Patent Application: US-201213692064-A

Abstract:
clostridium novyi is an obligate anaerobe that can infect hypoxic regions within experimental tumors . we found that mice bearing large , established tumors were often cured when treated with c . novyi plus a single dose of liposomal doxorubicin . the secreted factor responsible for this phenomenon was identified and , surprisingly , proved to be a member of the lipase family . the gene encoding this protein , called liposomase , has the potential to be incorporated into diverse therapeutic methods to deliver specifically a variety of chemotherapeutic agents to tumors .

Description:
the inventors have developed methods for treating tumors . the methods enhance the tumor specificity of treatments , thereby reducing systemic toxicities . using a combination of a protein made by c . novyi - nt and liposome encapsulated drugs or biological agents , the efficacy of tumor elimination is greatly increased . the protein , while having lipase enzymatic activity appears to function non - enzymatically as a liposome disrupter . the lipase enzyme activity is not necessary for the liposome disrupting activity . thus both wild - type and lipase - negative mutants can be used for this function . moreover , the liposome - disrupting function can be delivered by the protein as elaborated in situ by live c . novyi - nt , or by a cell - free preparation of the protein itself . furthermore , the protein can be delivered as part of a conjugate or fusion protein which provides additional desirable functionality to the liposomase protein . one of the potential advantages of the approach described here is that it is generally applicable to any chemotherapeutic drug or biological agent that can be encapsulated in a liposome . these include drugs of the following categories as examples : topoisomerase inhibitors , dna synthesis inhibitors , cell division inhibitors , angiogenesis inhibitors , and microtubule inhibitors . antibodies and antibody conjugates , such as rituxan , herceptin and erbitux , can also be encapsulated in a liposome . further , cytokines and other bioactive proteins which may enhance the patient &# 39 ; s endogenous tumor - fighting systems may be used . such cytokines can include , without limitation : il - 2 and interferon - alfa 2b and gm - csf . particular drugs , cytokines , and antibodies which can be used without limitation include : abarelix ; aldesleukin ; alemtuzumab ; alitretinoin ; allopurinol ; altretamine ; amifostine ; anakinra ; anastrozole ; arsenic trioxide ; asparaginase ; azacitidine ; bcg live ; bevacizumab ; bexarotene capsules ; bexarotene gel ; bleomycin ; bortezombi ; bortezomib ; busulfan ; calusterone ; capecitabine ; carboplatin ; carmustine ; celecoxib ; cetuximab ; chlorambucil ; cisplatin ; cladribine ; clofarabine ; cyclophosphamide ; cytarabine ; dacarbazine ; dactinomycin , actinomycin d ; dalteparin sodium ; darbepoetin alfa ; dasatinib ; daunorubicin ; daunomycin ; decitabine ; denileukin ; denileukin diftitox ; dexrazoxane ; dexrazoxane ; docetaxel ; doxorubicin ; dromostanolone propionate ; eculizumab ; elliott &# 39 ; s b solution ; epirubicin ; epirubicin hcl ; epoetin alfa ; erlotinib ; erlotinib ; estramustine ; etoposide phosphate ; etoposide , vp - 16 ; exemestane ; fentanyl citrate ; filgrastim ; floxuridine ; fludarabine ; fluorouracil , 5 - fu ; fulvestrant ; gefitinib ; gemcitabine ; gemcitabine hcl ; gemicitabine ; gemtuzumab ozogamicin ; goserelin acetate ; histrelin acetate ; hydroxyurea ; ibritumomab tiuxetan ; idarubicin ; ifosfamide ; imatinib mesylate ; interferon alfa - 2a ; interferon alfa - 2b ; irinotecan ; lapatinib ditosylate ; lenalidomide ; letrozole ; leucovorin ; leucovorin ; leucovorin ; leucovorin ; leuprolide acetate ; levamisole ; lomustine , ccnu ; meclorethamine , nitrogen mustard ; megestrol acetate ; melphalan , l - pam ; mercaptopurine , 6 - mp ; mesna ; methotrexate ; methoxsalen ; mitomycin c ; mitotane ; mitoxantrone ; nandrolone phenpropionate ; nelarabine ; nofetumomab ; oprelvekin ; oprelvekin ; oxaliplatin ; paclitaxel ; paclitaxel protein - bound particles ; palifermin ; pamidronate ; panitumumab ; pegademase ; pegaspargase ; pegfilgrastim ; peginterferon alfa - 2b ; pemetrexed disodium ; pentostatin ; pipobroman ; plicamycin , mithramycin ; porfimer sodium ; procarbazine ; quinacrine ; rasburicase ; rituximab ; sargramostim ; sorafenib ; streptozocin ; sunitinib ; sunitinib maleate ; talc ; tamoxifen ; temozolomide ; teniposide , vm - 26 ; testolactone ; thalidomide ; thioguanine , 6 - tg ; thiotepa ; topotecan ; topotecan hcl ; toremifene ; tositumomab ; tositumomab / 1 - 131 tositumomab ; trastuzumab ; tretinoin , atra ; uracil mustard ; valrubicin ; vinblastine ; vincristine ; vinorelbine ; vorinostat ; zoledronate ; and zoledronic acid . the gene encoding the liposomase has a sequence as shown in accession no . cp000382 of genbank ( seq id no : 2 ). the protein has a sequence as shown in accession no . abk60711 ( seq id no : 1 ). the first 35 amino acids are predicted to be cleaved as a signal sequence . mutants having amino acid substitutions in the highly - conserved gxsxg lipase motif ( residues 160 - 164 of seq id no : 1 ) also retain liposomase activity and can be used . for example , the s127g mutant ( mutation at serine 162 in seq id no : 1 ) can be used to provide liposomase activity . other lipase - defective mutants can be used as well . other substitutions of serine - 127 ( residue 162 in seq id no : 1 ) can be with amino acids a , c , d , e , f , g , h , i , k , l , m , n , p , q , r , t , v , w , or y . compositions according to the invention can be made before or after administration to a human or other mammal . thus , components of the compositions can be administered separately or mixed . if separately , the components can be administered in any order . the components may form the composition when they are within a patient or other mammal . the components of the compositions can be packaged together or separately in a kit . thus multiple vessels or containers within a single package may be provided to an end - user . the end user may administer the components separately or mixed , at one time or at more then one time . kits comprising the useful components for practicing the anti - tumor methods of the present invention can be packaged in a divided or undivided container , such as a carton , bottle , ampule , tube , etc . the spores , liposomase , and anti - tumor agents can be packaged in , e . g ., dried , lyophilized , or liquid form . additional components provided can include vehicles for reconsititution of dried components . preferably all such vehicles are sterile and apyrogenic so that they are suitable for injection into a mammal without causing adverse reactions . the anti - tumor agents other than the spores are also preferably sterile . the spores are preferably microbiologically pure , i . e ., containing no other bacteria other than the desired spore - forming anaerobe . methods for making liposomes are well known in the art . see for example , mozafari , cell mol biol lett . 2005 ; 10 ( 4 ): 711 - 9 ; andresen et al ., prog lipid res . 2005 january ; 44 ( 1 ): 68 - 97 ; jensen et al ., mol cancer ther . 2004 november ; 3 ( 11 ): 1451 - 8 ; pupo et al ., j control release . 2005 may 18 ; 104 ( 2 ): 379 - 96p ; brandl , biotechnol annu rev . 2001 ; 7 : 59 - 85 . any technique known in the art for making liposomes may be used . c . novyi spores can be prepared as is known in the art . preferably the spores will be from a toxin - defective strain . see for example , u . s . patent application 20050079157 , the contents of which are expressly incorporated herein . toxin can be eliminated by a process of curing of a bacteriophage or plasmid . a c . novyi useful as a starting material ( atcc 19402 ) can be obtained from the american type culture collection , 10801 university boulevard , manassas , va ., 20110 - 2209 . while strains need not be totally non - toxigenic , it is desirable that at least one of the toxin genes be mutated , deleted , or otherwise inactivated to render the bacteria less harmful to the host . if a toxin gene is episomal or on a phage , then curing of the episome or phage can be used to eliminate the toxin gene . techniques are well known in the art for mutagenesis and screening of mutants and for curing episomes . isolated and bacteriologically pure vegetative bacteria or spores , according to the invention are those which are not contaminated with other bacteria or spores . microbiological techniques for obtaining such pure cultures are will known in the art . typically single colonies are picked and spread upon an agar nutrient medium , separating colonies so that new colonies arise that are the progeny of single cells . this process is typically repeated to ensure pure cultures . alternatively , liquid cultures can be serially diluted and plated for single colony formation . serial repetition is desirable to ensure colony formation from single cells . see , e . g ., j . h . miller , experiments in molecular genetics , cold spring harbor laboratory , ny , 1972 . spores , liposomase , liposomase conjugates , and nucleic acids encoding liposomase or fusion proteins of it , can be administered to a tumor - bearing mammal by any means which will afford access to the tumor . spores can be injected intravenously , intradermally , subcutaneously , intramuscularly , intraperitoneally , intratrumorally , intrathecally , surgically , etc . preferred techniques are intravenous and intratumoral injections . tumor bearing mammals can be , for example , humans , pets , such as dogs and cats , agricultural animals such as cows , sheep , goats and pigs , and laboratory animals , such as rats , hamsters , monkeys , mice , and rabbits . the present delivery system is useful for protein therapy or gene therapy or a combination thereof . thus the liposomase can be provided as a protein or as a polynucleotide encoding lipsomase . similarly , the anti - tumor drug or biological agent may be a protein or a polynucleotide , as well as a small chemical entity , for example , not a biological polymer . polynucleotides can be provided in a viral or non - viral vector . any vectors which are known in the art can be used , without limitation . the vector constructs may contain a tumor - specific promoter to enhance the specificity of the treatment . examples of such promoters are known in the art . cxcr4 promoter is tumor - specific in melanomas ; hexokinase type ii promoter is tumor - specific in lung cancer ; trpm4 ( transient receptor potential - melastatin 4 ) promoter is preferentially active in prostate cancer . the anti - tumor drug may be one that directly or indirectly affects the tumor . thus , for example , it can be a protein which stimulates the native immune response to the tumor . alternatively , it can be an antibody which mobilizes other immune system components to destroy tumor cells . still other agents may be toxic to tumor cells . the choice of agent is well within the skill of the artisan . conjugate proteins are post - translationally linked proteins ; they may be linked by chemical linking of two polypeptides , directly or through an intermediate . alternatively , the proteins may be linked via co - translation of two coding regions within a single open reading frame . any means for joining two polypeptides that are not linked in nature may be used . one type of conjugate which can be used utilizes an antibody molecule or portion thereof as a second protein . thus , as an example , liposomase can be linked to a variable region of an antibody thus providing the liposomase with a means of targeting to a particular site in the body which expresses the antigen to which the antibody binds . linkers may be used between portions of the conjugate proteins as desired . isolated and purified liposomase protein according to the invention is a preparation in which the liposomase activity comprises at least 10 %, 25 %, 40 %, 55 %, 70 %, 85 %, or 95 % of the protein in a composition . isolated and purified liposomase - encoding polynucleotide is one which is separated from other genes of the c . novyi genome . thus it is separated from the genome - adjacent sequences encoding hypothetical proteins nt01cx — 2046 and nt01cx — 2048 . the complete genome of c . novyi - nt has been determined and is available at ncbi as nc — 008593 . it is a circular dna of 2 , 5478 , 720 nt . the methods and compositions of the present invention can be applied to any tumor type . the principles upon which the invention relies ( such as the selective ability of liposomes to penetrate through the fenestrated endothelium present in tumors and a few other organs ( 8 , 9 ), and the enhanced release of contents from a liposome in the presence of liposomase ,) apply to any tumor . thus the present invention can be used to treat tumors of the gastrointestinal tract , such as stomach , intestine , colon , rectum , esophagus , tumors of the kidney , breast , lung , liver , head and neck , and brain . the above disclosure generally describes the present invention . all references disclosed herein are expressly incorporated by reference . a more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only , and are not intended to limit the scope of the invention . hct116 ( ccl - 247 , human colorectal carcinoma ) and ct26 ( crl - 2638 , murine colorectal adenocarcinoma ) were purchased from the american type culture collection . both lines were grown in mccoy &# 39 ; s 5a medium ( invitrogen ) supplemented with 5 % fbs ( hyclone ) at 37 ° c . in 5 % co 2 reagents . doxorubicin was purchased from bedford laboratories , bedford , ohio . pegylated liposomal doxorubicin ( doxil ®) was purchased from tibotec therapeutics , raritan , n . j . chicken egg l - α - phosphatidylcholine ( epc ), hydrogenated chicken egg l - α - phosphatidylcholine ( hepc ), 1 , 2 - distearoyl - sn - glycero - 3 - phosphoethanolamine - n -[ methoxy ( polyethylene glycol )- 2000 ] ( dspe - peg 2000 ) and cholesterol ( chol ) were purchased from avanti polar lipids , alabaster , ala . irinotecan hcl ( camptosar ) was purchased from pharmacia & amp ; upjohn co ., kalamazoo , mich . calcimycin a23187 and triolein were obtained from sigma ( st . louis , mo .). 1 , 2 - dioleoyl - 3 - pyrenedecanoyl - rac - glycerol ( dpg ) was obtained from markergene technologies ( eugene , oreg .). a set of nine purified lipases were purchased from fluka , switzerland . c . novyi - nt spores were prepared as previously described ( 1 ). a mixture of hepc : chol : dspe - peg 2000 at a molar ratio of 50 : 45 : 5 was solubilized in chloroform and dried to a thin film under rotary evaporation then further dried under vacuum for 2 hours . the film was hydrated with 300 mm mnso 4 and submerged in a 65 ° c . sonication bath bransonic , danbury , conn .) to form large multilamellar vesicles ( mlvs ). this lipid suspension was extruded 10 times through a double stack of 0 . 1 um nuclepore filters ( whatman , florham park , n . j .) using a lipex thermobarrel extruder ( northern lipids , vancouver , bc , canada ). the resulting colloidal suspension of single unilamellar vesicles ( suv ) was filter - sterilized then dialysed against 300 mm sucrose at 4 ° c . to exchange the external milieu of the liposomes . the mean size of the suvs was 100 . 2 nm ( polydispersity index = 0 . 129 ) as determined by quasi - elastic light scattering using a malvern zetasizer 3000 ( malvern , worcestershire , uk ), cpt - 11 was actively loaded into liposomes using a mnso 4 ph gradient loading method ( 2 - 4 ). irinotecan was mixed with liposomes at a drug : lipid molar ratio of 1 : 3 and incubated at 65 ° c . for 10 min . calcimycin was then added at a ratio of 1 μg calcimycin : 10 μmol lipids and the suspension incubated at 65 ° c . for 45 min . the drug - loaded liposomes were then filter - sterilized and dialyzed against 300 mm sucrose at 4 ° c . to remove unencapsulated cpt - 11 . dialysis was performed in the dark to minimize photo - degradation of the drug . encapsulation efficiency was typically & gt ; 99 % as determined by disruption of liposomes with 1 - butanol and fluorometric measurement ( excitation at 390 nm , emission at 460 nm ) using a fluorescence plate reader ( fluostar galaxy , bmg labtech , gmbh ). concentrations were derived by reference to a cpt - 11 standard curve . all animal experiments were overseen and approved by the animal welfare committee of johns hopkins university and were in compliance with university standards . six - to 8 - week old mice purchased from harlan breeders , in , were used for tumor implantation studies . balb / c mice were used to establish ct26 tumors and athymic nu / nu mice were used to establish hct116 xenografts . a minimum of five animals were used for each experimental arm . five million tumor cells were injected subcutaneously into the right flank of each mouse and allowed to grow for ˜ 10 days before randomization and treatment . c . novyi - nt spores were administered as a bolus tail vein injection of 300 million spores suspended in 0 . 2 ml phosphate buffered saline . free and liposomal drugs in the relevant arms were administered 16 hours later via the same route . the doses of doxil , doxorubicin , liposomal irinotecan and irinotecan were 10 mg / kg and 25 mg / kg , respectively . tumor volume was calculated as length × width 2 × 0 . 5 . hct116 xenografts were established in athymic nu / nu mice and treated as described above . tissue samples were harvested at various times after treatment , suspended in 70 % ethanol , 0 . 3 n hcl and homogenized ( ultra - turrax ® t25 basic , ika , nc ) to extract doxorubicin . following centrifugation , doxorubicin fluorescence ( excitation at 470 nm , emission at 590 nm ) in the supernatant was measured with a fluorescence plate reader ( fluostar galaxy , bmg labtech , gmbh ). concentrations were derived by reference to a doxorubicin standard curve . samples were mixed with doxil ( 100 μl sample + 5 μl doxil in a 96 - well plate or 50 μl sample + 2 μl doxil in a 384 - well plate ). increase in fluorescence caused by the dequenching of released doxorubicin was kinetically measured over 30 - 60 minutes using excitation at 470 nm and emission at 590 nm ( 5 ). all measurements were performed at 37 ° c . in a fluorescence plate reader . a typical readout is shown in fig . s 1 . liposome - disrupting activity was defined as the maximum slope of the release curve . c . novyi - nt spores were inoculated into 20 ml bagadi medium ( 6 ) and incubated in an anaerobic chamber ( type a , coy labs , grass lake , mich .) at 37 ° c . for ˜ 16 hr . one ml of this starter culture was used to inoculate 100 ml bagadi medium that had been pre - equilibrated in the anaerobic chamber . this culture was grown to late log phase then centrifuged at 5 , 000 g to remove bacteria . the supernatant was precipitated with 50 % saturated ammonium sulfate at 4 ° c . for 1 hour , then centrifuged at 5 , 000 g . after discarding the supernatant , the pellet was solubilized in tn buffer ( 100 mm tris - hcl , ph 7 . 5 , 0 . 1 m nacl ) and filter - sterilized . all subsequent chromatography was performed on an äkta purifier fplc system ( amersham biosciences , piscataway , n . j .). the filtered sample was loaded onto a mono q 5 / 50 gl ( amersham ) column equilibrated in tn buffer . the proteins were eluted with a linear gradient of ten column volumes formed from tn buffer and 100 mm tris - hcl 7 . 5 , 1 m nacl . the fractions collected were assayed for doxorubicin releasing activity as described above . the two most active fractions from the mono q column were pooled and loaded onto a hiload 16 / 60 superdex 200 ( amersham ) column equilibrated in 100 mm tris - hcl ph 7 . 5 , 0 . 5 m nacl . the column was isocratically eluted with the same buffer over 1 . 5 column volumes . the fractions were assayed for doxorubicin disrupting activity as described above . proteins in the two most active fractions were separated by electrophoresis through an sds - polyacrylamide gel and silver - stained using the silversnap stain kit ii ( pierce , rockford , ill .). the single dominant band (≈ 45 kd ) was excised for analysis by lc / ms / ms . the excised protein band was digested in - gel with trypsin , and analyzed as previously described ( 7 ). in brief , purified tryptic fragments were injected onto a 150 × 0 . 3 mm vydac reverse phase column at 10 μl / min for 5 min in 5 % buffer b using an agilent 1100 series capillary - lc system . buffers a ( 0 . 1 % acetic acid ) and b ( 99 . 9 % acetonitrile and 0 . 1 % acetic acid ) were employed in the liquid chromatography ( lc ) step of the lc / ms / ms analysis . peptides were eluted from the column with a 10 - 65 % buffer b gradient over 90 minutes at a rate of 2 μl / min . eluted peptides were detected by a lcq deca xp mass spectrometer ( thermo , ma ), equipped with an electrospray ionization source , a low flow metal needle assembly operating in data dependent mode . the method consisted of two scan events , a full scan and a second data dependent ms / ms scan . the dynamic mass range of the full scan was set at 300 to 3000 m / z . the resulting ms / ms data dependent scan rejected known ‘ contaminant ’ masses of 371 . 0 , 391 . 0 , 445 . 0 , 462 . 0 , 1221 . 89 , 1321 . 9 , 1421 . 9 , 1521 . 8 , 1521 . 9 , 1621 . 9 , 1721 . 9 and 1821 . 9 m / z . other method settings included a default charge state set to 4 , dynamic exclusion with repeat count set to 2 , repeat duration 1 min , an exclusion list size of 25 and exclusion duration 3 min . all other method parameters were default values set by the xcaliber software v . 1 . 2 ( thermo , ma ). the coding sequence of nt01cx2047 , devoid of its n - terminal secretion signal ( as predicted by signalp 3 . 0 ) was pcr - amplified with phusion taq polymerase ( finnzymes , espoo , finland ) using forward primer 5 ′- tgcaccaccaccaccaccacaaagaaaatcaaaaagtatcacaaaataat tatcctataatactttgtcatgg ( seq id no : 3 ) and reverse primer 5 ′- ctgaccggtttattattcagttacaggaagattt ctaagcatttgagcc ( seq id no : 4 ). the ( cac ) 6 sequence in the forward primer added six histidine residues at the n - terminus of the encoded protein . this pcr product was digested with age ito create an insert with one sticky end . the cloning vector pcr2 . 1 / t7 - gfp ( invitrogen , carlsbad , calif .) was digested with nde i , blunted with t4 dna polymerase , then digested with age i to create a single sticky end . the vector and insert were ligated to create plip . the expected sequences of the inserts were verified by dna sequencing . the plip plasmid was digested with snab i to excise a 45 bp fragment containing the serine residue to be mutated . the plip ( s127g ) plasmid was generated by blunt - end ligating the digested plasmid with a replacement 45 bp double - stranded oligonucleotide of sequence 5 ′- gtaaagttcatttaataggacac ggt caa ggtggacaaactatac ( seq id no : 5 ). the plasmid plip ( s127x ) plasmid was generated in the same fashion using the double - stranded oligonucleotide 5 ′- gtaaagttcatttaataggaca c taataa ggtggacaaactatac ( seq id no : 6 ). the targeted alterations ( underlined above ) and orientation of the inserts were verified by dna sequencing . the expression vectors described above were transformed by heat shock into the rosetta - gami ( de3 ) plyss strain of e . coli ( novagen , madison , wis .). three clones per expression construct were picked and each was cultured in 20 ml hyperbroth ( athenaes , baltimore , md .) under antibiotic selection ( 100 μg / ml ampicillin + 34 μg / ml chloramphenicol ) at room temperature . as a negative control , bacteria carrying the plip plasmid were “ cured ” of their plasmid through growth in the absence of selective antibiotics . loss of plasmid was verified by pcr . expression was induced using iptg for 1 hour , reaching an od 600 ˜ 0 . 2 , after which the bacteria were pelleted by centrifugation and resuspended in 100 mm tris - hcl , ph 7 . 5 . lysates were prepared by sonication in a bioruptor sonicating bath ( diagenode , belgium ) and centrifugation at ˜ 14 , 000 g was performed to remove insoluble matter . western blotting using an α - polyhistidine mouse mab ( r & amp ; d systems , mn ) was used to confirm the presence of the expected proteins . epc : triolein : dpg in a molar ratio of 2 : 5 : 1 were mixed in chloroform and dried to a thin lipid film under rotary evaporation , then dried further under high vacuum for 2 hours . the film was hydrated with 100 mm glycine buffer , ph 9 . 5 , 19 mm sodium deoxycholate to yield a final dpg concentration of 1 mm . this suspension was vortexed vigorously to form an emulsion for use as a substrate . samples were mixed with this substrate ( 30 μl sample + 5 μl sample ) in a 384 - well plate ). increase in fluorescence caused by catalytic release of pyrenedecanoic acid was kinetically measured over 2 hours using excitation at 320 nm and emission at 405 nm . all measurements were performed at 37 ° c . in a fluostar galaxy fluorescence plate reader . lipase activity units were derived by reference to a pseudomonas cepacia lipase standard curve . 1 unit ( u ) corresponds to the equivalent amount of p . cepacia lipase activity which liberates 1 mmol per minute of oleic acid from triolein . purified nt01cx2047 enzyme and nine other purified lipases were dissolved in 100 mm tris - hcl , ph 7 . 5 to a concentration of 1 mg / ml . fifty μl of assay buffer ( 100 mm tris - hcl ph 7 . 5 + 0 . 125m nacl ) was pipetted into 384 - well plates , followed by 2 μl of the relevant lipase and 1 μl of doxil . samples were assayed in duplicate for liposome - disrupting activity as described above . we used syngeneic ct26 colorectal tumors in balb / c mice . c . novyi - nt spores were injected intravenously , and once germination had begun in the tumors (˜ 16 hr after injection ) a single dose of doxil , at 10 mg / kg , was administered through the tail vein . doxil is a liposomal formulation which encapsulates doxorubicin , a dna - damaging agent and widely used chemotherapeutic agent . liposome - encapsulated doxorubicin has been shown to result in improved outcomes compared to unencapsulated doxorubicin in a variety of experimental and clinical studies ( 18 - 21 ). the liposomes in doxil are surface modified by pegylation to increase their circulation time ( 18 ). as previously documented ( 11 , 12 ), treatment with c . novyi - nt spores alone resulted in germination and necrosis within the centrally hypoxic region of the tumor , but left a well - oxygenated viable rim that eventually regrew ( fig1 a ). neither doxorubicin nor doxil alone resulted in prolonged therapeutic effects in these mice . when doxil was combined with c . novyi - nt spores , however , the effects were remarkable , resulting in complete regression of tumors in all mice ( fig1 a ) and cures in more than half of them ( fig1 b ). notably , mice treated with c . novyi - nt and free doxorubicin at the same dose exhibited dramatic morbidity , with 100 % of mice dying within 2 weeks , emphasizing the crucial role of liposomal encapsulation in reducing systemic toxicity ( 18 ). to determine whether these pronounced anti - tumor effects could be observed in other tumor model systems , we treated human colorectal cancer xenografts [ hct116 ] growing in nude mice in the same way . as shown in fig1 c and d , c . novyi - nt spores , when used in combination with doxil , resulted in hct116 tumor regressions similar to those observed with ct26 tumors . the dose of liposomal doxorubicin used in these tumor models was matched to those currently used in the clinic to treat cancer patients ( 19 - 21 ). the synergistic effects observed in the experiments described above were presumably due to an increased concentration of doxorubicin in tumors as a result of c . novyi - nt infection . to substantiate this conjecture , the distribution of doxorubicin in mice receiving doxil alone was compared with that in mice treated with doxil plus c . novyi - nt spores . as shown in fig2 a , there was a remarkable increase in the intratumoral concentration of doxorubicin when doxil was administered in the presence of c . novyi - nt . in contrast , the levels of doxorubicin in the heart , liver , spleen , kidney and muscle were similar in the presence or absence of spores ( fig2 a ). the doxorubicin found in infected tumors had been released from liposomes and was bound to tumor cell nuclei , as revealed by immunofluorescence ( fig2 b ). note that the concentration of doxorubicin in conventionally - treated tumors was previously shown to be higher and more stable after administration of doxil than after administration of doxorubicin ( 22 ). in effect , the administration of c . novyi - nt spores plus doxil resulted in a greater than 100 - fold increase in tumor drug exposure compared to that achieved with an equivalent dose of free doxorubicin , without increasing drug concentrations in normal tissues . we next attempted to identify the mechanism underlying the ability of c . novyi - nt to release doxorubicin in tumors following injection of doxil . we found that culture medium conditioned by the growth of c . novyi - nt contained a robust liposome - disrupting factor and that the concentration of this factor was maximum in late log phase ( fig6 ). we anticipated that this factor would be a phospholipase , as these enzymes are known to disrupt the lipid bilayers of liposomes as well as those of erythrocytes ( 17 ). two phospholipase c enzymes have been purified from c . novyi and one of them possesses hemolytic activity ( 23 - 25 ). the c . novyi - nt genome contains four genes predicted to encode phospholipases ( 26 ). one of these four genes ( nt01cx0979 ) encoded an extracellular phospholipase c protein ( 23 , 25 ) which was expressed at high levels in growing bacteria ( 26 ). because c . novyi - nt has so far proved recalcitrant to transformation by exogenous dna , we resorted to a different strategy to test the hypothesis that the liposome - disrupting factor was the phospholipase c nt01cx0979 . following mnng - mediated mutagenesis , we plated ˜ 10 , 000 bacteria on blood - agar plates and identified one colony which reproducibly lacked hemolytic activity . this clone was demonstrated by dna sequencing to possess a 971g & gt ; a transition within the nt01cx0979 phospholipase c gene , resulting in a mutation from a well - conserved glycine to a glutamine ( 25 ). surprisingly , the growth media of this hemolysis - negative clone retained its liposome - disrupting activity , indicating that this activity was not the result of nt01cx0979 or indeed of any other enzyme sufficient for hemolysis . to identify the liposome - disrupting factor , we fractionated the growth medium from late log - phase c . novyi - nt via a combination of ammonium sulfate precipitation , ion exchange chromatography and gel filtration . a single , major peak of liposome - disrupting activity was observed ( fig3 a , b ). sds - polyacrylamide gel electrophoresis revealed a predominant silver - staining band in the active fractions ( fig3 c ). this band was purified , digested by trypsin , and analyzed by liquid chromatography - tandem mass spectrometry . using the c . novyi - nt genome as the reference , the polypeptide was found to be encoded by nt01cx2047 , a putative lipase . the two extracellular lipases identified in the c . novyi - nt genome ( nt01cx0630 and nt01cx2047 ) were not highly homologous to each other ( 47 % aa identity ) or to their closest counterparts in other bacteria ( 50 ˜ 55 % aa identity to a c . tetani lipase ). the identification of the liposome - disrupting factor as the product of nt01cx2047 was consistent with information from the genomic analysis of c . novyi - nt , which revealed that nt01cx2047 was preferentially expressed in late log phase , was predicted to be extracellular , and was highly expressed in tumors after infection with c . novyi - nt ( 26 ). to examine the properties of the protein encoded by nt01cx2047 , we cloned its orf into an inducible expression vector which was then introduced into a genetically modified e . coli strain that permitted expression of c . novyi - nt genes ( which have a codon usage very different than that of e . coli ). following induction of protein expression by iptg , the transformed e . coli cells grew poorly , presumably because the gene product was toxic . lysates from these cells were tested for lipase activity as measured by hydrolysis of 1 , 2 - dioleoyl - 3 - pyrenedecanoyl - rac - glycerol . the nt01cx2047 - expressing clones exhibited lipase activity , whereas clones cured of the vector ( described below ) did not ( fig4 b ). as a further control , a derivative was generated in which a stop codon was substituted for the serine residue at amino acid 127 ( s127x ). we also generated a mutant in which the serine at residue 127 was replaced by glycine ( s127g ). s127 is found within the highly - conserved gxsxg lipase motif and was predicted to be the essential catalytic serine responsible for lipase activity ( 27 ). accordingly , both the s127g missense mutant and the s127x truncation mutant were devoid of lipase activity though each produced similar amounts of nt01cx2047 polypeptide ( fig4 a , b ). interestingly , the non - catalytic s127g mutant exhibited the same poor growth as wild - type nt01cx2047 , whereas the s127x mutant grew much more robustly . the apparent toxicity of the s127g mutant , which had no lipase activity , was puzzling but was illuminated by the experiments described below . to test the liposome - disrupting activity of the lysates described above , we incubated them with liposomal doxorubicin . the strains containing the wild - type form of nt01cx2047 possessed potent activity in this assay ( fig4 c ). when this strain was cultured without selective antibiotics until the bacteria lost the expression vector by random segregation , the liposome - disrupting activity was lost ( fig4 c ). moreover , the cells that had lost the plasmid were able to grow as robustly as the parental e . coli and the s127x mutant . as expected , the s127x truncation mutant had no detectable liposome - disrupting activity ( fig4 c ). surprisingly , however , the s127g mutant , which was devoid of lipase activity , retained substantial liposome - disrupting activity ( fig4 c ). to determine whether the ability to disrupt liposomes was particular to the nt01cx2047 lipase of c . novyi - nt , we tested the liposome - disrupting activities of nine commercially available enzymes with well - defined lipase activity ; none had significant liposome - disrupting activity ( fig4 d ). accordingly , we tested liposomes containing cpt - 11 ( irinotecan ), a topoisomerase inhibitor that , like doxorubicin , is widely used in cancer therapy . single doses of liposomal cpt - 11 were injected alone or 16 hours after c . novyi - nt administration , just as in the doxil experiments . both tumor types ( ct26 in syngeneic mice and human colorectal cancer cell xenografts in nude mice ) were relatively resistant to liposomal cpt - 11 when injected alone . however , in the presence of spores , all tumors regressed and long - term cures were achieved in & gt ; 60 % of the mice ( fig5 ). it was also notable in these and the liposomal doxorubicin experiments that the liposomase strategy was effective in small as well as in large tumors , with cures observed in tumors as small as 136 mm 3 in volume . in previous studies , small tumors proved to be resistant to bacteriolytic therapies because of the relatively small regions of necrosis within them ( 11 ). we suspect that liposomal drugs and c . novyi - nt spores are effective in combination because they mutually reinforce one another : the cytotoxic drug accumulated in the tumor through the epr effect leads to necrosis and hypoxia , which leads to c . novyi - nt germination , which leads in turn to more release of the drug through liposomase . part of the release of doxorubicin from doxil in tumors ( fig2 ) could theoretically be due to other enzymes released from c . novyi - nt or infected tumor cells . however , the major doxil - disrupting factor secreted from c . novyi - nt is liposomase ( fig3 ) and its gene is expressed at high levels in infected tumors ( 26 ), suggesting that liposomase significantly contributes to the in vivo effects . moreover , now that liposomase has been identified and cloned , it can be incorporated into other anti - cancer strategies . these strategies include cancer gene therapy employing viral or non - viral delivery systems and antibody - directed enzyme prodrug therapy ( 3 , 4 , 31 , 32 ). in the past , such approaches have been limited by the small repertoire of prodrugs available and the necessity of getting the drug metabolized within a cell to exert a bystander effect on other tumor cells ( 31 , 32 ). with liposomase , a large variety of “ prodrugs ” are already commercially available , as any chemotherapeutic agent that can be encapsulated in a liposome could theoretically be used . multi - drug therapy , with each drug combined in a separate liposome , can also be envisaged . finally , because liposomase is secreted and liposomes are external to the tumor cells , substantial bystander effects are to be expected . 1 . d . d . von hoff , a .- r . hanauske , in cancer medicine d . w . kufe et al ., eds . 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