Patent Application: US-49511604-A

Abstract:
current chemotherapeutic approaches for cancer are in part limited by the inability of drugs to destroy neoplastic cells within poorly vascularized compartments of tumors . we have here systematically assessed anaerobic bacteria for their capacity to grow expansively within avascutar compartments of transplanted tumors . among 26 different strains tested , one appeared particularly promising . we created a strain of c . novyi devoid of its lethal toxin and showed that intravenously injected c . novyi - nt spores germinated within the avascular regions of tumors in mice and destroyed surrounding viable tumor cells . when c . novyi - nt spores were administered together with conventional chemotherapeutic drugs , extensive hemorrhagic necrosis of tumors often developed within 24 hours , resulting in significant and prolonged anti - tumor effects . this strategy , called combination bacteriolytic therapy , has the potential to add a valuablle dimension to the treatment of cancer .

Description:
it is a discovery of the present inventors that combination bacteriolytic therapy ( cobalt ) can result in rapid and dramatic regressions of experimental tumors in mice . even relatively large tumors could be treated successfully with cobalt , even though such tumors do not generally respond well to chemotherapeutic agents . the bacteria useful in the practice of the present invention are anaerobic , spore formers . suitable genera include bifidobacteria , lactobacilli , and clostridia . a number of species of these bacteria have been tested for their ability to grow in tumors in a robust and dispersed manner . clostridium novyi and clostridium sordelii were found to be the best of the strains we tested for these properties . other strains and species having suitable characteristics can be used as well . decreasing the natural production of toxins is desirable in using bacteria therapeutically . while strains need not be totally non - toxigenic , it is desirable that at least one of the toxin genes by mutated , deleted , or otherwise inactivated to render the bacteria less harmfull to the host . if a toxin gene is episomal or on a phage , then curing of the episome or phage can be used to delete the toxin gene . techniques are well known in the art for mutagenesis and screening of mutants . 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 cultnies can be serially diluted and plated for single colony formation . serial repitition is desirable to enure colony formation from single cells . see , e . g ., j . h . miller , experiments in molecular genetics , cold spring harbor laboratory , ny , 1972 . spores 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 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 tumors to be treated are preferably large enough to have outgrown their blood supply and contain necrotic regions . this factor should not be limiting for most human tumor situations , as the great majority of clinically apparent human tumors have large necrotic regions within them ( fig1 ). however , micrometastatic disease might not be susceptible to cobalt . combination treatment involves administering anaerobic spores as well as a second anti - tumor agent . together these agents synergize to produce a greater decerease in the growth of the tumor . second anti - tumor agents which can be used include any which are known in the art . such anti - tumor agents include but are not limited to dna damaging agents , agents which collapse tumor vasculature , radiation , and anti - tumor antigen antibodies . these anti - tumor agents are administered according to the conventional means used in the art of medical and radiation oncology . the agents can be administered in any order or simultaneously . it may be desirable , however , to administer the spores prior to administering the second anti - tumor agent . if agents are to be administered serially , they are preferably administered within a span of a month , more preferably within a span of a fortnight , and even more preferably within a span of a week . optimization of the time span is well within the skill of the art . moreover , multiple anti - tumor agents can be administered in conjunction with the spores . thus it may be desirable in order to achieve even greater reduction in tumor growth that a plurality of anti - tumor agents be used . anti - tumor agents from different categories or mechanisms may achieve superior results . thus a preferred combination includes spores , a tumor vasculature collapsing agent and a dna damaging agent . suitable anti - tumor agents which function to collapse tumor vessels are vinblastine , vincristine , colchicine , combrestatin a - 4 , dolastatin - 10 , and 5 , 6 dimethylxanthenone - 4 - acetic acid . others as are known or discovered with the same function can be used . suitable dna damaging chemotherapeutic drugs which can be used include but are not limited to mitomycin c and cytoxan . in order to mitigate the side - effects of the anti - tumor therapy various additional drugs or therapies can be utilized . these include allopurinol , hydration , uate oxidase , steroids such as prednisone , and hematopoietic factors such as granulocyte colony stimulating factor ( g - csf ). 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 and anti - tumor agents can be packaged in 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 . treatment of mice with large tumors was sometimes toxic . approximately 20 % of mice with 350 mm 3 tumors and 50 % of mice with 700 mm 3 tumors died within 24 - 72 hours of administration of spores plus d10 . no deaths were observed after treatment with c . novyi - nt spores alone or with d10 alone . though the basis for this toxicity is not yet known , it could have been due to efflux of toxic bacterial products from the tumors or due to “ tumor lysis syndrome .” it has previously been noted that the rapid lysis of very large tumor burdens is associated with systemic toxicity in humans treated with chemotherapy , perhaps due to the sudden efflux of tumor cell metabolites into the circulation ( altman , 2001 ). though tumor lysis syndrome can be controlled in humans , it is difficult to control in mice . any therapy which dramatically shrinks tumors may be subject to this side effect . treatments for tumor lysis syndrome which may be used in humans include allopurinol , urate osidase , and volume repletion ( hydration ). treatments to mitigate side - effects of anti - tumor agents such as bone marrow toxicity and neutropenia may also be desirable . such treatment are will known in the art and can be employed here in the known manner . the above disclosure generally describes the present invention . 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 . in the work described below we attempted to exploit the fact that necrotic regions exist only within tumors and in no normal tissues . we wished to develop a toxic agent that could be specifically delivered to these areas and , in theory , could kill surrounding viable tumor cells . we chose to investigate anaerobic bacteria for this purpose . we hoped that a systematic screen for appropriate anaerobic bacteria that could kill tumor cells adjacent to the poorly vascularized regions , rather than just localize to such regions , would rejuvenate interest in this approach . furthermore , we hoped that chemotherapeutic agents that killed the well - vascularized regions of tumors , when administered in conjunction with appropriate bacteria , would result in the destruction of a major proportion of neoplastic cells within the tumors . we used the following materials and methods in our studies . bacterial strains and growth . the bacterial strains tested in this study were purchased from the american type culture collection and listed in table 1 . they were grown anaerobically in liquid cultures at 37 ° c . in reinforced clostridial medium or lactobacilli mrs broth ( difco , detroit , mich .). drugs . d10 ( d10 ) was kindly provided by dr . george r . pettit ( cancer research institute , arizona state university , az ), dr . gregory p . kalemkerian ( department of internal medicine , wayne state university , mi ), and dr . robert j . schultz ( drug synthesis and chemistry branch , nci , bethesda , md .). combretastatin a - 4 was kindly provided by dr . robert j . schultz . cytoxan ( ctx ), mitomycin c ( mmc ), vincristine , colchicine , and vinblastine are commercially available chemotherapeutic agents ( sigma , st . louis , mo .). cell lines and animals . female athymic nude and c57bl / 6 mice 6 to 8 weeks of age were purchased from harlan . hct116 colon cancer cells and b16 melanoma cells were grown as monolayers in mccoy sa medium ( life technologies , rockville , md .) supplemented with 5 % fetal bovine serum and 1 % penicillin / streptomycin ( cat . no . 15140 - 122 ). sporulation and generation of nontoxigenic c . novyi strain . spores of both wild - type and nontoxigenic c . novyi strains were generated by growing the organisms anaerobically at 37 ° c ., ph 7 . 4 in a medium containing 5 g na 2 hpo 4 , 30 g peptone , 0 . 5 l - cysteine , 10 g maltose and 5 % w / v dried cooked meat particles ( difco , detroit , mich .) per 1 liter . after one week in this medium , spores settled in the cooked meat particle layer ( bagadi , 1973 ). spores were further purified from contaminating vegetative forms on a discontinuous percoll gradient . to remove the lethal toxin gene from the wild type c . novyi strain , c . novyi spores were heated at 70 ° c . for 15 minutes to inactivate the phage carrying the toxin ( eklund , 1974 )( eklund , 1976 ). the spores were then plated on rcm agar and incubated anaerobically at 37 ° c . for 48 hours . isolated colonies were cultured in liquid rcm for another 24 to 48 hours and then tested for the presence of the lethal toxin gene by pcr . in general , 1 % of the bacterial colonies were found to lose the phage carrying this gene . in vivo studies . six to eight week old female balb / c athymic nude or c57bl / 6 mice were implanted with subcutaneous tumors through the injection of 2 . 5 × 10 6 hct116 or b16 cells , respectively . after 8 to 12 days of tumor establishment , treatment was initiated with spores or drugs . screening of bacterial strains for their ability to populate tumor grafts was done by either intratumoral injection ( 100 ul volume , 1 × 10 7 bacteria ) or intravenous injection ( 500 ul volume , 5 × 10 7 bacteria or spores ) of the tail vein . c . novyi - nt spores and d10 were diluted to the appropriate concentration in 1 × dulbecco &# 39 ; s phosphate - buffered saline ph 7 . 4 ( pbs ) ( life technologies , rockville , md .) and then administered by intravenous injection in a volume of 500 ul . cix and mmc were diluted in pbs and then given by intraperitoneal injection in a volume of 500 ul . tumor growth was assessed by measuring the size of the major and minor axes of subcutaneous tumors every two and every four days for b16 and hct116 tumors , respectively , using calipers . tumor volume was then calculated using the equation length × width 2 × 0 . 5 . choice of bacterial species . from previous studies it was clear that species of anaerobic bacteria could grow within the hypoxic regions of tumors . an example is provided by b . longum , which , when injected intravenously into mice with subcutaneous tumors , grew specifically and robustly within the tumors but not within normal tissues ( yazawa , 2000 )( yazawa , 2001 ). gram stains of sections of the tumors , however , revealed that most bacteria were tightly clustered within colonies rather than distributed throughout the necrotic regions ( fig2 a , fig2 b ). as we considered dispersion of the bacteria essential to achieve the desired effects , numerous anaerobic species of three different genera were tested in an effort to find one ( s ) exhibiting this phenotype ( table 1 ). for this purpose , bifidobacterium and lactobacillus strains were injected intravenously , while clostodium strains , which are generally highly toxic when injected intravenously , were injected directly into tumors . among the 26 strains listed in table 1 , only two ( c . novyi and c . sordellii ) exhibited extensive spreading throughout the poorly vascularized portions of the tumors ( not shown ). though this spread was undoubtedly facilitated by the motile nature of these two species , other motile anaerobic bacteria , including other clostridium strains , did not exhibit this property when tested under identical conditions . infiltration of the tumor mass following intravenous injection of c . novyi spores . in order for an experimental therapy to represent a potentially viable tool for the treatment of disseminated cancers , it must have the capacity to be delivered systemically rather than through local , intratumoral injection . though live bacteria are often toxic when injected intravenously , it has been shown that bacterial spores are non - toxic to normal animals . accordingly , we found that large numbers ( up to 10 8 in a volume of 500 ul ) of c . novyi and c . sordellii spores could be injected intravenously into normal mice without causing any noticeable side effects . when intravenously injected into mice with subcutaneous b16 tumors , however , the c . novyi bacteria floridly germinated within the tumors within 16 hours ( fig2 c ). in contrast , no germinated bacteria were observed in the liver , spleen , kidney , lung , or brain of these mice ( not shown ). similar results were observed after iv injection of c . sordellii spores ( not shown ). genetic modification of c . novyi . though c . novyi and c . sordellii spores both had the capacity to grow within tumors and kill some surrounding tumor cells , there was at least one small problem encountered with this experimental treatment : 16 to 18 hours following the initiation of treatment , all the mice died . we suspected that the cause of death was the release of potent lethal toxins from the bacteria germinating within the tumors . indeed , other anaerobic bacterial spores have proved highly toxic to animals and humans following germination within the anaerobic environments present in tumors or wounds , and the resultant mortality shown to be due to specific secreted toxins ( boyd , 1972 )( boyd , 1972 )( bette , 1991 )( rood , 1991 )( bryant , 2000 ). to mitigate systemic toxicity , we attempted to eliminate the lethal toxin gene from c . novyi . we chose c . novyi rather than c . sordellii for this purpose because the latter has two homologous toxin genes ( martinez , 1992 ) rather than one and because the single c . novyi toxin gene is located within a phage episome ( eklund , 1974 )( eklund , 1976 )( hofmann , 1995 ). bacteria were heat treated to induce loss of the phage and inoculated onto agar plates . of 400 bacterial colonies screened , three were observed to have lost the toxin gene when assessed by pcr using toxin - gene specific primers ( examples in fig3 ). phospholipase c , a c . novyi gene contained within the bacterial rather than the phage genome ( tsutsui , 1995 ), served as control for this pcr experiment . one clone , named c . novyi - nt , that had lost the toxin gene , was selected for further analysis . destruction of tumor cells following injection of c . novyi - nt spores . c . novyi - nt spores devoid of the lethal toxin were injected intravenously into mice with tumors . these spores retained their capacity to germinate within tumors and resulted in greatly expanded areas of necrosis ( fig4 a vs . fig4 b ). however , these spores , unlike those of their parents , were non - toxic when injected alone , with no ill effects generally observed after injection of up to 10 8 spores into mice with tumors . in contrast , all mice died after injection of 5 × 10 7 parental c . novyi spores into mice with tumors . growing bacteria could be observed throughout the much - enlarged necrotic regions of tumors after injection of spores ( fig4 c , fig4 d ). the enlargement of the necrotic regions was apparently due to the destruction of viable tumor cells adjacent to the original necrotic regions by the bacteria . indeed , a bacterial “ film ” ( mcmanus , 1982 ) was routinely observed at the interface between the necrotic area and the remaining viable rim of the tumor , as if the bacteria were destroying the viable tumor cells and using its degradation products as nutrients ( fig4 d ). this tumor infiltration effect was similar to that observed with wild - type c . novyi bacteria ( fig2 d ). combination therapy . we hoped to combine a bacterial agent with more conventional chemotherapeutic agents in an effort to attack the tumors from both the inside and outside , respectively . following preliminary investigations with several such agents , we concentrated on two classes : ( i ) dna damaging agents , such as mmc and ctx , which selectively kill tumor cells , and ( ii ) agents that appear to partially collapse tumor vasculature , such as flavone acetic acid derivatives and microtubule binding agents ( chaplin , 1996 )( sweeney , 2001 ). the latter class of agents has been shown to be able to interfere with proper circulation through the tumors and thereby trap large molecules , such as antibodies or bacteria , that have gained access to the tumor tissue ( theys , 2001 )( pedley , 1999 )( pedley , 2001 ). among flavone acetic acid and the microtubule - binding agents tested ( including vinblastine , vincristine , colchicine , combretastatin a - 4 , and d10 ), d10 appeared to have the most pronounced effects and was chosen for further experimentation . xenografts of the colorectal cancer cell line hct116 were used to test the effects of this combination therapy in nude mice , as the tumors could easily be visualized under the hairless skin . as shown in fig5 , sequential treatment with c . novyi - nt spores , d10 and mmc resulted in dramatic effects on large subcutaneous tumors ( starting tumor volume ˜ 700 mm 3 ), easily observable through the skin . twenty four hours following the injection of c . novyi - nt spores , the tumor mass swelled and became edematous ( fig5 a ). six hours after receiving d10 , a black spot developed near the center of the tumor , representing an area of hemorrhagic necrosis . this spot expanded in size and within 24 hours often completely enveloped the tumor ( fig5 a , 1 day time point ). h & amp ; e staining of sections of these tumors revealed extensive destruction of the tumors , often accompanied by infiltration of inflammatory cells . these necrotic masses then shrank over a period of two to four weeks ( fig5 a , 14 - 30 day time points ). in many mice , these necrotic masses eventually dissolved and disappeared , leaving the animals tumor - free ( fig5 b ). similar , though less dramatic results , were observed following the sequential treatment with c . novyi - nt and d10 ( without mmc ), but never with d10 and mmc in the absence of c . novyi - nt and rarely with c . novyi - nt alone . the anti - neoplastic effects of this combination bacteriolytic therapy ( cobalt ) were further quantified in the experiments shown in fig6 . animals with relatively large subcutaneous hct116 tumors ( starting tumor volume ˜ 700 mm 3 ) were treated with drugs alone ( d10 plus mmc ) or c . novyi - nt spores plus the drugs . as can be seen in fig6 a , the drugs alone slowed the growth of the tumors , though the tumors continued to grow and the animals had to be sacrificed at ten to fourteen days , when tumor weights exceeded 10 % of body weight . the addition of c . novyi - nt spores dramatically enhanced the effects of treatment , with tumors actually shrinking rather than simply slowing . in the experiment shown in fig6 a , four of eight animals had complete tumor regressions after only one administration of cobalt . significant tumor shrinkage was also seen when mice were given sequential treatment with c . novyi - nt spores plus d10 ( fig6 b ). however , there was no long - term tumor - free survival and the treatment had to be repeated once every two weeks unless the full combination , with mmc , was included . systemic treatment with c . novyi - nt spores alone slowed tumor growth while d10 alone had no effect , clearly illustrating the value of the combination ( fig6 b ). to determine whether cobalt would affect other tumor types , we treated c57bl / 6 mice with large syngeneic b16 tumors . in this case , ctx was substituted for mmc , as b16 tumor cells were more sensitive to ctx than to mmc . as shown in fig6 c , the drugs alone had some anti - tumor effects , as expected , though the tumor continued to grow in size and the animals had to be sacrificed within a week after beginning therapy . c . novyi - nt spores considerably enhanced these effects , and the tumors remained small over the four week course of this experiment . as with hct116 human tumors , we found that d10 plus c . novyi - nt spores had significant anti - neoplastic effects on b16 tumors , but that the addition of a tumor cytotoxic agent ( ctx ) further enhanced the efficacy of cobalt ( fig6 c ). in the b16 tumor model , maintenance cobalt ( once weekly ) was required to keep tie tumors from regrowing while with hct116 cells , a single treatment cured ˜ half the mice . the results recorded above show that cobalt can result in rapid and dramatic regressions of experimental tumors in mice . even relatively large tumors could be treated successfully with cobalt , though tumors of the size used in our experiments don &# 39 ; t generally respond well to chemotherapeutic agents ( fig6 a - 6c ). it is also clear that many questions remain . for example , we don &# 39 ; t understand the basis for the potent tumor cell killing in the vicinity of the germinating bacteria . we found that many other bacterial strains could germinate within the necrotic regions of tumors but did not exhibit this potent cytotoxic activity . this killing is clearly not due to the lethal toxin gene of c . novyi , as this gene was deleted in the c . novyi - nt strain used in cobalt . it will be interesting in the future to determine which of the c . novyi - nt genes are responsible for these tumor cytolytic effects . another point of interest was that an agent acting on the vasculature ( d110 ) was synergistic with the c . novyi - nt spores in causing significant tumor shrinkage . presumably , the vascular collapse further lowered the oxygen tension near the trapped bacteria and thereby increased the potential for bacterial growth . d10 was given after the bacterial spores rather than before because we believed that partial vascular collapse prior to spore administration might have a deleterious effect on spore delivery . this belief was based on the fact that other vascular collapsing agents , such as dmxaa and combretastatin a - 4 , have been shown to exert their effects in combination with radioactively labeled antibodies only when administered after , and not before the antibodies ( theys , 2001 )( pedley , 1999 )( pedley , 2001 ). 2 . bagadi , h . o . & amp ; sewell , m . m . 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