Matrix Metalloproteinase-activated Anthrax Lethal Toxin Demonstrates High Potency in Targeting Tumor Vasculature

Anthrax lethal toxin (LT), a virulence factor secreted by Bacillus anthracis, is selectively toxic to human melanomas with the BRAF V600E activating mutation because of its proteolytic activities toward the mitogen-activated protein kinase kinases (MEKs). To develop LT variants with lower in vivo toxicity and high tumor specificity, and therefore greater potential for clinical use, we generated a mutated LT that requires activation by matrix metalloproteinases (MMPs). This engineered toxin was less toxic than wild-type LT to mice because of the limited expression of MMPs by normal cells. Moreover, the systemically administered toxin produced greater anti-tumor effects than wild-type LT toward human xenografted tumors. This was shown to result from its greater bioavailability, a consequence of the limited uptake and clearance of the modified toxin by normal cells. Furthermore, the MMP-activated LT had very potent anti-tumor activity not only to human melanomas containing the BRAF mutation but also to other tumor types, including lung and colon carcinomas regardless of their BRAF status. Tumor histology and in vivo angiogenesis assays showed that this anti-tumor activity is due largely to the indirect targeting of tumor vasculature and angiogenic processes. Thus, even tumors genetically deficient in anthrax toxin receptors were still susceptible to the toxin therapy in vivo. Moreover, the modified toxin also displayed lower immunogenicity compared with the wild-type toxin. All these properties suggest that this MMP-activated anti-tumor toxin has potential for use in cancer therapy. Anthrax lethal toxin (LT), a virulence factor secreted by Bacillus anthracis, is selectively toxic to human melanomas with the BRAF V600E activating mutation because of its proteolytic activities toward the mitogen-activated protein kinase kinases (MEKs). To develop LT variants with lower in vivo toxicity and high tumor specificity, and therefore greater potential for clinical use, we generated a mutated LT that requires activation by matrix metalloproteinases (MMPs). This engineered toxin was less toxic than wild-type LT to mice because of the limited expression of MMPs by normal cells. Moreover, the systemically administered toxin produced greater anti-tumor effects than wild-type LT toward human xenografted tumors. This was shown to result from its greater bioavailability, a consequence of the limited uptake and clearance of the modified toxin by normal cells. Furthermore, the MMP-activated LT had very potent anti-tumor activity not only to human melanomas containing the BRAF mutation but also to other tumor types, including lung and colon carcinomas regardless of their BRAF status. Tumor histology and in vivo angiogenesis assays showed that this anti-tumor activity is due largely to the indirect targeting of tumor vasculature and angiogenic processes. Thus, even tumors genetically deficient in anthrax toxin receptors were still susceptible to the toxin therapy in vivo. Moreover, the modified toxin also displayed lower immunogenicity compared with the wild-type toxin. All these properties suggest that this MMP-activated anti-tumor toxin has potential for use in cancer therapy. The symptoms of many bacterial diseases are due largely to the actions of toxic proteins released by the bacteria. Diphtheria toxin (DT) 3The abbreviations used are:DTdiphtheria toxinDMEMDulbecco's modified Eagle's mediumPBSphosphate-buffered salineLTlethal toxinMEKmitogen-activated protein kinase kinaseMMPmatrix metalloproteinaseCHOChinese hamster ovaryHUVEChuman umbilical vascular endothelial cellHMVEChuman microvascular endothelial cellVEGFvascular endothelial growth factorFGFfibroblast growth factorPAprotective antigenJNKc-Jun NH2-terminal kinaseILinterleukinBrdUrdbromodeoxyuridineDIVAAdirected in vivo angiogenesis assayERKextracellular signal-regulated kinaseRTreverse transcription 3The abbreviations used are:DTdiphtheria toxinDMEMDulbecco's modified Eagle's mediumPBSphosphate-buffered salineLTlethal toxinMEKmitogen-activated protein kinase kinaseMMPmatrix metalloproteinaseCHOChinese hamster ovaryHUVEChuman umbilical vascular endothelial cellHMVEChuman microvascular endothelial cellVEGFvascular endothelial growth factorFGFfibroblast growth factorPAprotective antigenJNKc-Jun NH2-terminal kinaseILinterleukinBrdUrdbromodeoxyuridineDIVAAdirected in vivo angiogenesis assayERKextracellular signal-regulated kinaseRTreverse transcription and Pseudomonas exotoxin A are two such well known toxins secreted by the pathogenic bacterium Coryne-bacterium diphtheriae and the opportunistic pathogen Pseudomonas aeruginosa (1Collier R.J. Toxicon. 2001; 39: 1793-1803Crossref PubMed Scopus (249) Google Scholar). After binding and entering mammalian cells, DT and Pseudomonas exotoxin A catalyze the ADP-ribosylation and inactivation of elongation factor 2, leading to protein synthesis inhibition and cell death (2Liu S. Leppla S.H. Mol. Cell. 2003; 12: 603-613Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 3Liu S. Milne G.T. Kuremsky J.G. Fink G.R. Leppla S.H. Mol. Cell. Biol. 2004; 24: 9487-9497Crossref PubMed Scopus (125) Google Scholar). The powerful lethal action of these toxins has been exploited extensively in the past 2 decades to target cancer cells by fusing the toxins with antibodies or growth factors that can selectively recognize antigens or receptors on cancer cells. These efforts have resulted in the first Food and Drug Administration-approved “immunotoxin,” DAB389IL2 (denileukin diftitox or Ontak), a fusion of DT catalytic and translocation domains and interleukin 2 (IL2), for treatment of persistent or recurrent T-cell lymphoma (4Olsen E. Duvic M. Frankel A. Kim Y. Martin A. Vonderheid E. Jegasothy B. Wood G. Gordon M. Heald P. Oseroff A. Pinter-Brown L. Bowen G. Kuzel T. Fivenson D. Foss F. Glode M. Molina A. Knobler E. Stewart S. Cooper K. Stevens S. Craig F. Reuben J. Bacha P. Nichols J. J. Clin. Oncol. 2001; 19: 376-388Crossref PubMed Scopus (559) Google Scholar). With the rapid progress in understanding the structures and functions of anthrax lethal toxin (LT), an important virulence factor secreted by Bacillus anthracis, LT has been identified as a bacterial toxin having a completely different mode of action that can be used for tumor targeting (5Liu S. Schubert R.L. Bugge T.H. Leppla S.H. Expert Opin. Biol. Ther. 2003; 3: 843-853Crossref PubMed Scopus (49) Google Scholar). diphtheria toxin Dulbecco's modified Eagle's medium phosphate-buffered saline lethal toxin mitogen-activated protein kinase kinase matrix metalloproteinase Chinese hamster ovary human umbilical vascular endothelial cell human microvascular endothelial cell vascular endothelial growth factor fibroblast growth factor protective antigen c-Jun NH2-terminal kinase interleukin bromodeoxyuridine directed in vivo angiogenesis assay extracellular signal-regulated kinase reverse transcription diphtheria toxin Dulbecco's modified Eagle's medium phosphate-buffered saline lethal toxin mitogen-activated protein kinase kinase matrix metalloproteinase Chinese hamster ovary human umbilical vascular endothelial cell human microvascular endothelial cell vascular endothelial growth factor fibroblast growth factor protective antigen c-Jun NH2-terminal kinase interleukin bromodeoxyuridine directed in vivo angiogenesis assay extracellular signal-regulated kinase reverse transcription LT consists of two proteins, the cell binding component protective antigen (PA, 83 kDa) and the enzymatic moiety lethal factor (LF, 89 kDa). To intoxicate mammalian cells, PA binds to the cell surface receptors tumor endothelium marker 8 (TEM8) and capillary morphogenesis gene 2 product (CMG2). PA is proteolytically activated by cell surface furin protease, leaving the carboxyl-terminal 63-kDa fragment (PA63) bound to the cell surface, resulting in the formation of the active The binds and the of the cell to its effects S.H. The of Scopus Google Scholar). is a and the mitogen-activated protein kinase kinases and Leppla S.H. Gordon K. PubMed Scopus Google Scholar, G. G. M. PubMed Scopus Google Scholar, G. L. G. M. J. PubMed Scopus Google and mitogen-activated protein kinase including the and c-Jun NH2-terminal kinase F. Full Text Full Text PDF PubMed Scopus Google Scholar). The that LT is selectively toxic to many human cell that LT be a for human melanomas M. Leppla S.H. S. A. PubMed Scopus Google Scholar). This of LT to human melanomas was to a mutation in the melanomas G.R. P. S. S. J. E. Y. K. S. J. A. A. Stevens S. S. Cooper J. D. K. G. G. A. A. A. PubMed Scopus Google Scholar). BRAF is a kinase of in the of the G.R. P. S. S. J. E. Y. K. S. J. A. A. Stevens S. S. Cooper J. D. K. G. G. A. A. A. PubMed Scopus Google that of human melanomas and a of other human cancer a BRAF to mutation This mutation of a with a that the of and in the activating and the in the S. D. D. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). melanomas with the BRAF V600E mutation are on the activation of the for Thus, was shown that human cell with the BRAF mutation are to the mutation are R.J. S. Leppla S.H. Frankel Mol. Ther. PubMed Scopus Google Scholar). The of LT was in vivo R.J. Leppla S.H. Y. B. Frankel Clin. 12: PubMed Scopus Google Scholar). a virulence factor of B. anthracis, has in vivo toxicity and not be to use in human cancer M. D. Leppla S.H. J. Clin. 2003; PubMed Scopus Google Scholar). the of an and of LT be The for PA proteolytic activation on the target cell surface a to this protein to its on that are in tumor To this we generated PA that can be activated by matrix metalloproteinases S. S. Leppla S.H. Google Scholar). MMPs are by tumor and in cancer cell and M. PubMed Scopus Google Scholar). is MMPs are to only a of normal cells. Thus, in MMP-activated LT have to tumors. this we that the MMP-activated LT not only lower toxicity than wild-type LT to mice but also toxicity to human tumors in tumor This is in to the greater of MMP-activated PA protein in Moreover, we that the MMP-activated LT has potent anti-tumor activity not only to human melanomas with the BRAF V600E mutation but also to other tumor types, regardless of the BRAF mutation status. identified that this potent anti-tumor activity is because of the indirect targeting of tumor vasculature and angiogenic processes. and were as S. Leppla S.H. PubMed Scopus Google Scholar). and human cancer cells and and lung cells were in with as S. Bugge T.H. Leppla S.H. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Leppla S.H. Bugge T.H. S. A. 2003; PubMed Scopus Google Scholar). umbilical vascular endothelial cells and human microvascular endothelial cells were from and were in endothelial cell growth and were from and mice as PubMed Google Scholar). cells were well in of PA proteins, with or were to the cells. was with the toxins for as S. S. Leppla S.H. Google Scholar). 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Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). mice were two and by of were in the The were was with and the was with the were The of was by and the was as only a was and of the progress of were by an that was as to the treatment of the MMP-activated Anthrax to with the BRAF V600E is a mutated PA protein with the furin by an S. S. Leppla S.H. Google Scholar). To the in anti-tumor activity of the MMP-activated LT were on BRAF tumor cell from the cell PubMed Scopus Google and in with BRAF wild-type and that was to and colon cancer cells having the BRAF mutation with of wild-type LT for these cells the tumor cells the BRAF V600E mutation were to and These that not only human cells but also human colon cancer cells with the BRAF mutation are to and To the that the of the tumor cells the BRAF mutation to the anthrax lethal toxins is because of a of expression of PA receptors on these cells, the tumor cells were also with and is a fusion protein of and the catalytic of Pseudomonas exotoxin cells by ADP-ribosylation and inactivation of is the of the cell in a S. S. Leppla S.H. Google Scholar). and showed a potent and to the human cancer cells regardless of their BRAF that these tumor cells PA receptors and These that MMP-activated LT be a for tumor in of the MMP-activated Anthrax the toxicity of in vivo. were with a with for 2 of or A of of PA protein to was used in the on the that PA can and to of cells J. K. M. R.J. S. A. PubMed Scopus Google Scholar). mice of of but not of of mice of of and of 2 of of with vascular had been identified as in mice M. D. Leppla S.H. J. Clin. 2003; PubMed Scopus Google Scholar). the mice as many as of of All the mice with of and of and of toxicity Full of the mice with of of not Furthermore, not in and including and and and not These that the MMP-activated LT has lower in vivo toxicity than wild-type the for is that of is and vivo toxicity of anthrax lethal toxins to for not in a not MMP-activated Anthrax in the anti-tumor activity of in can be in we human tumor in mice human cells and cells, containing the BRAF V600E and human cell lung cells, the BRAF After these tumors were well the mice were with of of of of or the two human melanomas with the BRAF mutation were very to with tumor and of the tumors with the the mice because of tumor in with the of of the tumors we or of these tumors to wild-type LT also toxicity to carcinomas that not have the BRAF resulting in the of of the tumors showed that treatment tumor not in the tumors Furthermore, a assay that cells were in the synthesis in the tumors was even in with cancer cells These that the MMP-activated LT has potent anti-tumor activity not only to human melanomas with the BRAF mutation but also to human tumor that the BRAF the of in two tumor and lung These two tumors a to mice melanomas and lung carcinomas were with of of and These tumors were also susceptible to the engineered with the of and tumors with the toxin and of with carcinomas and melanomas are to in the in assay and not but in the suggest that the potent anti-tumor of the modified LT be targeting of the tumor shown used the toxic displayed potent anti-tumor than we compared their the human colon cancer in The mice were with of or of or of even the was to the of only showed a anti-tumor on was lower than that of of of showed toxicity to mice we not the wild-type LT in mice in the with the to the anti-tumor of the MMP-activated and of the MMP-activated that has in vivo anti-tumor activity than were first because showed or in toxicity than in the cancer cells that the proteolytic and the of on cell are for the uptake and of PA in the S. 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D. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. M. J. Y. PubMed Scopus Google Scholar, S. J. J. J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). To LT can expression of angiogenic factors by cancer cells, we human angiogenic factor with human cancer cells, and the The effects of LT treatment on expression of well angiogenic factors were these cancer cells showed that was the only factor by LT treatment in cell that the expression of by these cancer cells was not by LT treatment not These with the from a that LT can expression in S. A. J. PubMed Scopus Google suggest that many cell a for is well that an important in tumor and that has been as an target in tumor therapy in A. D. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. M. J. Y. PubMed Scopus Google Scholar). therefore the of LT on for the potent anti-tumor activity of To we a human fragment the an LT S. A. J. PubMed Scopus Google Scholar). 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J. 2003; Full Text Full Text PDF PubMed Scopus Google by mice with containing and the mice were with of or the and of in vivo angiogenesis These with that the potent anti-tumor activity of the MMP-activated LT is due largely to the indirect targeting of tumor vasculature and angiogenic processes. To this we used tumor cells that were deficient in anthrax toxin receptors S. Leppla S.H. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the anthrax toxin Chinese hamster ovary cell PA proteins S. Leppla S.H. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google was xenografted to and the mice were with or with the anthrax toxin cell tumors to treatment MMP-activated Anthrax but and are on Furthermore, tumor angiogenesis is to important of angiogenesis J. PubMed Scopus Google Scholar). is such that is with J. PubMed Scopus Google Scholar). To the effects of on were in The mice were with or and the was were in from and mice mice displayed a in the that treatment but not a The the and by other identified the BRAF V600E mutation as in of human melanomas and less in other cancer types, such as and of human G.R. P. S. S. J. E. Y. K. S. J. A. A. Stevens S. S. Cooper J. D. K. G. G. A. A. A. PubMed Scopus Google Scholar, 2004; PubMed Scopus Google Scholar). BRAF is of in the kinase and is a that the BRAF mutation is in cancer with activating G.R. P. S. S. J. E. Y. K. S. J. A. A. Stevens S. S. Cooper J. D. K. G. G. A. A. A. PubMed Scopus Google Scholar, 2004; PubMed Scopus Google Scholar). the and the BRAF that mutation is to the kinase the tumors with these are for on their and that tumor cells with the but not the are to inhibition A. G. A. Y. J. PubMed Scopus Google Scholar). is not that tumors with activating are less to because can also the to tumor J. T. S. D. J. PubMed Scopus Google Scholar). targeting of the be to tumors with the BRAF that can and other by enzymatic is selectively toxic to human cell having the BRAF mutation but not to with the R.J. S. Leppla S.H. Frankel Mol. Ther. PubMed Scopus Google Scholar). This LT toxicity to human melanomas with BRAF V600E was in an therapy of in mice R.J. Leppla S.H. Y. B. Frankel Clin. 12: PubMed Scopus Google Scholar). the that anthrax LT is an important virulence factor in anthrax and has toxicity to mice M. D. Leppla S.H. J. Clin. 2003; PubMed Scopus Google that wild-type LT not be for use in human To the of in vivo toxicity of LT its anti-tumor we an of the toxin be by the but is activated by including and MMPs are in tumor and S. S. Leppla S.H. Google Scholar, S. Kuremsky J.G. M. A. Bugge T.H. Leppla S.H. PubMed Scopus Google Scholar). showed that the cancer cells MMPs and are to Furthermore, the cancer cells with the BRAF mutation are susceptible to and to the cancer cells BRAF V600E are to the Moreover, in to cells, colon cancer cells with the BRAF mutation are also to the that the to the activating BRAF mutation is not cell has lower toxicity than wild-type toxin in the mice of of can only to of and even 2 of of These that of the normal expression of MMPs and that is than used in vivo. The first in this from the in vivo anti-tumor that has a potent anti-tumor activity not only human melanomas with BRAF V600E but also other human tumor types, including colon and lung carcinomas and regardless of their BRAF status. that this potent anti-tumor activity is due largely to targeting of tumor vasculature and angiogenic processes. showed the the of human endothelial cells toward a of and angiogenic an for tumor can angiogenesis in tumor are largely in tumors with in with with and anthrax toxin tumor are susceptible to and A. D. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google showed that activation of in human cancer cells in of leading to of and in growth factors and angiogenic factors to tumor angiogenesis and showed that inhibition of by a or of can the growth of tumor A. D. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google to the of and in To the anti-tumor of was because of its to expression of we and we these tumor with are still very susceptible to has been for 2 decades that the from mice and are to LT in that these can be by the toxin in J. Biol. Full Text PDF PubMed Google Scholar). the of the has been to the a protein in the PubMed Scopus Google Scholar). is to to LT is still the that the potent anti-tumor of is because of the toxicity of the toxin to because from the of mice used for tumor are to from mice are by from and mice be even in this is that not only lower in vivo toxicity but also anti-tumor than the wild-type toxin. This is due in to the greater and of in as compared with showed that binding to its PA be proteolytically on cell for formation and of the PA the S. Leppla S.H. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the of on cell are to largely the clearance of PA proteins from M. Leppla S.H. PubMed Scopus Google Scholar). The that furin is MMPs are to a of normal cells has a and A. G. A. Y. J. PubMed Scopus Google that a potent is in inhibition of the growth of human tumor containing the BRAF but has limited tumors the BRAF that the action of the is targeting of the cancer cells. to targeting the LT also has activity the other enzymatic of and and and F. Full Text Full Text PDF PubMed Scopus Google an for that has anti-tumor activity than to the tumors with the BRAF we that the tumors the including from human as well as and even from the cells, are susceptible to in this has an in that can be to cancer cells PA proteins S. Bugge T.H. Leppla S.H. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Leppla S.H. Bugge T.H. S. A. 2003; PubMed Scopus Google Scholar, S. Kuremsky J.G. M. A. Bugge T.H. Leppla S.H. PubMed Scopus Google Scholar). of its catalytic be potent than in targeting the has anti-tumor activity the wild-type toxin with to and because of its inactivation of the indirect targeting of tumor vasculature and lower targeting of normal that and compared with wild-type toxin. The modified protective antigen also a therefore clinical of the MMP-activated anthrax lethal toxin in cancer many tumor are to to but with tumors containing the BRAF mutation because of the toxicity of the toxin to the cancer cells. for protein and and for with