title: Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo.
abstract: Spores of some species of the strictly anaerobic bacteria Clostridium naturally target and partially lyse the hypoxic cores of tumors, which tend to be refractory to conventional therapies. The anti-tumor effect can be augmented by engineering strains to convert a non-toxic prodrug into a cytotoxic drug specifically at the tumor site by expressing a prodrug-converting enzyme (PCE). Safe doses of the favored prodrug CB1954 lead to peak concentrations of 6.3 µM in patient sera, but at these concentration(s) known nitroreductase (NTR) PCEs for this prodrug show low activity. Furthermore, efficacious and safe Clostridium strains that stably express a PCE have not been reported. Here we identify a novel nitroreductase from Neisseria meningitidis, NmeNTR, which is able to activate CB1954 at clinically-achievable serum concentrations. An NmeNTR expression cassette, which does not contain an antibiotic resistance marker, was stably localized to the chromosome of Clostridium sporogenes using a new integration method, and the strain was disabled for safety and containment by making it a uracil auxotroph. The efficacy of Clostridium-Directed Enzyme Prodrug Therapy (CDEPT) using this system was demonstrated in a mouse xenograft model of human colon carcinoma. Substantial tumor suppression was achieved, and several animals were cured. These encouraging data suggest that the novel enzyme and strain engineering approach represent a promising platform for the clinical development of CDEPT.
authors.affiliation: Clostridia Research Group, Centre for Biomolecular Sciences, School of Life Sciences, The University of Nottingham, University Park, Nottingham, UK.
authors.forename: John T
authors.initials: JT
authors.lastname: Heap
authors.forename: Jan
authors.initials: J
authors.lastname: Theys
authors.forename: Muhammad
authors.initials: M
authors.lastname: Ehsaan
authors.forename: Aleksandra M
authors.initials: AM
authors.lastname: Kubiak
authors.forename: Ludwig
authors.initials: L
authors.lastname: Dubois
authors.forename: Kim
authors.initials: K
authors.lastname: Paesmans
authors.forename: Lieve
authors.initials: L
authors.lastname: Van Mellaert
authors.forename: Richard
authors.initials: R
authors.lastname: Knox
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Kuehne
authors.forename: Phillipe
authors.initials: P
authors.lastname: Lambin
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Minton
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000097:blood / Q000378:metabolism*; D001388:Aziridines / Q000097:blood / Q000378:metabolism*; D001691:Biological Therapy / Q000009:adverse effects; D002277:Carcinoma / Q000628:therapy*; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D003110:Colonic Neoplasms / Q000628:therapy*; D051379:Mice; D009345:Neisseria meningitidis / Q000201:enzymology / Q000235:genetics; D009601:Nitroreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D030781:Organisms, Genetically Modified; D010957:Plasmids; D011355:Prodrugs / Q000378:metabolism; D015202:Protein Engineering; D013171:Spores, Bacterial / Q000201:enzymology*; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D001388:Aziridines; D011355:Prodrugs; C100099:tretazicar; D009601:Nitroreductases
references.citation: Biochem Pharmacol. 1992 Dec 15;44(12):2297-301
references.pmid: 1472095
references.citation: Proc Natl Acad Sci U S A. 2005 Jan 18;102(3):755-60
references.pmid: 15644448
references.citation: Nature. 2011 Aug 31;477(7362):99-102
references.pmid: 21886163
references.citation: J Microbiol Methods. 2009 Jul;78(1):79-85
references.pmid: 19445976
references.citation: Cancer Res. 2008 Oct 1;68(19):7995-8003
references.pmid: 18829557
references.citation: Gene Ther. 2005 Jun;12(11):911-7
references.pmid: 15690061
references.citation: Biochem Pharmacol. 2010 Mar 1;79(5):678-87
references.pmid: 19852945
references.citation: Biochem Pharmacol. 2009 Jan 1;77(1):21-9
references.pmid: 18840409
references.citation: Nat Rev Microbiol. 2003 Dec;1(3):237-42
references.pmid: 15035028
references.citation: FEMS Microbiol Rev. 1995 Oct;17(3):357-64
references.pmid: 7576773
references.citation: Biochem Pharmacol. 2010 Jan 15;79(2):102-11
references.pmid: 19665450
references.citation: Biochem Pharmacol. 2012 Sep 15;84(6):775-83
references.pmid: 22796568
references.citation: Proc Natl Acad Sci U S A. 1975 Jun;72(6):1981-4
references.pmid: 806076
references.citation: Gene Ther. 2002 Feb;9(4):291-6
references.pmid: 11896468
references.citation: ACS Synth Biol. 2012 Oct 19;1(10):465-470
references.pmid: 23097750
references.citation: Hum Gene Ther. 2002 Jan 1;13(1):3-13
references.pmid: 11779411
references.citation: J Microbiol Methods. 2010 Jan;80(1):49-55
references.pmid: 19891996
references.citation: J Clin Invest. 2008 Sep;118(9):3132-42
references.pmid: 18688285
references.citation: Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):15155-60
references.pmid: 11724950
references.citation: Cancer Res. 2003 Sep 1;63(17):5532-7
references.pmid: 14500391
references.citation: FEMS Microbiol Lett. 1994 Dec 1;124(2):195-202
references.pmid: 7813889
references.citation: Front Biosci. 2007 May 01;12:3880-91
references.pmid: 17485344
references.citation: J Microbiol Methods. 2007 Sep;70(3):452-64
references.pmid: 17658189
references.citation: Clin Cancer Res. 2001 Sep;7(9):2662-8
references.pmid: 11555577
references.citation: Microbiology (Reading). 2002 Jan;148(Pt 1):297-306
references.pmid: 11782522
references.citation: J Mol Biol. 2007 Apr 27;368(2):481-92
references.pmid: 17350040
references.citation: Bioorg Med Chem Lett. 2005 Aug 1;15(15):3623-6
references.pmid: 15979874
references.citation: Biochem Pharmacol. 1988 Dec 15;37(24):4661-9
references.pmid: 3202902
references.citation: Cancer Res. 1955 Aug;15(7):473-8
references.pmid: 13240693
references.citation: Nat Biotechnol. 2006 Dec;24(12):1573-80
references.pmid: 17115055
references.citation: Anaerobe. 1998 Aug;4(4):183-8
references.pmid: 16887640
references.citation: Toxicol Sci. 2005 Dec;88(2):562-75
references.pmid: 16162850
references.citation: Br J Cancer. 2009 Jun 16;100(12):1903-11
references.pmid: 19455141
references.citation: Br J Cancer. 2006 Nov 6;95(9):1212-9
references.pmid: 17024128
references.citation: Methods Mol Med. 2000;35:413-8
references.pmid: 21390820
references.citation: Gene Ther. 1997 Aug;4(8):791-6
references.pmid: 9338007
references.citation: Biochemistry. 2009 Aug 18;48(32):7665-72
references.pmid: 19580253
references.citation: Nucleic Acids Res. 2012 Apr;40(8):e59
references.pmid: 22259038
references.citation: Nat Rev Cancer. 2010 Nov;10(11):785-94
references.pmid: 20944664
references.citation: Proc Natl Acad Sci U S A. 2007 Jun 12;104(24):10170-4
references.pmid: 17548809
references.citation: Gene Ther. 1999 May;6(5):764-70
references.pmid: 10505099
references.citation: Annu Rev Genet. 2004;38:1-35
references.pmid: 15568970
references.citation: Br J Cancer. 2003 Sep 1;89(5):944-50
references.pmid: 12942130
references.citation: Gene Ther. 2005 Jun;12(11):902-10
references.pmid: 15690062
references.citation: Cancer Res. 1964 Feb;24:212-6
references.pmid: 14115686
references.citation: Biochem Pharmacol. 1992 Dec 15;44(12):2289-95
references.pmid: 1472094