Protease inhibitor:protease sensitive expression system, composition and methods for improving the therapeutic activity and specificity of proteins delivered by bacteria
11219671 · 2022-01-11
Inventors
Cpc classification
C12N15/74
CHEMISTRY; METALLURGY
C12N9/22
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
C07K2319/33
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K48/005
HUMAN NECESSITIES
International classification
A61K48/00
HUMAN NECESSITIES
A61K38/16
HUMAN NECESSITIES
Abstract
Bacteria which co-express protease inhibitors and protease sensitive therapeutic agents, which are surface displayed, secreted and/or released and result in their localized production and maintenance within a target tissue and inactivation outside of the target tissue, thereby increasing therapeutic activity and reducing the systemic toxicity. The bacteria may be attenuated, non-pathogenic, low pathogenic or a probiotic. Protease sensitivity may be further accomplished by engineering protease degradation sites within the therapeutic agents, further enhancing the inactivation outside of the target tissue while retaining activity within the target tissue through co-expression of a protease inhibitor. Novel chimeric proteins secreted by bacteria, including chimeric toxins targeted to neoplastic cells, tumor matrix cells and cells of the immune system, and combination therapies of these protease inhibitor:chimeric toxin-expressing bacteria together with small-molecule and biologic agents are also described. Non-conjugative bacteria limiting exchange of genetic material, and antibody resistant bacteria are also provided.
Claims
1. A genetically engineered bacterium adapted for non-lethal administration to a human or animal, selected from the group consisting of Salmonella, E. coli, Vibrio, Shigella, Streptococcus, Listeria, Lactococcus, Bacteroides, Bifidobacterium, Bacillus, Enterococcus, Serratia, Yersinia, and Listeria, Clostridium, Proteus, Xenorhabdus, Photorhabdus, Lactobacillus, Lactococcus, Leuconostoc, and Pediococcus, targeted to a tumor tissue, the genetically engineered bacterium having at least one genetically engineered nucleic acid sequence that controls genetically engineered bacterium to co-express, within the target tissue: a heterologous protease-sensitive peptide molecule having a therapeutic effect to treat a tumor pathology of the human or animal; and a heterologous protease inhibitor, effective to reduce degradation of the heterologous protease-sensitive peptide molecule by at least one protease.
2. The genetically engineered bacterium according to claim 1, wherein the target tissue is bone marrow.
3. The genetically engineered bacterium according to claim 2, wherein the bone marrow comprises neoplastic cells selected from the group consisting of at least one of leukemia cells, lymphoma cells, and multiple myeloma cells.
4. The genetically engineered bacterium according to claim 1, wherein the heterologous protease-sensitive peptide molecule is selected from the group consisting of a toxin, chimeric toxin, cytokine, antibody, bispecific antibody, single chain antibody, chemokine, and as prodrug converting enzyme.
5. The genetically engineered bacterium according to claim 1, wherein the heterologous protease-sensitive peptide molecule comprises a peptide toxin selected from at least one of the group consisting of cytolethal distending toxin (cldt), cytotoxic necrotic factor (cnf), dermonecrotic factor (dmf), shiga toxin and shiga-like toxin, colicin colE3, colicin colE7, colicin colE8, colicin colE9, colicin col-Ia, membrane lytic peptide from Staphalococcus, leucotoxin, leuckotoxin:HlyA hybrid peptide, heat stable enterotoxin from enterobacteriaceae, heat stable enterotoxin from Escherichia coli, heat stable enterotoxin from Vibrio, Neisseria IgA protease autotransporter toxin, picU autotransporter toxin, espC autotransporter toxin, and sat autotransporter toxin, clostridium enterotoxin, aerolysin, typhoid toxin, subtilase, Bordetella adenylate cyclase toxin, pertussis toxin, and porB.
6. The genetically engineered bacterium according to claim 1, wherein the heterologous protease-sensitive peptide molecule comprises a cytolethal distending toxin (cldt) fused to a peptide containing a nuclear localization signal from apoptin.
7. The genetically engineered bacterium according to claim 1, wherein the heterologous protease-sensitive peptide molecule comprises a toxin fused to a nuclear localization signal from apoptin.
8. The genetically engineered bacterium according to claim 1, wherein the heterologous protease-sensitive peptide molecule comprises a colicin targeting sequence and a colicin translocation sequence.
9. The genetically engineered bacterium according to claim 8, wherein the colicin targeting sequence comprises an M13 pIII signal sequence fused to a targeting peptide.
10. The genetically engineered bacterium according to claim 1, wherein the heterologous protease-sensitive peptide molecule comprises C-terminal portion of the hemolysin A (hlyA).
11. The genetically engineered bacterium according to claim 10, wherein the heterologous protease inhibitor comprises a furin inhibitor.
12. The genetically engineered bacterium according to claim 11, wherein the furin inhibitor comprises GKRPRAKRA SEQ ID NO:11.
13. The genetically engineered bacterium according to the claim 1, wherein the heterologous protease inhibitor comprises a protease inhibitor selected from the group consisting of: PAAATVTKKVAKSPKKAKAAKPKKAAKSAAKAVKPK 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.
14. The genetically engineered bacterium according to claim 1, wherein the genetically engineered bacterium is conjugation deficient, the heterologous protease-sensitive peptide molecule comprises an RTX toxin.
15. A genetically engineered bacterium selected from the group consisting of Salmonella, E. coli, Vibrio, Shigella, Streptococcus, Listeria, Lactococcus, Bacteroides, Bifidobacterium, Bacillus, Enterococcus, Serratia, Yersinia, and Listeria, Clostridium, Proteus, Xenorhabdus, Photorhabdus, Lactobacillus, Lactococcus, Leuconostoc, and Pediococcus, and being adapted to target at least one solid tumor, having at least one genetically engineered nucleic acid sequence, that causes the genetically engineered bacterium to co-express: a heterologous protease-sensitive peptide molecule having a therapeutic effect to treat a tumor pathology of a human or animal; and a heterologous protease inhibitor, effective to reduce degradation of the heterologous protease-sensitive peptide molecule by at least one protease.
16. The genetically engineered bacterium according to claim 15, wherein the genetically engineered bacterium is conjugation deficient.
17. The genetically engineered bacterium according to claim 15, wherein a gene sequence corresponding to the heterologous protease-sensitive peptide molecule is in the same reading frame as a gene sequence corresponding to the heterologous protease inhibitor.
18. The genetically engineered bacterium according to claim 15, wherein the heterologous protease inhibitor comprises a plurality of protease inhibitor peptide sequences separated by at least one protease cleavage site.
19. The genetically engineered bacterium according to claim 1, wherein the heterologous targeting peptide portion and the heterologous protease inhibitor portion are part of a chimeric peptide, the chimeric peptide further comprising a targeting peptide.
20. A genetically engineered bacterium selected from the group consisting of Salmonella, E. coli, Vibrio, Shigella, Streptococcus, Listeria, Lactococcus, Bacteroides, Bifidobacterium, Bacillus, Enterococcus, Serratia, Yersinia, and Listeria, Clostridium, Proteus, Xenorhabdus, Photorhabdus, Lactobacillus, Lactococcus, Leuconostoc, and Pediococcus, comprising: a first gene sequence encoding a heterologous protease-sensitive peptide molecule having a therapeutic effect to effectively treat a solid tumor pathology of the human or animal; and a second gene sequence encoding a heterologous peptide protease inhibitor, effective to reduce degradation of the heterologous protease-sensitive peptide molecule by at least one protease present in a target tumor tissue of human or animal host, the genetically engineered bacterium having a tropism for the target tumor in the human or animal host, and being adapted for non-lethal administration to the human or animal host, and to co-express the heterologous protease-sensitive peptide molecule heterologous peptide protease inhibitor within the tumor target tissue.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
6. DETAILED DESCRIPTION OF THE INVENTION
(13) 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 relate to reducing or eliminating the bacteria's ability to undergo conjugation, further limiting incoming and outgoing exchange of genetic material.
(14) 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
(15) 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 sites 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.
(16) Protease-sensitivity may be enhanced 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], sulfhhydryl [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., Figure. 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.sup.+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.
(17) 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.
(18) TABLE-US-00002 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.sup.+sti/vfs/—/g (IEGR↓SV) SEQ ID NO: 153 Furin R/—/Kr/R.sup.+s/—/—/— (RXKR↓SX) SEQ ID NO: 154 Plasminogen activator —/—/—/R.sup.+R/iv/N/— (XXR↓RIN) SEQ ID NO: 155 Urokinase —/sg/Gs/Rk.sup.+—/r/—/— (XSGR↓XR) SEQ ID NO: 156 MMP1 —/pa/—/g.sup.+li/—/—/— (GPXG↓LXG) SEQ ID NO: 157 MMP8 g/Pas/—/g.sup.+l/—/g/— (GPQG↓LRG) SEQ ID NO: 158 MMP 13 g/P/—/g.sup.+l/—/ga/— (GPPG↓LXG) SEQ ID NO: 159 Membrane matrix —/p/—/—.sup.+l/—/—/— (LPAG↓LVLX) SEQ ID NO: 160 metalloprotease 1 PSA si/sq/—/yq.sup.+s/s/—/— (SSQY↓SSN) SEQ ID NO: 161 Kallikrein 2 g/—/—/R.sup.+—/—/—/gs (GGLR↓SGGG) SEQ ID NO: 162 Granzyme A t/—/—/RK.sup.+sa/—/—/— (TXXPR↓SX) SEQ ID NO: 163 Granzyme B v/—/—/D.sup.+—/—/—/— (VEXD↓SX) SEQ ID NO: 164 Granzyme M Ka/vaye/Pa/LM.sup.+—/—/—/— (KVPL↓X) SEQ ID NO: 165 Cathepsin B —/—/l/r.sup.+—/—/g/— (XLR↓XXGG) SEQ ID NO: 166 Cathepsin S —/—/flv/r.sup.+—/—/—/— (SGFR↓SXG) SEQ ID NO: 167 Thrombin —/—/pla/R.sup.+sag/—/—/— (AGPR↓SLX) SEQ ID NO: 168 Plasmin —/—/—/KR.sup.+—/—/—/— (AXLK↓SX) SEQ ID NO: 169 Plasminogen /—/—/KR.sup.+—/—/—/— (AXLK↓SX) SEQ ID NO: 170
(19) 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, e.g., 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 radiolabled peptides incubated with the target tumor can be used to identify which amino acids are being cleaved in a therapeutic protein, and therefore which protease inhibitors to use.
6.2 Protease Inhibitors
(20) 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.
(21) 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
(22) 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-Kirk) inhibitor 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).
(23) 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-a-trypsin inhibitors from mammalian serum, chelonianin from turtle egg white, soybean trypsin inhibitor (Kunitz), secretory trypsin inhibitors (Kazal) a.sub.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, collagenase 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.
(24) TABLE-US-00003 TABLE 3 Sequences of short peptide protease inhibitors Protease Protease(s) Inhibitor inhibited Protein/Peptide Name and/or Peptide Sequence Leupeptin calpain, plasmin, Leupeptin trypsin, papain, and cathepsin B Aprotinin Trypsin RPDFC LEPPY TGPCK ARIIR YFYNA KAGLC Plasmin QTFVY GGCRA KRNNF KSAED CMRTC GGA Tissue kallikrein 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 homologs Protease Trypsin Synthetic peptide: CFPGVTSNYLYWFK SEQ ID Inhibitor 15 NO: 6, corresponding to amino acids 245-258 of human protease inhibitor. Tissue protease Serine protease DSLGREAKCYNELNGCTKIYDPVCGTDGNTYPNE inhibitor inhibitor, Kazal CVLCFENRKRQTSILIQKSGPC type 1, mature SEQ ID NO: 7 Furin inhibitors Furin PAAATVTKKVAKSPKKAKAAKPKKAAKSAAKA VKPK 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 1-16 Pepsin LVKVPLVRKKSLRQNL SEQ ID NO: 19 Dunn et al., 1983 Biochem J 209: 355-362 Pepsinogen 1-12 Pepsin LVKVPLVRKKSL SEQ ID NO: 20 Dunn et al., 1983 Biochem J 209: 355-362 Pepsinogen 1-12 Pepsin LVKGGLVRKKSL (II) [Gly4,5] SEQ ID NO: 21 4-7 substitution LVKVPGGRKKSL (III) [Gly6,7] SEQ ID NO: 22 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 Trypsin AVNIPFKVHFRCKAAFC SEQ ID NO: 25 inhibitor Ascaris Chymtrypsin GQESCGPNEV WTECTGCEMK CGPDENTPCP chymotrypsin Elastase LMCRRPSCEC SPGRGMRRTN DGKCIPASQCP elastase inhibitor SEQ ID NO: 26 Ascaris trypsin Trypsin EAEKCBZZPG WTKGGCETCG CAQKIVPCTR inhibitor ETKPNPQCPR KQCCIASAGF VRDAQGNCIK FEDCPK SEQ ID NO: 27 Ascaris trypsin Trypsin EAEKCTKPNE QWTKCGGCEG TCAQKIVPCT inhibitor RECKPPRCEC IASAGFVRDA QGNCIKFEDC PK 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 MSSMEKKPEGVNIGAGDRQNQKTEWPELVGKSV chymotrypsin EEAKKVILQDK inhibitor 2 PAAQIIVLPVGTIVTMEYRIDRVRLFVDRLDNIAQ VPRVG 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 and Gelatinase CTTHWGFTLC SEQ IN NO: 111 endothelial cell Li et al., 2006. Molecular addresses of tumors: selection migration by in vivo phage display. Arch Immunol Ther Exp 54: inhibitor 177-181 Proteosome Proteosome inhibitors subunit 3 Chymostatin ‘chymotryptic- Clasto-tactastatin like’ (beta5), ‘tryptic-like’ (beta2) and ‘peptidyl- glutamyl peptide hydrolyzing’ (beta 1). Urokinase, Urokinase, Markowska et al., 2008, Effect of tripeptides on the thrombin, thrombin, amindolytic activities of urokinase, thrombin, plasmin plasmin and plasmin and and trypsin. Int. J. Peptide Research and Therapeutics 14: trypsin inhibitors trypsin 215-218.
6.3 Targeting Ligands
(25) 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 bombesin, in being amidated by a peptidylglycine-alpha-amidating monooxygenase 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.
(26) TABLE-US-00004 TABLE 4 Examples of targeting peptides Receptor or 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 signaling and intoxication of tumor cells by chimeric growth- factor toxins. In. J. Cancer 97: 349-356. SYAVALSCQCALCRR Rivero-Muller et al., CG-beta Molecular 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 Lymph node 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 through macropinocytosis 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: 31p521 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: 31p521 CD33 binding peptides CD-33 AML Myelodysplastic cells (MDS) CD37 binding peptides Leukemia and lymphoma CD40 binding peptides CD40 Multiple myeloma, non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), Hodgkin lymphoma and acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, 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 Kirchhofer et 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 .sub.vß.sub.3 integrin) 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. 6,743,893 SEQ ID NO: 57 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/Oncology Clinics of North America 11: 1007-1019, Lymphoma stem cell targeting peptides Hodgkin's Newcom et al., 1988, Inj. J. CD 20 and CD19 binding peptides; see lymphoma; Cell Cloning 6: 417-431; above Hodgkin Reed- Jones et al. 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 (Hen, 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, transferrin receptor), guanylyl receptors (Hen, 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 1-4GlcNAc. from Clostridium difficile binds to rabbit erythrocyte glycolipids with terminal Gal alpha 1-3Gal beta 1- 4GlcNAc 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 2002 carcinoma 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
(27) 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 (
(28) TABLE-US-00005 TABLE 5 Membrane lytic peptides useful in the invention Peptide and source Peptide Sequence or name Processed MAQDIISTISDLVKWIIDTVNKFTKK «short» active delta SEQ ID NO: 120 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 MAIVGTIIKIIKAIIDIFAK SEQ ID NO: 126 PSM-beta-1 MEGLFNAIKDTVTAAINNDGAKLGTSIVSIVENGVGLLGKLFGF SEQ ID NO: 127 PSM-beta-2 MTGLAEAIANTVQAAQQHDSVKLGTSIVDIVANGVGLLGKLFGF SEQ ID NO: 128 Actinoporins Lytic peptides from sea anemones and other coelenterates (e.g., SrcI, Equinatoxins FraC, Sticholysins StsI and StsII)
6.5 Antibody and Complement Deactivating Proteins
(29) 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
(30) 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).
(31) 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
(32) 6.6.2 In another version of chimeric colicins, the colicin targeting domain is replaced with a tumor-specific targeting domain (
(33) 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 (
(34) 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 (
(35) 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
(36) 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 (
(37) 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.
(38) 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.
(39) 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.
(40) 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.
(41) 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).
(42) 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.
(43) 6.6.13. Lytic chimeras. Lytic chimeras are shown in
6.7 Expression of Proteins without Generating Chimeras
(44) 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 (
6.8 Limiting Bacterial Conjugation
(45) 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 (
(46) 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 DNase Colicins as Active Extracellular Enzymes
(47) 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 DNase 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.
(48) In order to generate colicins with extracellular DNase activity capable of deactivating DNA from neutrophils capable of trapping bacteria (neutrophil nets), the present invention presents a novel combination of DNase colicin, such as colE9, co-expressed with a non-matching DNase 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 DNase activity.
(49) In another method of producing an immunity protein that dissociates extracellularly, thus activating the colicin such as a DNase 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 DNase 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 DNase 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
(50) 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, vascular 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
(51) 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 (
6.12 Characteristics of Therapeutic Bacteria Co-Expressing Protease Inhibitors with Chimeric Antigens, Lytic and Therapeutic Proteins
(52) 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.
(53) 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.
(54) 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. 0 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.
(55) 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.
(56) 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.
(57) Overall improvement is defined as an increase in effect, such as the ability to kill 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 be determined using the median effect analysis (Chou and Talaly 1981 Eur. J. Biochem. 115: 207-216) or other standard methods.
7. FIGURE LEGENDS
(58)
(59)
(60)
(61)
(62)
(63)
(64)
(65)
(66)
(67)
(68)
(69)
8. EXAMPLES
(70) 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
(71) 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. montevidio which have non-overlapping 0-antigen presentation (e.g., S. typhimurium is 0-1, 4, 5, 12 and S. typhi is Vi, S. montevideo is 0-6, 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
(72) 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.
(73) 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 (
(74) TABLE-US-00006 SEQ ID NO: 129 MGLKLDLTWFDKSTEDFKGEEYSKDFGDDGSVMESLGVPFKDNVNNGCFD VIAEWVPLLQPYFNHQIDISDNEYFVSFDYRDGDW
(75) 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.
(76) This example of a chimeric colicin follows the pattern shown in
(77) TABLE-US-00007 SEQ ID NO: 130 MKKLLFAIPLVVPFYSHSAGGGVVSHFNDCPDSHTQFCFHGTCRFLVQED KPACVCHSGYVGARCEHADLLAAETVESCLAKSHTENSFTNVWKDDKTLD RYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSE GGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEES QPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYD AYWNGKFRDCAFHSGFNEDLFVCEYQGQSSDLPQPPVNAGGGSGGGSGGG SEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADEN ALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQ VGDGDNSPLMNNFRQYLPSLPQSVECRFAHDPMAGGHRMWQMAGLKAQRA QTDVNNKQAAFDAAAKEKSDADAALSSAMESRKKKEDKKRSAENNLNDEK NKPRKGFKDYGHDYHPAPKTENIKGLGDLKPGIPKTPKQNGGGKRKRWTG DKGRKIYEWDSQHGELEGYRASDGQHLGSFDPKTGNQLKGPDPKRNIKKY L*
(78) 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
(79) The lytic peptide PSM-alpha-3 is inserted between the pIII signal sequence and the TGF-alpha (
(80) TABLE-US-00008 SEQ ID NO: 131 MKKLLFAIPLVVPFYSHSAMEFVAKLFKFFKDLLGKFLGNNVVSHFNDCP DSHTQFCFHGTCRFLVQEDKPACVCHSGYVGARCEHADLLAAETVESCLA KSHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYGTWVP IGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGT YPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGT DPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDLFVCEYQGQSSD LPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFD YEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGL ANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRFAHD PMAGGHRMWQMAGLKAQRAQTDVNNKQAAFDAAAKEKSDADAALSSAMES RKKKEDKKRSAENNLNDEKNKPRKGFKDYGHDYHPAPKTENIKGLGDLKP GIPKTPKQNGGGKRKRWTGDKGRKIYEWDSQHGELEGYRASDGQHLGSFD PKTGNQLKGPDPKRNIKKYL
8.4 Example: A Chimeric Colicin E7
(81) 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.
(82) The genetic construct of the first colicin E7 chimera follows the same pattern as shown in
(83) The genetic construct of the second colicin E7 chimera follows the same pattern as shown in
8.5 Example: A Chimeric Colicin Ia
(84) 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.
(85) The genetic construct of the first colicin Ia chimera follows the same pattern as shown in
(86) The genetic construct of the second colicin Ia chimera follows the same pattern as shown in
8.6 Example: Colicin TRC Fusions
(87) 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 (
8.7. Example: Selecting Protease Inhibitors
(88) 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
(89) 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
(90) 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;
(91) 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
(92) 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
(93) 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 monooxygenase (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:
—CHCONHCH.sub.2 COOH⊙—CHCONH.sub.2+glyoxylic acid
(94) is produced by the recombinant.
8.10 Example: Expression of Antitumor Lytic Peptides
(95) Examples of antitumor lytic peptides are shown in
8.11 Example: Expression of Antitumor Lytic Peptide Prodrugs
(96) Examples of antitumor lytic peptide prodrugs are shown in
8.12 Example: Cytolethal Distending Toxin cltdB Fusion with Apoptin (FIG. 6)
(97) 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 (
(98) TABLE-US-00009 SEQ ID NO: 139 MKKYIISLIVFLSFYAQADLTDFRVATWNLQGASATTESKWNINVRQLIS GENAVDILAVQEAGSPPSTAVDTGTLIPSPGIPVRELIWNLSTNSRPQQV YIYFSAVDALGGRVNLALVSNRRADEVFVLSPVRQGGRPLLGIRIGNDAF FTAHAIAMRNNDAPALVEEVYNFFRDSRDPVHQALNWMILGDFNREPADL EMNLTVPVRRASEIISPAAATQTSQRTLDYAVAGNSVAFRPSPLQAGIVY GARRTQISSDHFPVGVSRRGGGGGMNALQEDTPPGPSTVFRPPTSSRPLE TPHCREIRIGIAGITITLSLCGCANARAPTLRSATADNSESTGFKNVPDL RTDQPKPPSKKRSCDPSEYRVSELKESLITTTPSRPRTAKRRIRL
8.13 Example: Cytolethal Distending Toxin cltdB Fusion with a Truncated Apoptin
(99) 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 (
(100) TABLE-US-00010 SEQ ID NO: 140 MKKYIISLIVFLSFYAQADLTDFRVATWNLQGASATTESKWNINVRQLIS GENAVDILAVQEAGSPPSTAVDTGTLIPSPGIPVRELIWNLSTNSRPQQV YIYFSAVDALGGRVNLALVSNRRADEVFVLSPVRQGGRPLLGIRIGNDAF FTAHAIAMRNNDAPALVEEVYNFFRDSRDPVHQALNWMILGDFNREPADL EMNLTVPVRRASEIISPAAATQTSQRTLDYAVAGNSVAFRPSPLQAGIVY GARRTQISSDHFPVGVSRRGGGGGITPHCREIRIGIAGITITLSLCGCAN ARAPTLRSATADNSESTGFKNVPDLRTDQPKPPSKKRSCDPSEYRVSELK ESLITTTPSRPRTAKRRIRL
8.14 Example: Cytolethal Distending Toxin cltdB Fusion with a Truncated Apoptin
(101) 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 (
(102) TABLE-US-00011 SEQ ID NO: 141 MKKYIISLIVFLSFYAQADLTDFRVATWNLQGASATTESKWNINVRQLIS GENAVDILAVQEAGSPPSTAVDTGTLIPSPGIPVRELIWNLSTNSRPQQV YIYFSAVDALGGRVNLALVSNRRADEVFVLSPVRQGGRPLLGIRIGNDAF FTAHAIAMRNNDAPALVEEVYNFFRDSRDPVHQALNWMILGDFNREPADL EMNLTVPVRRASEIISPAAATQTSQRTLDYAVAGNSVAFRPSPLQAGIVY GARRTQISSDHFPVGVSRRGGGGGIRIGIAGITITLSL
8.15 Example: Cytolethal Distending Toxin cltdB Fusion with a Truncated Apoptin
(103) 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 (
(104) TABLE-US-00012 SEQ ID NO: 142 MKKYIISLIVFLSFYAQADLTDFRVATWNLQGASATTESKWNINVRQLIS GENAVDILAVQEAGSPPSTAVDTGTLIPSPGIPVRELIWNLSTNSRPQQV YIYFSAVDALGGRVNLALVSNRRADEVFVLSPVRQGGRPLLGIRIGNDAF FTAHAIAMRNNDAPALVEEVYNFFRDSRDPVHQALNWMILGDFNREPADL EMNLTVPVRRASEIISPAAATQTSQRTLDYAVAGNSVAFRPSPLQAGIVY GARRTQISSDHFPVGVSRRGGGGGTDQPKPPSKKRSCDPSEYRVSELKES LITTTPSRPRTAKRRIRL
8.16 Example: Exchange of the Variable Loop in cldtB to Enhance Activity
(105) 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 cytotoxicity.
8.17 Example: Expression of Repeat in Toxin (RTX) Family Members
(106) RTX family members, including E. coli hemolysin operon hlyCABD and Actinobacillus actinomycetemcomitans leucotoxin ltxCABD are expressed in coordination with protease inhibitors as shown in
8.18 Example: A Group B Streptococcus Expressing a Vascular-Targeted Lytic Peptide
(107) A low pathogenicity clyE.sup.− 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
(108) A low pathogenicity clyE.sup.− 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 (
(109) 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.