WO2006103452A2 - Enzymes nitroreductases ameliorees - Google Patents

Enzymes nitroreductases ameliorees Download PDF

Info

Publication number
WO2006103452A2
WO2006103452A2 PCT/GB2006/001180 GB2006001180W WO2006103452A2 WO 2006103452 A2 WO2006103452 A2 WO 2006103452A2 GB 2006001180 W GB2006001180 W GB 2006001180W WO 2006103452 A2 WO2006103452 A2 WO 2006103452A2
Authority
WO
WIPO (PCT)
Prior art keywords
nitroreductase
vector
ntr
isolated polynucleotide
prodrug
Prior art date
Application number
PCT/GB2006/001180
Other languages
English (en)
Other versions
WO2006103452A3 (fr
Inventor
Peter Francis Searle
Original Assignee
Innovata Plc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Innovata Plc filed Critical Innovata Plc
Publication of WO2006103452A2 publication Critical patent/WO2006103452A2/fr
Publication of WO2006103452A3 publication Critical patent/WO2006103452A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)

Definitions

  • the present invention relates to mutated nitroreductase enzymes and the DNA encoding them, and their use in the conversion of prodrugs for the treatment of cancer.
  • GDEPT gene- directed enzyme prodrug therapy
  • VDEPT virus-directed enzyme prodrug therapy
  • An example of an enzyme/prod rug system is nitroreductase and the aziridinyl prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) (Knox et al 1988).
  • CB1954 5-(aziridin-1-yl)-2,4-dinitrobenzamide)
  • the Walker rat carcinoma cell line was particularly sensitive to CB1954, it was shown that this was due to the expression of the rat nitroreductase DT diaphorase.
  • CB 1954 is a poor substrate for the human form of this enzyme, human tumour cells are far less sensitive to CB 1954.
  • GDEPT was conceived as a way of introducing a suitable nitroreductase, preferably with greater activity against CB1954, in order to sensitise targeted cells.
  • the Escherichia coli nitroreductase (EC1.6.99.7, alternatively known as the oxygen-insensitive NAD(P)H nitroreductase or dihydropteridine reductase, and often abbreviated to NTR) encoded by the NFSB gene (alternatively known as NFNB, NFSI, or DPRA) has been widely used for this purpose (Reviewed in Grove et al, 1999).
  • NTR ⁇ /FSB-encoded nitroreductase
  • FMN flavin mononucleotide
  • NTR reduces one or other of the two nitro-groups of CB 1954 to give either the highly toxic 4-hydroxylamine derivative or the relatively less toxic 2-hydroxylamine.
  • 5-(aziridin-1-yl)-4-hydroxylamino-2- nitrobenzamide probably via a further toxic metabolite, becomes very genotoxic (Knox et al, 1991).
  • the exact nature of the lesion caused is unclear, but is unlike that caused by other agents.
  • a particularly high rate of inter-strand cross-linking occurs and the lesions seem to be poorly repaired, with the result that CB 1954 is an exceptionally affective anti-tumour agent (Friedlos et al, 1992).
  • NFSB NTR The structure of the NFSB NTR has been analysed by X-ray crystallography (Parkinson et al 2000, Lovering et al, 2001). Each monomer consists of 217 amino acids forming a four-stranded beta sheet (a fifth parallel strand is contributed by the other subunit) and ten ⁇ helices (A-K) and comprises a large hydrophobic core (residues 2-91 and 131-217), a two helix domain (E and F, residues 92-130) that protrudes from the core region, and an extensive dimer interface formed by parts of helices A, B, G, J and K. (NB: the domain assignments are from Lovering et al, and differ slightly from the earlier structure solved by Parkinson et al).
  • Residues in what Parkinson et al designated as Helix G have been identified as being in or near the active site and are important in determining substrate specificity.
  • Lovering et al assigns residues 110-131 to helix F and 135-157 to helix G.
  • residues in this region form part of the opening to the substrate- and cofactor- binding pocket and that phenylalanine 124 is particularly important
  • the NFSB NTR has sequence homology to a number of other enzymes, in particular FRase I, a flavin reductase enzyme from Vibrio fischeri (Zenno et al 1996).
  • FRase I a flavin reductase enzyme from Vibrio fischeri
  • Zenno et al generated a number of nfsb mutants that had greatly increased flavin reductase activity. These mutants all had substitutions of phenylalanine 124 (F124), a crucial position in the ⁇ G helix.
  • F124 mutants having substitutions with serine, alanine, threonine, leucine, valine, isoleucine, aspartate, glutamine, arginine and histidine were generated, all of which had substantially increased flavin reductase activity.
  • the nitroreductase activity of these mutants was either broadly similar or substantially reduced, as judged with nitrofurazone and nitrofurantoin as substrates.
  • the histidine mutant (F124H) had approximately double the wild-type activity for these substrates.
  • these disclosures give no information as to what the effects on other substrates, such as CB1954, might be.
  • mutations of the F124 position have, at best, an unpredictable effect on nitroreductase activity and, in general, a deleterious effect.
  • the aim of GDEPT is to obtain efficient conversion of a prodrug such as CB 1954 in target cells in order to kill not only NTR-expressing cells but also bystander tumour cells that may not have been successfully transfected or transduced. It is therefore desirable to have efficient delivery of the NTR-encoding DNA, prodrugs with as high a therapeutic index as possible, and a nitroreductase enzyme that is as efficient as possible in the conversion of CB1954 and other nitro-based prodrugs to toxic DNA cross-linking products. To address the latter, it is desirable to develop modified nitroreductase enzymes, since these would allow more efficient therapy and/or lower systemic doses of the prodrug. Although prodrugs are of relatively low toxicity in comparison with their activated derivatives, it is nevertheless desirable to reduce the chances of adverse effects by minimising the required dose.
  • references to 'cancer' and treatment of cancer equally apply to a range of neoplastic, hyperplastic or other proliferative disorders including, but not limited to: carcinomas, sarcomas, melanomas, lymphomas, leukaemias and other lymphoproliferative or myeloproliferative conditions, and benign hyperplasias, (such as benign prostatic enlargement).
  • the present invention is based on efforts to produce a nitroreductase with improved activity in the reduction of prodrugs, especially CB1954.
  • the invention provides mutants of the E. coli nitroreductase enzyme (EC1.6.99.7, alternatively known as the oxygen insensitive NAD(P)H nitroreductase or dihydropteridine reductase) encoded by the NFSB gene (alternatively known as NFNB, NFSI, or DPRA) that have significantly greater nitroreductase activity than the wild-type enzyme when assayed with CB1954.
  • NTR adenovirally-delivered NTR and CB1954 is currently in clinical trials, following separate trials of the prodrug and virus.
  • the best mutant obtained, T41Q/N71S/F124T had an IC 50 of just 3 ⁇ M CB1954, about 50-fold lower than wild type and 3-fold lower than the second-best mutant, T41 N/N71S/F124T.
  • the T41Q substitution had not been obtained as a double mutant and must be detrimental as a single substitution.
  • the triple mutants it was usually combined with N71S, which resulted in considerably greater sensitivity than the single clone in which it was paired with F70E, together with restricted range of F124 substitutions. These residues cannot contact each other within the active site, and are likely to act cooperatively in an unpredicted way to improve substrate binding.
  • the invention therefore discloses mutant E.
  • coli nitroreductase enzymes with point mutations at three or more positions as compared with the wild type sequence.
  • Such improved enzymes are especially useful in directed enzyme prodrug therapy.
  • a polynucleotide comprising a sequence encoding the improved nitroreductase, together with a promoter and such other regulatory elements required to express said encoded nitroreductase, may be included in a vector suitable for gene therapy.
  • a vector may be a plasmid vector, whether intended to replicate episomally, to be transiently expressed, or to integrate into the target cell genome.
  • regulatory elements operably linked to the encoded enzyme may be elements facilitating tissue-specific expression, such as locus control regions (see US 5,736,359, which is incorporated herein by reference, or EP 0 332667) elements facilitating activation of transcription in most or all tissues, such as ubiquitous chromatin opening elements (see WO 00/05393, US application 09/358082, incorporated herein by reference).
  • tissue-specific promoter, enhancer or LCR, or combination thereof may allow targeted expression of an operably-linked gene, such as one encoding a prodrug- converting enzyme, in cells of a particular tissue type.
  • tumour cells may be targeted in a similar way, using promoters that allow expression only in, for example, foetal tissue and certain tumour types.
  • Use of such systems helps to prevent expression of therapeutic genes, such as prodrug-converting enzymes, in healthy tissue and so minimises adverse side-effects.
  • the vector may be administered to the patient systemically (parenterally or enterally), regionally (for instance by perfusion of an isolated limb, or peritoneal infusion), or locally as, for example, a direct intradermal, intramuscular, intraperitoneal, intracranial or intratumoral injection.
  • a suitable prodrug is administered, either locally (for instance around a tumour), regionally (for instance by perfusion of an isolated limb, or peritoneal infusion) or systemically.
  • any prodrug that is capable of being activated by means of reduction and, in particular reduction of nitro-groups may be suitable.
  • Such compounds include nitrobenzamides, in particular nitro- and dinitrobenzamide aziridines and mustards.
  • dinitrobenzamide aziridine 5- (aziridin-1-yl)-2, 4-dinitrobenzamide (CB1954) and the dinitrobenzamide mustard 5-[N, N-jb/s (2-chloroethyl) amino]-2, 4-dinitrobenzamide (SN23862), and functional and structural analogues thereof.
  • the recombinant mutant nitroreductase is encoded by a mutated equivalent of the wild-type E. coli NFSB gene.
  • the recombinant mutant nitroreductase is encoded by structurally homologous gene from another genus such as from Salmonella or Enterobacter, or from another species, such as the Salmonella typhimurium NFNB gene, or the Enterobacter cloacae NFNB gene.
  • the invention provides a recombinant mutant nitroreductase with an increased nitroreductase activity for CB1954 compared to the wild-type enzyme, encoded by a mutated equivalent of the E.coli NFSB gene (SEQ ID NO: 1), characterised in that said nitroreductase comprises substitutions at three or more amino acid positions.
  • the mutant comprises a substitution of threonine 41 , and in preferred embodiments threonine 41 is substituted by either glutamine or asparagine.
  • the mutated nitroreductase comprises a substitution of asparagine 71 , preferably by serine.
  • the mutated nitroreductase comprises a substitution of phenylalanine 124, preferably by threonine.
  • the mutant nitroreductase comprises substitutions of threonine 41 and asparagine 71 and phenylalanine 124.
  • threonine 41 is substituted by either glutamine or asparagine
  • asparagine 71 is substituted by serine
  • phenylalanine 124 is substituted by either threonine or asparagine.
  • vectors comprising isolated polynucleotides encoding one or more of the above-disclosed recombinant mutant nitroreductases.
  • these vectors may be replicating or non- replicating, episomal or integrating, designed for use in prokaryotic or eukaryotic cells. They may be expression vectors providing ubiquitous or tissue-specific expression of the encoded nitroreductase, which may be operably-linked to suitable promoters and other elements required for appropriate expression, such as locus control regions (LCRs) or ubiquitous chromatin opening elements (UCOEs) comprising extended, methylation-free CpG islands.
  • LCRs locus control regions
  • UCOEs ubiquitous chromatin opening elements
  • the nitroreductase is preferentially expressed in tumours.
  • the vector comprises a TCF-responsive element operably linked to a polynucleotide encoding nitroreductase. Also provided is a method of preparing such a vector.
  • said vector is a virus, and most preferably it is an adenovirus.
  • adenovirus vectors comprising a TCF-responsive tumour-selective promoter element operably linked to a nitroreductase gene is described in International application number PCT/GB01/00856, the whole of which is incorporated herein by reference. A copy of GB 01/00856 is filed with this application and its content is included in the present application but the copy is not included in the published specification of this application.
  • the vector may be any vector capable of transferring DNA to a cell.
  • the vector is an integrating vector or an episomal vector.
  • Preferred integrating vectors include recombinant retroviral vectors.
  • a recombinant retroviral vector will include DNA of at least a portion of a retroviral genome which portion is capable of infecting the target cells.
  • the term "infection" is used to mean the process by which a virus transfers genetic material to its host or target cell.
  • the retrovirus used in the construction of a vector of the invention is also rendered replication-defective to remove the effect of viral replication of the target cells. In such cases, the replication-defective viral genome can be packaged by a helper virus in accordance with conventional techniques.
  • any retrovirus meeting the above criteria of infectiousness and capability of functional gene transfer can be employed in the practice of the invention.
  • Suitable retroviral vectors include but are not limited to pLJ, pZip, pWe and pEM, well known to those of skill in the art.
  • Suitable packaging virus lines for replication- defective retroviruses include, for example, ⁇ Crip, ⁇ Cre, ⁇ 2 and ⁇ Am. Lentiviral vectors are particularly preferred.
  • vectors useful in the present invention include adenovirus, adeno- associated virus, SV40 virus, vaccinia virus, HSV and poxvirus vectors.
  • a preferred vector is the adenovirus.
  • Adenovirus vectors are well known to those skilled in the art and have been used to deliver genes to numerous cell types, including airway epithelium, skeletal muscle, liver, brain and skin (Hitt et a/ (1997); Anderson (1998)).
  • a further preferred vector is the adeno-associated (AAV) vector.
  • AAV vectors are well known to those skilled in the art and have been used to stably transduce human T-lymphocytes, fibroblasts, nasal polyp, skeletal muscle, brain, erythroid and haematopoietic stem cells for gene therapy applications (Philip et al (1994); Russell et al (1994); Flotte et al (1993); Walsh et al (1994); Miller et al (1994); Emerson (1996)).
  • International Patent Application WO 91/18088 describes specific AAV based vectors.
  • Preferred episomal vectors include transient non-replicating episomal vectors and self-replicating episomal vectors with functions derived from viral origins of replication such as those from EBV, human papovavirus (BK) and BPV-1.
  • viral origins of replication such as those from EBV, human papovavirus (BK) and BPV-1.
  • BK human papovavirus
  • BPV-1 BPV-1.
  • Mammalian artificial chromosomes can also be used as vectors in the present invention.
  • the use of mammalian artificial chromosomes is discussed by Calos (1996).
  • the vector of the present invention is a plasmid.
  • the plasmid may be a non-replicating, non-integrating plasmid.
  • plasmid refers to any nucleic acid encoding an expressible gene and includes linear or circular nucleic acids and double or single stranded nucleic acids.
  • the nucleic acid can be DNA or RNA and may comprise modified nucleotides or ribonucleotides, and may be chemically modified by such means as methylation or the inclusion of protecting groups or cap- or tail structures.
  • a non-replicating, non-integrating plasmid is a nucleic acid which when transfected into a host cell does not replicate and does not specifically integrate into the host cell's genome (i.e. does not integrate at high frequencies and does not integrate at specific sites).
  • Replicating plasmids can be identified using standard assays including the standard replication assay of Ustav et al (1991).
  • the present invention also provides a host cell transfected with the vector of the present invention.
  • the host cell may be any mammalian cell.
  • the host cell is a rodent or mammalian cell. Most preferably it is a human cell.
  • the host cell may be a bacterial cell, and such cells may be used for in vivo delivery to a mammal or for targeted delivery to a specific tissue or tumour (Theys et al, 2003, Curr Gene Ther 3: 207; Theys et al, 2001 , Cancer Gene Ther 8: 294).
  • nucleic acid condensing agents include the use of nucleic acid condensing agents, electroporation, complexing with asbestos, polybrene, DEAE cellulose, Dextran, liposomes, cationic liposomes, lipopolyamines, polyornithine, particle bombardment and direct microinjection (reviewed by Kucherlapati and Skoultchi (1984); Keown ef a/ (1990)).
  • a vector of the invention may be delivered to a host cell non-specifically or specifically (i.e., to a designated subset of host cells) via a viral or non-viral means of delivery.
  • Preferred delivery methods of viral origin include viral particle-producing packaging cell lines as transfection recipients for the vector of the present invention into which viral packaging signals have been engineered, such as those of adenovirus, herpes viruses and papovaviruses.
  • Preferred non- viral based gene delivery means and methods may also be used in the invention and include direct naked nucleic acid injection, nucleic acid condensing peptides and non-peptides, cationic liposomes and encapsulation in liposomes.
  • Nucleic acid condensing agents useful in the invention include spermine, spermine derivatives, histones, cationic peptides, cationic non-peptides such as polyethyleneimine (PEI) and polylysine.
  • 'Spermine derivatives' refers to analogues and derivatives of spermine and include compounds as set forth in International Patent Application WO 93/18759 (published September 30, 1993).
  • Disulphide bonds have been used to link the peptidic components of a delivery vehicle (Cotten et al., Meth. Enzymol. 217:618-644 (1992)); see also, Trubetskoy et al. (supra).
  • Delivery vehicles for delivery of DNA constructs to cells include D N A/poly-cation complexes which are specific for a cell surface receptor, as described in, for example, Wu and Wu (1988); Wilson et al (1992); and U.S. Patent No. 5,166,320).
  • nucleic acid condensing peptides which are particularly useful for condensing the vector and delivering the vector to a cell, are described in International Patent Application WO 96/41606.
  • Functional groups may be bound to peptides useful for delivery of a vector according to the invention, as described in WO 96/41606. These functional groups may include a ligand that targets a specific cell-type such as a monoclonal antibody, insulin, transferrin, asialoglycoprotein, or a sugar. The ligand thus may target cells in a non-specific manner or in a specific manner that is restricted with respect to cell type.
  • the functional groups also may comprise a lipid, such as palmitoyl, oleyl, or stearoyl; a neutral hydrophilic polymer such as polyethylene glycol (PEG), or polyvinylpyrrolidine (PVP); a fusogenic peptide such as the HA peptide of influenza virus; or a recombinase or an integrase.
  • the functional group also may comprise an intracellular trafficking protein such as a nuclear localisation sequence (NLS), an endosome escape signal such as a membrane disruptive peptide, or a signal directing a protein directly to the cytoplasm.
  • NLS nuclear localisation sequence
  • endosome escape signal such as a membrane disruptive peptide
  • a host cell comprising a polynucleotide encoding a recombinant mutant nitroreductase of the invention, or a host cell comprising a vector comprising such a polynucleotide.
  • a host cell may be a bacterial cell used to grow, manufacture, screen and test said vector, or a eukaryotic cell, preferably a mammalian cell and most preferably a human cell, in which the encoded nitroreductase is expressed.
  • the invention also provides a recombinant mutated nitroreductase as disclosed above, or a polynucleotide or vector encoding it, or a host cell containing such a polynucleotide or vector, for use as a medicament.
  • a medicament is of use in the treatment of cancer, more preferably by the conversion of a prodrug to an active cytotoxic compound, and further preferably the prodrug to be converted to an active cytotoxic compound is a nitrobenzamide aziridine or mustard, and most preferably it is CB1954.
  • any of the above-disclosed recombinant mutant nitroreductases and polynucleotides encoding them for the manufacture of a medicament, or in a process of the manufacture of a medicament is disclosed.
  • said medicament is for enzyme prodrug therapy.
  • Said medicament may take the form of naked DNA, a DNA-peptide, DNA-lipid or DNA-polymer conjugate or complex, or viral vector, comprising a polynucleotide encoding a recombinant mutant nitroreductase operably linked to a promoter with or without further elements such as enhancers and LCRs so arranged as to permit efficient tissue-specific expression of said nitroreductase in the appropriate cells following administration and transfection of said cells.
  • said medicament may comprise such a DNA-peptide, DNA-lipid or DNA-polymer conjugate or complex, or viral vector comprising a targeting moiety, such as an antibody or fragment thereof, or a peptide or carbohydrate ligand capable of binding specifically to a suitable cell surface receptor or other structure so as to allow efficient targeting to appropriate cell types.
  • a targeting moiety such as an antibody or fragment thereof, or a peptide or carbohydrate ligand capable of binding specifically to a suitable cell surface receptor or other structure so as to allow efficient targeting to appropriate cell types.
  • composition comprising any one of the above-disclosed recombinant mutant nitroreductases or polynucleotides encoding them, or viral or non-viral vectors or host cells comprising such polynucleotides, in an acceptable diluent or excipient.
  • the invention further provides an isolated polynucleotide encoding a nitroreductase of the invention, or a vector comprising such a polynucleotide, or a host cell comprising either said polynucleotide or vector for use in gene therapy.
  • gene therapy is of use in treating cancer.
  • a recombinant nitroreductase to aid in the design of, or screening for improved prodrugs.
  • Such a use comprises contacting said nitroreductase with candidate prodrugs and chemically measuring the kinetics of conversion to a reduced product.
  • an in vitro assay may be used where the ability of a disclosed recombinant mutant nitroreductase to convert candidate prodrugs to cytotoxic products is assayed by the inhibition of growth of bacterial host cells in the presence of various concentrations said prodrugs, or by the killing of eukaryotic cells cultured in the presence of various concentrations of said prodrugs.
  • Also provided is a method of treating cancer in a mammalian subject comprising administering any of the isolated polynucleotides or vectors described above, allowing a suitable time for expression of the encoded nitroreductase to occur, and administering a prodrug capable of being activated by said expressed nitroreductase.
  • Table 2 NTR mutants harvested after 10 rounds of selection.
  • Figure 1 Increasing doses of CB1954 trigger phage expressing NTR into the lytic cycle of replication. Cultures of lysogens lacking NTR and lysogens expressing either WT NTR or F124N NTR were exposed to a range of CB1954 concentrations (0-500 ⁇ M) for 15 minutes. Following removal of prodrug, cultures were incubated for a further hour to allow for lytic replication. Cultures were then assayed to determine both the number of viable cells remaining and the phage titre.
  • Figure 1A effect of increasing prodrug concentration on cell viability.
  • Figure 1B effect of increasing prodrug concentration on phage release.
  • Lysogens were generated from mixtures of empty vector phage (90%) and NTR- expressing phage (10%). Selection was carried out in the presence of 100 ⁇ M CB1954 for 15 minutes alongside no prodrug controls. A further incubation of 60 minutes allowed time for lytic replication before harvesting phage. Grids of 50 plaques were plated out on an E.coli lawn and blot analysis using a NTR DNA probe used to determine the percentage of NTR-expressing phage. Strips on the left side of the blots show 5 control plaques of NTR + and NTR - phage in the order + - + - + (top to bottom).
  • Figures 2A and B show plaques from mixtures containing F124N NTR
  • C and D show plaques from mixtures containing WT NTR.
  • No prodrug controls with no amplification of NTR-positive plaques are shown in A and C (8% and 6% NTR-positive plaques respectively).
  • Selection at 100 ⁇ M CB1954 is shown in B and D (with 78% and 18% NTR-positive plaques respectively).
  • Figure 3 Bacterial colony forming assays of test libraries. Colony forming assays carried out on cultures of lysogens during the selection rounds. Graphs show percentage survival of lysogens from each library at a range of CB1954 concentrations.
  • Figure 3A survival curves for the F124 library.
  • Figure 3B survival curves for the N71 library.
  • Figure 4 Bacterial colony forming assays of multiple mutant library.
  • Figure 4A percentage survival of E.coli lysogens expressing NTR mutants with multiple mutations at a range of CB1954 concentrations.
  • Figure 4B percentage survival of E.coli lysogens expressing selected NTR mutants identified after several rounds of selection, at a range of CB1954 concentrations.
  • Lysogens were grown in LB/kanamycin. Expression of NTR from the ptac promoter was induced through addition of 0.1mM IPTG at an OD600 of 0.1 and growth continued to an OD600 of 0.5. 1ml of culture was added to 4ml of LB/ kanamycin containing final concentrations of 0.1mM IPTG, 5OmM Tris (pH 7.5) and 0-500 ⁇ M CB1954. After 15 minutes incubation 0.01 ml of culture was added to 10ml fresh LB to dilute out the CB1954. After 1hour incubation the phage titre was determined by means of a plaque assay.
  • Plaques obtained from phage titre plates were picked and spotted onto a fresh E.coli lawn to create a grid of plaques for each set of experimental conditions. Plaques were transferred to nitrocellulose and a NTR DNA probe was used for the detection of NTR-encoding phage plaques. Detection was carried out with the Amersham ECL ® direct labelling kit according to the manufacturer's instructions.
  • the generated library contained up to 1.05x10 6 different altered codon combinations encoding for up to 3.2x10 4 different NTR mutants. After packaging, 6.8x10 5 phage particles were recovered. The library was then amplified up giving 4ml of a 9x10 8 pfu/ml stock used for the first round of selection. 1x10 8 pfu of phage was used for the first round of selection, this would equate to the generation of around 1x10 6 lysogens. Subsequent rounds of selection used between 1x10 7 and 1x10 8 pfu of phage depending on the amount harvested between each round.
  • Phage libraries containing modified NTR were used to infect E. coli UT5600 cells. Lysogens were selected by addition of kanamycin. Expression of NTR was induced through addition of 0.1mM IPTG at an OD600 of 0.1 and growth continued to an OD600 of 0.5. 1ml of culture was added to 4ml of LB/kanamycin containing final concentrations of 0.1mM IPTG, 5OmM Tris (pH 7.5) and 0.03 - 0.1 imM CB1954. Prodrug was diluted out through addition of 45ml LB and centrifugation of bacteria at 2,500rpm.
  • Lysogen pellets were resuspended in 5ml fresh LB and incubated for one hour following which phage were harvested (usually by filtration through a 0.2 ⁇ m filter, to remove remaining lysogens and bacterial debris) and analysed. Harvested phage was used to infect fresh UT5600 cells for subsequent rounds of selection. During the selection process the overall sensitivity of the population to CB1954 was determined through use of E. coli colony forming assays.
  • Lysogens grown to an OD600 of 0.5 were diluted to 1000 cell/ml and 0.1 ml were spread onto a series if Tris-buffered LB/kanamycin plates containing 0.1mM IPTG and 0 - 0.4 or 0.5 mM CB1954.
  • Plaques obtained through selection with CB1954 were picked for analysis. Primers binding to the lambda genome flanking the NTR insert were used to amplify the NTR DNA for each plaque; sequencing analysis was used to determine the presence of mutations within this sequence. Phage shown to contain novel NTR variants were used to generate lysogens and their sensitivity to CB1954 was determined through a lysogen CB1954 sensitivity assay (Grove et al (2003).
  • Lysogens that showed the greatest sensitivity were analysed further in an E. coli colony forming assay to determine an IC50 value.
  • the titre of NTR-expressing phage was similar to the empty vector immediately following the exposure to prodrug, but increased more rapidly over the following 60 minutes, to reach a 28-fold excess over empty vector. This confirmed that the NTR-dependent activation of CB1954 led to activation of the lambda lytic cycle, leading to phage release on the expected timescale.
  • the phage titres from the cultures without CB1954 treatment were 5.1 x 10 4 and 4.1 x 10 4 pfu/ml for the WT and F124N mixtures respectively, and the proportion of NTR-containing phage recovered (3 - 4 out of 50 plaques, Fig. 2a, c) was close to the expected 10% as present in the original phage mixtures.
  • This concentration of CB 1954 is suboptimal for induction of lysogens expressing WT NTR.
  • NTR genes from a number of phage harvested after 7 and 10 rounds of selection were sequenced and the amino acid substitutions relative to WT NTR are listed in Tables 1 and 2.
  • Figure 4b compares the plating efficiency of E. coli lysogens expressing either WT NTR, or examples of the selected NTR mutants, at a range of concentrations of CB1954, demonstrating that the enhanced sensitisation to the prodrug conferred by these mutants.
  • NTR mutants were analysed by replica plating at different concentrations of CB1954 to estimate sensitivity to CB1954.
  • the only single mutant isolated was F124N, in two separate clones after 7 rounds of selection. This showed significant growth reduction at 30 ⁇ M CB1954, and its IC 50 was previously determined as 25 ⁇ M.
  • Double and triple mutants made up 37 and 79 of the analysed clones respectively, and there were 10 quadruple mutants (Tables 1 & 2).
  • T41 L/N71S T41 L/N71S
  • S40A/F124M S40A/F124K
  • N71S/F124N T41L/N71S was especially noteworthy, as when tested in SKOV3 human ovarian carcinoma cells it required ⁇ 30-fold fewer adenovirus particles expressing the enzyme to achieve equivalent sensitisation to CB1954, compared with WT NTR.
  • T41/N71/F124 triple mutants many incorporated T41Q, a substitution which was not selected as a double mutant and must be detrimental as a single mutant since it was not identified by Grove et al.
  • T41Q/N71S/F124T was the best mutant identified, with an IC 50 of ⁇ 3 ⁇ M CB 1954.
  • Other noteworthy triple mutants include T41Q/N71S/F124N (IC 50 13 ⁇ M), T41N/N71S/F124T (IC 50 9 ⁇ M) and T41N/N71S/F124N (IC 50 12 ⁇ M CB1954).
  • the quadruple mutants each incorporated one or two mutations at non-targeted sites, presumably due to PCR errors. The best of these was D17N/T41 N/N71S/F124T with an IC 50 of 12 ⁇ M CB1954, less good than the corresponding T41N/N71S/F124T triple mutant (IC 50 9 ⁇ M).

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne de nouveaux mutants de nitroréductase présentant une activité de conversion améliorée pour le promédicament aziridinyl CB1954 (5-(aziridine-1-yl)-2,4-dinitrobenzamide). Les mutants ont des mutations ponctuelles à trois ou plusieurs sites, comparativement au E coli nitroréductase type sauvage, et sont utilisés pour une thérapie à promédicament d'enzyme à orientation virale ou génique, pour le traitement de maladies telles que le cancer. L'invention concerne en outre des polynucléotides et des vecteurs codant les nitroréductases améliorées de l'invention.
PCT/GB2006/001180 2005-04-01 2006-03-30 Enzymes nitroreductases ameliorees WO2006103452A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0506642A GB0506642D0 (en) 2005-04-01 2005-04-01 Improved nitroreductase enzymes
GB0506642.8 2005-04-01

Publications (2)

Publication Number Publication Date
WO2006103452A2 true WO2006103452A2 (fr) 2006-10-05
WO2006103452A3 WO2006103452A3 (fr) 2006-12-14

Family

ID=34586548

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2006/001180 WO2006103452A2 (fr) 2005-04-01 2006-03-30 Enzymes nitroreductases ameliorees

Country Status (2)

Country Link
GB (1) GB0506642D0 (fr)
WO (1) WO2006103452A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010048976A1 (fr) * 2008-10-31 2010-05-06 Universita' Degli Studi Di Pavia Nitroréductase nfnb provenant de mycobacterium smegmatis
WO2011026898A3 (fr) * 2009-09-02 2011-11-10 Bangor University Système d'activation de médicament
WO2015075475A1 (fr) * 2013-11-22 2015-05-28 The University Of Nottingham Traitement contre le cancer
US10357577B2 (en) 2010-07-16 2019-07-23 Auckland Uniservices Limited Bacterial nitroreductase enzymes and methods relating thereto
EP4299736A2 (fr) 2022-04-15 2024-01-03 Vilnius University Hydrolases et leurs utilisations

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003018788A2 (fr) * 2001-08-21 2003-03-06 Ml Laboratories Plc Enzymes de nitroreductase ameliorees

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003018788A2 (fr) * 2001-08-21 2003-03-06 Ml Laboratories Plc Enzymes de nitroreductase ameliorees

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GROVE JANE I ET AL: "Generation of Escherichia coli nitroreductase mutants conferring improved cell sensitization to the prodrug CB1954." CANCER RESEARCH, vol. 63, no. 17, 1 September 2003 (2003-09-01), pages 5532-5537, XP002399200 ISSN: 0008-5472 cited in the application *
SEARLE PETER F ET AL: "Nitroreductase: A prodrug-activating enzyme for cancer gene therapy" CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, vol. 31, no. 11, November 2004 (2004-11), pages 811-816, XP002399201 ISSN: 0305-1870 cited in the application *
ZENNO S ET AL: "Conversion of NfsB, a minor Escherichia coli nitroreductase, to a flavin reductase similar in biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri, by a single amino acid substitution" JOURNAL OF BACTERIOLOGY, WASHINGTON, DC, US, vol. 178, no. 15, August 1996 (1996-08), pages 4731-4733, XP002227224 ISSN: 0021-9193 cited in the application *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010048976A1 (fr) * 2008-10-31 2010-05-06 Universita' Degli Studi Di Pavia Nitroréductase nfnb provenant de mycobacterium smegmatis
WO2011026898A3 (fr) * 2009-09-02 2011-11-10 Bangor University Système d'activation de médicament
US9089613B2 (en) 2009-09-02 2015-07-28 Bangor University Drug activation system
US10357577B2 (en) 2010-07-16 2019-07-23 Auckland Uniservices Limited Bacterial nitroreductase enzymes and methods relating thereto
WO2015075475A1 (fr) * 2013-11-22 2015-05-28 The University Of Nottingham Traitement contre le cancer
US10183062B2 (en) 2013-11-22 2019-01-22 The University Of Nottingham Treatment for cancer
EP4299736A2 (fr) 2022-04-15 2024-01-03 Vilnius University Hydrolases et leurs utilisations

Also Published As

Publication number Publication date
GB0506642D0 (en) 2005-05-11
WO2006103452A3 (fr) 2006-12-14

Similar Documents

Publication Publication Date Title
Bridgewater et al. Expression of the bacterial nitroreductase enzyme in mammalian cells renders them selectively sensitive to killing by the prodrug CB1954
US6207150B1 (en) Variants of thymidine kinase, nucleic acids encoding them, and methods of using them
KR20200083550A (ko) ACE-tRNA에 의한 유전자 재지정을 통해 정지 코돈을 구조하는 방법
US6642043B1 (en) Double mutants of dihydrofolate reductase and methods of using same
US20060034817A1 (en) Cloning, overexpression and therapeutic use of bioactive histidine ammonia lyase
US20200216818A1 (en) Vaccinia virus for gene-directed enzyme prodrug therapy
WO2006103452A2 (fr) Enzymes nitroreductases ameliorees
AU2009281723C1 (en) Purine nucleoside phosphorylase as enzymatic activator of nucleoside prodrugs
CA2373121A1 (fr) Diffusion de sequences de transposon integratreur par des vecteurs
US6518062B1 (en) Enzyme combinations for destroying proliferative cells
JP5091669B2 (ja) チミジンキナーゼ
CA2458226A1 (fr) Enzymes de nitroreductase ameliorees
CA2500336A1 (fr) Immunotherapie amelioree
AU2002330602A1 (en) Improved nitroreductase enzymes
JP2003530855A (ja) 遺伝子治療のためのベクター
Black Enzyme and pathway engineering for suicide gene therapy
MXPA98007181A (en) Combinations of enzymes for the destruction of proliferati cells
Tzeng et al. Oxide Synthase Expression

Legal Events

Date Code Title Description
NENP Non-entry into the national phase in:

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

NENP Non-entry into the national phase in:

Ref country code: RU

WWW Wipo information: withdrawn in national office

Country of ref document: RU

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 06726586

Country of ref document: EP

Kind code of ref document: A2

122 Ep: pct application non-entry in european phase

Ref document number: 06726586

Country of ref document: EP

Kind code of ref document: A2

WWW Wipo information: withdrawn in national office

Ref document number: 6726586

Country of ref document: EP