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Use of an optimised enzyme/prodrug combination for Clostridia directed enzyme prodrug therapy induces a significant growth delay in necrotic tumours

Cancer Gene Therapy volume 29, pages 178–188 (2022)Cite this article

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Abstract

Necrosis is a typical histological feature of solid tumours that provides a selective environment for growth of the non-pathogenic anaerobic bacterium Clostridium sporogenes. Modest anti-tumour activity as a single agent encouraged the use of C. sporogenes as a vector to express therapeutic genes selectively in tumour tissue, a concept termed Clostridium Directed Enzyme Prodrug Therapy (CDEPT). Here, we examine the ability of a recently identified Neisseria meningitidis type I nitroreductase (NmeNTR) to metabolise the prodrug PR-104A in an in vivo model of CDEPT. Human HCT116 colon cancer cells stably over-expressing NmeNTR demonstrated significant sensitivity to PR-104A, the imaging agent EF5, and several nitro(hetero)cyclic anti-infective compounds. Chemical induction of necrosis in human H1299 xenografts by the vascular disrupting agent vadimezan promoted colonisation by NmeNTR-expressing C. sporogenes, and efficacy studies demonstrated moderate but significant anti-tumour activity of spores when compared to untreated controls. Inclusion of the pre-prodrug PR-104 into the treatment schedule provided significant additional activity, indicating proof-of-principle. Successful preclinical evaluation of a transferable gene that enables metabolism of both PET imaging agents (for vector visualisation) and prodrugs (for conditional enhancement of efficacy) is an important step towards the prospect of CDEPT entering clinical evaluation.

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Fig. 1: Evaluation of prodrug activity in an E. coli SOS-R2 strain over-expressing NmeNTR and NfsB_Ec.
Fig. 2: NmeNTR can be stably expressed in HCT116 cells.
Fig. 3: NmeNTR demonstrates in vitro anti-proliferative activity with PR-104A in human cell lines.
Fig. 4: NmeNTR is a multi-functional reductase.
Fig. 5: Construction and in vitro characterisation of the NmeNTR-expressing C. sporogenes strain N2.
Fig. 6: Modulation of necrosis promotes colonisation of spores and produces significant anti-tumour efficacy.

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References

  1. Richards CH, Mohammed Z, Qayyum T, Horgan PG, McMillan DC. The prognostic value of histological tumour necrosis in solid organ malignant disease: a systematic review. Future Oncol. 2011;7:1223–35.

    Article  CAS  PubMed  Google Scholar 

  2. Mose JR, Mose G. Activity of Clostridium butyricum (M-55) and other non pathogenic clostridia against the Erlich carcinoma. Cancer Res. 1964;24:212–6.

    Google Scholar 

  3. Minton NP, Mauchline ML, Lemmon MJ, Brehm JK, Fox M, Michael NP, et al. Chemotherapeutic tumour targeting using clostridial spores. FEMS Microbiol Rev. 1995;17:357–64.

    Article  CAS  PubMed  Google Scholar 

  4. Liu SC, Minton NP, Giaccia AJ, Brown JM. Anticancer efficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis. Gene Ther. 2002;9:291–6.

    Article  CAS  PubMed  Google Scholar 

  5. Liu SC, Ahn GO, Kioi M, Dorie MJ, Patterson AV, Brown JM. Optimized clostridium-directed enzyme prodrug therapy improves the antitumor activity of the novel DNA cross-linking agent PR-104. Cancer Res. 2008;68:7995–8003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Theys J, Pennington O, Dubois L, Anlezark G, Vaughan T, Mengesha A, et al. Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo. Br J Cancer. 2006;95:1212–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Heap JT, Pennington OJ, Cartman ST, Minton NP. A modular system for Clostridium shuttle plasmids. J Microbiol Methods. 2009;78:79–85.

    Article  CAS  PubMed  Google Scholar 

  8. Keese P. Risks from GMOs due to horizontal gene transfer. Environ Biosaf Res. 2008;7:123–49.

    Article  CAS  Google Scholar 

  9. Heap JT, Ehsaan M, Cooksley CM, Ng YK, Cartman ST, Winzer K, et al. Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker. Nucleic Acids Res. 2012;40:e59.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Heap JT, Theys J, Ehsaan M, Kubiak AM, Dubois L, Paesmans K, et al. Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumours in vivo. Oncotarget. 2014;5:1761–9.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Helsby NA, Ferry DM, Patterson AV, Pullen SM, Wilson WR. 2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT. Br J Cancer. 2004;90:1084–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wilson WR, Pullen SM, Hogg A, Helsby NA, Hicks KO, Denny WA. Quantitation of bystander effects in nitroreductase suicide gene therapy using three-dimensional cell cultures. Cancer Res. 2002;62:1425–32.

    CAS  PubMed  Google Scholar 

  13. Patterson AV, Ferry DM, Edmunds SJ, Gu Y, Singleton RS, Patel K, et al. Mechanism of action and preclinical antitumor activity of the novel hypoxia-activated DNA crosslinking agent PR-104. Clin Cancer Res. 2007;13:3922–32.

    Article  CAS  PubMed  Google Scholar 

  14. Singleton DC, Li D, Bai SY, Syddall SP, Smaill JB, Shen Y, et al. The nitroreductase prodrug SN 28343 enhances the potency of systemically administered armed oncolytic adenovirus ONYX-411(NTR). Cancer Gene Ther. 2007;14:953–67.

    Article  CAS  PubMed  Google Scholar 

  15. Wilson WR, Hicks KO, Pullen SM, Ferry DM, Helsby NA, Patterson AV. Bystander effects of bioreductive drugs: potential for exploiting pathological tumor hypoxia with dinitrobenzamide mustards. Radiat Res. 2007;167:625–36.

    Article  CAS  PubMed  Google Scholar 

  16. Gu Y, Patterson AV, Atwell GJ, Chernikova SB, Brown JM, Thompson LH, et al. Roles of DNA repair and reductase activity in the cytotoxicity of the hypoxia-activated dinitrobenzamide mustard PR-104A. Mol Cancer Therapeutics. 2009;8:1714–23.

    Article  CAS  Google Scholar 

  17. Prosser GA, Copp JN, Mowday AM, Guise CP, Syddall SP, Williams EM, et al. Creation and screening of a multi-family bacterial oxidoreductase library to discover novel nitroreductases that efficiently activate the bioreductive prodrugs CB 1954 and PR-104A. Biochemical Pharmacol. 2013;85:1091–103.

    Article  CAS  Google Scholar 

  18. Yang S, Atwell GJ, Denny WA. Synthesis of asymmetric halomesylate mustards with aziridineethanol/alkali metal halides: application to an improved synthesis of the hypoxia prodrug PR-104. Tetrahedron. 2007;63:5470–6.

    Article  CAS  Google Scholar 

  19. Atwell GJ, Denny WA. Synthesis of 3H and 2H4-labelled versions of the hypoxia-activated pre-prodrug 2-[(2-bromoethyl)-2,4-dinitro-6-[[[2-(phosphonooxy)ethyl]amino]carbonyl]anilino]ethyl methanesulfonate (PR-104). J Label Compd Radiopharm. 2007;50:7–12.

  20. Atwell GJ, Yang S, Denny WA. An improved synthesis of 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Eur J Medicinal Chem. 2002;37:852–8.

    Article  Google Scholar 

  21. Su J, Guise CP, Wilson WR. FSL-61 is a 6-nitroquinolone fluorogenic probe for one-electron reductases in hypoxic cells. Biochemical J. 2013;452:79–86.

    Article  CAS  Google Scholar 

  22. Jin C-Z, Nagasawa H, Shimamura M, Uto Y, Inayama S, Takeuchi Y, et al. Angiogenesis inhibitor TX-1898: syntheses of the enantiomers of sterically diverse haloacetylcarbamoyl-2-nitroimidazole hypoxic cell radiosensitizers. Bioorg Med Chem. 2004;12:4917–27.

    Article  CAS  PubMed  Google Scholar 

  23. Copp JN, Mowday AM, Williams EM, Guise CP, Ashoorzadeh A, Sharrock AV, et al. Engineering a multifunctional nitroreductase for improved activation of prodrugs and PET probes for cancer gene therapy. Cell Chem Biol. 2017;24:391–403.

    Article  CAS  PubMed  Google Scholar 

  24. Prosser GA, Copp JN, Syddall SP, Williams EM, Smaill JB, Wilson WR, et al. Discovery and evaluation of Escherichia coli nitroreductases that activate the anticancer prodrug CB 1954. Biochem Pharmacol. 2010;79:678–87.

    Article  CAS  PubMed  Google Scholar 

  25. Helsby NA, Atwell GJ, Yang S. Aziridinyldinitrobenzamides: synthesis and structure-activity relationships for activation by E. coli nitroreductase. J Medicinal Chem. 2004;47:3295–307.

    Article  CAS  Google Scholar 

  26. Koch CJ. Importance of antibody concentration in the assessment of cellular hypoxia by flow cytometry: EF5 and pimonidazole. Radiat Res. 2008;169:677–88.

    Article  CAS  PubMed  Google Scholar 

  27. Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM, Scott JC, et al. The ClosTron: mutagenesis in Clostridium refined and streamlined. J Microbiol Methods. 2010;80:49–55.

    Article  CAS  PubMed  Google Scholar 

  28. Knox RJ, Boland MP, Friedlos F, Coles B, Southan C, Roberts JJ. The nitroreductase enzyme in Walker cells that activates 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is a form of NAD(P)H dehydrogenase (quinone) (EC 1.6.99.2). Biochem Pharm. 1988;37:4671–7.

    Article  CAS  PubMed  Google Scholar 

  29. Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP. The ClosTron: a universal gene knock-out system for the genus Clostridium. J Microbiol Methods. 2007;70:452–64.

    Article  CAS  PubMed  Google Scholar 

  30. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82.

    Article  CAS  PubMed  Google Scholar 

  31. Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, et al. New guidelines to evaluate the response to treatment in solid tumours. J Natl Cancer Inst. 2000;92:205–16.

    Article  CAS  PubMed  Google Scholar 

  32. Mowday AM, Copp JN, Syddall SP, Dubois LJ, Wang J, Lieuwes NG, et al. E. coli nitroreductase NfsA is a reporter gene for non-invasive PET imaging in cancer gene therapy applications. Theranostics. 2020;10:10548–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Serganova I, Blasberg RG. Molecular imaging with reporter genes: has its promise been delivered? J Nucl Med. 2019;60:1665–81.

    Article  CAS  PubMed  Google Scholar 

  34. Koch CJ, Hahn SM, Rockwell JrK, Covery JM, McKenna WG, Evans SM. Pharmacokinetics of EF5 [2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide] in human patients: implications for hypoxia measurements in vivo by 2-nitroimidazoles. Cancer Chemother Pharmacol. 2001;48:177–87.

  35. Baguley BC, Wilson WR. Potential of DMXAA combination therapy for solid tumors. Expert Rev Anticancer Ther. 2002;2:593–603.

    Article  CAS  PubMed  Google Scholar 

  36. Zwi LJ, Baguley BC, Gavin JB, Wilson WR. The morphological effects of the anti-tumor agents flavone acetic acid and 5,6-dimethyl xanthenone acetic acid on the colon 38 mouse tumor. Pathology (Phila, Pa). 1994;26:161–9.

    CAS  Google Scholar 

  37. Dang LH, Bettegowda C, Huso DL, Kinzler KW, Vogelstein B. Combination bacteriolytic therapy for the treatment of experimental tumors. Proc Natl Acad Sci (USA). 2001;98:15155–60.

    Article  CAS  Google Scholar 

  38. Theys J, Landuyt W, Nuyts S, van Mellaert L, Bosmans E, Rijnders A, et al. Improvement of Clostridium tumour targeting vectors evaluated in rat rhabdomyosarcomas. FEMS Immunol Med Microbiol. 2001;30:37–41.

    Article  CAS  PubMed  Google Scholar 

  39. Divine MR, Katiyar P, Kohlhofer U, Quintanilla-Martinez L, Pichler BJ, Disselhorst JA. A population-based gaussian mixture model incorporating 18F-FDG PET and diffusion-weighted MRI quantifies tumor tissue classes. J Nucl Med. 2016;57:473–9.

    Article  CAS  PubMed  Google Scholar 

  40. Beddy P, Genega EM, Ngo L, Hindman N, Wei J, Bullock A, et al. Tumor necrosis on magnetic resonance imaging correlates with aggressive histology and disease progression in clear cell renal cell carcinoma. Clin Genitourin Cancer. 2014;12:55–62.

    Article  PubMed  Google Scholar 

  41. Egeland TA, Gaustad JV, Galappathi K, Rofstad EK. Magnetic resonance imaging of tumor necrosis. Acta Oncol. 2011;50:427–34.

    Article  CAS  PubMed  Google Scholar 

  42. Mowday AM, Guise CP, Ackerley DF, Minton NP, Lambin P, Dubois L, et al. Advancing Clostridia to clinical trial: past lessons and recent progress. Cancers. 2016;8:63–76.

    Article  PubMed Central  Google Scholar 

  43. Xu K, Wang F, Pan X, Liu R, Ma J, Konga F, et al. High selectivity imaging of nitroreductase using a near-infrared fluorescence probe in hypoxic tumour. Chem Commun. 2013;49:2554–6.

    Article  CAS  Google Scholar 

  44. Li Z, He X, Wang Z, Yang R, Shi W, Ma H. In vivo imaging and detection of nitroreductase in zebrafish by a new near-infrared fluorescence on-off probe. Biosens Bioelectron. 2015;63:112–6.

    Article  CAS  PubMed  Google Scholar 

  45. Thorne SH, Barak Y, Liang W, Bachmann MH, Rao J, Contag CH, et al. CNOB/ChrR6, a new prodrug enzyme cancer chemotherapy. Mol Cancer Therapeutics. 2009;8:333–41.

    Article  CAS  Google Scholar 

  46. Ntziachristos V, Ripoli J, Weissleder R. Would near-infrared fluorescence signals propagate through large human organs for clinical studies? Opt Lett. 2002;27:527–9.

    PubMed  Google Scholar 

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Acknowledgements

This work was funded by the Health Research Council of New Zealand (Grant 14/289), the ZonMW TGO program (Grant 43400009), the Dutch Cancer Society (KWF Alpe d’HuZes Unieke Kansen #8025), the UK Biotechnology and Biological Sciences Research Council (BBSRC; grant number BB/L013940/1) and by a scholarship from the University of Auckland (awarded to AMM).

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Authors and Affiliations

  1. Auckland Cancer Society Research Centre, University of Auckland, Auckland, New Zealand

    Alexandra M. Mowday, Christopher P. Guise, Amir Ashoorzadeh, Jeff B. Smaill & Adam V. Patterson

  2. Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland, New Zealand

    Alexandra M. Mowday, Christopher P. Guise, Amir Ashoorzadeh, David F. Ackerley, Jeff B. Smaill & Adam V. Patterson

  3. The M-Lab, Department of Precision Medicine, Maastricht University, Maastricht, Netherlands

    Alexandra M. Mowday, Ludwig J. Dubois, Aleksandra M. Kubiak, Philippe Lambin & Jan Theys

  4. BBSRC/EPSRC Synthetic Biology Research Centre, University of Nottingham, Nottingham, UK

    Aleksandra M. Kubiak & Nigel P. Minton

  5. School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand

    Jasmine V. E. Chan-Hyams & David F. Ackerley

  6. NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham, UK

    Nigel P. Minton

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Contributions

AMM, AMK, CPG, DFA, PL, NPM, JBS and AVP conceived and designed the experiments; AMM, JVEC and AMK performed the experiments; AMM, LD, JVEC, AMK, JT and AVP analyzed the data; AA, JBS, NPM contributed reagents/materials/analysis tools; AMM, LD, JBS, JT and AVP wrote the paper; JVEC, AMK, CPG, AA, DFA, PL and NPM reviewed and edited the paper.

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Correspondence to Adam V. Patterson.

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Mowday, A.M., Dubois, L.J., Kubiak, A.M. et al. Use of an optimised enzyme/prodrug combination for Clostridia directed enzyme prodrug therapy induces a significant growth delay in necrotic tumours. Cancer Gene Ther 29, 178–188 (2022). https://doi.org/10.1038/s41417-021-00296-7

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