101: 967-974) by addition of rare codons to the hlyA gene, each of which is expressly incorporated by reference in their entirety herein. Fusion to the gene ClyA (Galen et al., 2004, Infection and Immunity, 72: 7096-7106 and Type III secretion proteins have also been used. Surface display has been used to export proteins outside of the bacteria. For example, fusion of the Lpp protein amino acids 1-9 with the transmembrane region B3-B7 of OmpA has been used for surface display (Samuelson et al., 2002, Display of proteins on bacteria, J. Biotechnology 96: 129-154, expressly incorporated by reference in its entirety herein). The autotransporter surface display has been described by Berthet et al., WO/2002/070645, expressly incorporated by reference herein. Other heterologous protein secretion systems utilizing the autotransporter family can be modulated to result in either surface display or complete release into the medium (see Henderson et al., 2004, Type V secretion pathway: the autotransporter story, Microbiology and Molecular Biology Reviews 68: 692-744; Jose, 2006 Applied Microbiol. Biotechnol. 69: 607-614; Jose J, Zangen D (2005) Autodisplay of the protease inhibitor aprotinin in Escherichia coli. Biochem Biophys Res Commun 333:1218-1226 and Rutherford and Mourez 2006 Microbial Cell Factories 5: 22). For example, Veiga et al. (2003 Journal of Bacteriology 185: 5585-5590 and Klauser et al., 1990 EMBO Journal 9: 1991-1999) demonstrated hybrid proteins containing the □-autotransporter domain of the immunoglobulin A (IgA) protease of Nisseria gonorrhea. Fusions to flagellar proteins have been demonstrated. The peptide, usually of 15 to 36 amino acids in length, is inserted into the central, hypervariable region of the FliC gene such as that from Salmonella muenchen (Verma et al. 1995 Vaccine 13: 235-24; Wu et al., 1989 Proc. Natl. Acad. Sci. USA 86: 4726-4730; Cuadro et al., 2004 Infect. Immun. 72: 2810-2816; Newton et al., 1995, Res. Microbiol. 146: 203-216, expressly incorporated by reference in their entirety herein). Multihybrid FliC insertions of up to 302 amino acids have also been prepared (Tanskanen et al. 2000, Appl. Env. Microbiol. 66: 4152-4156, expressly incorporated by reference in its entirety herein). Trimerization of antigens can be achieved using the T4 fibritin foldon trimerization sequence (Wei et al. 2008 J. Virology 82: 6200-6208) and VASP tetramerization domains (Kühnel et al., 2004 PNAS 101: 17027-17032), expressly incorporated by reference in their entirety herein. The multimerization domains are used to create, bi-specific, tri-specific, and quad-specific targeting agents, whereby each individual agent is expressed with a multimerization tag, each of which may have the same or separate targeting peptide, such that following expression, surface display, secretion and/or release, they form multimers with multiple targeting domains.

Surprisingly, although bacteria have been used vectors for neoplastic disease and several authors have suggested delivering cytotoxins and other agents, no means of conferring safety and specificity of the actual cytotoxic agent has been developed within the delivery platform itself. Therapeutic protein activity proximal to the delivery vector at the target site, such as a solid tumor, lymphoma or leukemic bone marrow, and inactivation distal to the delivery vector, has remained to be achieved.

3. SUMMARY OF THE INVENTION

3.1 Therapeutic Molecules and Protease Inhibitors

The present invention consists of known and/or novel chimeric proteins, or combination of proteins, that are expressed, secreted, surface displayed and/or released by bacteria and result in anti-cancer activity or have direct inhibitory or cytotoxic anti-neoplastic activity. The bacterial delivery vector may be attenuated, non-pathogenic, low pathogenic (including wild type), or a probiotic bacterium. The bacteria are introduced either systemically (e.g., parenteral, intravenous (IV), intramuscular (IM), intralymphatic (IL), intradermal (ID), subcutaneously (sub-q), local-regionally (e.g., intralesionally, intratumorally (IT), intraperitoneally (IP), topically, intrathecally (intrathecal), by inhaler or nasal spray) or to the mucosal system through oral, nasal, pulmonary intravessically, enema or suppository administration where they are able to undergo limited replication, express, surface display, secrete and/or release the anti-cancer inhibitory proteins or a combination thereof, and thereby provide a therapeutic benefit by reducing or eliminating the disease, malignancy and/or neoplasia.

The present invention further consists of the co-expression by a bacterial expression system, or a combination of bacterial expression systems, of one or more protease inhibitors together with one or more protease sensitive therapeutic agent. The therapeutic agent may be inherently sensitive to proteases, or engineered to have enhanced sensitivity. Within the local high-concentration of the targeted tissue or cells such as the confines of a solid tumor, lymph node or lumen of a bone, the protease inhibitor prevents the degradation of the therapeutic agent that is therapeutically active against the target tissue such as colon cancer cells within a tumor, lymphoma cells within a lymph node, or leukemic cells within the lumen of a bone. Upon egress from the confined space of the targeted tissue, the inhibitor falls below the inhibitory concentration, and the therapeutic agent which is protease sensitive is freely degraded, thus deactivating it outside the target site, resulting in cell or tissue-specific activity as well as increased activity and inactivation in non-target cell or tissues (Table I). A schematic diagram comparing the relative effect of co-expression is shown in FIG. 1. This surprising solution to the problem of off target toxicity by a tumor targeting vector stems from the unique localized production of therapeutic agents by tumor-targeted vectors, wherein the active agent is produced locally and subsequently diffuses out resulting in systemic exposure rather than being injected peripherally with intent to treat distal sites.

TABLE 1

Projected relative effects of toxin forms with and

without protease sensitivity and protease inhibitor(s).

Tumor
Systemic

Composition
Efficacy
Toxicity

Unmodified toxins
+++
+++

Protease activated and/or insensitive toxin
+++
++

e.g., prostate protease activated Aerolysin

Protease sensitive (i.e., deactivated) toxin
+
—

Protease sensitive toxin + protease inhibitor
+++++
—

The types of cancers or neoplasias to which the present invention is directed include all neoplastic malignancies, including solid tumors such as those of colon, lung, breast, prostate, sarcomas, carcinomas, head and neck tumors, melanoma, as well as hematological, non-solid or diffuse cancers such as leukemia and lymphomas, myelodysplastic cells, plasma cell myeloma, plasmacytomas, and multiple myelomas. Specific types of cancers include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma adrenocortical carcinoma, aids-related cancers, aids-related lymphoma, anal cancer, appendix cancer, astrocytomas, childhood, teratoid/rhabdoid tumor, childhood, central nervous system tumors, basal cell carcinoma, bile duct cancer, extrahepatic bladder cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain stem glioma, brain tumor, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, brain tumor, astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymal tumors, supratentorial primitive neuroectodermal tumors and pineoblastoma, spinal cord tumors, breast cancer (female), breast cancer (male), bronchial tumors, burkitt lymphoma, carcinoid tumor, gastrointestinal, nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system lymphoma, primary cervical cancer, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, embryonal tumors, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, ewing sarcoma family of tumors, extracranial germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, eye cancer, retinoblastoma gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), gastrointestinal stromal cell tumor, germ cell tumor, extracranial germ cell tumor, extragonadal germ cell tumor, ovarian gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, primary hepatocellular (liver) cancer, histiocytosis, langerhans cell, hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors (endocrine pancreas), kaposi sarcoma, kidney (renal cell) cancer, kidney cancer, langerhans cell histiocytosis, laryngeal cancer, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, adult (primary) liver cancer, (primary) lung cancer, non-small cell lung cancer, small cell lymphoma, aids-related lymphoma, burkitt lymphoma, cutaneous T-cell lymphoma, hodgkin lymphoma, non-hodgkin lymphoma, primary central nervous system lymphoma, macroglobulinemia, Waldenström malignant fibrous histiocytoma of bone and osteosarcoma, medulloblastoma, melanoma, intraocular (eye) melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, childhood multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, myelogenous leukemia, chronic myeloid leukemia, adult acute myeloid leukemia, childhood acute myeloma, multiple myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, lip and oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, islet cell tumors, papillomatosis, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pineal parenchymal tumors of intermediate differentiation, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell cancer, respiratory tract carcinoma involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, ewing sarcoma family of tumors, kaposi sarcoma, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, skin cancer (nonmelanoma), melanoma, skin carcinoma, merkel cell, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, see skin cancer (nonmelanoma), squamous neck cancer with occult primary, metastatic stomach (gastric) cancer, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumors, T-cell lymphoma, cutaneous T-cell lymphoma, mycosis fungoides and Sézary syndrome, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, (gestational), unknown primary site, carcinoma of, unknown primary site carcinoma, ureter and renal pelvis, transitional cell cancer, urethral cancer, uterine cancer, endometrial uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor.

The therapeutic agent can be a peptide or protein, toxin, chimeric toxin, cytokine, antibody, bispecific antibody including single chain antibodies, camel antibodies and nanobodies chemokine, prodrug converting enzyme or metabolite-degrading enzyme such as thiaminase, methionase (methioninase, L-methionine γ-lyase) or asparaginase. In a preferred embodiment the therapeutic agent is a toxin, or modified toxin.

Toxins, therapeutic cytokines and other molecules, homologs or fragments thereof useful in conjunction with the present invention include small lytic peptides, larger lytic peptides, pore-forming toxins, protein inhibitors, extracellular DNAases (DNase), intracellular DNAases, apoptosis inducing peptides, cytokines, prodrug converting enzymes, metabolite destroying enzymes, ribonucleases, antibody inactivating toxins and other anticancer peptides. In a preferred embodiment, the toxins include those that are naturally secreted, released and/or surface displayed, or heterologously secreted, released and/or surface displayed, and that can be modified uniquely to suit the delivery by a bacterium and may be further engineered to have the tumor, lymphoma, leukemic bone marrow or proximity-selective targeting system described herein, including but not limited to the proteins azurin, carboxyesterase Est55 (a prodrug converting enzyme from Geobacillus that activates CPT-11 to SN-38), thiaminase (e.g., from Bacillus), methionase (methioninase), asparaginase, apoptin, bax, bim, p53, BAK, BH3 peptide (BCL2 homology domain 3), cytochrome C, thrombospondin, platelet factor 4 (PF4) peptide, Bacillus sp. cytolysins, Bacillus sp. nheABC toxins, cytolethal distending toxins (cldt), typhoid toxins (pertussis like toxin) (pltAB), pertussis toxin, cldt:plt hybrids, actAB, cytotoxic nectrotic factor (cnf), dermonecrotic factor (dnf), shiga toxins and shiga-like toxins, bacteriocins, (colicins and microcins; Hen and Jack, Chapter 13 Microcins, in Kastin (ed), 2006, Handbook of Biologically Active Peptides, Academic Press; Nes et al., Chapter 17, The nonlantibiotic heat-stable bacteriocins in gram-positive bacteria, in Kastin (ed), 2006, Handbook of Biologically Active Peptides, Academic Press; Sharma et al., Chapter 18 in Kastin (ed), 2006, Handbook of Biologically Active Peptides, Academic Press) including membrane depolarizing (or pore-forming), DNAases (including colicin DNase, Staphylococcal Nuclease A:OmpA fusions (Takahara et al., 1985 J Biol. Chem 260: 2670-2674), Serratia marcescens DNase (Clegg and Allen, 1985, FEMS Microbiology Letters 27: 257-262; Vibrio DNase Newland et al., 1985 Infect Immun 47: 691-696) or other bacterial DNase), RNAases, and tRNAases, including but not limited colicin A, colicin D, colicin E5, colicin E492, microcin M24, colE1, colE2, colE3, colE5 colE7, coleE8, colE9, col-Ia, colicin N and colicin B, membrane lytic peptides from Staphalococcus (listed below) and sea anemones, P15 peptide and other TGF-beta mimics, repeat in toxin (RTX) family members (together with the necessary acylation and secretion genes) including Actinobacillus leucotoxins, a leuckotoxin: E. coli HlyA hybrid, E. coli HlyA hemolysin, Bordetella adenylate cyclase toxin, heat stable enterotoxins from E. coli and Vibrio sp. (Dubreuil 2006, Chapter 48, Eschericia coli, Vibrio and Yersinia species heat stable enterotoxins, Alouf and Popoff (eds), 2006, Comprehensive Sourcebook of Bacterial Protein Toxins, Third Edition, Academic Press), autotransporter toxins including but not limited to IgA protease, picU espC, and sat, Staphalococcus protein A, clostridium enterotoxin, Clostridium difficile toxin A, scorpion chlorotoxin, aerolysin, subtilase, cereolysin, Staphalococcus leukotoxins (e.g. LukF-PV, LukF-R, LukF-I, LukM, HlgB) and the other, to class S (e.g. LukS-PV, LukS-R, LukS-I, HlgA, HlgC). Best known are the toxins produced by S. aureus: γ-haemolysins, HlgA/HlgB and HlgC/HlgB and leukocidin Panton-Valentine, LukS-PV/LukF-PV (Luk-PV, PVL)) TRAIL, fasL, IL-18, CCL-21, human cytokine LIGHT, agglutinins (Maackia amurensis, wheat germ, Datura stramonium, Lycopersicon (tomato) plant lectin, leukoagglutinin (L-PHA, Helix pomatia) saporin, ricin, pertussis toxin, and porB, as well as other toxins and peptides (Kastin (ed), 2006, Handbook of Biologically Active Peptides, Academic Press; Alouf and Popoff (eds), 2006, Comprehensive Sourcebook of Bacterial Protein Toxins, Third Edition, Academic Press; each of which is expressly incorporated by reference in their entirety herein).

Metabolite toxins such as the Chromobacterium violacium dipsepeptides (Shigeatsu et al., 1994, FR901228, a novel antitumor bicyclic depsipeptide produced by Chromobacterium violaceum No. 968. II. Structure determination. J Antibiot (Tokyo) 47(3):311-4) or those from Serratia are also of use in the present invention.

Bacterial collagenases (Harrington, 1996, Infect. Immun. 64: 1885-1891) useful in the invention include but not limited to those from Actinobacillus actinomycetemcomitans, Acinomadura (Streptomyces) madurae, Bacillus cereus, Bacteroides spp., Bifidobacterium sp., Bruecella melitensis, Capnocytophaga ochracea, Clostridium spp., Enterococcus faecalis, Echerichia coli, Eubacterium alactolyticum, Flavobacterium meningosepticum, Fusobacterium nucleatum, Peptococcus sp., Peptostreptococcus spp., Porphyromoas (Bacteroides) spp., Prevotella (Bacteroides) spp., Proteus mirabilis, Pseudomaons aeruginosa, Serratia marsescensm Serratia, spp., Staphalococcus spp., Streptococcus agalactiae (group B streptococcus), Streptococcus mutans, Streptococcus sobrinus (S. mutans 6715), Treponema spp. and Vibro vulnificus), and those described in the MEROPS Database (Rawlings et al., 2010, MEROPS: The Peptidase Database, Nucleic Acids Res. 2010 (Database issue):D227-33.) including but not limited to those from Clostridium histolyticum (bacterial collagenase G/A), Cytophaga (cytophagalysin), Empedobacter collagenolyticum (Empdeobacter collagenase), Helicobacter (Helicobacter-type collagenase), Porphyromonas (Porphyromonase-type collagenase), Geobacillus sp. MO-1 (collagenolytic endopeptidase) and Salmonella sp. (Salmonella-type collegenase, including the collagenase from Salmonella DT-104), Alternatively, an endogenous collagenase may be activated by a transactivator, such as SlyA. (Carlson 2006, Microbial Pathogenesis 38: 181-187).

The chimeras may be further modified by addition of one or more multimerization domains, such as the T4 foldon trimerization domain (Meier et al., 2004, Journal of Molecular Biology, 344: 1051-1069; Bhardwaj et al., Protein Sci. 2008 17: 1475-1485) or tetramerization domains such as VASP (Kühnel et al., 2004 PNAS 101: 17027-17032). Chimeric toxins may be further modified by the addition of known cell penetrating (ferry) peptide which further improves their entry into target cells. Cell penetrating peptides include those derived from the HIV TAT protein (e.g., TAT-apoptin, TAT-bim, TAT-p53), the antennapedia homeodomain (penetraxin), Kaposi fibroblast growth factor (FGF) membrane-translocating sequence (MTS), herpes simplex virus VP22, hexahistidine, hexalysine, hexaarginine or “Chariot” (Active Motif, Carlsbad, Calif.; U.S. Pat. No. 6,841,535). Nuclear localization signals (NLSs) may also be added, including but not limited to that from herpes simplex virus thymidine kinase, the SV40 large T antigen (PPKKKRKV SEQ ID NO:1) monopartite NLS, or the nucleoplamin bipartite NLS (KR[PAATKKAGQA]KKKK SEQ ID NO:2, or more preferably, the NLS from apoptin, a tumor associated (tumor-selective) NLS. The tumor-selective nuclear export signal from apoptin may be used alone or together with NLS from apoptin (Heckl et al., 2008, Value of apoptin's 40-amino-acid C-terminal fragment for the differentiation between human tumor and non-tumor cells, Apoptosis 13: 495-508; Backendor et al., 2008, Apoptin: Therapeutic potential of an early sensor of carcinogenic transformation, Ann Rev Pharmacol Toxicol 48: 143-69).

The toxin may be further modified by addition of one or more protease cleavage sites that enhance its degradation outside of the tumor. Preferred protease cleavage sites are those for proteases that are under-expressed within the tumor compared to normal tissues (rather than over-expressed within the tumor as utilized for aerolysin activation). However, the expression levels of many proteases are elevated within tumors (e.g., Edwards et al., (eds) 2008, The Cancer Degradome: Proteases and Cancer Biology, Springer, expressly incorporated in its entirety herein). Preferred examples of proteases for which inhibitory peptides may be coexpressed including but not limited to furin, tissue plasminogen activator, activated protein C, factor Xa, granzymes (A, B & M), cathepsins (A, B, C, D, E, F, G, H, K, L, S, W & X), thrombin, plasmin, urokinase, matrix metalloproteases (1-28) membrane matrix metalloproteases (1-4), prostate specific antigen (PSA) and kallikrein 2. Furin, for example, recognizes a number of specific cleavage sites, including RXKR□SX SEQ ID NO:3. In accordance with the present invention, the presence of this cleavage site, whether naturally occurring or introduced through genetic modification, may be compensated for within the target tissue by co-expression of a furin inhibitor, stabilizing its activity unless it escapes the target tissue such as a tumor, lymph node or lumen of a bone whereupon the inhibitor concentration drops and the effector protein is degraded. Use of protease inhibitors alone or in combination by bacterial delivery vectors has not previously been suggested. Indeed, Wang et al. 2008 (Acta Biochim Biophys Sin (Shanghai). 2008 October; 40(10):848-54) suggested furin inhibitors could be used as antibiotics to suppress bacterial infection which would thereby interfere with delivery by a bacterial vector. Therefore, it has not been considered desirable to use a furin inhibitor or other protease inhibitors to have a positive effect on the bacteria and/or the therapeutics they release.

The peptide inhibitors are engineered to be secreted from the gram negative bacteria secretion signals known to those skilled in the arts, including E. coli cytolethal distending toxin, Shiga toxin, LPP:OmpA, M13pIII, M13pVIII, zirS (Finlay et al., 2008, PLoS Pathogens 4 (4), e100003), heat-stable (ST; thermostable) toxins from Escherichia and Vibrio (U.S. Pat. No. 5,399,490), E. coli enterotoxin II (Kwon et al., U.S. Pat. No. 6,605,697) N-terminal signal sequences, or hlyA C-terminal signal sequence (requires addition of hlyBD and TolC), or by colicin fusions together with colicin lysis proteins, or using autotransporter (autodisplay) fusions. Fusion to the M13 pIX may also be used (WO 2009/086116) or fusions to type III secretion system of Salmonella or other bacteria (Wilmaier et al., 2009 Mol Sys Biol 5: 309. The inhibitors can be further modified to have the protease cleavage signal of the protease that they inhibit or for a different protease. Secretion signal from gram positive bacteria include that from listerialysin O (LLO), alkaline phosphatase (phoZ) (Lee et al., 1999, J Bacteriol. 181: 5790-5799), ClTase gene (Shiroza and Kuramitsu 1998, Methods in Cell Science, 20: 127-136) or the twin arginine translocation system (Berks et al., 2005, Protein targeting by the bacterial twin-arginine translocation (Tat) pathway, Current Opinion in Microbiology 8: 174-181). Enhanced secretion may be achieved as described in U.S. Pat. No. 7,358,084, WO/2009/139985 Methods and materials for gastrointestinal delivery of a pathogentoxin binding agent; van Asseldonk, M et al. 1990, Cloning of usp45, a gene encoding a secreted protein from Lacotococcs lactis subsp. lactis MG1363 Gene 95, 15-160; Kim et al., Display of heterologous proteins on the surface of Lactococcus lactis using the H and W domain of PrtB from Lactobacillus delburueckii subsp. bulgaricus as an anchoring matrix J Appl Microbiol. 2008 June; 104(6):1636-43. Epub 2008 Feb. 19).

The chimeric proteins may have one or more additional features or protein domains known to those skilled in the arts which are designed to be active or catalytic domains that result in the death of the cell, allow or facilitate them being secreted or released by autolytic peptides such as those associated with colicins or bacteriophage release peptides have targeting peptides that direct them to the target cells, and protease cleavage sites for activation (e.g., release from parent peptide), and thioredoxin or glutathione S-transferase (GST) fusions that improve solubility.

The present invention also provides in accordance with some embodiments, unique chimeric modifications of the above listed toxins that contain specific combinations of components resulting in secretion by selective anti-tumor activity. The invention also provides extracellular protease sensitivity (deactivation) that may include the addition of protease cleavage sites and may be co-expressed with a protease inhibitor. The chimeric proteins may have one or more additional features or protein domains known to those skilled in the arts which are designed to 1) be active or catalytic domains that result in the death of the cell or make them susceptible to other known anticancer agents, 2) allow or facilitate them being secreted or released by autolytic peptides such as colicin release peptides, 3) membrane protein transduction (ferry) peptides, 4) autotransporter domains, 5) have targeting peptides that direct them to the target cells, and 6) protease cleavage sites for activation (e.g., release from parent peptide). However, the specific organization and combination of these domains is unique and specific to the invention.

Bombesin and gastrin are amidated peptides. Amidation of these peptides would not be expected to occur in gram-negative bacteria. A unique composition in accordance with one embodiment of the present invention is the co-expression of the C-terminal amidating enzyme, which results in amidating these peptides in order for them to confer their targeting specificity.

Small lytic peptides (less than 50 amino acids) are used to construct chimeric proteins for more than one purpose. The chimeric proteins containing lytic peptides may be directly cytotoxic for the cancer cells, and/or other cells of the tumor including the tumor matrix cells and immune cells which may diminish the effects of the bacteria by eliminating them. Furthermore, the lytic peptides are useful in chimeric proteins for affecting release from the endosome. Small lytic peptides have been used in the experimental treatment of cancer. However, it is evident that most, if not all, of the commonly used antitumor small lytic peptides have strong antibacterial activity, and thus are not compatible with delivery by a bacterium (see Table 1 of Leschner and Hansel, 2004 Current Pharmaceutical Design 10: 2299-2310, the entirety of which is expressly incorporated herein by reference). Small lytic peptides useful in the invention are those derived from Staphaloccus aureus, S. epidermidis and related species, including the phenol-soluble modulin (PSM) peptides and delta-lysin (Wang et al., 2007 Nature Medicine 13: 1510-1514, expressly incorporated herein by reference). Larger lytic peptides that may be used includes the actinoporins from sea anemones or other coelenterates, such as SrcI, FraC equinatoxin-II and sticholysin-II (Anderluh and Macek 2002, Toxicon 40: 111-124). The selection of the lytic peptide depends upon the primary purpose of the construct, which may be used in combination with other constructs providing other anticancer features. Construct designed to be directly cytotoxic to cells employ the more cytoxic peptides, particularly PSM-alpha-3 and actinoporins. Constructs which are designed to use the lytic peptide to affect escape from the endosome use the peptides with the lower level of cytotoxicity, such as PSM-alpha-1, PSM-alpha-2 or delta-lysin.

Promoters, i.e., genetic regulatory elements that control the expression of the genes encoding the therapeutic molecules described above that are useful in the present invention include constitutive and inducible promoters. A preferred constitutive promoter is that from the vector pTrc99a (Promega). Preferred inducible promoters include the tetracycline inducible promoter (TET promoter), SOS-response promoters responsive to DNA damaging agents such as mitomycin, alkylating agents, X-rays and ultraviolet (UV) light such as the recA promoter, colicin promoters, sulA promoters and hypoxic-inducible promoters including but not limited to the PepT promoter (Bermudes et al., WO 01/25397), the arabinose inducible promoter (Ara_BAD) (Lossner et al., 2007, Cell Microbiol. 9: 1529-1537; WO/2006/048344) the salicylate (Aspirin) derivatives inducible promoter (Royo et al., 2007, Nature Methods 4: 937-942; WO/2005/054477), or a tumor-specific promoter (Arrach et al., 2008, Cancer Research 68: 4827-4832; WO/2009/152480). A single promoter may be used to drive the expression of more than one gene, such as a protease sensitive toxin and a protease inhibitor. The genes may be part of a single synthetic operon (polycistronic), or may be separate, monocistronic constructs, with separate individual promoters of the same type used to drive the expression of their respective genes. The promoters may also be of different types, with different genes expressed by different constitutive or inducible promoters. Use of two separate inducible promoter for more than one cytotoxin or other effector type peptide allows, when sufficient X-ray, tetracycline, arabinose or salicylic acid is administered following administration of the bacterial vector, their expression to occur simultaneously, sequentially, or alternatingly (repeated).

3.2 Non-Conjugative, Bacteria

The present invention provides, according to some embodiments, a composition that would minimize the effect of bacteria released into the environment by eliminating the ability of the bacteria to exchange genetic information with related bacteria, as well as provide a delivery enhancing bacteria resulting in a greater therapeutic effect. Conjugative transfer is a major genetic exchange mechanism that may occur between Salmonella and the normal commensal gut bacterium E. coli, requiring the presence of an F′ factor. The present invention provides gram-negative bacteria including E. coli, Vibrio, Shigella and Salmonella that are genetically modified in one or more ways to eliminate conjugative transfer of DNA with closely related species including E. coli. One of the modifications works on both male (F′+) and female (F′−) bacteria. These modifications facilitate the safety of a bacteria carrying expressing chimeric toxins. The F′ factor provides functions which may be undesirable in conjunction with aspects of the present invention, including mating stabilization and DNA transfer. The present invention therefore provides, according to one aspect, a composition lacking these features by their genetic disruption on the F′ factor or by the cloning of the pilus factor genes into the tumor-targeted bacterium in the absence of the other factors, and hence, resulting in a strain which is non-conjugative and significantly less likely to transfer DNA to other bacteria. The invention may also incorporate entry exclusion into the bacteria and the fertility inhibition complex (finO and finP) and/or TraO, alone or in combination, and thus, even in tumor-targeted bacterial strains in which the pilus factors are not incorporated (i.e., F−), the bacterial strain will remain resistant to mating with F′ bacteria.

3.3. Novel Methods for Testing the Efficacy of Engineered Effector Proteins Using Strains with Low-Level Tumor Targeting and/or Lower Antitumor Effects.

As cited above, the primary shortcoming of tumor-targeted bacteria investigated in the human clinical trials (Salmonella strain VNP20009 and its derivative TAPET-CD; Toso et al., 2002, Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma, J. Clin, Oncol. 20: 142-152; Meir et al., 2001, Phase 1 trial of a live, attenuated Salmonella Typhimurium (VNP20009) administered by direct Intra-tumoral (IT) injection, Proc Am Soc Clin Oncol 20: abstr 1043; Nemunaitis et al., 2003, Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients, Cancer Gene Therapy 10: 737-744). However, one of the main differences between the murine studies (e.g., Pawelek et al., 1997, Tumor-targeted Salmonella as a novel anticancer agent, Cancer Research 57: 4537-4544; Low et al., 1999, Lipid A mutant salmonella with suppressed virulence and TNF-alpha induction retain tumor-targeting in vivo, Nature Biotechnol. 17: 37-41), is that in most patients, the levels of the bacteria were significantly lower. For example, whereas in the murine models the bacteria frequently achieved levels of 1×10⁹per gram of tumor tissue, in humans the levels were significantly lower, e.g., 1×10⁶was achieved in 3 patients (Meir et al., 2001). Generally, it has been perceived that the murine studies should precede using bacteria with the greatest amount of tumor targeting. For example, Pawelek et al., WO/1996/040238 selected “super infective” bacteria by cycling through tumors. The novel cycling and selection procedure they employed selected for increased targeting numbers which was correlated with a greater antitumor effect. A similar study was performed by Zhao et al., 2005, (Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proc Natl Acad Sci USA. 102: 755-760).

In the isolation of the Salmonella strain A1-R by re-isolation form a tumor, as described by the same group in a later study (Hayashi et al., 2009, Cancer metastasis directly eradicated by targeted therapy with a modified Salmonella Typhimurium, Journal of Cellular Biochemistry 106: 992-998). “The idea was to increase the tumor targeting capability of the bacteria.” Thus, developing and testing bacteria with enhanced tumor targeting has been a focus within the field. However, while it is desirable to find ways to improve the levels of bacteria within tumors, including the present invention, the importance of selecting an appropriate model to assess the contribution that an effector system might have in a human, or how it might improve tumor colonization levels, wherein the model should provide lower (rather than higher) levels of tumor colonization, has not been appreciated. It has not been understood that to evaluate how an effector system such as the herpes simplex thymidine kinase or cytosine deaminase described by Pawelek et al., WO/1996/040238, or those provided in the present invention, would function in humans where lower targeting numbers might be expected (at least at the outset; greater number could be achieved if the effector system is effective), such that the murine system where the tumor-targeting level is similar to the level achieved in humans represents an appropriate model.

As described by Pawelek et al., fir A is a mutation within the gene that encodes the enzyme UDP-3-O(R-30 hydroxymyristoyl)-glycocyamine N-acyltransferase, that regulates the third step in endotoxin biosynthesis (Kelley et al., 1993, J. Biol. Chem. 268:19866-19874). Salmonella typhimurium and E. coli strains bearing this type of mutation produce a lipid A that differs from wild type lipid A in that it contains a seventh fatty acid, a hexadecanoic acid (Roy and Coleman, 1994, J. Bacteriol. 176:1639-1646). Roy and Coleman demonstrated that in addition to blocking the third step in endotoxin biosynthesis, the firA′ mutation also decreases enzymatic activity of lipid A 4′ kinase that regulates the sixth step of lipid A biosynthesis. Salmonella typhimurium strain SH5014 and its firA′ derivative SH7622 are described in Hirvas et al, 1991, EMBO J. 10:1017-1023. The genotypes of these strains are as follows: strain SH5014 ilv-1178 thr-914 {acute over (η)}is-6116 metA22 metE551 trpB2 xyl-404 HI-b H2-e, n, x flaA66 rpsL120 rfaJ4041; strain SH7622 ilv-1178 thr-914 his-6116 metA22 metE551 trpB2 xyl-404 H1-b H2-e, n, x flahββ rpsL120 rfaJ4041, ssc-1 (firA^ts). A derivative of Salmonella typhimurium firA′ strain SH7622 was picked, designated SH7622-64, and used as the firA′ strain for the experiments. SH7622-64 was selected for its supersensitivity to the antibiotic novobiocin and temperature-sensitive growth, characteristics of the firA′ SH7622 strain. When studies in two different tumor models, Pawelek et al. found Salmonella/g tissue: Primary Tumor of M27 lung cancer, 2.9×10⁶per gram and in B16 melanoma, 3.2×10⁵per gram, yet retaining a similar 3200:1 tumor to liver targeting ratio. This strain, while never used in any subsequent studies represents a surprising solution to translating murine to human studies, wherein both systems tend to have the same number of bacteria per gram of target tissue.

In an alternative approach, as opposed to selecting bacteria with optimal antitumor effects as is commonly applied (Zhao et al., 2005 (Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proc Natl Acad Sci USA. 102: 755-760).), bacterial mutants are selected for suboptimal or low antitumor effects. The bacterial mutants can be generated by any standard method of mutation (e.g., UV, nitrosoguanadine, Tn10, Tn5), or can be a spontaneous mutation such as a suppressor mutation (e.g., Murray et al., 2001, Extragenic suppressors of growth defects in msbB Salmonella, J. Bacteriol. 183: 5554-5561).

4. OBJECTS OF THE INVENTION

The present invention provides, according to one embodiment, live attenuated therapeutic bacterial strains that express one or more therapeutic molecules together with one or more protease inhibitor polypeptides that inhibit local proteases that could deactivate the therapeutic molecules. In particular, one aspect of the invention relates to live attenuated tumor-targeted bacterial strains that may include Salmonella, group B Streptococcus or Listeria vectoring chimeric anti-tumor toxins to an individual to elicit a therapeutic response against cancer. Another aspect of the invention relates to live attenuated tumor-targeted bacterial strains that may include Salmonella, group B Streptococcus or Listeria vectoring chimeric anti-tumor toxin molecules to an individual to elicit a therapeutic response against cancer including cancer stem cells. The toxins may also be targeted to tumor matrix cells, and/or immune cells.

Whereas the prior strains of Salmonella studied in human clinical trials used either no heterologous antitumor protein (i.e., VNP20009) or an antitumor protein located within the cytoplasm of the bacterium (i.e., cytosine deaminase expressed by TAPET-CD), or secreted proteins (Bermudes et al., WO 2001/025397) the invention provides, according to some embodiments, methods and compositions comprising bacterial vectors that express, secrete, surface display and/or release protease inhibitors that protect coexpressed protease sensitive antitumor molecules that are also secreted, surface displayed and/or released into the tumor, lymphoma-containing lymph node, leukemic bone lumen, or proximally or topically on a carcinoma or precancerous lesion for the treatment of the neoplasia.

The bacteria according to a preferred embodiment of the present invention include those modified to have little or no ability to undergo bacterial conjugation, limiting incoming and outgoing exchange of genetic material, whereas the prior art fails to limit exchange of genetic material. In addition, certain of the therapeutic molecules have co-transmission requirements (e.g., colicin proteins and colicin immunity) that are distal (i.e., genetically dissected and separated) to the therapeutic molecule location further limiting known forms of genetic exchange.

Aspects of the present invention also provide bacteria with antibody deactivating proteins that minimize the neutralizing effect of any vector specific antibodies and/or complement at the time of injection, or thereafter. The neutralizing proteins may be induced prior to injection into the host using known inducible promoters such that the bacteria are only temporarily antibody resistant, and may be optionally continuously produced thereafter at low level. Upon reaching the tumor site where the antibody penetration is poor, the bacteria no longer make the protein in sufficient quantity to have substantial spread to other tissues, except other tumor tissues and are controlled by neutralizing antibodies systemically, maintaining the safety of the bacteria.

Aspects of the present invention also provide novel chimeric bacterial toxins particularly suited for expression by gram-negative bacteria. The toxins may have added targeting ligands that render them selectively cytotoxic for tumor cells, tumor stem cells and/or matrix and tumor-infiltrating immune cells. The invention also provides means to determine optimal toxin combinations which are preferably additive or more preferably synergistic. The invention also provides means to determine the optimal combination of protein toxin with conventional cancer chemotherapeutics, liposomal agents or biologics, including immunosuppressive anti-complement agents (e.g., anti-C5B). Accordingly, administration to an individual, of a live Salmonella bacterial vector, in accordance with an aspect of the present invention, that is genetically engineered to express one or more protease inhibitors as described herein co-expressed with one or more cytotoxic proteins has the ability to establish a population in the tumor, kill tumor cells, tumor stem cells as well as tumor matrix and immune infiltrating cells, resulting in a therapeutic benefit.

Aspects of the present invention also provide novel methods to test the efficacy of the protease inhibitor and effector gene combinations described herein. The methods employ bacteria with low tumor colonization capability in order to establish the ability of low numbers of tumor-targeted bacteria to result in the desired effect, and bacteria with low inherent antitumor activity, such that the innate antitumor activity of a bacterial strain is minimized, and therefore less likely to mask the results of the effector systems.

A preferred composition will contain, for example, a sufficient amount of live bacteria expressing the protease inhibitors and cytotoxin(s) or effector proteins/peptides to produce a therapeutic response in the patient. Accordingly, the attenuated Salmonella strains described herein are both safe and useful as live bacterial vectors that can be orally administered to an individual to provide therapeutic benefit for the treatment of cancer.

Although not wishing to be bound by any particular mechanism, an effective antitumor response in humans by administration of genetically engineered, attenuated strains of Salmonella strains as described herein may be due to the ability of such mutant strains to persist within the tumor, lymphoma or leukemic bone marrow and to supply their own nutrient needs by killing tumor cells, tumor matrix and or immune infiltrating cells and further expanding the zone of the tumor that they occupy. Bacterial strains useful in accordance with a preferred aspect of the invention may carry the ability to produce a therapeutic molecule expressing plasmid or chromosomally integrated cassette that encodes and directs expression of one or more therapeutic molecules together with one or more protease inhibitors, as described herein. The protease inhibitors serve to prevent the destruction of the therapeutic molecule while within the tumor. The protease inhibitor may also have an anticlotting effect, wherein a blood clot may prevent spread of the bacteria throughout the tumor. The protease inhibitor may also have direct or indirect anticancer effects. If the cytotoxin and protease inhibitor diffuse outside of the tumor, lymph node, bone lumen, proximity to a carcinoma or other neoplasia-localized distribution, they fall below the protease inhibitory concentration, no longer inhibit proteolysis of the cytotoxins or effector genes, and are then inactivated. Thus the protease inhibitor system both increases activity and provides tumor specificity.

The serovars of S. enterica that may be used as the attenuated bacterium of the live compositions described in accordance with various embodiments herein include, without limitation, Salmonella enterica serovar Typhimurium (“S. typhimurium”), Salmonella montevideo, Salmonella enterica serovar Typhi (“S. typhi”), Salmonella enterica serovar Paratyphi B (“S. paratyphi 13”), Salmonella enterica serovar Paratyphi C (“S. paratyphi C”), Salmonella enterica serovar Hadar (“S. hadar”), Salmonella enterica serovar Enteriditis (“S. enteriditis”), Salmonella enterica serovar Kentucky (“S. kentucky”), Salmonella enterica serovar Infantis (“S. infantis”), Salmonella enterica serovar Pullorum (“S. pullorum”), Salmonella enterica serovar Gallinarum (“S. gallinarum”), Salmonella enterica serovar Muenchen (“S. muenchen”), Salmonella enterica serovar Anatum (“S. anatum”), Salmonella enterica serovar Dublin (“S. dublin”), Salmonella enterica serovar Derby (“S. derby”), Salmonella enterica serovar Choleraesuis var. kunzendorf (“S. cholerae kunzendorf”), and Salmonella enterica serovar minnesota (S. minnesota). A preferred serotype for the treatment of bone marrow related diseases is S. dublin.

By way of example, live bacteria in accordance with aspects of the invention include known strains of S. enterica serovar Typhimurium (S. typhimurium) and S. enterica serovar Typhi (S. typhi) which are further modified as provided by the invention to form vectors for the prevention and/or treatment of neoplasia. Such Strains include Ty21a, CMV906, CMV908, CMV906-htr, CMV908-htr, Ty800, aroA−/serC−, holavax, M01ZH09, VNP20009. These strains contain defined mutations within specific serotypes of bacteria. The invention also includes the use of these same mutational combinations contained within alternate serotypes or strains in order to avoid immune reactions which may occur in subsequent administrations. In a preferred embodiment, S. Typhimurium, S. montevideo, and S. typhi which have non-overlapping O-antigen presentation (e.g., S. typhimurium is O-1, 4, 5, 12 and S. typhi is Vi, S. montevideo is O-6, 7) may be used. Thus, for example, S. typhimurium is a suitable serotype for a first injection and another serotype such as S. typhi or S. montevideo are used for a second injection and third injections. Likewise, the flagellar antigens are also selected for non-overlapping antigenicity between different injections. The flagellar antigen may be H1 or H2 or no flagellar antigen, which, when combined with the three different O-antigen serotypes, provides three completely different antigenic profiles.

Novel strains of Salmonella are also encompassed that are, for example, attenuated in virulence by mutations in a variety of metabolic and structural genes. The invention therefore may provide a live composition for treating cancer comprising a live attenuated bacterium that is a serovar of Salmonella enterica comprising an attenuating mutation in a genetic locus of the chromosome of said bacterium that attenuates virulence of said bacterium and wherein said attenuating mutation is a combinations of other known attenuating mutations. Other attenuating mutation useful in the Salmonella bacterial strains described herein may be in a genetic locus selected from the group consisting of phoP, phoQ, edt, cya, crp, poxA, rpoS, htrA, nuoG, pmi, pabA, pts, damA, met, cys, pur, purA, purB, purl, purF, leu, ilv, arg, lys, zwf, aroA, aroB, aroC, aroD, serC, gua, cadA, rfc, rjb, rfa, ompR, msbB, pfkAB, crr, glk, ptsG, ptsHl, manXYZ and combinations thereof. The strain may also contain a mutation known as “Suwwan”, which is an approximately 100 kB deletion between two IS200 elements. The strain may also carry a defective thioredoxin gene (trxA−; which may be used in combination with a TrxA fusion), a defective glutathione oxidoreductase (gor−) and optionally, overexpress a protein disulfide bond isomerase (DsbA). The strain may also be engineered to express invasion and/or escape genes tlyA, tlyC patI and pld from Rickettsia, whereby the bacteria exhibit enhanced invasion and/or escape from the phagolysosome (Witworth et al., 2005, Infect. Immun. 73: 6668-6673), thereby enhancing the activity of the effector genes described below. The strain may also be engineered to be deleted in an avirulence (anti-virulence) gene, such as .zirTS, grvA and/or pcgL, or express the E. coli lac repressor, which is also an avirulence gene in order to compensate for over-attenuation. The strain may also express SlyA, a known transcriptional activator. In a preferred embodiment, the Salmonella strains are msbB mutants (msbB−). In a more preferred embodiment, the strains are msbB- and Suwwan. In a more preferred embodiment the strains are msbB−, Suwwan and zwf−. Zwf has recently been shown to provide resistance to CO2, acidic pH and osmolarity (Karsten et al., 2009, BMC Microbiology August 18; 9:170). Use of the msbB zwf genetic combination is also particularly preferred for use in combination with administered carbogen (an oxygen carbon dioxide mixture that may enhance delivery of therapeutic agents to a tumor). In a more preferred embodiment, the strains are msbB−, Suwwan, zwf− and trxA−. In a most preferred embodiment, the strains are msbB−, Suwwan, zwf−, trxA− and gor−.

The invention also encompasses according to a preferred embodiment, gram-positive bacteria. Preferred bacteria of the invention are group B Streptococcus including S. agalaciae, and Listeria species including L. monocytogenes. It is known to those skilled in the arts that minor variations in molecular biology techniques such as use of gram-positive origins of replication, gram-positive signal sequences gram-positive promoters (e.g., Lactococcus expression, Mohamadzadeh et al., PNAS Mar. 17, 2009 vol. 106 no. 11 4331-4336; Geertsma and Poolman, 2007, High-throughput cloning and expression in recalcitrant bacteria, Nature Methds 4: 705-707; Prudhomme et al., 2006, Antibiotic stress induces genetic transformability in the human pathogen Streptococcus pneumoniae, Science 313: 89-92; WO/2009/139985 Methods and materials for gastrointestinal delivery of a pathogentoxin binding agent; van Asseldonk, M et al. 1990, Cloning of usp45, a gene encoding a secreted protein from Lacotococcs lactis subsp. lactis MG1363 Gene 95, 15-160; Kim et al., J Appl Microbiol. 2008 June; 104(6):1636-43. Epub 2008 Feb. 19. Display of heterologous proteins on the surface of Lactococcus lactis using the H and W domain of PrtB from Lactobacillus delburueckii subsp. bulgaricus as an anchoring matrix; Lee et al., 1999, Characterization of Enterococcus faecalis alkaline phosphatase and use in identifying Streptococcus agalactiae secreted proteins, J. Bacteriol 181(18):5790-9.) are required and substituted as needed.

Mutational backgrounds of Listeria vectors include those previously isolated, including the delta-actA strain 142 (Wallecha et al., 2009, Construction and characterization of an attenuated Listera monocytogenes strain for clinical use in cancer immunotherapy, Clin Vaccine Immunol 16: 96-103), the double D-alanine (D-ala) strain described by Jiang et al., 2007, Vaccine 16: 7470-7479, Yoshimura et al., 2006, Cancer Research 66: 1096-1104, Lenz et al., 2008, Clinical and Vaccine Immunology 15: 1414-1419, Roberts et al., 2005, Definition of genetically distinct attenuation mechanisms in naturally virulent Listeria monotytogenes by comparative cell culture and molecular characterization, Appl. Environ. Microbiol 71: 3900-3910, the actA, prfA strain by Yan et al., Infect Immun 76: 3439-3450, and those described by Portnoy et al., EP1513924 and Portnoy et al., WO/2003/102168.

Mutational backgrounds of the group B Streptococcus, S. agalactiae, include wild type (no mutations), of any of the nine serotypes that depend on the immunologic reactivity of the polysaccharide capsule and among nine serotypes, preferably types Ia, Ib, II, III, and V capable of being invasive in humans. The strain may be deleted in the beta-heolysin/cytolysin (beta-H/C), including any member of the cly operon (clyXDGZAEFLJK SEQ ID NO:4), preferably the clyE gene, or the CspA protease associated with virulence (Shelver and Bryan, 2008, J Bacteriol. 136: 129-134), or the hyaluronate lyse C5a peptidase CAMP factor, oligopeptidase (Liu and Nizet 2004, Frontiers in Biosci 9: 1794-1802; Doran and Nizet 2004, Mol Microbiol 54: 23-31; Herbert et al., 2004, Curr Opin Infect Dis 17: 225-229). The strains may further have mutations in metabolic genes pur, purA, aroA, aroB, aroC, aroD, pgi (glucose-6-phosphate isomerase), fructose-1,6-bisphosphatase, ptsH, ptsI, and/or one or more amino acid transporters and/or amino acid permeases. In a preferred embodiment, the strain is clyE deficient.

Other bacterial strains are also encompassed, including non-pathogenic bacteria of the gut such as E. coli strains, Bacteriodies, Bifidobacterium and Bacillus, attenuated pathogenic strains of E. coli including enteropathogenic and uropathogenic isolates, Enterococcus sp. and Serratia sp. as well as attenuated Shigella sp., Yersinia sp., Streptococcus sp. and Listeria sp. Bacteria of low pathogenic potential to humans such as Clostridium spp. and attenuated Clostridium spp., Proteus mirabilis, insect pathogenic Xenorhabdus sp., Photorhabdus sp. and human wound Photorhabdus (Xenorhabdus) are also encompassed. Probiotic strains of bacteria are also encompassed, including Lactobacillus sp., Lactococcus sp., Leuconostoc sp., Pediococcus sp., Streptococcus sp., Streptococcus agalactiae, Lactococcus sp., Bacillus sp., Bacillus natto, Bifidobacterium sp., Bacteroides sp., and Escherichia coli such as the 1917 Nissel strain.

The invention also provides, according to one embodiment, a process for preparing genetically stable therapeutic bacterial strains comprising genetically engineering the therapeutic genes of interest into a bacterially codon optimized expression sequence within a bacterial plasmid expression vector, endogenous virulence (VIR) plasmid (of Salmonella sp), or chromosomal localization expression vector for any of the deleted genes or IS200 genes, defective phage or intergenic regions within the strain and further containing engineered restriction endonuclease sites such that the bacterially codon optimized expression gene contains subcomponents which are easily and rapidly exchangeable, and the bacterial strains so produced. Administration of the strain to the patient is therapeutic for the treatment of cancer.

The present invention provides, for example, and without limitation, live bacterial compositions that are genetically engineered to express one or more protease inhibitors combined with antitumor effector molecules for the treatment of cancers or neoplasias.

According to various embodiments, the invention provides pharmaceutical compositions comprising pharmaceutically acceptable carriers and one or more bacterial mutants. The invention also provides pharmaceutical compositions comprising pharmaceutically acceptable carriers and one or more bacterial mutants comprising nucleotide sequences encoding one or more therapeutic molecules. The pharmaceutical compositions of the invention may be used in accordance with the methods of the invention for the prophylaxis or treatment of neoplastic disease. Preferably, the bacterial mutants are attenuated by introducing one or more mutations in one or more genes in the lipopolysaccharide (LPS) biosynthetic pathway (for gram-negative bacteria), and optionally one or more mutations to auxotrophy for one or more nutrients or metabolites.

In one embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more bacterial mutants, wherein said attenuated bacterial mutants are facultative anaerobes or facultative aerobes. In another embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated bacterial mutants, wherein said attenuated bacterial mutants are facultative anaerobes or facultative aerobes and comprise one or more nucleic acid molecules encoding one or more therapeutic molecules where the therapeutic molecule is chimeric toxin.

In a specific embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated bacterial mutants, wherein the bacterial mutants are a Salmonella sp. In another specific embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated stress-resistant gram-negative bacterial mutants, wherein the attenuated stress-resistant gram-negative bacterial mutants are a Salmonella sp., and the attenuated stress-resistant gram-negative bacterial mutants comprise one or more nucleic acid molecules encoding one or more therapeutic molecules, prodrug converting enzymes, metabolite degrading enzymes, lytic peptides, DNAases or anti-cancer peptides.

In a specific embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated bacterial mutants, wherein the bacterial mutants are a Streptococcus sp. In another specific embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated gram-positive bacterial mutants, wherein the attenuated gram-positive bacterial mutants are a Streptococcus sp., and the attenuated gram-positive bacterial mutants comprise one or more nucleic acid molecules encoding one or more therapeutic molecules, prodrug converting enzymes, metabolite degrading enzyme, lytic peptides, DNAases or anti-cancer peptides.

In a specific embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated bacterial mutants, wherein the bacterial mutants are a Listeria sp. In another specific embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more attenuated bacterial mutants, wherein the attenuated gram-positive bacterial mutants are a Listeria sp., and the attenuated gram-positive bacterial mutants comprise one or more nucleic acid molecules encoding one or more therapeutic molecules, prodrug converting enzymes, metabolite degrading enzyme, lytic peptides, DNAases or anti-cancer peptides.

The present invention encompasses treatment protocols that provide a better therapeutic effect than current existing anticancer therapies. In particular, the present invention provides methods for prophylaxis or treatment of neoplastic diseases in a subject comprising administering to said subject and one or more bacterial mutants. The present invention also provides methods for the prophylaxis or treatment of neoplastic diseases in a subject comprising administering to said subject one or more bacterial mutants, wherein said bacterial mutants comprise one or more nucleic acid molecules encoding one or more therapeutic molecules together with one or more protease inhibitors.

The methods of the present invention permit lower dosages and/or less frequent dosing of the bacterial mutants to be administered to a subject for prophylaxis or treatment of neoplastic disease to achieve a therapeutically effective amount of one or more therapeutic molecules. In a preferred embodiment, the genetically modified bacteria are used in animals, including humans, dogs, cats, and/or horses for protection or treatment against neoplastic diseases.

Accordingly, when administered to an individual, a live Salmonella, Listeria or Streptococcus bacterial vector or therapeutic, in accordance with the present invention, that is genetically engineered to express one or more anti-neoplastic disease molecules or molecules against other cells within the neoplastic milieu in combination with a protease inhibitor and have improved stability due to the presence of the protease inhibitor and result in anti-neoplastic activity.

5. DEFINITIONS

In order that the invention may be more fully understood, the following terms are defined.

As used herein, “attenuated”, “attenuation”, and similar terms refer to elimination or reduction of the natural virulence of a bacterium in a particular host organism, such as a mammal.

“Virulence” is the degree or ability of a pathogenic microorganism to produce disease in a host organism. A bacterium may be virulent for one species of host organism (e.g., a mouse) and not virulent for another species of host organism (e.g., a human). Hence, broadly, an “attenuated” bacterium or strain of bacteria is attenuated in virulence toward at least one species of host organism that is susceptible to infection and disease by a virulent form of the bacterium or strain of the bacterium.

As used herein, the term “genetic locus” is a broad term and comprises any designated site in the genome (the total genetic content of an organism) or in a particular nucleotide sequence of a chromosome or replicating nucleic acid molecule (e.g., a plasmid), including but not limited to a gene, nucleotide coding sequence (for a protein or RNA), operon, regulon, promoter, inducible promoters (including tetracycline, arabinose, (EP1,655,370 A1, expressly incorporated in its entirety herein), salicylic acid, hypoxic, tumor cell specific inducible promoters) regulatory site (including transcriptional terminator sites, ribosome binding sites, transcriptional inhibitor binding sites, transcriptional activator binding sites), origin of replication, intercistronic region, and portions therein. It is understood that all protein expression constructs require a stop signal. A genetic locus may be identified and characterized by any of a variety of in vivo and/or in vitro methods available in the art, including but not limited to, conjugation studies, crossover frequencies, transformation analysis, transfection analysis, restriction enzyme mapping protocols, nucleic acid hybridization analyses, polymerase chain reaction (PCR) protocols, nuclease protection assays, and direct nucleic acid sequence analysis.

The terms “oral”, “enteral”, “enterally”, “orally”, “non-parenteral”, “non-parenterally”, and the like, refer to administration of a compound or composition to an individual by a route or mode along the alimentary canal. Examples of “oral” routes of administration of a vaccine composition include, without limitation, swallowing liquid or solid forms of a vaccine composition from the mouth, administration of a vaccine composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a vaccine composition, and rectal administration, e.g., using suppositories that release a live bacterial vaccine strain described herein to the lower intestinal tract of the alimentary canal.

The term “recombinant” is used to describe non-naturally altered or manipulated nucleic acids, cells transformed, electroporated, or transfected with exogenous nucleic acids, and polypeptides expressed non-naturally, e.g., through manipulation of isolated nucleic acids and transformation of cells. The term “recombinant” specifically encompasses nucleic acid molecules that have been constructed, at least in part, in vitro using genetic engineering techniques, and use of the term “recombinant” as an adjective to describe a molecule, construct, vector, cell, polypeptide, or polynucleotide specifically excludes naturally existing forms of such molecules, constructs, vectors, cells, polypeptides, or polynucleotides.

Cassette, or expression cassette is used to describe a nucleic acid sequence comprising (i) a nucleotide sequence encoding a promoter, (ii) a first unique restriction enzyme cleavage site located 5′ of the nucleotide sequence encoding the promoter, and (iii) a second unique restriction enzyme cleavage site located 3′ of the nucleotide sequence encoding the promoter. The cassette may also contain a multiple cloning site (MCS) and transcriptional terminator within the 5′ and 3′ restriction endonuclease cleavage sites. The cassette may also contain cloned genes of interest.

As used herein, the term “salmonella” (plural, “salmonellae”) and “Salmonella” refers to a bacterium that is a serovar of Salmonella enterica. A number of serovars of S. enterica are known. Particularly preferred salmonella bacteria useful in the invention are attenuated strains of Salmonella enterica serovar Typhimurium (“S. typhimurium”) and serovar Typhi (“S. typhi”) as described herein.

As used herein, the terms “strain” and “isolate” are synonymous and refer to a particular isolated bacterium and its genetically identical progeny. Actual examples of particular strains of bacteria developed or isolated by human effort are indicated herein by specific letter and numerical designations (e.g. strains Ty21a, CMV906, CMV908, CMV906-htr, CMV908-htr, Ty800, holavax, M01ZH09, VNP20009).

The definitions of other terms used herein are those understood and used by persons skilled in the art and/or will be evident to persons skilled in the art from usage in the text.

As used herein, the terms “homology” and “identity” are used interchangeably, but homology for proteins can include conservative amino acid changes. In general, to identify corresponding positions, the sequences of amino acids are aligned so that the highest order match is obtained (see, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer. Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carillo et al. (1988) SIAM J Applied Math 48:1073).

As use herein, “sequence identity” refers to the number of identical amino acids (or nucleotide bases) in a comparison between a test and a reference polypeptide or polynucleotide. Homologous polypeptides refer to a pre-determined number of identical or homologous amino acid residues. Homology includes conservative amino acid substitutions as well identical residues. Sequence identity can be determined by standard alignment algorithm programs used with default gap penalties established by each supplier. Homologous nucleic acid molecules refer to a pre-determined number of identical or homologous nucleotides. Homology includes substitutions that do not change the encoded amino acid (i.e., “silent substitutions”) as well identical residues. Substantially homologous nucleic acid molecules hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid or along at least about 70%, 80% or 90% of the full-length nucleic acid molecule of interest. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule. (For determination of homology of proteins, conservative amino acids can be aligned as well as identical amino acids; in this case, percentage of identity and percentage homology vary). Whether any two nucleic acid molecules have nucleotide sequences (or any two polypeptides have amino acid sequences) that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can be determined using known computer algorithms such as the “FAST A” program, using for example, the default parameters as in Pearson et al. Proc. Natl. Acad. Sci. USA 85: 2444 (1988) (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J. Molec. Biol. 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego (1994), and Carillo et al. SIAM J Applied Math 48: 1073 (1988)). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.)). Percent homology or identity of proteins and/or nucleic acid molecules can be determined, for example, by comparing sequence information using a GAP computer program (e.g., Needleman et al. J. MoI. Biol. 48: 443 (1970), as revised by Smith and Waterman (Adv. Appl. Math. 2: 482 (1981)). Briefly, a GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non identities) and the weighted comparison matrix of Gribskov et al. Nucl. Acids Res. 14: 6745 (1986), as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

Therefore, as used herein, the term “identity” represents a comparison between a test and a reference polypeptide or polynucleotide. In one non-limiting example, “at least 90% identical to” refers to percent identities from 90 to 100% relative to the reference polypeptides. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared, no more than 10% (i.e., 10 out of 100) of amino acids in the test polypeptide differs from that of the reference polypeptides. Similar comparisons can be made between a test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g., 10/100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. At the level of homologies or identities above about 85-90%, the result should be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often without relying on software.

The phrase or term “substantially identical” or “homologous” or similar varies with the context as understood by those skilled in the relevant art and generally means at least 60% or 70%, preferably means at least 80%, more preferably at least 90%, and most preferably at least 95%, 96%, 97%, 98%, 99% or greater identity. As used herein, substantially identical to a product means sufficiently similar so that the property of interest is sufficiently unchanged so that the substantially identical product can be used in place of the product.

As used herein, Salmonella encompasses all Salmonella species, including: Salmonella typhi, Salmonella choleraesuis, and Salmonella enteritidis. Serotypes of Salmonella are also encompassed herein, for example, typhimurium, a subgroup of Salmonella enteritidis, commonly referred to as Salmonella typhimurium.

As used herein, the term “analog” refers to a polypeptide that possesses a similar or identical function as a primary or secondary effector molecule but does not necessarily comprise a similar or identical amino acid sequence of a primary or secondary effector molecule, or possess a similar or identical structure of a primary or secondary effector molecule. A polypeptide that has a similar amino acid sequence refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide having an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least .sup.85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of a primary or secondary effector molecule described herein; (b) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a primary or secondary effector molecule described herein of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 200 contiguous amino acid residues, at least 300 contiguous amino acid residues, at least 500 contiguous amino acid residues, or at least 1000 contiguous amino acid residues; and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the nucleotide sequence encoding a primary or secondary effector molecule described herein. A polypeptide with similar structure to a primary or secondary effector molecule described herein refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of primary or secondary effector molecule described herein. The structure of a polypeptide can be determined by methods known to those skilled in the art, including but not limited to, peptide sequencing, X-ray crystallography, nuclear magnetic resonance, circular dichroism, and crystallographic electron microscopy.

The phrase “anti-angiogenic factor” relates to any proteinaceous molecule which has anti-angiogenic activity, or a nucleic acid encoding such a proteinaceous molecule. In a preferred embodiment, the anti-angiogenic factor is a peptide fragment or cleavage fragment of a larger protein.

The term “attenuation” relates to a modification so that a microorganism or vector is less pathogenic. The end result of attenuation is that the risk of toxicity as well as other side-effects is decreased, when the microorganism or vector is administered to the patient.

The term “bacteriocin” relates to a bacterial proteinaceous toxin with selective activity, in that the bacterial host is immune to the toxin. Bacteriocins may be encoded by the bacterial host genome or by a plasmid, may be toxic to a broad or narrow range of other bacteria, and may have a simple structure comprising one or two subunits or may be a multi-subunit structure. In addition, a host expressing a bacteriocin has immunity against the bacteriocin. Bacteriocins include a number of bacterial antibiotics, including colicins and microcins.

The phrase “chelating agent sensitivity” is defined as the effective concentration at which bacteria proliferation is affected, or the concentration at which the viability of bacteria, as determined by recoverable colony forming units (c.f.u.), is reduced.

As used herein, the term “derivative” in the context of a “derivative of a polypeptide” refers to a polypeptide that comprises an amino acid sequence of a polypeptide, such as a primary or secondary effector molecule, which has been altered by the introduction of amino acid residue substitutions, deletions or additions, or by the covalent attachment of any type of molecule to the polypeptide.

The term “derivative” as used herein in the context of a “derivative of a primary or a secondary effector molecule” refers to a primary or secondary effector molecule which has been so modified, e.g., by the covalent attachment of any type of molecule to the primary or secondary molecule. For example, but not by way of limitation, a primary or secondary effector molecule may be modified, e.g., by proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative of a primary or secondary effector molecule may be modified by chemical modifications using techniques known to those of skill in the art (e.g., by acylation, phosphorylation, carboxylation, glycosylation, selenium modification and sulfation). Further, a derivative of a primary or secondary effector molecule may contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as a primary or secondary effector molecule described herein.

The term “derivative” in the context of a “derivative of an msbB⁻ attenuated tumor-targeted Salmonella mutant” refers to a modified msbB Salmonella mutant as defined in International Publication No. WO 99/13053 at page 17, incorporated herein by reference in its entirety.

As used herein, the term “fragment” refers to a peptide or polypeptide comprising an amino acid sequence of at least 2 contiguous amino acid residues, at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least contiguous 80 amino acid residues, at least contiguous 90 amino acid residues, at least contiguous 100 amino acid residues, at least contiguous 125 amino acid residues, at least 150 contiguous amino acid residues, at least contiguous 175 amino acid residues, at least contiguous 200 amino acid residues, at least contiguous 250 amino acid residues, at least contiguous 300 amino acid residues, at least contiguous 500 amino acid residues, or at least contiguous 1000 amino acid residues of the amino acid sequence of a primary or secondary effector molecule.

As used herein, the term “functional fragment” refers to a fragment of a primary or secondary effector molecule that retains at least one function of the primary or secondary effector molecule (e.g., enzymatic activity, anti-angiogenic activity, or anti-tumor activity of the effector molecule).

As used herein, the term “fusion protein” refers to a polypeptide that comprises an amino acid sequence of primary or secondary effector molecule, or functional fragment or derivative thereof, and an amino acid sequence of a heterologous polypeptide (e.g., a non-primary or non-secondary effector molecule).

As used herein, “purified” attenuated tumor-targeted bacterial strain is substantially free of contaminating proteins or amino acids (e.g., debris from dead bacteria), or media. An attenuated tumor-targeted bacterial strain that is substantially free of contaminating proteins or amino acids includes preparations of attenuated tumor-targeted bacteria having less than about 30%, 20%, 10%, or 5% (by dry weight) of contaminating protein or amino acid.

As used herein, a “release factor” includes any protein, or functional portion thereof which enhances release of bacterial components. In one embodiment a release factor is a bacteriocin release protein. Release factors include, but are not limited to, the bacteriocin release protein (BRP) encoded by the colicin D13 plasmid, the BRPs encoded by the colicin E1-E9 plasmids, or BRPs encoded by the colicin A, N or D plasmids.

“Septic shock” is a state of internal organ failure due to a complex cytokine cascade, initiated by TNF-α. The relative ability of a microorganism or vector to elicit TNF-α. is used as one measure to indicate its relative ability to induce septic shock.

“Tumor-targeted” is defined as the ability to preferentially localize to a cancerous or neoplastic target cell or tissue relative to a non-cancerous counterpart cell or tissue and replicate. Thus, a tumor-targeted bacteria such as Salmonella preferentially attaches to, infects and/or remains viable in the cancerous target cell or the tumor, carcinoma, lymphoma or leukemic bone marrow environment.

“Virulence” is a relative term describing the general ability to cause disease, including the ability to kill normal cells or the ability to elicit septic shock (see specific definition below).

As used herein, the strain designations VNP20009 (International Publication No. WO 99/13053), YS1646 and 41.2.9 are used interchangeably and each refer to the strain deposited with the American Type Culture Collection and assigned Accession No. 202165. As used herein, the strain designations YS1456 and 8.7 are used interchangeably and each refer to the strain deposited with the American Type Culture Collection and assigned Accession No. 202164.5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a tumor-protease activated toxin and FIG. 1B shows a tumor protease inhibitor and protease sensitive toxin expression system.

FIG. 2 shows secreted protease inhibitors.

FIG. 3 shows chimeric colicins.

FIG. 4 shows colicin TRC chimeras.

FIG. 5 shows lytic peptide chimeras.

FIG. 6 shows protease activated lytic peptide chimera prodrugs.

FIG. 7 shows apoptotic peptide and toxic peptide chimeras.

FIG. 8 shows cytolethal distending toxin subunit B (cldtB) chimeras.

FIG. 9 shows repeat in toxin (RTX) family members and hybrid operons.

FIG. 10 shows cytoplasmic expressed proteins, hlyA fusions thereof, and non-chimeric surface (autotransporter) expressed proteins.

FIG. 11 shows a non-conjugative bacterium with and without the F′ factor.

FIG. 12 shows segregation of required colicin toxin and immunity factors.

6. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, according to various embodiments, live attenuated therapeutic bacterial strains that express one or more therapeutic molecules together with one or more protease inhibitor polypeptides that inhibit local proteases that could deactivate the therapeutic molecules. In particular, one aspect of the invention relates to live attenuated tumor-targeted bacterial strains that may include Salmonella, Streptococcus or Listeria vectoring novel chimeric anti-tumor toxins to an individual to elicit a therapeutic response against cancer. The types of cancer may generally include solid tumors, carcinomas, leukemias, lymphomas and multiple myelomas. In addition, certain of the therapeutic molecules have co-transmission requirements that are genetically unlinked to the therapeutic molecule(s), limiting certain forms of genetic exchange, i.e., distal to rather than adjacent to). Another aspect of the invention relates to live attenuated tumor-targeted bacterial strains that may include Salmonella, Streptococcus, and Listeria that encode anti-neoplastic molecules to an individual to elicit a therapeutic response against cancers including cancer stem cells, immune infiltrating cells and or tumor matrix cells. The therapeutic agents also relates to reducing or eliminating the bacteria's ability to undergo conjugation, further limiting incoming and outgoing exchange of genetic material.

For reasons of clarity, the detailed description is divided into the following subsections: protease sensitivity; protease inhibitors; targeting ligands; lytic peptides; antibody deactivating proteins; chimeric bacterial toxins; expression of proteins without generating chimeras; limiting bacterial conjugation; expression of DNase, or colicin DNase as active extracellular enzymes; co-expression of protease inhibitors with bacterial toxins; co-expression of protease inhibitors with bacterial toxins; segregation of required colicin cofactors; characteristics of therapeutic bacteria.

6.1. Protease Sensitivity.

The therapeutic proteins of the invention are sensitive to extracellular proteases (in contrast pro-aerolysin or urokinase chimeric toxins that are activated by proteases). Proteases may be classified by several different systems, for example, into six groups: serine proteases, threonine proteases, cysteine proteases, aspartate proteases, metalloproteases and glutamic acid proteases. Alternatively, proteases may be classified by the optimal pH in which they are active: acid proteases, neutral proteases, and basic proteases (or alkaline proteases). Protease digestion sites may be added to the therapeutic agent to enhance protease sensitivity when coexpressed with a corresponding protease inhibitor as discussed below within the localized confines of the bacteria and its surroundings, e.g., within a solid tumor, carcinoma, lymphoma or leukemic bone marrow, the extracellular protease sensitive protein is protected from degradation whereas if it and its protective inhibitor leak outside the confines, the inhibitor falls below the level necessary to cause inhibition and the effector molecule is degraded. Preferred proteases for conferring greater sensitivity are those that are under-expressed in tumors and over-expressed in normal tissues. However, many proteases are over-expressed within tumors. Proteases for which sensitivity sights may be added and for which protease inhibitors may be co-expressed include but are not limited to those described by Edwards et al. (eds) 2008 (The Cancer Degradome: Proteases and Cancer Biology, Springer, 926 pp). as well as proteases of lysosomes and the gut such as tissue plasminogen activator, activated protein C, factor Xa, granzyme (A, B, M), cathepsins (e.g., cathepsin B and S), thrombin, plasmin, urokinase, matrix metaloproteaes (types 1-28) membrane matrix metalloproteases (types 1-4), prostate specific antigens (PSA; kallikrein 3-related peptidase), kallikrein 2, elastin, trypsin, chymotrypsin. A variety of protease assays are known to those skilled in the arts. Many protease assays are commercially available, such as the QuantiCleave Fluorescent Protease Assay Kit, and QuantiCleave Protease Assay Kit II (Thermo/Fisher, Rockford, Ill.), Protease Assay Kit (G Biosciences, Maryland Heights, Mo.), PepTag Protease Assay (Promega, Madison, Wis.; 1993 Promega Notes Magazine 44: 2), Viral Protease Assay Kits (AnaSpec, Fremont, Calif.), Protease Assay Kit from Calbiochem (Calbiochem, San Diego, Calif.). Standard laboratory techniques to measure protease activity, and thus the reduced activity of protease inhibitors, include densiometric, spectrophotometric, colorimetric and fluorometric assays, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), two dimensional SDS-PAGE, high pressure liquid chromatography (HPLC) and mass spectroscopy (mass-spec). High sensitivity methods have also been described US Patent Pub. 2009/0294288.

Protease sensitivity may be enhance either by the complete addition of protease cleavage sites, or minor alteration of the amino acid sequence by making amino acid changes that are “conservative” or “tolerated”, resulting in addition or enhancement of a cleavage site. Determination of conservative or tolerated amino acids is generally known to those skilled in the arts by their chemistry, whereby amino acids are grouped into hydrophilic [ala, pro, gly, glu, asp, gln, asn, ser, thr], sulfhydryl [cys], aliphatic [val, ile, leu, met], basic [lys, arg, his], and aromatic [phe, tyr, trp] (French and Robson, What is a conservative substitution? J. Mol. Evol. 19: 171-175), but may also be determined by methods such as SIFT (Ng and Henikoff 2003, SIFT: predicting amino acids changes that affect protein function, Nucleic Acids Research 31: 3812-3814; Kumar et al., 2009, Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm, Nat Protoc 4: 1073-1081; Altschul 1991, Amino acid substitutions matrices from an information theoretic perspective, Journal of Molecular Biology 219: 555-665; Henikoff and Henikoff, 1992, Amino acid substitution matrices from protein blocks, Proceedings of the National Academy of Sciences USA 89: 10915-10919). PAM (percent absent mutations), PMB (probability matrix from blocks) and BLOSUM (blocks of amino acid substitution matrix) matrices are well known and may be used. Addition of cleavage sites by minor sequence alteration is conducted preferably in knowledge of the protein 3 dimensional crystal structure, and/or based on multiple sequence alignments that establish protein domains and variable regions between domains such that it is understood that those changes in the amino acid sequence might normally occur and/or be tolerated, in addition to SIFT or other analyses. Protein domain information is used to select interdomain regions. 3D information is also used to select regions of the protein that are exposed externally, and thus more sensitive to proteases. For example, the crystal structure of a number of colicins are known (e.g., colicin E3, Soelaiman et al., 2001, Molecular Cell 8: 1053-1062). Colicins have also been the subject of multiple sequence alignments (e.g., FIG. 18.2 in Sharma et al., Chapter 18 in Kastin (ed), 2006, Handbook of Biologically Active Peptides, Academic Press), and distinct protein domains have been established which correlate with the crystal structure (Sharma et al., 2006, Handbook of Biologically Active Peptides, Chapter 18, Colicins: Bacterial/Antibiotic Peptides, pp 115-123). In colicin E3, there are 3 domains, an N-terminal “T”, or translocation domain, an internal “R” or receptor domain, and C-terminal “C” or catalytic domain. Examination of the “hinge” sequence between domains R and C of colE3, amino acids 451 to 456 (NKPRKG SEQ ID NO:147), shows that these amino acids are variable compared to other homologous colicins such as colE7 (KRNKPG SEQ ID NO:148), colE2 (KRNKPG SEQ ID NO:148) and are thus identified as candidates for sequence alteration. For example, a furin cleavage sequence (designated R/−/Kr/R⁺s/−/−/−; also designated RXKR↓SX SEQ ID NO:149 can be added by conservative changes. Thus for example, the sequence NKPRGK SEQ ID NO:150 within colE3 can be conservatively changed to NKPRKs SEQ ID NO:151 which adds weak furin site, and further modified conservatively to NrPRKs SEQ ID NO:152 which results in a strong furin site which, using the ProP algorithm (e.g., ProP 1.0, Duckert et al., 2004, Prediction of proprotein convertase cleavage sites, Protein Engineering Design and Selection 17: 107-122) is predicted to be cleaved by furin. Biochemical confirmation can be conducted by standard techniques such as 1D and 2D SDS-PAGE gel electrophoresis on the secreted proteins in the media in the presence of furin.

Protease cleavage sites are defined in the Merops database (Rawlings et al., 2010, MEROPS: The Peptidase Database, Nucleic Acids Res. 2010 (Database issue):D227-33. It will be understood to those skilled in the arts that many proteases do not have strict sequence recognition sites, but rather have sequence preferences and/or frequencies. The MEROPS site depicts the preferences with a weighted pictogram and a table which lists frequencies of occurrence within a cleavage sequence. The table a non-limiting list proteases of tumors, the MEROPS sequence specification, and a simplified representative of an amino acid one letter code recognition sequence (where X is any amino acid) and the cleavage signal is given by a downward arrow) is presented in Table 2.

TABLE 2

Examples of protease cleavage sequences usable to guide

protease sensitivity modifiction of effector proteins.

MEROPS Sequence
Simplified Representative

Protease
Designation
Sequence Designation

Factor Xa
ia/e/Gfp/R⁺sti/vfs/-/g
(IEGR↓SV) SEQ ID NO:153

Furin
R/-/Kr/R⁺s/-/-/-
(RXKR↓SX) SEQ ID NO:154

Plasminogen
-/-/-/R⁺R/iv/N/-
(XXR↓RIN) SEQ ID NO:155

activator

Urokinase
-/sg/Gs/Rk⁺-/r/-/-
(XSGR↓XR) SEQ ID NO:156

MMP1
-/pa/g⁺li/-/-/-
(GPXG↓LXG) SEQ ID NO:157

MMP8
g/Pas/-/g⁺1/-/g/-
(GPQG↓LRG) SEQ ID NO:158

MMP 13
g/P/-/g⁺1/-/ga/-
(GPPG↓LXG) SEQ ID NO:159

Membrane
-/p/-/-⁺1/-/-/-
(LPAG↓LVLX) SEQ ID NO:160

matrix

metallo-

protease 1

PSA
si/sq/-/yq⁺s/s/-/-
(SSQY↓SSN) SEQ ID NO:161

Kallikrein 2
g/-/-/R⁺-/-/-/gs
(GGLR↓SGGG) SEQ ID NO:162

Granzyme A
t/-/-/RK⁺sa/-/-/-
(TXXPR↓SX) SEQ ID NO:163

Granzyme B
v/-/-/D⁺-/-/-/-
(VEXD↓SX) SEQ ID NO:164

Granzyme M
Ka/vaye/Pa/LM⁺-/-/-/-
(KVPL↓X) SEQ ID NO:165

Cathepsin B
-/-/l/r⁺-/-/g/-
(XLR↓XXGG) SEQ ID NO:166

Cathepsin S
-/-/flv/r⁺-/-/-/-
(SGFR↓SXG) SEQ ID NO:167

Thrombin
-/-/pla/R⁺sag/-/-/-
(AGPR↓SLX) SEQ ID NO:168

Plasmin
-/-/-/KR⁺-/-/-/-
(AXLK↓SX) SEQ ID NO:169

Plasminogen
/-/-/KR⁺-/-/-/-
(AXLK↓SX) SEQ ID NO:170

The MEROPS database can be used to identify which proteases to inhibit, by analysis of a particular effector protein and the cleavage sites it contains. Comparison with the target tissue, eg Edwards et al. (eds) 2008, The Cancer Degradome: Proteases and Cancer Biology, Springer, 926 pp is also used to inform the choice. Alternatively, 2-dimensional gel electrophoresis and protein sequencing of radiolabeled peptides incubated with the target tumor can be used to identify which aminoacids are being cleaved in a therapeutic protein, and therefore which protease inhibitors to use.

6.2 Protease inhibitors

Protease inhibitors of the invention are preferably based on known polypeptide inhibitors. The inhibitors include both synthetic peptides and naturally occurring, endogenous peptides. Classes of protease inhibitors include: cysteine protease inhibitors, serine protease inhibitors (serpins), trypsin inhibitors, Kunitz STI protease inhibitor, threonine protease inhibitors, aspartic protease inhibitors, metalloprotease inhibitors. Protease inhibitors can also be classified by mechanism of action as suicide inhibitors, transition state inhibitors, protein protease inhibitor (see serpins) and chelating agents. The protease inhibitors of the invention are protein or polypeptide inhibitors encoded by DNA contained within the bacteria.

To result in the desired activity, the peptides should be surface displayed, released or secreted outside of the bacteria. Accordingly, the peptides are modified by fusing them to secretion signals. The secretion signals may be either N-terminal (LPP:OmpA, M13pIII, M13pVIII, zirS (Finlay et al., 2008, PLoS Pathogens 4 (4), e100003), heat-stable (ST; thermostable) toxins from Escherichia and Vibrio (U.S. Pat. No. 5,399,490), E. coli enterotoxin II (Kwon et al., U.S. Pat. No. 6,605,697), or by colicin fusions together with colicin lysis proteins, or using autotransporter fusions, fusion to the M13 pIX may also be used (WO 2009/086116). or hlyA C-terminal signal sequence last 60 amino acids of the E. coli HlyA hemolysin, together with the required HlyBD supplied in trans and endogenous tolC as shown in FIG. 2. The N-terminal signal sequences are well known and characterized by the presence of a protease cleavage site for an endogenous bacterial protease. Thus, N-terminal signal sequences provide free protease inhibitors, free from the signal sequence. The C-terminal signal sequence may be further engineered to have a protease cleavage site in between the protease inhibitory peptide and the signal sequence. The cleavage site may be for the same protease that the peptide inactivates. Thus, the protease activates its own inhibitor. The protease cleavage site may also be for a protease other than for the protease inhibitor, thus deactivating another protease. Multiple protease inhibitor peptides may be used in-frame with multiple protease cleavage signals (polymeric protease activated protease inhibitors), where the inhibitors alternate with cleavage sites. The polymeric protease activated protease inhibitors can be homo- or hetero-inhibitor polymers (i.e., have inhibitors for the same or different proteases, respectively), and/or homo- or hetero-protease cleavage polymers (i.e., have the same or different protease cleavage sites). Proteases upregulated within tumors for which protease cleavage sites may be engineered include: tissue plasminogen activator, activated protein C, factor Xa, granzyme (A, B, M), cathepsin, thrombin, plasmin, urokinase, matrix metaloproteaes, prostate specific antigen (PSA) and kallikrein 2 (e.g., Edwards et al. (eds) 2008, The Cancer Degradome: Proteases and Cancer Biology, Springer, 926 pp), as well as proteases of lysosomes and the gut.

Protease inhibitors have been reviewed by Laskowski and Kato, 1980, (Annual Review of Biochemistry 49: 593-626), expressly incorporated by reference herein. Serine proteases inhibitors, the largest group, include 1) bovine pancreatic trypsin inhibitor (Kunitz) family, 2) pancreatic secretory trypsin inhibitor (Kazal) family, 3) Streptomyces subtilisin inhibitor family, 4) soybean trypsin inhibitor (Kunitz) family, 5) soybean proteinase inhibitor (Bowman-Birk) family 6) potato I inhibitor family, 7) potato II inhibitor family, 8) Ascaris trypsin inhibitor family, and 9) others. Protease inhibitors have also been grouped within the MEROPS peptidase database (Rawlings et al., 2008 Nucleic Acids Res. 36 Database issue, D320-325).

Specific examples of protease inhibitors that may be expressed as complete proteins or peptide fragments corresponding to the active inhibitory site include but are not limited to aprotinin, autodisplay aprotinin (Jose J, Zangen D (2005) Autodisplay of the protease inhibitor aprotinin in Escherichia coli. Biochem Biophys Res Commun 333:1218-1226; Jose, 2006, Autodisplay: efficient bacterial surface display of recombinant proteins, Appl Microbiol Biotechnol 69: 607-614). cathepsin inhibitor peptide sc-3130, Neisseria protease inhibitor, lymphocyte protease inhibitor, maspin, matrix metalloprotease inhibitors, macroglobulins, antithrombin, equistatin, Bowman-Kirk inhibitor family, ovomucoid, ovoinhibitor-proteinase inhibitors from avian serum, dog submandibular inhibitors, inter-α-trypsin inhibitors from mammalian serum, chelonianin from turtle egg white, soybean trypsin inhibitor (Kunitz), secretory trypsin inhibitors (Kazal) a_i-proteinase inhibitor, Streptomyces subtilisin inhibitor, plasminostreptin, plasmin inhibitor, factor Xa inhibitor, coelenterate protease inhibitors, protease inhibitor anticoagulants, ixolaris, human Serpins (SerpinA1(alpha 1-antitrypsin), SerpinA2, SerpinA3, SerpinA4, SerpinA5, SerpinA6, SerpinA7, SerpinA8, SerpinA9, SerpinA10, SerpinA11, SerpinA12, SerpinA13, SerpinB1, SerpinB2, SerpinB3, SerpinB4, SerpinB5, SerpinB6, SerpinB7, SerpinB8, SerpinC1 (antithrombin), SerpinD1, SerpinE1, SerpinE2, SerpinF1, SerpinF2, SerpinG1, SerpinNI1, SerpinNI2), cowpea trypsin inhibitor, onion trypsin inhibitor, alpha 1-antitrypsin, Ascaris trypsin and pepsin inhibitors, lipocalins, CI inhibitor, plasminogen-activator inhibitor, collegenase inhibitor, Acp62F from Drosophila, bombina trypsin inhibitor, bombyx subtilisin inhibitor, von Willebrand factor, leukocyte secretory protease inhibitor. Short peptide inhibitors of protease are preferred. Many protease inhibitors have one or more disulfide bonds. Fusion to thioredoxin (trxA) is known to improve protease inhibitor activity (e.g., Furuki et al., 2007, Fukuoka University Science Reports 37: 37-44). Fusion to glutathione-S transferase (GST) and co-expression with disulfide bond isomerase (DsbA) or nusA (Harrison 2000, Expression of soluble heterologous proteins via fusion with NusA protein. inNovations 11: 4-7) are also known to improve solubility. Methods to isolate novel protease inhibitors using M13 phage display have been described by Roberts et al., 1992 (Gene 121: 9-15). Examples of the peptide sequences of short peptide inhibitors is shown in Table 3.

TABLE 3

Sequences of short peptide protease inhibitors

Protease
Protease(s)

Inhibitor
inhibited
Protein/Peptide Name and/or Peptide Sequence

Leupeptin
calpain,
Leupeptin

plasmin,

trypsin, papain,

and cathepsin

B

Aprotinin
Trypsin
RPDFC LEPPY TGPCK ARIIR YFYNA KAGLC QTFVY

Plasmin
GGCRA KRNNF KSAED CMRTC GGA

Tissue kallikrei
SEQ ID NO:5

Jose J, Zangen D (2005) Autodisplay of the protease inhibitor

aprotinin in Escherichia coli. Biochem Biophys Res Commun

333:1218-1226

Aprotinin
Variable
Brinkmann et al, 1991 Eur J. Biochem 202: 95-99

homologues

Protease
Trypsin
Synthetic peptide: CFPGVTSNYLYWFK SEQ ID NO:6,

Inhibitor 15

corresponding to amino acids 245-258 of human protease

inhibitor.

Tissue
Serine protease
DSLGREAKCYNELNGCTKIYDPVCGTDGNTYPNECVL

protease
inhibitor,
CFENRKRQTSILIQKSGPC

inhibitor
Kazal type 1,
SEQ ID NO:7

mature

Furin
Furin
PAAATVTKKVAKSPKKAKAAKPKKAAKSAAKAVKPK

inhibitors

SEQ ID NO:8

TKKVAKRPRAKRAA
SEQ ID NO:9

TKKVAKRPRAKRDL
SEQ ID NO:10

GKRPRAKRA
SEQ ID NO:11

CKRPRAKRDL
SEQ ID NO:12

CVAKRPRAKRDL
SEQ ID NO:13

CKKVAKRPRAKRDL
SEQ ID NO:14

RRRRRR L6R (hexa-L-arginine)
SEQ ID NO:15

Kallikrein
Kallikrein 2
SRFKVWWAAG
SEQ ID NO:16

Inhibitors

AARRPFPAPS
SEQ ID NO:17

PARRPFPVTA
SEQ ID NO:18

Pepsinogen
Pepsin
LVKVPLVRKKSLRQNL
SEQ ID NO:19

1-16

Dunn et al., 1983 Biochem J 209: 355-362

Pepsinogen
Pepsin
LVKVPLVRKKSL
SEQ ID NO:20

1-12

Dunn et al., 1983 Biochem J 209: 355-362

Pepsinogen
Pepsin
LVKGGLVRKKSL (II) [Gly4,5]
SEQ ID NO:21

1-12 4-7

LVKVPGGRKKSL (III) [Gly6,7]
SEQ ID NO:22

substitution

LVKGGGGRKKSL (IV) [GIy4-7]
SEQ ID NO:23

Dunn et al., 1983 Biochem J 209: 355-362

Sunflower
Trypsin
GRCTKSIPPICFPD
SEQ ID NO:24

trypsin

inhibitor

SFTI-1

Odorrana
Trypsin
AVNIPFKVHFRCKAAFC SEQ ID NO:25

trypsin

inhibitor

Ascaris
Chymtrypsin
GQESCGPNEV WTECTGCEMK CGPDENTPCP

chymotrypsin
Elastase
LMCRRPSCEC SPGRGMRRTN DGKCIPASQCP

elastase

SEQ ID NO:26

inhibitor

Ascaris
Trypsin
EAEKCBZZPG WTKGGCETCG CAQKIVPCTR

trypsin

ETKPNPQCPR KQCCIASAGF VRDAQGNCIK FEDCPK

inhibitor

SEQ ID NO:27

Ascaris
Trypsin
EAEKCTKPNE QWTKCGGCEG TCAQKIVPCT

trypsin

RECKPPRCEC IASAGFVRDA QGNCIKFEDC PK

inhibitor

SEQ ID NO:28

Onion trypsin
Trypsin
MKAALVIFLL IAMLGVLAAE AYPNLRQVVV

inhibitor

TGDEEEGGCC DSCGSCDRRA PDLARCECRD

VVTSCGPGCK

RCEEADLDLN PPRYVCKDMS FHSCQTRCSI L

SEQ ID NO:29

Barley
Chymotrypsin
MSSMEKKPEGVNIGAGDRQNQKTEWPELVGKSVEEA

chymotrypsin

KKVILQDK

inhibitor 2

PAAQIIVLPVGTIVTMEYRIDRVRLFVDRLDNIAQVPRV

G

SEQ ID NO:30

Thrombin
Thrombin
IQPR
SEQ ID NO:31

inhibitors

GSAVPR
SEQ ID NO:32

Feng et al., (WO 2004/076484) PEPTIDE INHIBITORS OF

THROMBIN AS POTENT ANTICOAGULANTS)

Tumor cell
Gelatinase
CTTHWGFTLC
SEQ IN NO:111

and

Li et al., 2006. Molecular addresses of tumors: selection by in

endothelial

vivo phage display. Arch Immunol Ther Exp 54: 177-181

cell

migration

inhibitor

Proteosome
Proteosome

inhibitors
subunit 3

Chymostatin
‘chymotryptic-

Clasto-
like’ (beta5),

tactastatin
‘tryptic-like’

(beta2) and

‘peptidyl-

glutamyl

peptide

hydrolyzing’

(beta1).

Urokinase,
Urokinase,
Markowska et al., 2008, Effect of tripeptides on the

thrombin,
thrombin,
amindolytic activities of urokinase, thrombin, plasmin and

plasmin and
plasmin and
trypsin. Int. J. Peptide Research and Therapeutics 14: 215-

trypsin
trypsin
218.

inhibitors

6.3 Targeting Ligands

Targeting ligands have specificity for the target cell and are used to both confer specificity to chimeric proteins, and to direct attachment and/or internalization into the target cell. The ligands are known ligands or may be novel ligands isolated through standard means such as phage display (Barbass III et al., 2004, Phage Display, A Laboratory Manual, Cold Spring Harbor Press) including the use of commercially available kits (Ph.D-7 Phage Display Library Kit, New England Biolabs, Ipswich, Mass.; Li et al., 2006. Molecular addresses of tumors: selection by in vivo phage display. Arch Immunol Ther Exp 54: 177-181,). The ligands of various aspects of the present invention are peptides that can be expressed as fusions with other bacterially-expressed proteins. The peptides may be further modified, as for gastrin and bombisin, in being amidated by a peptidylglycine-alpha-amidating monoxygenase or C-terminal amidating enzyme, which is co-expressed in the bacteria that use these peptides using standard molecular genetic techniques. Examples of targeting peptides are shown in Table 4.

TABLE 4

Examples of targeting peptides

Receptor

Peptide sequence or ligand name
Target
Reference

MNSDSECPLSHDGYCLHDGVCMYIEA
EGFR

LDKYACNCVVGYIGERCQYRDLKWW

ELR

SEQ ID NO:172

ERRP

Marciniak et al., 2004,

Epidermal growth factor receptor related

Molecular Cancer

peptide

Therapeutics 3: 1615-1621

Wu et al., 1989, J. Biol.

Chem 246: 17469-17475.

Marciniak et al., 2003,

Gastroenterology 124:

1337-1347.

TGF-alpha
EGFR
Schmidt and Wells 2002,

Replacement of N-terminal

portions of TGF-alpha with

corresponding heregulin

sequences affects ligand-

induced receptor signalling

and intoxication of tumor

cells by chimeric growth-

factor toxins. In. J. Cancer

97: 349-356.

SYAVALSCQCALCRR

Rivero-Muller et al.,

CG-beta

Moleclar and Cellular

SEQ ID NO:33

Endocrinology 2007: 17-25

Morbeck et al., 1993

HAVDI and INPISGQ and dimeric versions
N-cadherin
Williams et al., 2002,

prostate
Journal of Biological

Chemistry 277: 4361-4367.

laminin-411 binding peptides
Brain
Ding et al., (2010) Proc.

neovasculature
Natl. Acad. Sci. U. S. A.

107:18143-18148

Pertussis toxin S3 subunit
cancer cells

Peptides described by Li et al., 2006.
Tumor
Li et al., 2006. Molecular

Molecular addresses of tumors: selection by
vasculature,
addresses of tumors:

in vivo phage display. Arch Immunol Ther
VEGF-R (Flt-1),
selection by in vivo phage

Exp 54: 177-181
VCAM, EphA2,
display. Arch Immunol Ther

Aminopeptidase
Exp 54: 177-181

DUP-1 peptide FRPNRAQDYNTN
Prostate cancer
Zitzmann et al., Clinical

SEQ ID NO:173

Cancer Research January

2005 11; 139

DARPins
HER2
Stumpp and Amstutz 2007,

SEQ ID NO:34

DARPins: a true alternative

to antibodies,

Curr Opin Drug Discov

Devel. 10:153-159.

AVALSCQCALCRR

Jia et al., Journal of

CG-beta (ala truncation)

Pharmacy and

SEQ ID NO:35

Pharmacology 2008; 60:

1441-1448

Leuteinizing hormone-releasing hormone
LHRH receptor

(LHRH)

pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-

Pro-Gly CONH2

SEQ ID NO:36

IL2
IL2R
Frankel et al. 2000, Clinical

Cancer Research 6: 326-

334.

Tf
TfR
Frankel et al. 2000, Clinical

Cancer Research 6: 326-

334.

IL4
IL4R
Frankel et al. 2000, Clinical

Cancer Research 6: 326-

334.

IL13
IL13R
Kawakami et al., 2002, J.

Immunol., 169: 7119-7126

GM-CSF
GM-CSFR
Frankel et al. 2000, Clinical

Cancer Research 6: 326-

334.

CAYHRLRRC
Lymphnode
Nishimura et al., 2008 JBC

SEQ ID NO:174
homing and cell
283: 11752-11762

Lymph node homing Cys-Ala-Tyr and cell
penetrating

penetrating Arg-Leu-Arg-Arg, proceeds

thorugh macripinocytosis

A33 antigen-binding peptide
A33
U.S. Pat. No. 5,712,369

CLTA-4 (CD152)
Melanoma
Specific antibodies and

antibody fragments

U.S. Pat. No. 6,207,156

CD19 binding peptides
specific for
Pamejer et al., 2007, Cancer

12-mer peptide (Bpep)
alpha(v) beta(6)
Gene Therapy 14: 91-97.

integrin (αvβ6)

non-Hodgkin

lymphoma,

chronic

lymphocytic

leukemia (CLL)

and acute

lymphocytic

leukemia (ALL)

CD20 binding peptides
CD-20; B-cell
WO/2004/103404 Watkins

malignancies
et al. “CD-20 binding

molecules”

CD22 binding peptides
B lymphocytes;
Pearson et al. Int. J. Peptide

hairy cell leukemia
Research and Therapeutics

14: 237-246.

CD25 binding peptides
Chemotherapy-
Saito et al., 2010, Science

resistant human
Translational Medicine 2:

leukemia stem
17ra9; Jordan

cells.
Sci Transl Med 12 May

2010 2:31ps21

TRU-015
CD-20
Hayden-Ledbetter et al.,

2009 Clin Cancer Res 15:

2739-2746; Burge et al..

2008, Clin Ther. 30:1806-

16.

CD30 binding peptides
CD-30

Hodgkin

lymphoma

CD32 binding peptides
Chemotherapy-
Saito et al., 2010, Science

resistant human
Translational Medicine 2:

leukemia stem
17ra9; Jordan

cells.
Sci Transl Med 12 May

2010 2:31ps21

CD33 binding peptides
CD-33

AML

Myelodysplastic

cells (MDS)

CD37 bnding peptides
Leukemia and

lymphoma

CD40 binding peptides
CD40

Multiple myeloma,

non-Hodgkin

lymphoma, chronic

lymphocytic

leukemia (CLL),

Hodgkin lymphocytic

and acute

lymphoblastic

leukemia (ALL),

diffuse large B-cell

lymphome,

refractory non-

Hodgkin Lymphoma,

including follicular

lymphoma

CD52 binding peptides
CLL

CD55 binding peptides

CD70 binding peptides
Hematological

malignancies,

Non-Hodgkin's

lymphoma

Also, killing

activated T and B

immune cells that

would eliminate

the bacterial vector

CD123 binding peptides
AML

RGD-containing peptides

De Villiers et al., 2008,

e.g., GRDGS SEQ ID NO:132,

Nanotechnology in drug

ACDCRGDCFCG (RGD4C)

delivery, Springer.

SEQ ID NO:174

Nanobodies derived from camels and llamas
Cancer
Rothbauer, et al. 2006. Nat.

(camelids), including humanized

Methods 3: 887-889;

nanobodies and VHH recognition domains

Kirchhoferet al. 2010. Nat.

Struct. Mol. Biol. 17:133-

138

Bombesin
Gastrin releasing
Dyba et al., 2004 Current

peptide receptor
Pharmaceutical Design 10:

2311-2334

Gastrin releasing peptide
Gastrin releasing

peptide receptor

somatostatin octapeptide RC-121

(D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-

NH2

SEQ ID NO:37

somatostatin

Vasoactive intestinal peptide (VIP

Neurotensin)

Parathyroid hormone-related protein PTHrP
Parathyroid

N-terminal 36 residues also has nuclear
hormone receptor

targeting
G-protein coupled

receptor

KLAKLAKKLALKLA
Proapoptotic

SEQ ID NO:38
peptide

EGFR binding peptides
EGFR

Mesothelin binding peptides
Mesothelin

Heat stable enterotoxin (ST)
Guanylyl cyclase

NSSNYCCELCCNPACTGCY
C

Mature peptide

SEQ ID NO:39

VLSFSPFAQD AKPVESSKEK
Heat stable
Sieckman et al.,

ITLESKKCNI AKKSNKSDPE
enterotoxin
WO/2003/072125

SMNSSNYCCE LCCNPACTGC
unprocessed

SEQ ID NO:40

CM-CSF
AML

Alfa(V)Beta(3)

integrin

STEAP-1 (six

transmembrane

antigen of the

prostate)

CDCRGDCFC
RGD 4C: active
Line et al. 46 (9): 1552.

SEQ ID NO:41
peptide targeting
(2005) Journal of Nuclear

the _vβ₃integrin)
Medicine

LGPQGPPHLVADPSKKQGP
bind to the gastrin

WLEEEEEAYGWMDF (gastrin-34) or big
receptor, also

gastrin
known in the art as

SEQ ID NO:42
the

cholecystokinin B

(CCKB) receptor

MGWMDF N-terminal truncation of gastrin

SEQ ID NO:43

VPLPAGGGTVLTKM
Gastrin releasing

SEQ ID NO:44
peptide

YPRGNHWAVGHLM

SEQ ID NO:45

CAYHLRRC
AML
Nishimra et al., 2008. J Biol

SEQ ID NO:46

Chem 283: 11752-11762

CAY (cys-ala-tyr)
Lymph node
Nishimra et al., 2008. J Biol

SEQ ID NO:47
homing
Chem 283: 11752-11762

RLRR (arg-leu-arg-arg)
Cell penetrating
Nishimra et al., 2008. J Biol

SEQ ID NO:48

Chem 283: 11752-11762

VRPMPLQ
Colonic dysplasia
Hsi u ng et al, Natre

SEQ ID NO:49

Medicin 14: 454-458

HVGGSSV
2622 Radiation-
International Journal of

SEQ ID NO:50
Induced
Radiation

Expression of Tax-
OncologyBiologyPhysics,

Interacting Protein
Volume 66, Issue 3, Pages

1 (TIP-1) in
S555-S556

Tumor
H. Wang, A. Fu, Z. Han, D.

Vasculature
Hallahan

Binds irradiated

tumors i.e., ones

responding to

therapy

CGFECVRQCPERC
Lung vasculature -
Mori 2004, Current

SEQ ID NO:171
MOSE
Pharmaceutical Design 10:

Binds membrane
2335-2343

dipeptidase (MDP)

SMSIARL
MURINE
Mori 2004, Current

SEQ ID NO:51
PROSTATE
Pharmaceutical Design 10:

VASCULATURE
2335-2343

VSFLEYR
MURINE
Mori 2004 Current

SEQ ID NO:52
PROSTATE
Pharmaceutical Design 10:

VASCULATURE
2335-2343

Fragment 3 of the high mobility group

(HMG)N2

CKDEPQRRSARLSAKPAPP

KPEPKPKKAPAKK

SEQ ID NO:53

H-VEPNCDIHVMW
VEGF BINDING
(WO/2006/116545)

EWECFERL-NH2
PEPTIDE
SPATIAL CONTROL OF

SEQ ID NO:54

SIGNAL

TRANSDUCTION

RLLDTNRPLLPY
L-PEPTIDE
Let al., 2004. Cancer

SEQ ID NO:55
Nasopharyngeal
Research 64: 8002-8008.

Phage derived -

causes

internalization of

phage

RGDLATL truncated
Alfa(v) beta (6)
Shunzi et al. (Kathyll C

RGDLATLRQLAQEDGVVGVR
integrin
Brown

SEQ ID NO:56

HAIYPRH
Transferrin
U.S. Pat. No.

SEQ ID NO:57

6,743,893

and

THRPPMWSPVWP

SEQ ID NO:58

Peptide 1 CKASQSVTNDVAC (CDR1)
CD-22
Pearson et al., 2008, Int J

SEQ ID NO:59

Pept Res Ther (2008)

Peptide 2 CYASNRYTC (CDR2)

14:237-246

SEQ ID NO:60

Peptide 3 CQQDYRSPLTFC (CDR3)

SEQ ID NO:61

Peptide 4 CSDYGVNWVC (CDR1)

SEQ ID NO:62

Peptide 5 CLGIIWGDGRTDYNSALKSRC

(CDR2)

SEQ ID NO:63

Cancer stem cell targeting peptides
Cancer stem cells
Cripe et al., 2009,

Molecular Therapy (2009)

17 10,1677-1682

Short and Curiel 2009,

Molecular Cancer

Therapeutics 8: 2096-2102

Chronic Lymphocytic leukemia binding
CLL
Takahashi et al., Cancer

peptides

Research 63: 5213-5217

LTVXPWY
Breast cancer
Shadidi and Sioudm 2003,

SEQ ID NO:64

The FASEB Journal 17:

256-258

Leukemia binding peptides
Leukemia
Fairlie et al., 2003,

Biochemistry 42: 13193-

13202

Jaalouk et al.,

WO/2006/010070

“Compositions and methods

related to peptides that

selectively bind leukemia

cells”

Adebahr et al.,

CPLDIDFYC
AML
Jager et al., Leukemia 21:

SEQ ID NO:65

411-420

Lymphoma binding peptides
Lymphoma
Lam and Zhao, 1997

Targeted Therapy for

Lymphoma with Peptides,

Hematology/Onocology

Clinincs of North America

11: 1007-1019,

Lymphoma stem cell targeting peptides
Hodgkins
Newcom et al., 1988, Inj. J.

CD 20 and CD19 binding peptides; see
lymphoma;
Cell Cloning 6: 417-431;

above
Hodgkin Reed-
Jones et at. 2009, Blood,

Sternberg (HRS)
113: 5920-5926.

cells

Leukemia stem cell targeting peptides

ADGACLRSGRGCGAAK
Hematological
Berntzen et al., 2006,

SEQ ID NO:66
malignancies
Protein Engineering ,

Design and Selection, doi:

10.1093/protein/gzj011

Somatostatin receptor-binding peptide
Renal cell
Shih et al., 2004, J. Nucl.

metastasis
Med. Technol 32: 19-21

GFLGEDPGFFNVE
Lymphoma
Tang et al., 2000,

SEQ ID NO:67

Bioconjugate Chem 11:

363-371

The cysteine modified F3-peptide sequence
Tumor
Henke et al., 2008, Nature

is 5'-
neovasculature
Biotechnology 26: 91-100.

CKDEPQRRSARLSAKPAPPKPEPKPKK

APAKK-3'.

SEQ ID NO:68

Transferrin
Transferrin

receptor

Binding peptides for tumor-specific
for tumor-specific
Dyba et al, 2004, Current

receptors
receptors
Pharmaceutical Design 10:

PTHrP, LHRH, alpha V Beta 3 integrin,
PTHrP, LHRH,
2311-2334; Tarasova et al.,

STEAP, Mesothelin, Endoglin (CD105),
alpha V Beta 3
WO/2003;072754

KCNK9, EGF receptors (Her1, Her2, Her3,
integrin, STEAP,

Her4), human mucin (CD19, CD22, CD25,
Mesothelin,

CD33, IL2R, CD2, CD3, CD5, CD7, CD30,
Endoglin (CD105),

GM-CSFR, IL4R IL6R, urokinase receptor,
KCNK9, EGF

IL13R, transferring receptor), guanylyl
receptors (Her1,

cyclase C
Her2, Her3, Her4),

human mucin

(CD19, CD22,

CD25, CD33,

IL2R, CD2, CD3,

CD5, CD7, CD30,

GM-CSFR, IL4R

IL6R, urokinase

receptor, IL13R,

transferrin

receptor), guanylyl

cyclase C

Transferrin
Transferrin

receptor

P15 peptide
Type II receptor
Bhatnagar et al., U.S.

GTPGPQGIAGQRGVV

Pat. No. 6,638,912

SEQ ID NO:69

ANVAENA peptide

SEQ ID NO:70

CQTIDGKKYYFN

Kushnaryov et al.,

SEQ ID NO:71

U.S. Pat. No. 5,466,672

Peptide from Clostridium

Clostridium difficile toxin A
Gal alpha 1-3Gal
Clark et al., 1987, Toxin A

beta 1-4G1cNAc.
from Clostridium difficile

binds to rabbit erythrocyte

glycolipids with terminal

Gal alpha 1-3Gal beta 1-

4G1cNAc sequences Arch

Biochem Biophys 15: 257:

217-229

KNGPWYAYTGRO
Surface idiotype of
Reviewed by Aina et al.

SEQ ID NO:72
SUP-88 human B-
Therapeutic Cancer

NWAVWXKR,
cell lymphoma
Targeting Peptides,

SEQ ID NO:73

Biopolymers 66: 184-199

YXXEDLRRR

SEQ ID NO:74

XXPVDHGL

SEQ ID NO:75

LVRSTGQFV, LVSPSGSWT
Surface idiotype of
Reviewed by Aina et al.

ALRPSGEWL, AIMASGQWL
human chronic
2002,

QILASGRWL, RRPSHAMAR
lymphocytic
Therapeutic Cancer

DNNRPANSM, LQDRLRFAT
lymphoma (CLL)
Targeting

PLSGDKSST

Peptides, Biopolymers

SEQ ID NO:76

66: 184-199

FDDARL SEQ ID NO:77,
Human multiple
Reviewed by Aina et al.

FSDARL SEQ ID NO:78,
myeloma M
2002

FSDMRL SEQ ID NO:79,
protein

FVDVRL SEQ ID NO:80,

FTDIRL SEQ ID NO:81,

FNDYRL SEQ ID NO:82

FSDTRL SEQ ID NO:83,

PIHYIF SEQ ID NO:84,

YIHYIF SEQ ID NO:85,

RIHYIF SEQ ID NO:86

IELLQAR SEQ ID NO:87
HL 60 human
Reviewed by Aina et al.

lymphoma & B-16
2002

mouse melanoma

CVFXXXYXXC SEQ ID NO:88,
Prostate-specific
Reviewed by Aina et al.

CXFXXXYXYLMC SEQ ID NO:89
antigen (PSA)
2002

CVXYCXXXXCYVC SEQ ID NO:90

CVXYCXXXXCWXC SEQ ID NO:91

DPRATPGS
LNCaP prostate
Reviewed by Aina et al.

SEQ ID NO:92
cancer
2002

HLQLQPWYPQIS
WAC-2 human
Reviewed by Aina et al.

SEQ ID NO:93
neuroblastoma
2002

VPWMEPAYQRFL
MDA-MB435
Reviewed by Aina et al.

SEQ ID NO:94
breast cancer
2002

TSPLNIHNGQKL
Head and neck
Reviewed by Aina et al.

SEQ ID NO:95
cancer lines
2002

SPL W/F,R/K,N/H,S, V/H, L
ECV304
Reviewed by Aina et al.

endothelial cell
2002

line

RLTGGKGVG
HEp-2 human
Reviewed by Aina et al.

SEQ ID NO:96
laryngeal carcinoma
2002

CDCRGDCFC (RGD-4C)
Tumor vasculature
Reviewed by Aina et al.

SEQ ID NO:97

2002

ACDCRGDCFCG
Tumor vasculature
Reviewed by Aina et al.

SEQ ID NO:98

2002

CNGRCVSGCAGRC
Aminopeptidase N
Reviewed by Aina et al.

SEQ ID NO:99

2002

CVCNGRMEC SEQ ID NO:100,
Vasculature of
Reviewed by Aina et al.

NGRAHA SEQ ID NO:101,
various tumors
2002

TAASGVRSMH SEQ ID NO:102,

LTLRWVGLMS SEQ ID NO:103

LRIKRKRRKRKKTRK SEQ ID NO:104,
IC-12 rat trachea
Reviewed by Aina et al.

NRSTHI SEQ ID NO:105

2002

SMSIARL SEQ ID NO:106,
Mouse prostate
Reviewed by Aina et al.

VSFLEYR SEQ ID NO:107

2002

CPGPEGAGC SEQ ID NO:108
Aminopeptidase P
Reviewed by Aina et al.

2002

ATWLPPR SEQ ID NO:109,
VEGF
Reviewed by Aina et al.

RRKRRR SEQ ID NO:110

2002

CTTHWGFTLC
Gelatinase
Reviewed by Aina et al.

SEQ ID NO:111

2002

-WYD- SEQ ID NO:112,
idiotype of WEHI-
Reviewed by Aina et al.

-WYDD- SEQ ID NO:113,
231 murine
2002

-WYT- SEQ ID NO:114,
lymphoma cell line

-WYV- SEQ ID NO:115

RWID SEQ ID NO:116,
idiotype of WEHI-
Reviewed by Aina et al.

RWFD SEQ ID NO:117
279 murine
2002

lymphoma cell line

LNNIVSVNGRHX SEQ ID NO:118,
Alpha-6-beta 1
Reviewed by Aina et al.

DNRIRLQAKXX SEQ ID NO:119
integrin of
2002

DU145 prostate

cancer

cell line

Leukemia stem cell binding peptides
Stem cells

Leukemia and lymphoma stem cell binding

Barbass III et al., 2004,

peptides isolated by phage display

Phage Display, A

Laboratory Manual, Cold

Spring Harbor Press; Ph.D-

7 Phage Display Library

Kit, New England Biolabs,

Ipswich, MA).

Macrophage cell binding peptides

Macrophage cell binding peptides isolated

Barbass III et al., 2004,

by phage display

Phage Display, A

Laboratory Manual, Cold

Spring Harbor Press; Ph.D-

7 Phage Display Library

Kit, New England Biolabs,

Ipswich, MA).

T-cell binding peptides

T-cell binding peptides isolated by phage

Barbass III et al., 2004,

display

Phage Display, A

Laboratory Manual, Cold

Spring Harbor Press; Ph.D-

7 Phage Display Library

Kit, New England Biolabs,

Ipswich, MA).

Neutrophil binding peptides

Neutrophil binding peptides isolated by

Barbass III et al., 2004,

phage display

Phage Display, A

Laboratory Manual, Cold

Spring Harbor Press; Ph.D-

7 Phage Display Library

Kit, New England Biolabs,

Ipswich, MA).

Tumor stromal matrix cell binding peptides

Tumor stromal matrix cell binding peptides

Barbass III et al., 2004,

isolated by phage display

Phage Display, A

Laboratory Manual, Cold

Spring Harbor Press; Ph.D-

7 Phage Display Library

Kit, New England Biolabs,

Ipswich, MA).

6.4 Lytic Peptides

The desirability of combining protease inhibitors with lytic peptides has not previously been recognized as a means of improving both activity and specificity of proteins delivered by targeted bacteria. Small lytic peptides (less than 50 amino acids) are used to construct chimeric proteins for more than one purpose. The chimeric proteins containing lytic peptides may be directly cytotoxic for neoplasias. In order to be cytotoxic they must be released, surface displayed and/or secreted (FIG. 3) and may be provided with cell specificity by the addition of a targeting ligand. Small lytic peptides have been proposed for use in the experimental treatment of neoplastic diseases. However, it is evident that most, if not all, of the commonly used small lytic peptides have strong antibacterial activity, and thus are not compatible with delivery by a bacterium (see Table 1 of Leschner and Hansel, 2004 Current Pharmaceutical Design 10: 2299-2310, expressly incorporated herein by reference). Small lytic peptides useful in the invention are those derived from Staphylococcus aureus, S. epidermidis and related species, including the phenol-soluble modulin (PSM) peptides and delta-lysin (Wang et al., 2007 Nature Medicine 13: 1510-1514, expressly incorporated herein by reference). The selection of the lytic peptide depends upon the primary purpose of the construct, which may be used in combination with other constructs providing other anticancer features. That is, the therapies provided in accordance with aspects of the present invention need not be provided in isolation, and the bacteria may be engineered to provide additional therapies or advantageous attributes. Constructs designed to be directly cytotoxic to cells employ the more cytotoxic peptides, particularly PSM-alpha-3. Constructs which are designed to use the lytic peptide to affect escape from the endosome use the peptides with the lower level of cytotoxicity, such as PSM-alpha-1, PSM-alpha-2 or delta-lysin. Larger lytic peptides that may be used includes the actinoporins and equinatoxins from sea anemones or other coelenterates such as FraC, Sticholysins StsI and StsII (Anderluh and Macek 2002, Toxicon 40: 111-124), are generally more potent than the bacterially-derived peptides, and are selected for use in being directly cytotoxic to parasites. Assay of lytic peptides is known to those skilled in the arts. Examples of lytic peptides useful in the invention are shown in Table 5.

TABLE 5

Membrane lytic peptides useful in the invention

Peptide and source
Peptide Sequence or name

Processed
MAQDIISTISDLVKWIIDTVNKFTKK

<<short>> active
SEQ ID NO:120

delta lysin

S aureus

Delta lysin processed
MMAADIISTI GDLVKWIIDTVNKFKK

S epidermitidis

SEQ ID NO:121

Delta lysin from CA-
MAQDIISTISDLVKWIIDTVNKFTKK

MRSA
SEQ ID NO:122

PSM-alpha-1
MGIIAGIIKVIKSLIEQFTGK

SEQ ID NO:123

PSM-alpha-2
MGIIAGIIKFIKGLIEKFTGK

SEQ ID NO:124

PSM-alpha-3
MEFVAKLFKFFKDLLGKFLGNN

SEQ ID NO:125

PSM-alpha-4
MAI VGTIIKIIKAIIDIFAK

SEQ ID NO:126

PSM-beta-1
MEGLFNAIKDTVTAAINNDGAKLG-

TSIVSIVENGVGLLGKLFGF

SEQ ID NO:127

PSM-beta-2
MTGLAEAIANTVQAAQQHDSVKLG-

TSIVDIVANGVGLLGKLFGF

SEQ ID NO:128

Actinoporins
Lytic peptides from sea anemones and

Equinatoxins
other coelenterates (e.g., SrcI,

FraC, Sticholysins StsI and StsII)

6.5 Antibody and Complement Deactivating Proteins.

Antibody deactivating proteins are useful for limiting the effective immune response against the bacteria vector such that the vector is not eliminated prior to its effective treatment of the neoplastic disease, or during (i.e., following administration but prior to arrival at the target site) and after multiple injections of the same vector at later points in time when an adaptive immune response may have occurred. Antibody deactivating proteins have been suggested to be potentially useful therapeutics for treatment of antibody-based diseases, such as autoimmunity (Nandakumar and Holmadh. 2008, Trends in Immunology 29: 173-178). However, it has not been recognized that expression of these proteins would be desirable in a tumor-targeting bacterial vector as an alternative to serotype variation (as described above), which does not require the generation of multiple strains, each of which require separate testing alone as well as in combination (i.e., succession). The IgG-degrading enzyme of S. pyogenes IdeS is a cysteine endopeptidase, secreted by group A streptococcal strains during infection. It cleaves the heavy chains of IgG with a unique specificity by binding and cleaving in the hinge region, thus generating an Fc and a F(ab′)2 fragment that can be detected by protein G capture and mass spectrometry. By removing the Fc section from the antigen recognizing Fab, immune responses such as complement deposition and Fc-mediated phagocytosis are blocked. This IgG proteolytic degradation disables opsonophagocytosis and interferes with the killing of group A Streptococcus. IdeS bestows a local protective effect for the bacteria. Another IgG degrading enzyme of Streptococcus pyogenes is endo-b-N-acetylglucosaminidase (EndoS) which cleavage sites on the IgG molecule. Protein G, the aforementioned protein used in biochemical purification, has IgG antibody deactivation properties Bjork and Kronvall 1984 J Immunol 133: 969-974). Other antibody deactivating proteins include Shistosome IgE proteases and the antibody binding protein A peptides from Staphalococcus (e.g., spa gene). The IgA protease of Neisseria sp. is an autotrasporter protein. Streptococcus PspA inhibits complement activation (Anh-Hue, T et al., 1999. Infect. Immun 67: 4720-4724). Each of these proteins may be expressed individually or in combination in tumor-targeting strains of bacteria.

6.6 Chimeric Bacterial Toxins

Chimeric toxins are toxins that may contain combinations of additional elements including targeting peptides, lytic peptides, nuclear localization signals, blocking peptides, protease cleavage (deactivation) sites, N- or C-terminal secretion signals, autotransporter constructs, used to adapt the proteins to provide therapeutic molecules that are effective in treating neoplastic cells, stromal cells, neoplastic stem cells as well as immune infiltrating cells. Targeting to a particular cell type uses the appropriate ligand from the Table 2 above or from other known sources. Toxin activity is determined using standard methods known to those skilled in the arts such as Aktories (ed) 1997 (Bacterial Toxins, Tools In Cell Biology and Pharmacology, Laboratory Companion, Chapman & Hall).

6.6.1 Chimeric colicins with phage proteins. Colicins lack tumor cell targeting. In the present invention, the colicin targeting and translocation domains are replaced with an M13pIII-derived signal sequence and truncated membrane anchor together with a targeting ligand. A lytic peptide may also be added. Examples of the unique organization for chimeric colE3, colE7 and col-Ia are shown in FIG. 3.

6.6.2 In another version of chimeric colicins, the colicin targeting domain is replaced with a tumor-specific targeting domain (FIG. 4).

6.6.3 In another version of chimeric colicins, the targeting domain is attached to the C-terminus. Further C-terminal modification can include the addition of a NLS, preferably from apoptin, and/or a lytic peptide (FIGS. 3 and 4). The tumor-selective nuclear export signal of apoptin may also be used alone or in combination with the NLS.

6.6.4 Chimeric cytolethal distending toxin. Cytolethal distending toxin (cldt) is a three component toxin of E. coli, Citrobacter, Helicobacter and other genera. Cldt is an endonuclease toxin and has a nuclear localization signal on the B subunit. Chimeric toxins are provided that utilize fusion to apoptin, a canary virus protein that has a tumor-specific nuclear localization signal, a normal cell nuclear export signal (FIG. 8). The cytolethal distending toxin B and chimeric cltdB may be expressed as a polycistronic construct consisting of cldtABC. The cytolethal distending toxin B and chimeric cltdB may be expressed as a polycistronic construct consisting containing the typhoid pertussis-like toxin (plt) AB genes.

6.6.5 RTX toxins and hybrid operons. E coli HlyA(s) operon hlyCABD (+TolC), Actinobacillus actinomycetemcomitans leukotoxin ltxCABD, and a hybrid CABD operon are shown in FIG. 9. In addition to direct antitumor activity, they may activate/release liposomal agents when used in combination. The ltxA may be generated as a chimera wherein it contains the C-terminal 60 amino acids of the E. coli HlyA. The ltx genes and chimeras may be expressed together with prtF and/or cyaE. Hybrid RTX toxins may be further constructed from known RTX toxins to confer species specificity to multiple species, such as both mouse and human, such that the safety and efficacy testing can occur in multiple species. Multiple sequence alignments of RTX toxins with various species specificities (Ludwig and Goebel, Chapter 29, Structure and mode of action of RTX toxins, Alouf and Popoff (eds), 2006, Comprehensive Sourcebook of Bacterial Protein Toxins, Third Edition, Academic Press; Frey Chapter 30, Genetics and phylogeny of RTX cytolysins, in Kastin (ed), 2006, Alouf and Popoff (eds), 2006, Comprehensive Sourcebook of Bacterial Protein Toxins, Third Edition, Academic Press) are used in the analysis using computer based algorithms such as ClustalW, Muscle 3DCoffee and others (Larkin et al., 2007, ClustalW and ClustalX version 2. Bioinformatics 2007 23(21): 2947-2948 MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5: 113; MUSCLE: multiple sequence alignment with high accuracy and high throughput Nucleic Acids Research 32(5): 1792-1797; 3DCoffee: Combining Protein Sequences and Structures within Multiple Sequence Alignments, Journal of Molecular Biology 340: 385-395; Notredame e al., 2000, T-Coffee: A novel method for multiple sequence alignments, Journal of Molecular Biology 302: 205-217; Zdobnov E. M. and Apweiler R. (2001), InterProScan—an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17(9): 847-848; Lassmann T. and Sonnhammer E. L. L. (2006), Kalign, Kalignvu and Mumsa: web servers for multiple sequence alignment, Nucleic Acids Research 34: W596-W59).

6.6.6 Saporin and ricin chimeras. Saporin and ricin can be replaced for the active portion of the colicin chimeras. It can also be generated as a targeting peptide, saporin, HlyA C-terminus (FIG. 10).

6.6.7 Cytotoxic necrotic factor (cnf) and Bordetella dermonecrotic factor (dnf) chimeras. Cnf and dnf can be expressed as chimeras, where the N-terminal binding domain (amino acids 53 to 190 of cnf) is replaced with a tumor cell binding ligand, such as TGF-alpha.

6.6.8 Shiga toxin (ST) and shiga-like toxin (SLT) chimeras. ST and SLT chimeras are generated wherein the GB3-binding domain is replaced with a tumor cell binding ligand, such as TGF-alpha.

6.6.9 Subtilase toxin chimeras. Subtilase chimeras are generated by replacing the binding domain with a tumor cell binding ligand, such as TGF-alpha.

6.6.10 Nhe (non-hemolytic toxins from Bacillus) chimeras are generated by replacing the targeting domain with a tumor cell binding ligand and may be made protease sensitive by addition of a protease cleavage site.

6.6.11 Clostridium Tox A binding domain replacements (Rupnik and Just, Chapter 21 in: Alouf and Popoff (eds), 2006, Comprehensive Sourcebook of Bacterial Protein Toxins, Third Edition, Academic Press).

6.6.12 Collagenase chimeras. Collagenase is fused with a targeting peptide that directs its activity towards tumor cells, and may be made protease sensitive by the addition of a protease cleavage site.

6.6.13. Lytic chimeras. Lytic chimeras are shown in FIGS. 5 and 6.

6.7 Expression of Proteins without Generating Chimeras.

Certain proteins of the invention augment the effector gene and protease inhibitor combination without requiring chimeric modification. These proteins include the Geobacter carboxyesterase, the bacillus thiaminase and the Neisseria IgA protease. The carboxyesterase and thiaminase may also be expressed as hlyA fusion proteins. These proteins may be expressed using constitutive or inducible promoters (FIG. 10).

6.8 Limiting Bacterial Conjugation.

The fertility inhibition complex (finO and finP), are cloned onto the chromosome using standard genetic techniques such that strains either with or without the pilus resistant to mating with F′ bacteria (FIG. 11). Other known inhibitory factors may also be used.

The F′ pilus factors in a Salmonella strain needed for phage to be able to infect the cell are provided by the F′ plasmid using standard mating techniques from an F′ E coli. The F′ factor provides other functions such as traD and the mating stabilization which are deleted using standard techniques.

6.9 Expression of DNAase Colicins as Active Extracellular Enzymes.

Colicins have innate potential to harm the host that produces them. In order to protect the host, colicins are naturally co-produced with an “immunity” protein which protects it from the action of the colicin. The immunity proteins are generally specific for each individual colicin, and each has a high affinity for the colicin. When colicins are expressed by the bacteria, the immunity protein immediately binds to the colicin preventing it from harming the host. When colicins are released, the immunity protein may remain bound. Thus, a DNAase colicin may not be expected to have extracellular activity. When colicins are internalized into the target cell the immunity protein remains extracellular, and the colicin thus becomes activated inside the target cell.

In order to generate colicins with extracellular DNAase activity capable of deactivating DNA from neutrophils capable of trapping bacteria (neutrophil nets), the present invention presents a novel combination of DNAase colicin, such as colE9, co-expressed with a non-matching DNAase colicin immunity protein, such as that from colE2, colE7, or colE8, which have higher dissociation constants for colE9 (James et al., 1996, Microbiology 142: 1569-1580). In order to compensate for the reduced amount of protection expected to occur, multiple copies of the non-cognate immunity protein are expressed. Thus, when the colicin E9 is released, the immunity proteins partially dissociate, resulting in extracellular DNAase activity.

In another method of producing an immunity protein that dissociates extracellularly, thus activating the colicin such as a DNAase colicin, the immunity protein, such as colE9 immunity, is subjected to error-prone PCR (e.g., Cirino et al., 2003, Generating mutant libraries using error-prone PCR, Methods in Molecular Biology 231: 3-9; Arnold and Georgiou (eds) 2003, Directed Evolution Library Creation, Humana Press). The library is then cloned into a DNAase colicin-containing plasmid, such as the colE9 colicin, and transformed into a suitable E. coli or Salmonella. The bacteria are plated to appropriate nutrient agar plates containing DNA. After an incubation period the plates are stained for DNA, e.g., ethidium bromide, and viewed under fluorescent light for “halos”; clear or lighter regions around colonies where the DNA has been digested. Such colonies will contain the colE9 colicin, and an immunity protein that is sufficiently stable intracellularly such that it protects the bacterial cell, allowing it to grow, and is capable of dissociating under extracellular conditions, allowing the DNAase colicin to degrade extracellular DNA. The assay may be further modified to alter the agar plate conditions to match conditions of the target site, such as lower pH that is known to occur in solid tumors. The, the process would then select for functional immunity proteins that dissociate under acidic pH, such as occurs in solid tumors, allowing the degradation of extracellular DNA, such as may occur from infiltrating neutrophils.

6.10 Co-Expression of Protease Inhibitors with Bacterial Toxins, Chemotherapeutic Agents, Clodronate, Carbogen, and Determinations of Combination Effects, Antagonism, Additivity and/or Synergy.

Each of the bacterial toxins and therapeutic peptides and proteins listed herein may be improved in its therapeutic activity by co-expression with a protease inhibitor. Inhibitors are expressed as secreted proteins as described above. The effect of the protease inhibitor on in vitro cytotoxicity is determined using standard cell culture techniques and cytotoxicity assays such as MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazol; Mosmann 1983; J. Immunol Methods 65:55-63) known to those skilled in the arts. The contribution of the protein cytotoxin and protease inhibitors is determined individually and in combination. Purified protease of types known to occur in the target tissue, such as a solid tumor, lymphoma, myeloma, or the lumen of a leukemic bone, may be added to the assay. Combination effects, including antagonism, additivity or synergy may be determined using the median effect analysis (Chou and Talaly 1981 Eur. J. Biochem. 115: 207-216) or other standard methods (White et al., 1996, Antimicrobial Agents and Chemotherapy 40: 1914-1918; Brenner, 2002, Annals of Oncology 13: 1697-1698; Berenbaum M C. 1989. What is synergy? Pharmacol Rev. 41(2): 93-141; Greco W R, Bravo G, Parsons J C. 1995. The search for synergy: a critical review from a response surface perspective. Pharmacol Rev. 47(2): 331-85); Zhao et al., 2004, Evaluation of Combination Chemotherpy, Clin Cancer Res 10: 7994-8004; Loewe and Muischnek, 1926. Effect of combinations: mathematical basis of the problem, Arch. Exp. Pathol. Pharmakol. 114: 313-326). The assay may also be used to determine synergy, additivity or antagonism of two or more bacterial cytotoxins. The assay may also be used to determine synergy, additivity or antagonism a bacterial cytotoxin together with a conventional small molecule cytotoxin (e.g., cisplatin, doxorubicin, irinotecan, paclitaxel or vincristine), targeted therapeutic (e.g., imatinib, irissa, cetuximab), proteosome inhibitors (e.g., bortezomib), mTOR inhibitors or PARP inhibitors. Treatment with drugs such as imatinib prior to injection of Salmonella may also enhance bacterial tumor targeting (Vlahovic et Br J Cancer 2007, 97 735-740). In vivo studies may also be performed with antiangiogenic inhibitors such as Avastin, combrettastatin, thalidomide. In vivo studies with reticuloendothelial system (RES) blocker such as chlodronate which have the potential to improve the circulation time of the bacteria, vacular permeability inducing agents such as bradykinin, hyperthermia or carbogen which have the potential to improve the permeability of the tumor enhancing entry of the bacteria or aldose reductase inhibitors. Preferred genetic backgrounds for msbB mutant Salmonella in combination with carbogen (carbon dioxide oxygen mixture) includes zwf, which confers resistance to CO₂(Karsten et al., 2009, BMC Microbiol. BMC Microbiol. 2009 Aug. 18; 9:170).

6.11 Segregation of Required Colicin Toxin Cofactors.

The chimeric colicin toxins have active colicin components that require their respective immunity proteins, which are usually genetically linked. By unlinking the two genes and separating them on the chromosome, a single fragment or phage transduction is highly unlikely to contain both elements. In order to separate the elements from co-transmission by a transducing phage such as P22, separation by 50 kB or greater is preferred. Without both elements, the toxin portion cannot be carried and will kill most bacteria. Any additional genes such as other chimeric therapeutic molecules genetically linked to the colicin will also be inhibited from being transferred to other bacteria (FIG. 12)

6.12 Characteristics of Therapeutic Bacteria Co-Expressing Protease Inhibitors with Chimeric Antigens, Lytic and Therapeutic Proteins

The primary characteristic of the bacteria of the invention is the enhanced effect of the effector molecule such as a toxin, lytic peptide etc. relative to the parental strain of bacteria without expressing one or more protease inhibitors. In one embodiment, the percent increase in effect is approximately 2% to approximately 95%, approximately 2% to approximately 75%, approximately 2% to approximately 50%, approximately 2% to about 40%, approximately 2% to about 30%, approximately 2% to about 25%, approximately 2% to about 20% or about 2% to approximately 10% greater than the parental strain of bacteria without expressing one or more protease inhibitors under the same conditions.

A second characteristic of the bacteria of the invention is that they carry novel chimeric proteins that prevent their elimination by antibodies compared to other chimeric protein expression systems. In one embodiment, the percent improvement is approximately 2% to approximately 95%, approximately 2% to approximately 75%, approximately 2% to approximately 50%, approximately 2% to about 40%, approximately 2% to about 30%, approximately 2% to about 25%, approximately 2% to about 20% or about 2% to approximately 10% that of another expression system under the same conditions.

A third characteristic of the bacteria of the invention is that they carry novel chimeric proteins that improve their function compared to other chimeric protein expression systems. In one embodiment, the percent improvement is approximately 2% to approximately 95%, approximately 2% to approximately 75%, approximately 2% to approximately 50%, approximately 2% to about 40%, approximately 2% to about 30%, approximately 2% to about 25%, approximately 2% to about 20% or about 2% to approximately 10% that of another expression system under the same conditions.

A fourth characteristic of the bacteria of the invention is that they carry heterologous proteins that suppress features of the immune system that include antibody binding and/or deactivating proteins, targeted peptides against activated T and B cells, extracellular DNases that prevent destruction by neutrophil nets, and antitumor toxins with cross-over anti-neutrophil activity (dual antitumor and anti-neutrophil activity). The Yersinia pestis secreted protein LcrV that triggers the release of interleukin 10 (IL-10) by host immune cells and suppresses proinflammatory cytokines such as tumor necrosis factor alpha and gamma interferon as well as innate defense mechanisms required to combat the pathogenesis of plague.

The immunosuppressive features together with the antibody and complement deactivation proteins allow repeated injections of the bacteria without elimination form the immune system, where improvement is defined as the percentage of bacteria present at the target site after between 1 to 21 days compared to the parental strain in a murine model. In one embodiment, the percent improvement is approximately 2% to approximately 95%, approximately 2% to approximately 75%, approximately 2% to approximately 50%, approximately 2% to about 40%, approximately 2% to about 30%, approximately 2% to about 25%, approximately 2% to about 20% or about 2% to approximately 10% that of another expression system under the same conditions.

Overall improvement is defined as an increase in effect, such as the ability to kill a neoplastic cells in vitro by the bacteria, or inhibit or reduce the volume or cell number of a solid tumor, carcinoma, lymphoma or leukemia in vivo following administration with the bacteria expressing a therapeutic molecule, with and without the protease inhibitor, and/or with and without an antibody inhibiting peptide. The effect of the protease inhibitor on protein therapeutic activity is determined using standard techniques and assays known to those skilled in the arts. Inhibitors are expressed as secreted, surface displayed and/or released proteins as described above. Likewise, the effect of the antibody inhibitory protein on therapeutic activity is determined using standard techniques and assays known to those skilled in the arts. Antibody inhibitors are expressed as native proteins (e.g., IgA protease in gram negative bacteria for vectors such as those using Salmonella, or spa, IdeS and EndoS in gram positive bacteria for vectors such as those using Streptococcus) or as secreted protein chimeras as described above such as a fusion with hlyA. The contribution of the therapeutic protein, protease inhibitors and/or antibody inhibitory proteins is determined individually and in combination. Additivity, synergy or antagonism may determined using the median effect analysis (Chou and Talaly 1981 Eur. J. Biochem. 115: 207-216) or other standard methods.

7. FIGURE LEGENDS

FIG. 1. Comparison of tumor-protease activated toxin with tumor protease inhibitor and protease sensitive toxin expression. A) Intravenously injected tumor protease activated toxin remains active if it diffuses out of the tumor. B) Intratumoral bacteria co-expressing a protease inhibitor and a protease sensitive toxin achieve high intratumoral activity and degradation following diffusion out of the tumor. The co-expression system results in high intratumoral activity, achieving a therapeutic benefit with low toxicity.

FIG. 2. Secreted protease inhibitors (PIs). A) A PI followed by the hlyA C-terminal signal sequence. B) A PI followed by an intervening protease cleavage site (downward arrow) and the hlyA C-terminal signal sequence. B′). Multiple protease inhibitor peptides may be used in-frame with multiple protease cleavage signals (polymeric protease activated protease inhibitors), where the inhibitors alternate with cleavage sites. The polymeric protease activated protease inhibitors can be homo- or hetero-inhibitor polymers (i.e., have multiple inhibitors for the same or different proteases, respectively), and/or homo- or hetero-protease cleavage polymers (i.e., have multiple of the same or different protease cleavage sites). Thus, protease inhibitors 1, 2 and 3 can be the same protease inhibitor or different protease inhibitors, and the protease cleavage sites (downward arrows) can be the same protease cleavage side or different protease cleavage sites. C) A blocking peptide followed by an intervening protease cleavage site (downward arrow) and then the hlyA C-terminal signal sequence. D) The LPP:OmpA signal sequence followed by a protease inhibitor. E) The M13 pIII signal sequence (amino acids 1-18) followed by a protease inhibitor. F) An autotransporter cassette consisting of an autotransporter signal peptide, a protease inhibitor (passenger) followed by the autotransporter linker and β-barrel. G) A pINIIIompA leader with a protease inhibitor (Longstaff et al., Biochemistry 1990 29: 7339-7347). H) A colicin N-terminal domain with a protease inhibitor. I) A thioredoxin (TrxA) fusion with a PI followed by the hlyA C-terminal signal sequence. J) A thioredoxin (TrxA) fusion with a PI followed by an intervening protease cleavage site (downward arrow) and the hlyA C-terminal signal sequence. K) A blocking peptide followed by a thioredoxin (TrxA) fusion with an intervening protease cleavage site (downward arrow) and then the hlyA C-terminal signal sequence. L) The LPP:OmpA signal sequence followed by a thioredoxin (TrxA) fusion with a protease inhibitor. M) The M13 pIII signal sequence followed by a thioredoxin (TrxA) fusion with a protease inhibitor. N) An autotransporter cassette consisting of an autotransporter signal peptide, a thioredoxin (TrxA) fusion and a protease inhibitor (passengers) followed by the autotransporter linker and β-barrel. O) A pINIIIompA leader with a thioredoxin (TrxA) fusion with a protease inhibitor. P) A colicin N-terminal domain with a thioredoxin (TrxA) fusion with a protease inhibitor. Q) F) A colicin lysis protein that may be co-expressed in trans with any of the above.

FIG. 3. Chimeric phage pIII protein colicins. A) An M13 pIII signal sequence with amino acids 1 to 18 followed by a targeting peptide (TGF-alpha), a membrane anchor truncated M13 pIII amino acids 19 to 372 and the C-terminus of ColE3 (ribonuclease). The colicin is secreted, the signal sequence cleaved and the targeting peptide targets the EGFR-expressing cancer cell. B) A lytic peptide is added between the signal sequence and the targeting peptide. Following cleavage of the signal sequence, the targeting peptide localizes to the EFGF-expressing cancer cell and the lytic peptide assists in its release from the endosome. C) A ColE7 (DNAase) chimera. Optionally, a NLS, preferably from apoptin, may be added at the C-terminus. D) A ColE7 chimera with a lytic peptide. Optionally, a NLS, preferably from apoptin, may be added at the C-terminus. E) A Col-Ia (membrane channel forming peptide) chimera. F) A Col-Ia chimera with a lytic peptide.

FIG. 4. Colicin TRC fusions. Colicin TRC fusions utilize the entire colicin with its three domains, T (translocation), R (receptor), and C (catalytic), and fuse active moieties to the C-terminal catalytic domain. A) TRC of a colicin, such as colE3, and a targeting domain, such as TGF-alpha. B) TRC of a colicin, such as colE3, a lytic peptide such as PSM-alpha-1, and a targeting domain, such as TGF-alpha. The lytic peptide may be engineered to have protease cleavage sites, such as those from cathepsin, that effect its release and aid in escape from an endosome. C) TRC of a DNAase colicin, such as colE9 where it is desirable to direct the DNAase activity to the tumor, a nuclear localization domain (NLS), preferably that of Apoptin, and a targeting domain such as a CD22 binding peptide. The CD22 peptide is disulfide bonded (S—S) loop. Alternatively, a peptide library such as are used in phage display, including those using disulfide bonding may be used. Such libraries are may be first selected using phage, or may alternatively first be selected by screening of colicins for target cell specificity and then transferred to the colicin receptor region. In addition, a library of the “tol box” penta peptide (DGSGW SEQ ID NO:133) variations and/or extended tolB box (DGSGWSSENNPWGGGSGSIHW SEQ ID NO:134; Hands et al., 2005, Interactions of TolB with the translocation domain of colicin E9 require and extended tolB box, J Bacteriol. 187: 6733-6741) variations may be screened alone or in combination with individual receptor peptides such as the CD22 binding peptide, or a library of receptor peptides and a library of tol box or extended tol box peptides may be screened in combination. D) TRC of a DNAase colicin, such as colE9 where it is desirable to direct the DNAase activity to the tumor, a nuclear localization domain (NLS), preferably that of Apoptin, a lytic peptide such as PSM-alpha-1, and a targeting domain, such as a CD22 binding peptide. The lytic peptide may be engineered to have protease cleavage sites, such as those from cathepsin, that affects its release and aids in escape of the colicin chimera from an endosome. E) TRC of colicin E3, a ribonuclease colicin active in the cytoplasm, where a targeting peptide, such as CD22 binding peptide, is inserted into the targeting domain, between amino acids 374 and 391. F) TRC of colicin E3, a ribonuclease colicin active in the cytoplasm, where a targeting peptide, such as CD22 binding peptide, is inserted into the targeting domain between amino acids 374 and 391 and a lytic peptide is engineered in-frame. The lytic peptide may be engineered to have a protease cleavage site, such as those from a cathepsin, that affects its release and aids in escape of the colicin chimera from an endosome. G) TRC of colicin E9, a DNAase colicin active in the nucleus, where a targeting peptide, such as CD22 binding peptide, is inserted into the targeting domain. H) TRC of colicin E9, a DNAase colicin active in the nucleus, where a targeting peptide, such as CD22 binding peptide, is inserted into the targeting domain. and a lytic peptide is engineered in-frame. The lytic peptide may be engineered to have a protease cleavage site, such as those from a cathepsin, that affects its release and aids in escape of the colicin chimera from an endosome.

FIG. 5. Lytic effector peptide chimeras. A) A lytic peptide followed by the hlyA signal sequence. B) A lytic peptide, targeting peptide (TGF-alpha), hlyA signal peptide chimera. C) The M13 pIII signal sequence followed by a lytic peptide, the membrane anchor truncated M13 pIII amino acids 19 to 372 and a targeting peptide (TGF-alpha). D) The M13 pIII signal sequence followed by a lytic peptide and a targeting peptide (TGF-alpha).

FIG. 6. Protease activated lytic peptide chimera prodrugs (for which the active portion is not sensitive and for which a protease inhibitor is not being co-expressed). A) A blocking peptide followed by a tumor protease cleavage site, a lytic peptide followed by the hlyA signal sequence. The bracket underneath shows the active portion of the chimera following proteolytic cleavage. B) A blocking peptide followed by a tumor protease cleavage site, a lytic peptide, targeting peptide (TGF-alpha) followed by a second tumor protease cleavage site and the hlyA signal peptide. C) The M13 pIII signal sequence followed by a blocking peptide with a tumor protease cleavage site, a lytic peptide, the membrane anchor truncated M13 pIII amino acids 19 to 372 and a targeting peptide (TGF-alpha). D) The M13 pIII signal sequence followed by a blocking peptide with a tumor protease cleavage site, a lytic peptide, a targeting peptide (TGF-alpha) with a tumor protease cleavage site and the membrane anchor truncated M13 pIII amino acids 19 to 372.

FIG. 7. Proapoptotic and cytotoxic peptide fusions. A) Proapoptotic (“apop”) peptides, such as BH3 peptide, BAX, BIM, BAD, p53 peptide, or apoptin are engineered with signal sequence such as that from ZirT and a ferry peptide, such as the HIV TAT peptide. The chimeras may be expressed together with a release factor such as a colicin lysis protein. B) Proapoptitic (apop) peptides, such as BH3 peptide, BAX, BIM, BAD, p53 peptide, or apoptin) are engineered with an N-terminal signal sequence (e.g., ZirT) and a C-terminal targeting peptide such as TGF-alpha. May be expressed together with a release factor such as a colicin lysis protein. C) Proapoptitic (apop) peptides, such as BH3 peptide, BAX, BIM, BAD, p53 peptide, or apoptin) are engineered with an N-terminal signal sequence, a lytic peptide and a targeting peptide such as TGF-alpha. The lytic peptide may be engineered to have protease cleavage sites, such as those from a cathepsin, that affects its release and aids in escape of the apoptotic peptide chimera from an endosome. May be expressed together with a release factor such as a colicin lysis protein. D) An N-terminal ferry peptide such as HIV TAT peptide is engineered in-frame with an apoptotic peptide with a C-terminal signal sequence from an RTX toxin such as HlyA. E) A targeting peptide such as TGF-alpha engineered in-frame with an apoptotic peptide and a C-terminal signal sequence from an RTX toxin such as HlyA. F) A targeting peptide such as TGF-alpha engineered in-frame with a lytic peptide and an apoptotic peptide with a C-terminal signal sequence from an RTX toxin such as HlyA. The lytic peptide may be engineered to have protease cleavage sites, such as those from a cathepsin, that affects its release and aids in escape of the colicin chimera from an endosome. G) An N-terminal signal sequence, such as that from ZirT, a toxic peptide (e.g., ricin, soporin), and a targeting peptide such as TGF-alpha. H) An N-terminal signal sequence, such as that from ZirT, a toxic peptide (e.g., ricin, soporin), a lytic peptide and a targeting peptide such as TGF-alpha. The lytic peptide may be engineered to have protease cleavage sites, such as those from a cathepsin, that affects its release and aids in escape of the colicin chimera from an endosome. I) A targeting peptide such as TGF-alpha, a toxic peptide, and the hlyA C-terminal signal sequence. J) A targeting peptide such as TGF-alpha, a lytic peptide, a toxic peptide, and the hlyA C-terminal signal sequence. The lytic peptide may be engineered to have protease cleavage sites, such as those from a cathepsin, that affects its release and aids in escape of the colicin chimera from an endosome.

FIG. 8. Cytolethal distending toxin subunit B (cldtB) chimeras. It is understood that full functionality requires cltdA and cltdC. A) CldtB followed by apoptin 1 to 121. B) CldtB followed by apoptin 33 to 121. C) CldtB followed by apoptin 33-46. D) CldtB followed by apoptin 81-121.

FIG. 9. Repeat in toxin (RTX) family members and hybrid operons. A) HlyCABD from E. coli. B) LtxCABD from Actinobacillus. C) A hybrid CABD of E coli (HlyBD) and Actinobacillus (HlyCA). D) A hybid ltxCA with E. coli BD where the ltxA contains the C-terminal 60 amino acids of HlyA.

FIG. 10. Non-chimeric effector proteins which may be optionally expressed as secretion fusions in combination with protease inhibitors. A) An inducible promoter (e.g., TET) which drives the expression of a metabolic degrading enzyme such as thiaminase, methionase (methioninase L-methionine γ-lyase) or asparaginase. B) An inducible promoter (e.g., TET) which drives the expression of a metabolic enzyme such as thiaminase (or methionase or asparaginase) expressed as a HlyA fusion. C) An inducible promoter (e.g., TET) which drives the expression of a prodrug converting enzyme (e.g., carboxyesterase; CE). D) An inducible promoter (e.g., TET) which drives the expression of a prodrug converting enzyme (e.g., carboxyesterase; CE) expressed as a HlyA fusion. E) An inducible promoter (e.g., TET) which drives the expression of an antibody degrading autotransporter (IgA protease from Neisseria).

FIG. 11. A non-conjugative bacterium with and without the F′ factor. The bacterial chromosome contains a secreted protease inhibitor construct (PI) that results in a secreted protease inhibitor. The chromosome also contains the FinO and FinP genes in order to inhibit conjugation. When present, the F′ factor containing the pilus genes with deletions relating to conjugation in traD and the mating stabilization (MS) results in a pilus expressed by the bacterium.

FIG. 12. Segregation of colicin toxin and required immunity factor(s). The bacterial chromosome has a colicin immunity protein integrated into a neutral sight (e.g., attenuating mutation or IS200 element). The colicin, or colicin hybrid is not linked to the immunity protein, but is distal to it. Other therapeutic molecules may be in the same proximity, such as in a polycistronic organization. Based on this organization, a random DNA fragment, or a portion of the genome packaged by a transducing phage, could not contain the immunity protein. If such a fragment were transferred to another bacterium, expression of the colicin without the immunity protein would kill the cell. A second colicin set, with another distal colicin immunity protein and a corresponding colicin on the other flanking side of the effector system may also be used.

8. EXAMPLES

In order to more fully illustrate the invention, the following examples are provided.

8.1. Example: Methods for Obtaining Bacterial Strains with Suitable Genetic Backgrounds

A first step in selection of an appropriate strain based upon the known species specificity (e.g, S. typhi is human specific and S. typhimurium has broad species specificity including humans, birds, pigs and many other vertebrates). Thus, if the target species for treatment were limited to humans, S. typhi would be appropriate. If more species are desired to be treated including humans, cats, dogs, horses and many other vertebrates, then other serotypes may be used. For example, S. typhimurium and S. montevideo which have non-overlapping O-antigen presentation (e.g., S. typhimurium is O-1, 4, 5, 12 and S. typhi is Vi, S. montevideo is O-6, 7) are representative examples. Methods to genetically alter the serotype within a single strain are known to those skilled in the arts, including Favre et al., 1997 WO 97/14782 Methods for delivering heterologous 0-antigens; and Roland, 2000, WO/2000/004919). Thus, S. typhimurium is a suitable serotype for a prime/boost strategy where S. typhimurium is either the primary vaccine, or the booster vaccine where the primary vaccine is another serotype such as S. typhi or S. montevideo. Furthermore, both S. typhimurium and S. montevideo are suitable for humans, cats, dogs, or horses. A second step follows serotype selection where the first genetic mutation is introduced which may involve the use of antibiotic resistance markers and where any antibiotic resistance makers are then eliminated, followed by a third step where a second genetic mutation is introduced which may involve the use of antibiotic resistance markers and where any antibiotic resistance makers are then also eliminated. Reiteration of genetic deletion and antibiotic marker elimination can be used to supply additional mutations. Methods for reiterative chromosomal deletion and elimination of antibiotic resistance markers are known to those skilled in the arts, including Tn10 transposon deletion followed by “Bochner” selection (Bochner et al., 1980, J Bacteriol. 143: 926-933) for elimination of the tetracycline antibiotic resistance marker, lamda red recombinase deletion followed by flip recombinase elimination of the antibiotic resistance marker (Lesic and Rahme, 2008, BMC Molecular Biology 9:20), and suicide vectors such as those containing sucrase gene (e.g., pCVD442, Donnenberg and Kaper, 1991 Infect Immun 59: 4310-4317). Spontaneous mutations may also be rapidly and accurately selected for, such as the “Suwwan”, a large IS200-mediated deletion (Murray et al., 2004, Journal of Bacteriology, 186: 8516-8523). Thus, the starting strain can be a wild type Salmonella such as ATCC 14028, and the Suwwan, IS200 deletion selected for using chlorate (Murray et al., 2004, Journal of Bacteriology, 186: 8516-8523). A second mutation in msbB can be introduced using pCVD442 as described by Low et al., 2004, Methods Mol Med. 2004; 90:47-60). A third mutation can be generated in zwf as described by Karsten et al., 2009, BMC Microbiol. BMC Microbiol. 2009 Aug. 18; 9:170. Thus, the strain generated has deletions in the Suwwan region, msbB and zwf. In S. montevideo, where the Suwwan mutation is not known to occur, a pCVD442 vector is used to generate the equivalent mutation, together with the same procedures above (altered as necessary for DNA sequence variations in the DNA portions used for homologous recombination), resulting in a pair of strains having the same mutational background together with different bacterial antigens. These strains, alone or used for alternating doses, form a basic platform into which the effector genes and protease inhibitor gene constructs are inserted.

8.2 Example: A Targeted Colicin E3 (colE3) Chimera

Chimeric cytotoxins are generated using standard molecular genetic techniques, including synthetic biology (e.g., chemically synthesized oligonucleotides annealed into larger constructs forming entire genes based on the nucleic acid and/or amino acid sequence selected, including codon optimization) and expressed in bacteria using methods known to those skilled in the arts, operably linking a promoter, ribosomal binding site and initiating methionine if not provided by the first portion of the construct. The upstream and downstream regions may contain a transcriptional termination signal (terminator). The construct may be inserted into an exogenous plasmid or a chromosomal or virulence (VIR; pSLT) plasmid integration vector, for which many different integration sites exist, including but not limited to any of the attenuation mutations or any defective (incomplete) phage elements, intergenic regions or the IS200 elements. The constructs may also be polycistronic, having multiple genes and/or gene products separated by ribosomal binding sites.

The colicin colE3 immunity protein is first synthesized as an expression cassette and cloned into a chromosomal localization vector for an integration site distal to the that of the intended site for the chimeric effector gene vector (FIG. 12) as described below, e.g., an IS200 deletion vector at location. The amino acid sequence of the immunity protein is given as:

SEQ ID NO: 129

MGLKLDLTWFDKSTEDFKGEEYSKDFGDDGSVMESLGVPFKDNVNNGCFD

VIAEWVPLLQPYFNHQIDISDNEYFVSFDYRDGDW

The sequence is reverse translated using codons optimal for Salmonella. The entire chimeric effector protein and expression cassette components are synthesized using standard DNA synthesis techniques at a contract DNA synthesis facility and integrated into the chromosome (Donnenberg and Kaper, 1991 Infect Immun 59: 4310-4317, Low et al., 2004, Methods in Molecular Medicine 90: 47-60, each of which is expressly incorporated herein by reference). The recipient stain can be any tumor-targeted bacterium.

This example of a chimeric colicin follows the pattern shown in FIG. 3A. This chimera is targeted to cancer cells over-expressing EGFR via a TGF-alpha ligand. The chimera consists of the M13 filimenous phage pIII protein 18 amino acid signal sequence, followed by the natural alanine and a 3 glycine spacer. The spacer is followed by the mature 50 amino acid peptide for TGF-alpha, the remaining pIII protein truncated after amino acid 372 of pIII, followed by the enzymatically active (ribonuclease) C-terminus of colicin E3, followed by a stop signal. The complete amino acid sequence is:

SEQ ID NO: 130

MKKLLFAIPLVVPFYSHSAGGGVVSHFNDCPDSHTQFCFHGTCRFLVQED

KPACVCHSGYVGARCEHADLLAAETVESCLAKSHTENSFTNVWKDDKTLD

RYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSE

GGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEES

QPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYD

AYWNGKFRDCAFHSGFNEDLFVCEYQGQSSDLPQPPVNAGGGSGGGSGGG

SEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADEN

ALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQ

VGDGDNSPLMNNFRQYLPSLPQSVECRFAHDPMAGGHRMWQMAGLKAQRA

QTDVNNKQAAFDAAAKEKSDADAALSSAMESRKKKEDKKRSAENNLNDEK

NKPRKGFKDYGHDYHPAPKTENIKGLGDLKPGIPKTPKQNGGGKRKRWTG

DKGRKIYEWDSQHGELEGYRASDGQHLGSFDPKTGNQLKGPDPKRNIKKY

L*

The entire chimeric effector protein and expression cassette components are synthesized using standard DNA synthesis techniques, for example, at a contract DNA synthesis facility, and cloned into a chromosomal localization vector, e.g., an IS200 deletion vector, and integrated into the chromosome (Donnenberg and Kaper, 1991, Low et al., 2003, each of which is expressly incorporated herein by reference).

8.3 Example: A Targeted Colicin Chimera Containing a Lytic Peptide Resulting in Endosomal Release and/or Increased Anti-Cancer Cell Cytotoxicity

The lytic peptide PSM-alpha-3 is inserted between the pIII signal sequence and the TGF-alpha (FIG. 3B). The complete sequence of the construct is as follows:

SEQ ID NO: 131

MKKLLFAIPLVVPFYSHSAMEFVAKLFKFFKDLLGKFLGNNVVSHFNDCP

DSHTQFCFHGTCRFLVQEDKPACVCHSGYVGARCEHADLLAAETVESCLA

KSHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYGTWVP

IGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGT

YPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGT

DPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDLFVCEYQGQSSD

LPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFD

YEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGL

ANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRFAHD

PMAGGHRMWQMAGLKAQRAQTDVNNKQAAFDAAAKEKSDADAALSSAMES

RKKKEDKKRSAENNLNDEKNKPRKGFKDYGHDYHPAPKTENIKGLGDLKP

GIPKTPKQNGGGKRKRWTGDKGRKIYEWDSQHGELEGYRASDGQHLGSFD

PKTGNQLKGPDPKRNIKKYL

8.4 Example: A Chimeric Colicin E7

As for the other colicin E3 constructs, the colicin colE7 immunity protein is synthesized as an expression cassette and cloned into a chromosomal localization vector for an integration site distal to the that of the chimeric effector gene vector described below, e.g., an IS200 deletion vector at location.

The genetic construct of the first colicin E7 chimera follows the same pattern as shown in FIG. 3A, except that the ColE3 C-terminus is replaced with the colE7 (a DNAase) C-terminus comprising amino acids 444 to 576 (FIG. 3 C). Nuclear localization signals (NLSs) may also be added, including but not limited to that from herpes simplex virus thymidine kinase, the SV40 large T antigen (PPKKKRKV SEQ ID NO:1) monopartite NLS, or the nucleoplamin bipartite NLS (KR[PAATKKAGQA]KKKK SEQ ID NO:2, or more preferably, the NLS from apoptin, a tumor associated (tumor-selective) NLS. The tumor-selective nuclear export signal from apoptin may be used alone or together with NLS from apoptin.

The genetic construct of the second colicin E7 chimera follows the same pattern as shown in FIG. 3C, except that the lysis peptide is inserted between the M13pIII signal sequence and the targeting peptide (TGF-alpha) (FIG. 3D). Nuclear localization signals (NLSs) may also be added, including but not limited to that from herpes simplex virus thymidine kinase, the SV40 large T antigen (PPKKKRKV SEQ ID NO:1) monopartite NLS, or the nucleoplamin bipartite NLS (KR[PAATKKAGQA]KKKK SEQ ID NO:2, or more preferably, the NLS from apoptin, a tumor associated (tumor-selective) NLS. The tumor-selective nuclear export signal from apoptin may be used alone or together with NLS from apoptin.

8.5 Example: A Chimeric Colicin Ia

As for the other colicin E3 constructs, the colicin Ia immunity protein is synthesized as an expression cassette and cloned into a chromosomal localization vector for an integration site distal to the that of the chimeric effector gene vector described below, e.g., an IS200 deletion vector at location.

The genetic construct of the first colicin Ia chimera follows the same pattern as shown in FIG. 3A, except that the ColE3 C-terminus is replaced with the Ia (pore forming) C-terminus comprising amino acids 450 to 626 (FIG. 3 E).

The genetic construct of the second colicin Ia chimera follows the same pattern as shown in FIG. 3B, except that the lysis peptide is inserted between the M13pIII signal sequence and the targeting peptide (TGF-alpha) (FIG. 3F).

8.6 Example: Colicin TRC Fusions

Colicin TRC fusions utilize the entire colicin with its three domains, T (translocation), R (receptor), and C (catalytic), and fuse active moieties to the C-terminal catalytic domain (FIG. 4). A) TRC of a colicin, such as colE3, and a targeting domain, such as TGF-alpha. B) TRC of a colicin, such as colE3, a lytic peptide such as PSM-alpha-1, and a targeting domain, such as TGF-alpha. The lytic peptide may be engineered to have protease cleavage sites, such as those from cathepsin, that effect its release and aid in escape from an endosome. C) TRC of a DNAase colicin, such as colE9 where it is desirable to direct the DNAase activity to the tumor, a nuclear localization domain (NLS), preferably that of Apoptin, and a targeting domain such as a CD22 binding peptide. The CD22 peptide is disulfide bonded (S—S) loop. Alternatively, a peptide library such as are used in phage display, including those using disulfide bonding may be used. Such libraries are may be first selected using phage, or may alternatively first be selected by screening of colicins for target cell specificity and then transferred to the colicin receptor region. In addition, a library of the “tol box” penta peptide (DGSGW SEQ ID NO:133) variations and/or extended tolB box (DGSGWSSENNPWGGGSGSIHW SEQ ID NO:134; Hands et al., 2005, Interactions of TolB with the translocation domain of colicin E9 require and extended tolB box, J Bacteriol. 187: 6733-6741) variations may be screened alone or in combination with individual receptor peptides such as the CD22 binding peptide, or a library of receptor peptides and a library of tol box or extended tol box peptides may be screened in combination. D) TRC of a DNAase colicin, such as colE9 where it is desirable to direct the DNAase activity to the tumor, a nuclear localization domain (NLS), preferably that of Apoptin, a lytic peptide such as PSM-alpha-1, and a targeting domain, such as a CD22 binding peptide. The lytic peptide may be engineered to have protease cleavage sites, such as those from cathepsin, that affects its release and aids in escape of the colicin chimera from an endosome. E) TRC of colicin E3, a ribonuclease colicin active in the cytoplasm, where a targeting peptide, such as CD22 binding peptide, is inserted into the targeting domain, between amino acids 374 and 391. F) TRC of colicin E3, a ribonuclease colicin active in the cytoplasm, where a targeting peptide, such as CD22 binding peptide, is inserted into the targeting domain between amino acids 374 and 391 and a lytic peptide is engineered in-frame. The lytic peptide may be engineered to have a protease cleavage site, such as those from a cathepsin, that affects its release and aids in escape of the colicin chimera from an endosome. G) TRC of colicin E9, a DNAase colicin active in the nucleus, where a targeting peptide, such as CD22 binding peptide, is inserted into the targeting domain. H) TRC of colicin E9, a DNAase colicin active in the nucleus, where a targeting peptide, such as CD22 binding peptide, is inserted into the targeting domain. and a lytic peptide is engineered in-frame. The lytic peptide may be engineered to have a protease cleavage site, such as those from a cathepsin, that affects its release and aids in escape of the colicin chimera from an endosome.

8.7. Example: Selecting Protease Inhibitors

Protease inhibitors are generated using knowledge of the predicted proteolytic cleavage of the effector molecule (e.g., ProP 1.0, Duckert et al., 2004, Prediction of proprotein convertase cleavage sites, Protein Engineering Design and Selection 17: 107-122; ExPASy PeptideCutter tool, Gasteiger et al. Protein Identification and Analysis Tools on the ExPASy Server, In: John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press, 2005), and may be used to test the predicted proteolytic sensitivity of the effector molecule. Using the colicin lytic peptide TGF fusion described above, the Duckert et al., method predicts a furin cleavage at amino acid 509. Thus, since cleavage of the effector molecule has the potential to occur, furin represent a protease target for which inhibition could improve the effectiveness or activity of a co-expressed molecule by inhibiting its destruction by proteolytic degradation, whereas Factor Xa is identified by ProP as a cleavage site that is not present, does not need to be inhibited, and who's cleavage recognition site could be added between protein domains where removal of a domain by proteolysis is desirable.

8.8. Example: Secreted Protease Inhibitors

Secreted protease inhibitors are generated using standard molecular genetic techniques and expressed in bacteria using methods known to those skilled in the arts, operably linking a promoter, ribosomal binding site and initiating methionine if not provided by the first portion of the construct. The construct may either be a plasmid or a chromosomal virulence (VIR) plasmid integration vector, for which many different integration sites exist, including but not limited to any of the attenuation mutations, intergenic regions or any of the IS200 elements. The constructs may also be polycistronic, having multiple genes and/or gene products separated by ribosomal binding sites. The different forms of the protease inhibitor constructs are shown in FIG. 2. The constructs used have multiple forms, for example:

1) An N-terminal signal sequence, such as that from M13pIII MKKLLFAIPLVVPFYSHS SEQ ID NO:135, followed by a protease inhibitor such as the furin inhibitor GKRPRAKRA SEQ ID NO:11;

2) a protease inhibitor such as the furin inhibitor GKRPRAKRA SEQ ID NO:11 followed by the C-terminal signal sequence of hlyA STYGSQDYLNPLINEISKIISAAGNLDVKEERSAASLLQLSGNASDFSYGRNSITLTASA SEQ ID NO:136, or

3) a protease inhibitor such as the furin inhibitor GKRPRAKRA SEQ ID NO:11, followed by a furin cleavage signal RXKR↓SX SEQ ID NO:137 followed by the C-terminal signal sequence of hlyA STYGSQDYLNPLINEISKIISAAGNLDVKEERSAASLLQLSGNASDFSYGRNSITLTASA SEQ ID NO:138

8.9 Example 6: Expression of a C-Terminal Amidating Enzyme Required to Post-Translationally Modify Gastrin and Bombesin Targeting Peptides

A C-terminal amidating enzyme composition known form serum or plasma which comprises a C-terminal amidating enzyme capable of amidating a C-terminal glycine which amidates the carboxy terminus of the C-terminal glycine of a peptide terminating in Gly-Gly. The enzyme participating in such amidation is called peptidylglycine-α-amidating monoxygenase (C-terminal amidating enzyme) (EC.1.14.17.3) (Bradbury et al, Nature, 298, 686, 1982: Glembotski et al, J. Biol, Chem., 259, 6385, 1984; and U.S. Pat. No. 5,354,675, expressly incorporated herein by reference), is considered to catalyze the following reaction:

—CHCONHCH₂COOH—CHCONH₂+glyoxylic acid

- is produced by the recombinant.

8.10 Example: Expression of Antitumor Lytic Peptides

Examples of antitumor lytic peptides are shown in FIG. 5. It is understood that those peptides utilizing the hlyA signal sequence requires hlyBD in trans together with a functional tolC. The lytic peptide constructs consist of A) lytic peptide joined to the HlyA signal sequence, B) lytic peptide, targeting peptide, signals sequence, C) M13 pIII signal sequence, lytic peptide, M13 pIII amino acids 19 to 372, targeting peptide, D) M13 signal sequence, lytic peptide, targeting peptide, M13 pIII amino acids 19 to 372.

8.11 Example: Expression of Antitumor Lytic Peptide Prodrugs

Examples of antitumor lytic peptide prodrugs are shown in FIG. 6. It is understood that those peptides utilizing the hlyA signal sequence requires hlyBD in trans together with a functional tolC. The lytic peptide prodrug constructs consist of A) a neutral (e.g., beta sheet) blocking peptide of 50 amino acids, a protease cleavage site shown by downward arrow (for a protease not being blocked by a protease inhibitor), a lytic peptide, and the hlyA signal sequence, which may contain the same protease cleavage site shown by a downward arrow, B) a neutral (e.g., beta sheet) blocking peptide of 50 amino acids, a lytic peptide, a targeting peptide (e.g., TGF-alpha), a protease cleavage site shown by downward arrow (for a protease not being blocked by a protease inhibitor), and the hlyA signal sequence, which may contain the same protease cleavage site shown by a downward arrow, C) the M13 pIII signal sequence, a blocking peptide, a protease cleavage sequence, a lytic peptide, M13 pIII amino acids 19 to 372, and a targeting peptide (e.g., TGF-alpha), and D) the M13 pIII signal sequence, a blocking peptide, a protease cleavage sequence, a lytic peptide, a targeting peptide (e.g., TGF-alpha), and M13 pIII amino acids 19 to 372.

8.12 Example: Cytolethal Distending Toxin cltdB Fusion with Apoptin (FIG. 6)

A cytolethal distending toxin subunit B with tumor-specific nuclear localization and normal cell nuclear export is generated by a fusion with apoptin containing a five glycine linker in between (FIG. 6A). The complete sequence of the construct is as follows:

SEQ ID NO: 139

MKKYIISLIVFLSFYAQADLTDFRVATWNLQGASATTESKWNINVRQLIS

GENAVDILAVQEAGSPPSTAVDTGTLIPSPGIPVRELIWNLSTNSRPQQV

YIYFSAVDALGGRVNLALVSNRRADEVFVLSPVRQGGRPLLGIRIGNDAF

FTAHAIAMRNNDAPALVEEVYNFFRDSRDPVHQALNWMILGDFNREPADL

EMNLTVPVRRASEIISPAAATQTSQRTLDYAVAGNSVAFRPSPLQAGIVY

GARRTQISSDHFPVGVSRRGGGGGMNALQEDTPPGPSTVFRPPTSSRPLE

TPHCREIRIGIAGITITLSLCGCANARAPTLRSATADNSESTGFKNVPDL

RTDQPKPPSKKRSCDPSEYRVSELKESLITTTPSRPRTAKRRIRL

8.13 Example: Cytolethal Distending Toxin cltdB Fusion with a Truncated Apoptin

A cytolethal distending toxin subunit B with tumor-specific nuclear localization and normal cell nuclear export is generated by a fusion with a truncated apoptin amino acids 33 to 121 containing a five glycine linker in between (FIG. 6B). The complete sequence of the construct is as follows:

SEQ ID NO: 140

MKKYIISLIVFLSFYAQADLTDFRVATWNLQGASATTESKWNINVRQLIS

GENAVDILAVQEAGSPPSTAVDTGTLIPSPGIPVRELIWNLSTNSRPQQV

YIYFSAVDALGGRVNLALVSNRRADEVFVLSPVRQGGRPLLGIRIGNDAF

FTAHAIAMRNNDAPALVEEVYNFFRDSRDPVHQALNWMILGDFNREPADL

EMNLTVPVRRASEIISPAAATQTSQRTLDYAVAGNSVAFRPSPLQAGIVY

GARRTQISSDHFPVGVSRRGGGGGITPHCREIRIGIAGITITLSLCGCAN

ARAPTLRSATADNSESTGFKNVPDLRTDQPKPPSKKRSCDPSEYRVSELK

ESLITTTPSRPRTAKRRIRL

8.14 Example: Cytolethal Distending Toxin cltdB Fusion with a Truncated Apoptin

A cytolethal distending toxin subunit B with tumor-specific nuclear retention signal is generated by a fusion with a truncated apoptin amino acids 33 to 46 containing a five glycine linker in between (FIG. 6C). The complete sequence of the construct is as follows:

SEQ ID NO: 141

MKKYIISLIVFLSFYAQADLTDFRVATWNLQGASATTESKWNINVRQLIS

GENAVDILAVQEAGSPPSTAVDTGTLIPSPGIPVRELIWNLSTNSRPQQV

YIYFSAVDALGGRVNLALVSNRRADEVFVLSPVRQGGRPLLGIRIGNDAF

FTAHAIAMRNNDAPALVEEVYNFFRDSRDPVHQALNWMILGDFNREPADL

EMNLTVPVRRASEIISPAAATQTSQRTLDYAVAGNSVAFRPSPLQAGIVY

GARRTQISSDHFPVGVSRRGGGGGIRIGIAGITITLSL

8.15 Example: Cytolethal Distending Toxin cltdB Fusion with a Truncated Apoptin

A cytolethal distending toxin subunit B with a normal cell nuclear export signal is generated by a fusion with a truncated apoptin amino acids 81 to 121 containing a five glycine linker in between (FIG. 6D). The complete sequence of the construct is as follows:

SEQ ID NO: 142

MKKYIISLIVFLSFYAQADLTDFRVATWNLQGASATTESKWNINVRQLIS

GENAVDILAVQEAGSPPSTAVDTGTLIPSPGIPVRELIWNLSTNSRPQQV

YIYFSAVDALGGRVNLALVSNRRADEVFVLSPVRQGGRPLLGIRIGNDAF

FTAHAIAMRNNDAPALVEEVYNFFRDSRDPVHQALNWMILGDFNREPADL

EMNLTVPVRRASEIISPAAATQTSQRTLDYAVAGNSVAFRPSPLQAGIVY

GARRTQISSDHFPVGVSRRGGGGGTDQPKPPSKKRSCDPSEYRVSELKES

LITTTPSRPRTAKRRIRL

8.16 Example: Exchange of the Variable Loop in cldtB to Enhance Activity

The amino acid sequence FRDSRDPVHQAL SEQ ID NO:143 which is associated with dimerization and inactivation can be exchanged for the loop NSSSSPPERRVY SEQ ID NO:144 from Haemophilus which is associated with stabile retention of cytotoxicty.

8.17 Example: Expression of Repeat in Toxin (RTX) Family Members

RTX family members, including E. coli hemolysin operon hlyCABD and Actinobacillus actinomycetemcomitans leucotoxin ltxCABD are expressed in coordination with protease inhibitors as shown in FIG. 7. E coli hemolysin operon hlyCABD is expressed as a non-chimera (FIG. 7A). Actinobacillus actinomycetemcomitans leucotoxin ltxCABD operon is expressed as either a non-hybrid (FIG. 7B) or as a hybrid (FIG. 7C). It is understood that a functional tolC gene is required in the gram-negative bacterial strain for functional expression of each of these operons, or that homologs such as prtF and/or cyaE may be used.

8.18 Example: A Group B Streptococcus Expressing a Vascular-Targeted Lytic Peptide

A low pathogenicity clyE⁻ group B Streptococcus expressing a gram positive secretion signal from alkaline phosphatase (Lee et al., 1999 J. Bacteriol, 181: 5790-5799) in frame with the vascular targeting peptide F3 CKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK SEQ ID NO:145 in frame with the lytic peptide PSM-α-3.

8.19 Example: A Group B Streptococcus Expressing a Vascular-Targeted Toxin

A low pathogenicity clyE⁻ group B Streptococcus expressing a gram positive secretion signal from alkaline phosphatase (Lee et al., 1999 J. Bacteriol, 181: 5790-5799) in frame with saporin and the vascular targeting peptide F3 CKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK SEQ ID NO: 146 (FIG. 7).

It will be understood that the foregoing is only illustrative of the principles of the invention, and that various modifications can be made by those skilled in the art without departing from the scope and spirit of the invention.

Claims

1. A method of treating a human or animal for a neoplasia, in need of treatment, comprising: administering a genetically engineered Salmonella bacterium to a human or animal, that co-expresses a heterologous protease sensitive therapeutic molecule for the neoplasia, which is distinct from a protease inhibitor, and a heterologous protease inhibitor; andpermitting the genetically engineered bacterium to grow within a common target tissue of the human or animal,wherein the heterologous protease sensitive therapeutic molecule is effective to treat the human or animal for the neoplasia of the common target tissue, andthe protease inhibitor is effective to delay degradation of the heterologous protease sensitive therapeutic molecule in the common target tissue.
2. The method according to claim 1, wherein the common target tissue is a solid tumor.
3. The method according to claim 1, wherein the common target tissue is bone marrow.
4. The method according to claim 3, wherein the common target tissue is selected from the group consisting of at least one of leukemia cells, lymphoma cells, and multiple myeloma cells.
5. The method according to claim 1 where the heterologous protease sensitive therapeutic molecule is selected from the group consisting of a chimeric toxin, a cytokine, an antibody, a bispecific antibody, a single chain antibody, a chemokine, and a prodrug converting enzyme.
6. The method according to claim 1, wherein the heterologous protease sensitive therapeutic molecule comprises a toxin selected from the group consisting of cytolethal distending toxin (cldt), cytotoxic nectrotic factor (cnf), dermonecrotic factor (dmf), shiga toxin, shiga-like toxin, colicins, membrane lytic peptides from Staphalococcus, leucotoxin, a leuckotoxin:HlyA hybrid, heat stable enterotoxin from enterobacteriaceae, an autotransporter toxin, chlostridium enterotoxin, aerolysin, typhoid toxin, subtilase, Bordetella adenylate cyclase toxin, pertussus toxin, and porB.
7. The method according to claim 1, wherein the heterologous protease sensitive therapeutic molecule comprises at least one of a cytolethal distending toxin (cldt) and a typhoid toxin fused to a peptide containing a nuclear localization signal from apoptin.
8. The method according to claim 1, wherein the protease inhibitor is secreted from the genetically engineered Salmonella bacterium, effective for achieving a targeting of the heterologous protease sensitive therapeutic or molecule to the common target tissue having the neoplasia, with respect to non-target tissues of the human or animal.
9. The method according to claim 1, wherein the heterologous protease sensitive therapeutic molecule comprises a colicin fused to a targeting peptide.
10. The method according to claim 1, wherein the heterologous protease sensitive therapeutic molecule comprises a targeting peptide effective for targeting cells within the common target tissue having the neoplasia.
11. The method according to the claim 1, wherein the heterologous protease inhibitor is a furin inhibitor selected from one or more of the group consisting of: PAAATVTKKVAKSPKKAKAAKPKKAAKSAAKAVKPK SEQ ID NO:8TKKVAKRPRAKRAA SEQ ID NO:9TKKVAKRPRAKRDL SEQ ID NO:10GKRPRAKRA SEQ ID NO:11CKRPRAKRDL SEQ ID NO:12CVAKRPRAKRDL SEQ ID NO:13CKKVAKRPRAKRDL SEQ ID NO:14RRRRRR SEQ ID NO:15.
12. A method of treating a disease selected from one or more of the group consisting of a neoplasia and a cancer, in a human or mammal, comprising administering a pharmaceutically acceptable formulation to the human or mammal, comprising a Salmonella genetically engineered live bacterium that co-expresses:(a) a heterologous protease sensitive therapeutic molecule which is distinct from a protease inhibitor, and(b) a heterologous protease inhibitor,within a target tissue after administration to the human or mammal, andpermitting the Salmonella genetically engineered live bacterium to replicate within the target tissue of the human or mammal associated with the disease selected from one or more of the group consisting of the neoplasia and the cancer which produces a protease effective for degrading the heterologous protease sensitive therapeutic molecule in an absence of an effective amount of a protease inhibitor, to produce in situ the heterologous protease sensitive therapeutic molecule in the effective amount and the heterologous protease inhibitor.
13. The method according to claim 12, wherein the heterologous protease sensitive therapeutic molecule has antineoplastic effects, and the target tissue comprises a tumor.
14. The method according to claim 12, wherein the heterologous protease sensitive therapeutic molecule is selected from the group consisting of a chimeric toxin, cytokine, antibody, bispecific antibody, single chain antibody, chemokine, and prodrug converting enzyme.
15. The method according to claim 12, wherein the heterologous protease sensitive therapeutic molecule is a chimeric toxin formed of at least a toxin moiety and a secretion peptide.
16. The method engineered bacterium according to claim 12, wherein the heterologous protease sensitive therapeutic molecule is colicin col-Ia or colE7 in which targeting and translocation sequences are replaced with an M13 pIII signal sequence fused to a targeting peptide, followed by M13, and a membrane anchor-truncated M13 pIII, fused to the C-terminal catalytic portion of col-Ia or colE7, respectively, the targeting peptide is the mature form of TGF-alpha, and a Staphalococcus lytic peptide is inserted between the M13 pIII signal sequence and the targeting peptide.
17. A method of treating a human having a pathological tissue comprising a neoplastic tissue, comprising: administering a Salmonella genetically engineered live bacteria to the human; andpermitting the Salmonella genetically engineered live bacteria to selectively replicate within the pathological tissue, and to produce within the pathological tissue both a heterologous protease-sensitive cytotoxic molecule distinct from a protease inhibitor, and a heterologous protease inhibitor,wherein endogenous proteases within the pathological tissue are delayed from degrading the heterologous protease sensitive cytotoxic molecule within the pathological tissue by a presence of a sufficient amount of the heterologous protease inhibitor produced by the Salmonella genetically engineered live bacteria, and the heterologous protease sensitive cytotoxic molecule exerts a cytotoxic effect against the pathological tissue, andwherein endogenous proteases within a tissue surrounding the pathological tissue are exposed to a sufficiently low concentration of the heterologous protease inhibitor produced by the Salmonella genetically engineered live bacteria, to permit degradation of the heterologous protease sensitive cytotoxic molecule produced by the Salmonella genetically engineered live bacteria, to thereby decrease cytotoxicity and increase a selectivity of action of the heterologous protease-sensitive cytotoxic molecule against the pathological tissue with respect to the tissue surrounding the pathological tissue.
18. The method according to claim 17, wherein the heterologous protease sensitive cytotoxic molecule comprises a fusion protein.
19. The method according to claim 17, wherein the Salmonella genetically engineered live bacteria comprises a genetically engineered gene which encodes and causes the Salmonella genetically engineered live bacterium to produce a chimeric peptide comprising: the heterologous protease sensitive cytotoxic molecule,the heterologous protease inhibitor,at least one of a secretion peptide and a targeting peptide, andat least one intervening protease cleavage site distinct from the heterologous protease sensitive cytotoxic molecule, the heterologous protease inhibitor, and the at least one of a secretion peptide and a targeting peptide.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Division of U.S. patent application Ser. No. 14/016,407, filed Sep. 3, 2013, now U.S. Pat. No. 9,068,187, issued Jun. 30, 2015, which is a Division of U.S. patent application Ser. No. 13/024,172, filed Feb. 9, 2011, now U.S. Pat. No. 8,524,220, issued Sep. 3, 2013, which is a non-provisional of U.S. Provisional Patent Application No. 61/302,938, filed Feb. 9, 2010, each of which is expressly incorporated herein by reference in its entirety.

US Referenced Citations (450)

Number	Name	Date	Kind
4436727	Ribi	Mar 1984	A
4906567	Connelly	Mar 1990	A
5021234	Ehrenfeld	Jun 1991	A
D320325	Barfield	Oct 1991	S
5087569	Gabay et al.	Feb 1992	A
5126257	Gabay et al.	Jun 1992	A
5143830	Holland et al.	Sep 1992	A
5223409	Ladner et al.	Jun 1993	A
5278049	Baker et al.	Jan 1994	A
5281530	Sick et al.	Jan 1994	A
5318900	Habuka et al.	Jun 1994	A
5338724	Gabay et al.	Aug 1994	A
5344762	Karapetian	Sep 1994	A
5354675	Iida et al.	Oct 1994	A
5399490	Balganesh et al.	Mar 1995	A
5403484	Ladner et al.	Apr 1995	A
5466463	Ford	Nov 1995	A
5466672	Kushnaryov et al.	Nov 1995	A
5495001	McGrogan et al.	Feb 1996	A
5506139	Loosmore et al.	Apr 1996	A
5569597	Grimsley et al.	Oct 1996	A
5571698	Ladner et al.	Nov 1996	A
5604201	Thomas et al.	Feb 1997	A
5651965	Payne	Jul 1997	A
5656436	Loosmore et al.	Aug 1997	A
5665353	Loosmore et al.	Sep 1997	A
5705151	Dow et al.	Jan 1998	A
5712369	Old et al.	Jan 1998	A
5824538	Branstrom et al.	Oct 1998	A
5830702	Portnoy et al.	Nov 1998	A
5837500	Ladner et al.	Nov 1998	A
5869302	Loosmore et al.	Feb 1999	A
5877159	Powell et al.	Mar 1999	A
5935573	Loosmore et al.	Aug 1999	A
5939297	Loosmore et al.	Aug 1999	A
5945102	de Faire et al.	Aug 1999	A
5958406	de Faire et al.	Sep 1999	A
5962430	Loosmore et al.	Oct 1999	A
5981503	Loosmore et al.	Nov 1999	A
5997881	Powell et al.	Dec 1999	A
6004562	Campagnari	Dec 1999	A
6020183	Loosmore et al.	Feb 2000	A
6022855	Thomas et al.	Feb 2000	A
6025342	Loosmore et al.	Feb 2000	A
6030612	de Faire et al.	Feb 2000	A
6030780	Vinkemeier et al.	Feb 2000	A
6037526	Grimsley et al.	Mar 2000	A
6051237	Paterson	Apr 2000	A
6080849	Bermudes et al.	Jun 2000	A
6114125	Loosmore et al.	Sep 2000	A
6143551	Goebel	Nov 2000	A
6147057	Loosmore et al.	Nov 2000	A
6150170	Powell et al.	Nov 2000	A
6153580	Loosmore et al.	Nov 2000	A
6177083	Lubitz	Jan 2001	B1
6190657	Pawelek et al.	Feb 2001	B1
6207156	Kuchroo et al.	Mar 2001	B1
6232110	Pallas et al.	May 2001	B1
6245892	Oaks et al.	Jun 2001	B1
6251406	Haefliger et al.	Jun 2001	B1
6277379	Oaks et al.	Aug 2001	B1
6348344	Ayal-Hershkovitz et al.	Feb 2002	B1
6355790	Rosenblatt et al.	Mar 2002	B1
6376234	Grimsley et al.	Apr 2002	B1
6410012	Sizemore et al.	Jun 2002	B1
6447777	Terman et al.	Sep 2002	B1
6447784	Bermudes et al.	Sep 2002	B1
6475482	Bermudes et al.	Nov 2002	B1
6475763	Ayal-Hershkovitz et al.	Nov 2002	B1
6537558	Kaniga	Mar 2003	B2
6548287	Powell et al.	Apr 2003	B1
6605286	Steidler et al.	Aug 2003	B2
6605697	Kwon et al.	Aug 2003	B1
6638912	Bhatnagar et al.	Oct 2003	B2
6680374	Oaks et al.	Jan 2004	B2
6685935	Pawelek et al.	Feb 2004	B1
6743893	Engler et al.	Jun 2004	B2
6746671	Steidler et al.	Jun 2004	B2
6841535	Divita et al.	Jan 2005	B2
6863894	Bermudes et al.	Mar 2005	B2
6923972	Bermudes et al.	Aug 2005	B2
6962696	Bermudes et al.	Nov 2005	B1
6979538	Ladner et al.	Dec 2005	B2
7001884	Komiyama et al.	Feb 2006	B2
7033991	Lindberg et al.	Apr 2006	B2
7118879	Ladner et al.	Oct 2006	B2
7208293	Ladner et al.	Apr 2007	B2
7258863	Oaks et al.	Aug 2007	B2
7354592	Bermudes et al.	Apr 2008	B2
7358084	Kolkman	Apr 2008	B2
7390646	Andino-Pavlovsky et al.	Jun 2008	B2
7413877	Collier et al.	Aug 2008	B2
7452531	Bermudes et al.	Nov 2008	B2
7514089	Bermudes et al.	Apr 2009	B2
7569547	Lindberg et al.	Aug 2009	B2
7635682	Denmeade et al.	Dec 2009	B2
7666627	Gal et al.	Feb 2010	B2
7691599	Rubin	Apr 2010	B2
7696173	Collier et al.	Apr 2010	B2
7700349	Romaine et al.	Apr 2010	B2
7700830	Corbin et al.	Apr 2010	B2
7705195	French et al.	Apr 2010	B2
7718618	Gallo et al.	May 2010	B2
7776823	Gallo et al.	Aug 2010	B2
7803918	van der Hoek	Sep 2010	B2
7846678	Pepe et al.	Dec 2010	B2
7850970	Shapiro	Dec 2010	B2
7887794	Luquet et al.	Feb 2011	B2
7888321	Cooper et al.	Feb 2011	B2
7892803	Tanner et al.	Feb 2011	B2
7892825	Barr et al.	Feb 2011	B2
7893007	Ladner et al.	Feb 2011	B2
7943754	Bentwich et al.	May 2011	B2
7947822	Nabel et al.	May 2011	B2
7964362	Lee et al.	Jun 2011	B2
7989202	Mach et al.	Aug 2011	B1
8030447	Motin et al.	Oct 2011	B2
8030542	Corbin et al.	Oct 2011	B2
8062885	Mach et al.	Nov 2011	B2
8101349	Garcia et al.	Jan 2012	B2
8101826	Romano	Jan 2012	B2
8119354	Katanaev	Feb 2012	B2
8128922	Wu et al.	Mar 2012	B2
8153414	Caplan et al.	Apr 2012	B2
8173397	Gal et al.	May 2012	B2
8206700	Horwitz et al.	Jun 2012	B2
8231878	Colonna et al.	Jul 2012	B2
8236315	Lazarides et al.	Aug 2012	B2
8241623	Bermudes	Aug 2012	B1
8244484	Lee et al.	Aug 2012	B2
8246945	Caplan et al.	Aug 2012	B2
8283319	Schulte et al.	Oct 2012	B2
8323961	Nabel et al.	Dec 2012	B2
8349570	Pepe et al.	Jan 2013	B2
8372620	Sibbesen et al.	Feb 2013	B2
8440207	Bermudes	May 2013	B2
8445650	Simpson et al.	May 2013	B2
8507249	Finlay et al.	Aug 2013	B2
8524220	Bermudes	Sep 2013	B1
8603824	Ramseier et al.	Dec 2013	B2
8609358	Sebastian et al.	Dec 2013	B2
8623350	Bermudes	Jan 2014	B1
8628782	Berkower	Jan 2014	B2
8633305	Shapiro	Jan 2014	B2
8647642	Bermudes	Feb 2014	B2
8685392	Helmerhorst et al.	Apr 2014	B2
8686218	Romaine et al.	Apr 2014	B2
8741313	Sable et al.	Jun 2014	B2
8748373	Chai et al.	Jun 2014	B2
8758771	Finlay et al.	Jun 2014	B2
8759086	Mach et al.	Jun 2014	B2
8771669	Bermudes	Jul 2014	B1
8771671	Spencer et al.	Jul 2014	B2
8791237	Paterson et al.	Jul 2014	B2
8795730	Vachon	Aug 2014	B2
8815251	Caplan et al.	Aug 2014	B2
8821893	Dattwyler et al.	Sep 2014	B2
8835107	Van Der Hoek	Sep 2014	B2
8853362	Tissot et al.	Oct 2014	B2
8906662	Nataro et al.	Dec 2014	B2
8920809	Dirienzo	Dec 2014	B2
8926993	Dubensky, Jr. et al.	Jan 2015	B2
8951992	Nathan et al.	Feb 2015	B2
8956859	Bermudes	Feb 2015	B1
8962816	Ertl et al.	Feb 2015	B2
8969542	Buyse et al.	Mar 2015	B2
8981061	Colonna et al.	Mar 2015	B2
8999949	Spencer et al.	Apr 2015	B2
9068187	Bermudes	Jun 2015	B1
9109229	Ramseier et al.	Aug 2015	B2
9161974	Dubensky et al.	Oct 2015	B2
9187523	Motin et al.	Nov 2015	B2
9187762	Albert et al.	Nov 2015	B2
9198960	Dubensky, Jr. et al.	Dec 2015	B2
9200251	Bermudes	Dec 2015	B1
9200289	Bermudes	Dec 2015	B1
9206456	Lenormand	Dec 2015	B2
9486513	Bermudes	Nov 2016	B1
20010006642	Steidler et al.	Jul 2001	A1
20010009957	Oaks et al.	Jul 2001	A1
20010029024	Kodadek	Oct 2001	A1
20010029043	Haefliger et al.	Oct 2001	A1
20020016982	Romaine et al.	Feb 2002	A1
20020026655	Bermudes et al.	Feb 2002	A1
20020106380	Hung et al.	Aug 2002	A1
20020107374	Pallas et al.	Aug 2002	A1
20020123053	Luo et al.	Sep 2002	A1
20020150881	Ladner et al.	Oct 2002	A1
20020197276	Oaks et al.	Dec 2002	A1
20030059400	Szalay	Mar 2003	A1
20030082219	Warren et al.	May 2003	A1
20030087827	Lindberg et al.	May 2003	A1
20030092066	Vinkemeier et al.	May 2003	A1
20030106096	Barry	Jun 2003	A1
20030109026	Bermudes et al.	Jun 2003	A1
20030113293	Bermudes et al.	Jun 2003	A1
20030113717	Ladner et al.	Jun 2003	A1
20030115630	Romano	Jun 2003	A1
20030124561	Mach et al.	Jul 2003	A1
20030131372	Copenhaver et al.	Jul 2003	A1
20030131376	Okubara et al.	Jul 2003	A1
20030144490	Edwards et al.	Jul 2003	A1
20030165875	Colonna et al.	Sep 2003	A1
20030166140	Chen et al.	Sep 2003	A1
20030170276	Bermudes et al.	Sep 2003	A1
20030186416	Pallas et al.	Oct 2003	A1
20030188336	Corbin et al.	Oct 2003	A1
20030203377	Milne Edwards et al.	Oct 2003	A1
20030211476	O'Mahony et al.	Nov 2003	A1
20030219722	Ladner et al.	Nov 2003	A1
20030219886	Ladner et al.	Nov 2003	A1
20040005539	Ladner et al.	Jan 2004	A1
20040013658	Fulton et al.	Jan 2004	A1
20040023205	Ladner et al.	Feb 2004	A1
20040023282	Luo et al.	Feb 2004	A1
20040038307	Lee et al.	Feb 2004	A1
20040096426	Chen et al.	May 2004	A1
20040101531	Curtiss et al.	May 2004	A1
20040110939	Dumas Milne Edwards et al.	Jun 2004	A1
20040115788	Zheng et al.	Jun 2004	A1
20040133930	Cooper et al.	Jul 2004	A1
20040180380	Lee et al.	Sep 2004	A1
20040191787	Tanner et al.	Sep 2004	A1
20040219169	Bermudes et al.	Nov 2004	A1
20040229338	King	Nov 2004	A1
20040234956	Kabat et al.	Nov 2004	A1
20040234998	Sibbesen et al.	Nov 2004	A1
20040266674	Mills et al.	Dec 2004	A1
20050013822	Oaks et al.	Jan 2005	A1
20050032157	Gal et al.	Feb 2005	A1
20050036987	Pawelek et al.	Feb 2005	A1
20050055746	Michaud et al.	Mar 2005	A1
20050063994	Caplan et al.	Mar 2005	A1
20050069532	Weinrauch et al.	Mar 2005	A1
20050069911	Lee et al.	Mar 2005	A1
20050070007	Romaine et al.	Mar 2005	A1
20050074463	Autran et al.	Apr 2005	A1
20050079573	Sibbesen	Apr 2005	A1
20050084972	Barr et al.	Apr 2005	A1
20050106151	Shapiro	May 2005	A1
20050112139	Karp	May 2005	A1
20050118193	Andino-Pavlovsky et al.	Jun 2005	A1
20050148504	Katunuma et al.	Jul 2005	A1
20050158295	Swiercz et al.	Jul 2005	A1
20050166274	French et al.	Jul 2005	A1
20050180963	Adams et al.	Aug 2005	A1
20050202535	Collier et al.	Sep 2005	A1
20050203007	Komiyama et al.	Sep 2005	A1
20050208033	Luquet et al.	Sep 2005	A1
20050227917	Williams et al.	Oct 2005	A1
20050241015	Mach et al.	Oct 2005	A1
20050241016	Mach et al.	Oct 2005	A1
20050249706	Bermudes et al.	Nov 2005	A1
20050251885	Michaud et al.	Nov 2005	A1
20050255088	Bermudes et al.	Nov 2005	A1
20050257282	Mach et al.	Nov 2005	A1
20050260670	Colonna et al.	Nov 2005	A1
20050266560	Preuss et al.	Dec 2005	A1
20050268359	Mach et al.	Dec 2005	A1
20050273882	Romano	Dec 2005	A1
20050281828	Bowdish et al.	Dec 2005	A1
20060009633	Dimas et al.	Jan 2006	A9
20060014212	Benkovic et al.	Jan 2006	A1
20060024668	Hoek	Feb 2006	A1
20060035270	Lee et al.	Feb 2006	A1
20060035320	Tissot et al.	Feb 2006	A1
20060035371	Zheng et al.	Feb 2006	A1
20060084113	Ladner et al.	Apr 2006	A1
20060088910	Nguyen	Apr 2006	A1
20060110747	Ramseier et al.	May 2006	A1
20060156440	Michaud et al.	Jul 2006	A1
20060160152	Vinkemeier et al.	Jul 2006	A1
20060174357	Velander et al.	Aug 2006	A1
20060182762	Irene Martina Maas et al.	Aug 2006	A1
20060223142	Dumas Milne Edwards et al.	Oct 2006	A1
20060229336	Kazmierski et al.	Oct 2006	A1
20060241050	Cameron et al.	Oct 2006	A1
20060269561	Paterson et al.	Nov 2006	A1
20060275823	Kodadek	Dec 2006	A1
20060275897	Nabel et al.	Dec 2006	A1
20060286639	Dumas Milne Edwards et al.	Dec 2006	A1
20070009489	Bermudes et al.	Jan 2007	A1
20070020327	Fikes et al.	Jan 2007	A1
20070028324	Corbin et al.	Feb 2007	A1
20070037744	Gallo et al.	Feb 2007	A1
20070041997	Finlay et al.	Feb 2007	A1
20070059799	Sette et al.	Mar 2007	A1
20070065908	Gallo et al.	Mar 2007	A1
20070071773	Hanski et al.	Mar 2007	A1
20070143871	French et al.	Jun 2007	A1
20070192905	Piller et al.	Aug 2007	A1
20070254329	Rubin	Nov 2007	A1
20070259417	Ladner et al.	Nov 2007	A1
20070275423	Sebastian et al.	Nov 2007	A1
20070298012	King et al.	Dec 2007	A1
20080019994	Brunham et al.	Jan 2008	A1
20080038296	Brahmbhatt et al.	Feb 2008	A1
20080070255	Tanner et al.	Mar 2008	A1
20080089862	Benhar et al.	Apr 2008	A1
20080090770	Belmares et al.	Apr 2008	A1
20080124355	Bermudes	May 2008	A1
20080181892	Ledbetter et al.	Jul 2008	A1
20080261869	Shapiro	Oct 2008	A1
20080269070	Ramseier et al.	Oct 2008	A1
20080286306	Nabel et al.	Nov 2008	A1
20080288264	Mach et al.	Nov 2008	A1
20080311081	Fruehauf et al.	Dec 2008	A1
20090011974	Bocharov et al.	Jan 2009	A1
20090019609	Romano	Jan 2009	A1
20090023157	Lee et al.	Jan 2009	A1
20090042248	Gal et al.	Feb 2009	A1
20090042278	Barr et al.	Feb 2009	A1
20090069248	Motin et al.	Mar 2009	A1
20090081199	Colonna et al.	Mar 2009	A1
20090111160	Collier et al.	Apr 2009	A1
20090123426	Li et al.	May 2009	A1
20090162356	Lookeren Campagne	Jun 2009	A1
20090169517	Bermudes et al.	Jul 2009	A1
20090169566	Rawlin et al.	Jul 2009	A1
20090209749	Mach et al.	Aug 2009	A1
20090214506	Hardy et al.	Aug 2009	A1
20090217396	Kyrkaniders et al.	Aug 2009	A1
20090232804	Lazarides et al.	Sep 2009	A1
20090234101	Ladner et al.	Sep 2009	A1
20090239797	Cooper et al.	Sep 2009	A1
20090240073	Barry	Sep 2009	A1
20090246220	Ertl et al.	Oct 2009	A1
20090258935	Zheng et al.	Oct 2009	A1
20090294288	May et al.	Dec 2009	A1
20090297560	Dattwyler et al.	Dec 2009	A1
20090305296	Bengtsson et al.	Dec 2009	A1
20090317418	Catanzaro et al.	Dec 2009	A1
20100022584	Kenyon et al.	Jan 2010	A1
20100086546	Lee et al.	Apr 2010	A1
20100111998	Nabel et al.	May 2010	A1
20100135961	Bermudes	Jun 2010	A1
20100136048	Bermudes	Jun 2010	A1
20100137162	Retallack et al.	Jun 2010	A1
20100137192	Shapiro	Jun 2010	A1
20100166802	Caplan et al.	Jul 2010	A1
20100169988	Kohli et al.	Jul 2010	A1
20100184613	Lee et al.	Jul 2010	A1
20100189774	Lenormand	Jul 2010	A1
20100215679	Horwitz et al.	Aug 2010	A1
20100215682	Berkower	Aug 2010	A1
20100239546	Fruehauf et al.	Sep 2010	A1
20100247544	Vachon	Sep 2010	A1
20100247560	Simpson et al.	Sep 2010	A1
20100261201	Katanaev	Oct 2010	A1
20100272750	Buyse et al.	Oct 2010	A1
20100278819	Bossuyt et al.	Nov 2010	A1
20100279923	Schulte et al.	Nov 2010	A1
20100286251	Rubin	Nov 2010	A1
20100305306	Colonna et al.	Dec 2010	A1
20100310560	Colonna et al.	Dec 2010	A1
20100319087	Corbin et al.	Dec 2010	A1
20100333235	Mach et al.	Dec 2010	A1
20110014701	Ghosh	Jan 2011	A1
20110021416	Shapiro	Jan 2011	A1
20110027349	Sable et al.	Feb 2011	A1
20110028397	Tozser et al.	Feb 2011	A1
20110038917	Kappers et al.	Feb 2011	A1
20110065091	Van Der Hoek	Mar 2011	A1
20110104146	Faraday	May 2011	A1
20110152176	Horswill	Jun 2011	A1
20110189774	Mach et al.	Aug 2011	A1
20110190234	Nathan et al.	Aug 2011	A1
20110195423	Selinfreund et al.	Aug 2011	A1
20110201109	Zwaka et al.	Aug 2011	A1
20110223241	Tardi et al.	Sep 2011	A1
20110257080	Chai et al.	Oct 2011	A1
20110262474	Du et al.	Oct 2011	A1
20110274721	Nabel et al.	Nov 2011	A1
20110275585	Brahmbhatt et al.	Nov 2011	A1
20110277180	Romano	Nov 2011	A1
20110287037	Gentschev et al.	Nov 2011	A1
20110293608	Jaffee et al.	Dec 2011	A1
20110305724	Paterson et al.	Dec 2011	A1
20120027785	Dirienzo	Feb 2012	A1
20120042413	Albert et al.	Feb 2012	A1
20120045474	Motin et al.	Feb 2012	A1
20120064062	Goguen et al.	Mar 2012	A1
20120064572	Finlay et al.	Mar 2012	A1
20120071545	Shapiro	Mar 2012	A1
20120088314	Katanaev	Apr 2012	A1
20120093773	Li et al.	Apr 2012	A1
20120142080	Bermudes	Jun 2012	A1
20120142623	Lagunoff et al.	Jun 2012	A1
20120164687	Bereta et al.	Jun 2012	A1
20120219545	Ayuso et al.	Aug 2012	A1
20120230976	Helmerhorst et al.	Sep 2012	A1
20120271036	Smith et al.	Oct 2012	A1
20120276132	Feng et al.	Nov 2012	A1
20120308575	Guo et al.	Dec 2012	A1
20130017173	Nataro et al.	Jan 2013	A1
20130023472	Bristow	Jan 2013	A1
20130028901	Colonna et al.	Jan 2013	A1
20130028924	Ertl et al.	Jan 2013	A1
20130102017	Pfaendler et al.	Apr 2013	A1
20130122043	Guimaraes et al.	May 2013	A1
20130129713	Rescigno et al.	May 2013	A1
20130150559	Colonna et al.	Jun 2013	A1
20130171109	Helmerhorst et al.	Jul 2013	A1
20130196432	Poehlmann et al.	Aug 2013	A1
20130202557	Li et al.	Aug 2013	A1
20130209405	Curtiss et al.	Aug 2013	A1
20130269057	Fosu-Nyarko et al.	Oct 2013	A1
20130276168	Romaine et al.	Oct 2013	A1
20130344033	Vergnolle et al.	Dec 2013	A1
20140005108	Bristow	Jan 2014	A1
20140056841	Vachon	Feb 2014	A1
20140057940	Mankowski et al.	Feb 2014	A1
20140093528	Berkower	Apr 2014	A1
20140150134	Li et al.	May 2014	A1
20140155343	Brahmbhatt et al.	Jun 2014	A1
20140162279	Ramseier et al.	Jun 2014	A1
20140162952	Katagiri et al.	Jun 2014	A1
20140173774	Pareddy et al.	Jun 2014	A1
20140173780	Pareddy et al.	Jun 2014	A1
20140194346	Aebi et al.	Jul 2014	A1
20140220661	Bermudes	Aug 2014	A1
20140227286	Jaffee et al.	Aug 2014	A1
20140234310	Shapiro	Aug 2014	A1
20140287419	Althoff et al.	Sep 2014	A1
20140289906	Althoff et al.	Sep 2014	A1
20140296480	Sanchez Garcia et al.	Oct 2014	A1
20140322790	Sebastian et al.	Oct 2014	A1
20140370036	Shapiro	Dec 2014	A1
20150004705	Lu et al.	Jan 2015	A1
20150017138	Fruehauf et al.	Jan 2015	A1
20150017204	Bermudes	Jan 2015	A1
20150030573	Fruehauf et al.	Jan 2015	A1
20150044256	Dattwyler et al.	Feb 2015	A1
20150050308	van der Hoek	Feb 2015	A1
20150057191	Tissot et al.	Feb 2015	A1
20150071957	Kelly et al.	Mar 2015	A1
20150125849	Yeh et al.	May 2015	A1
20150132330	Garcia-Sastre et al.	May 2015	A1
20150139940	Bermudez Humaran et al.	May 2015	A1
20150153358	Ayuso et al.	Jun 2015	A1
20150184220	Sebastian et al.	Jul 2015	A1
20150197748	Liu et al.	Jul 2015	A1
20150216965	Diamond et al.	Aug 2015	A1
20150225692	Bhatia et al.	Aug 2015	A1
20150231207	Kaspar	Aug 2015	A1
20150246137	Guo et al.	Sep 2015	A1
20150291667	Dirienzo	Oct 2015	A1
20150337321	Mach et al.	Nov 2015	A1
20150351390	Castle et al.	Dec 2015	A1
20150355172	Kraus et al.	Dec 2015	A1

Foreign Referenced Citations (53)

Number	Date	Country
0973911	Jan 2000	EP
1513924	Mar 2005	EP
1655370	May 2006	EP
2009269922	Nov 2009	JP
WO1991000014	Jan 1991	WO
WO9638159	Dec 1996	WO
WO9640238	Dec 1996	WO
WO1996040238	Dec 1996	WO
WO9714782	Apr 1997	WO
WO1997014782	Apr 1997	WO
WO9833923	Aug 1998	WO
WO1999010014	Mar 1999	WO
WO1999010485	Mar 1999	WO
WO0004919	Feb 2000	WO
WO2000004919	Feb 2000	WO
WO2001014579	Mar 2001	WO
WO0125397	Apr 2001	WO
WO2001025397	Apr 2001	WO
WO02070645	Sep 2002	WO
WO2002070645	Sep 2002	WO
WO03072125	Sep 2003	WO
WO2003072125	Sep 2003	WO
WO03102168	Dec 2003	WO
WO2003102168	Dec 2003	WO
WO2004076484	Sep 2004	WO
WO2004103404	Dec 2004	WO
WO2005014618	Feb 2005	WO
WO2005018332	Mar 2005	WO
WO2005054477	Jun 2005	WO
WO2006010070	Jan 2006	WO
WO2006013441	Feb 2006	WO
WO2006048344	May 2006	WO
WO2006116545	Nov 2006	WO
WO2007083193	Jul 2007	WO
WO2008073148	Jun 2008	WO
WO2008091375	Jul 2008	WO
WO2008156702	Dec 2008	WO
WO2009006450	Jan 2009	WO
WO2009006453	Jan 2009	WO
WO2009014650	Jan 2009	WO
WO2009086116	Jul 2009	WO
WO2009126189	Oct 2009	WO
WO2009139985	Nov 2009	WO
WO2009145956	Dec 2009	WO
WO2009150433	Dec 2009	WO
WO2009152480	Dec 2009	WO
WO2010036391	Apr 2010	WO
WO2010057009	May 2010	WO
WO2011017137	Feb 2011	WO
WO2011086172	Jul 2011	WO
WO2012104025	Aug 2012	WO
WO2012150269	Nov 2012	WO
WO2013067185	May 2013	WO

Non-Patent Literature Citations (36)

Entry
Callaway, E; www.newscientist.com, 2008 “Counting in a Bacterial World”.
Soghomonyan, Surren A., et al. “Positron emission tomography (PET) imaging of tumor-localized Salmonella expressing HSV1-TK.” Cancer gene therapy 12.1 (2005): 101-108.
Honée, Guy, Wim Vriezen, and Bert Visser. “A translation fusion product of two different insecticidal crystal protein genes of Bacillus thuringiensis exhibits an enlarged insecticidal spectrum.” Applied and environmental microbiology 56.3 (1990): 823-825.
Gumbmann, M. R., et al. “The USDA trypsin inhibitor study. IV. The chronic effects of soy flour and soy protein isolate on the pancreas in rats after two years.” Plant Foods for Human Nutrition 35.3 (1985): 275-314.
Bacillus thuringiensis, Wikipedia, Mar. 21, 2016.
Hu, Yan, et al. “Bacillus subtilis strain engineered for treatment of soil-transmitted helminth diseases.” Applied and environmental microbiology 79.18 (2013): 5527-5532.
Bermudes, David, et al. “Tumour-selective Salmonella-based cancer therapy.” Biotechnology and Genetic Engineering Reviews 18.1 (2001): 219-233.
Pawelek, John M., K. Brooks Low, and David Bermudes. “Tumor-targeted Salmonella as a novel anticancer vector.” Cancer research 57.20 (1997): 4537-4544.
Carroll, J., J. Li, and D. J. Ellar. “Proteolytic processing of a coleopteran-specific δ-endotoxin produced by Bacillus thuringiensis var. tenebrionis.” Biochemical Journal 261.1 (1989): 99-105.
Friedlos, Frank, et al. “Attenuated Salmonella targets prodrug activating enzyme carboxypeptidase G2 to mouse melanoma and human breast and colon carcinomas for effective suicide gene therapy.” Clinical Cancer Research 14.13 (2008): 4259-4266.
Efficacy, Wikipedia, Mar. 21, 2016.
Bacillus thuringiensis, University of Califonia San Diego, Mar. 21, 2016; http://www.bt.ucsd.edu/how—bt—work.html.
Gaofu, Qi, et al. “In vitro assessment of plant lectins with anti-pinwood nematode activity.” Journal of invertebrate pathology 98.1 (2008): 40-45.
Clairmont, C., et al. “Biodistribution and Genetic Stability of the Novel Antitumor Agent VNP20009, a Genetically Modified Strain of Salmonella typhimuvium.” Journal of Infectious Diseases 181.6 (2000): 1996-2002.
Murray, Sean R., et al. “Extragenic Suppressors of Growth Defects inmsbB Salmonella.” Journal of bacteriology 183.19 (2001): 5554-5561.
Larsson, Helena, et al. “A novel anti-angiogenic form of antithrombin with retained proteinase binding ability and heparin affinity.” Journal of Biological Chemistry 276.15 (2001): 11996-12002.
Sznol, Mario, et al. “Use of preferentially replicating bacteria for the treatment of cancer.” The Journal of clinical investigation 105.8 (2000): 1027-1030.
Wang, Suming, et al. “Design of peptide inhibitors for furin based on the C-terminal fragment of histone H1.2.” Acta biochimica et biophysica Sinica 40.10 (2008): 848-854.
Laskowski Jr, Michael, and Ikunoshin Kato. “Protein inhibitors of proteinases.” Annual review of biochemistry 49.1 (1980): 593-626.
Shiga, Yasuhiro, et al. “Characterization of an extracellular protease inhibitor of Bacillus brevis HPD31 and nucleotide sequence of the corresponding gene.” Applied and environmental microbiology 58.2 (1992): 525-531.
Taguchi, Seiichi, Izumi Kumagai, and Kin-ichiro Miura. “Comparison of secretory expression in Escherichia coli and Streptomyces of Streptomyces subtilisin inhibitor (SSI) gene.” Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression 1049.3 (1990): 278-285.
MacIntosh, Susan C., et al. “Potentiation of Bacillus thuringiensis insecticidal activity by serine protease inhibitors.” Journal of Agricultural and Food Chemistry 38.4 (1990): 1145-1152.
Wasilewski, Margaret M., et al. “Cysteine protease inhibitors block schistosome hemoglobin degradation in vitro and decrease worm burden and egg production in vivo.” Molecular and biochemical parasitology 81.2 (1996): 179-189.
Deist, Benjamin R et al. “Bt toxin modification for enhanced efficacy.” Toxins 6.10 (2014): 3005-3027.
Thaul, Susan. “How FDA approves drugs and regulates their safety and effectiveness.” (2012).
Oppert, Brenda. “Protease interactions with Bacillus thuringiensis insecticidal toxins.” Archives of insect biochemistry and physiology 42 (1999): 1-12.
Mourão, Caroline BF, and Elisabeth F. Schwartz. “Protease inhibitors from marine venomous animals and their counterparts in terrestrial venomous animals ” Marine drugs 11.6 (2013): 2069-2112.
Wei, Jun-Zhi, et al. “Bacillus thuringiensis crystal proteins that target nematodes.” Proceedings of the National Academy of Sciences 100.5 (2003): 2760-2765.
Hu, Yan, et al. “Mechanistic and single-dose in vivo therapeutic studies of Cry5B anthelmintic action against hookworms.” PLoS Negl Trop Dis 6.11 (2012): e1900.
Urban Jr, Joseph F., et al. “Bacillus thuringiensis-derived Cry5B has potent anthelmintic activity against Ascaris suum.” PLoS Negl Trop Dis 7.6 (2013): e2263.
Suming Wang, Jinbo Han, Yanfang Wang, Wuyuan Lu, and Chengwu Chi, “Design of peptide inhibitors for furin based on the C-terminal fragment of histone H1.2”, Acta Biochim Biophys Sin (2008), vol. 40, Issue 10, p. 848-854.
http://en.wikipedia.org/wiki/Neutrophil (accesed Jul. 1, 2014).
http://en.wikipedia.org/wiki/Macrophage (Acessed Jul. 1, 2014).
http://en.wikipedia.org/wild/T-cell (Accessed Jul. 1, 2014).
http://www.uniprot.org/uniprot/Q4GWU5, Q4GWU5 (SFTI1.sub.—Helan) Reviewed, UniProtKB/Swiss-Prot, Trypsin inhibitor 1 (SFTI-1) (Acessed Jul. 1, 2014).
http://www.ebi.ac.uk/pdbe-site/pdbemotif/sequence?accessionCode=1o8y (Acessed Jul. 1, 2014) Enzymatic Cyclization of a Potent Bowman-Birk Protease Inhibitor, Sunflower Trypsin Inhibitor-1, and Solution Structure of an Acyclic Precursor Peptide, Marx, U.C.search; Korsinczky, M.search; Schirra, H.search; Jones, A. search; Condie, B.search; Otvos, L.; Craik, D.J., J.Biol.Chem.search vol. 278, pag:21782 (2003), PubMed ID (U.S. Appl. No. 12/621,047) DOI (10.1074/jbc.M212996200).

Provisional Applications (1)

	Number	Date	Country
	61302938	Feb 2010	US

Divisions (2)

	Number	Date	Country
Parent	14016407	Sep 2013	US
Child	14754113		US
Parent	13024172	Feb 2011	US
Child	14016407		US

Protease inhibitor: protease sensitive expression system composition and methods improving the therapeutic activity and specificity of proteins delivered by bacteria

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