AU2451100A - Nitroreductase enzymes - Google Patents

Description

WO 00/47725 PCT/GBOO/00431 NITROREDUCTASE ENZYMES The present invention relates to polypeptides and proteins having nitroreductase activity, to DNA and genes encoding these nitroreductases 5 and to methods of obtaining such enzymes, DNA and genes. A number of cancer therapies are based upon or exploit the conversion of a non-toxic prodrug into a toxic derivative. 10 One example concerns the monofunctional alkylating agent CB1 954, which exhibits extreme toxicity towards the Walker 256 rat carcinoma as a result of the presence of a DT-diaphorase enzyme (DTD) which reduces the 4 nitro group of CB1 954 to give a highly cytotoxic 4-hydroxylamine (4HX) derivative. CB1 954 does not have the same effect on human carcinomas 15 because human cells lack this enzyme but would be effective against human tumours if an enzyme such as DTD were externally supplied, e.g. in a Directed-Enzyme Prodrug Therapy (DEPT). The rat DTD, however, has a relatively poor specific activity for CB1 954. The E.co/i B nitroreductase enzyme (NfnB) was isolated as a more effective alternative and is the 20 subject of EP-A-0540263. It exhibits a higher specific activity for CB1 954, compared with the rat enzyme and is, therefore, currently the preferred enzyme in anti-cancer DEPT strategies. Whilst the known E. co/i enzyme receives widespread attention from cancer 25 biologists seeking to develop gene based DEPT strategies, it has a number of drawbacks. These mostly relate to its activity against the preferred prodrug, CB1 954 - it has a relatively high Km and low Kcat and converts CB1 954 into equimolar amounts of a relatively innocuous 2-hydroxylamino derivative (2HX) in addition to the highly cytotoxic 4-hydroxylamino species 30 (4HX). In relation to this specific prodrug, it is hence desired to provide an WO 00/47725 PCT/GBOO/00431 -2 alternative to the known E.co/i enzyme. Additionally, and more generally, analogues of CB1 954 and prodrugs other than CB1954 are known and further such precursors of potential toxic 5 agents may become the focus of future therapies. In relation to all of these it is desired to provide further enzymes capable of use in converting prodrugs into drugs, e.g. for clinical uses. It is an object of the present invention to provide nitroreductase enzymes, 10 in particular nitroreductase enzymes for converting CB1 954 and analogues thereof into drugs. It is a further object of the present invention to provide DNA and genes encoding nitroreductases, which DNA and genes in particular are incorporated into pharmaceutical compositions for prodrug therapies. 15 The present invention is based upon the discovery, purification, gene sequencing and/or expression of nitroreductases in bacteria and other microorganisms with hitherto unknown properties in converting prodrugs such as CB1954 into toxic derivatives. These nitroreductases posses 20 properties which alone or in combination offer potential improvements compared with the known enzymes in this technology. The nitroreductases of the invention may be divided into different families based upon such characteristics as activity, product spectrum and/or amino acid sequence, and each given nitroreductase may fall into more than one of these 25 families. The present invention provides, in a first aspect, a nitroreductase enzyme, characterised in that it preferentially reduces CB1 954 to a product that is a cytotoxic 4-hydroxylamine (4HX) derivative. 30 The enzymes of this aspect of the present invention confer the advantage that the product they generate from CB1 954 contains a greater proportion WO 00/47725 PCT/GBO0/00431 -3 of the cytotoxic 4HX derivative then the non-cytotoxic 2-hydroxylamino derivative. In preferred embodiments of the invention, the product is substantially entirely the cytotoxic derivative. The enzymes may hence be more efficient that those of the art as the enzymes of the invention 5 produce more cytotoxic product for a given amount of pro-drug. The present invention further provides, in a second aspect, a nitroreductase enzyme, characterised in that it reduces a prodrug to a toxic derivative with a Km of less 700 micromolar, wherein the prodrug is selected from CB1 954 10 and analogues thereof or other bioreductive drugs (Denny et al, B.J. Cancer, 1996, 74, pp S32-S38). The enzymes of the second aspect of the invention offer an advantage over the known E. co/i- derived enzyme in that they have a lower Km (Km of E.coli NfnB for CB1954 is around 862 micromolar) and thus have a higher affinity for substrate. Twenty nitrogen 15 mustard analogues of CB1 954 are described by Friedlos et al (J Med Chem, 1997, 40, 1270-1275). More preferably, the Km of the enzymes of the second aspect of the invention is less than 300 micromolar. 20 In a third aspect, the present invention provides a nitroreductase enzyme characterised in that it reduces a prodrug to a toxic derivative with a Kcat of at least 8, wherein the prodrug is selected from CB1 954 and analogues thereof. 25 The enzymes of this aspect of the invention offer an improvement over that of the art, specifically the E. co/i enzyme, in that they have an improved Kcat - i.e a higher value than for E.coli NfnB indicating a higher turnover of substrate by the enzyme. In preferred embodiments of this aspect of the 30 invention, the Kcat of the enzymes is at least 10. In a fourth aspect of the invention, there is provided a nitroreductase WO 00/47725 PCT/GBOO/00431 -4 enzyme characterised in that it reduces CB1 954 to a toxic derivative, it reduces SN23862 to a toxic derivative, it can use NADH and/or NADPH as electron donor and in that it shares no more than 50% sequence identity with the E.coli NfnB sequence. Preferably, the sequence identity is about 5 25% or less, this sequence identity being measured using the MEGALIGN (registered trade mark) software. It has already been discussed how the known E.coli nitroreductase is well characterised and is fully sequenced. The nitroreductases of the fourth 10 aspect thus represent a class of enzymes having nitroreductase activity, or being nitroreductase-like, which nevertheless are so different in amino acid sequence from the E.coli enzyme as to represent a separate family of nitroreductases. 15 This aspect of the invention thus advantageously provides a further class of nitroreductase enzymes for use e.g. in prodrug therapies. The invention still further provides, in a fifth aspect, a nitroreductase enzyme characterised in that it reduces CB1 954 or an analogue thereof to 20 a toxic derivative, in that it shares at least 50% sequence identity with the rat DTD sequence and in that it does not contain a domain that is the same as or corresponds to amino acids 51 to 82 of the rat DTD sequence. Sequence identity is suitably measured in the same way as described above 25 in relation to the fourth aspect. To determine whether a given nitroreductase contains a domain that is the same as or corresponds to amino acids 51 to 82 of the rat DTD sequence, the amino acid sequence of the given nitroreductase and of the rat DTD 30 sequence are aligned using a conventional sequence alignment program, such as MEGALIGN (registered trade mark) made by DNASTAR, Inc.

WO 00/47725 PCT/GBOO/00431 -5 If the alignment program indicates that there are no amino acids in the given sequence that, following the algorhythm of the program, are held to correspond to those at positions 51-82 of the rat DTD sequence then it is concluded that the rat domain is lacking from the given sequence. 5 This aspect of the invention thus provides a further class of nitroreductase enzymes for conversion e.g. of prodrugs into drugs. A nitroreductase in this class may also be obtained by deleting amino acid residues that correspond to residues 51-82 of the rat DTD from a known mammalian 10 enzyme. The nitroreductases of the invention may also be NADPH dependant. This property further distinguishes some enzymes of the invention from the known E.coli enzyme and the rat DTD. 15 It has been found that enzymes having one or more of the properties described may be obtained from bacteria of the family Bacillus, in particular a Bacillus selected from B. amyloliquefaciens, B. subtilis, B. pumilis, B. lautus, B. thermoflavus, B. licheniformis and B. alkophilus. This finding is 20 of surprise in that at least three nitroreductase enzymes have been found in some species, in particular B.subtiis, B.lautus and B.pumilis, and as nitroreductases having the advantageous properties of the invention have not hitherto been identified in these bacteria, the currently used nitroreductase being obtained from E.coli. 25 In specific embodiments of the invention described in more detail below, a nitroreductase has a sequence selected from SEQ ID Nos 2, 4, 6, 8, 10, 12, 14, 16, 17, 18, 19, 20, 21, 23, 25, 27 and 29. 30 It has further been found that nitroreductases according to the invention may fall into more than one aspects of the invention. It is hence preferred that a nitroreductase of the invention possesses the properties of at least WO 00/47725 PCT/GBOO/00431 -6 two aspects of the invention, and more preferably at least three aspects of the invention. A specific embodiment of the invention is a nitroreductase of SEQ ID NO:2 5 obtained from B. amylo/iquefaciens this enzyme converts CD194 into substantially only the cytotoxic derivative, hence falling into the first aspect of the invention, but also has a Km that is improved compared to the E. co/i enzyme, hence falling also into the second aspect of the invention. 10 A further specific embodiment of the invention is a nitroreductase from B.subtilis, SEQ ID NO:9. This enzyme has a better Kct than the E.coli enzyme, its Kcat being about 15 compared with about 6 for the E.coli enzyme, and hence falls into the third aspect of the invention. Additionally, this enzyme falls into the fourth aspect of the invention in that it reduces 15 both CB1 954 and SN23862 but shares less than 30% sequence identity with the E.co/i sequence. Another B.subtilis enzyme, SEQ ID NO:1 1 is similarly in both the third and fourth aspects of the invention, having a Kca, of about 15. 20 From the examples set out below it will be apparent how the further specific embodiments of the invention fall into at least two and even three aspects of the invention. The enzymes of the invention are of use in enzyme directed prodrug 25 therapy. Accordingly, it is preferred that they are provided in purified form. A sixth aspect of the invention provides a pharmaceutical composition comprising a nitroreductase enzyme according to any of the first to fifth aspects of the invention in combination with a pharmaceutically acceptable 30 carrier. As mentioned above, the nitroreductase of the invention are of use in WO 00/47725 PCT/GBOO/00431 -7 therapies such as directed-enzyme prodrug therapies. In these therapies, it is required to deliver the nitroreductase to the target site. This delivery can be achieved by delivering the enzyme itself or by delivering a DNA or gene coding for the enzyme. 5 In an example of the enzyme of the invention in use, a pharmaceutical composition is designed for a directed-enzyme prodrug therapy, and comprises a pharmaceutically acceptable carrier and a compound for converting a prodrug into a drug, wherein a compound is composed of at 10 least a nitroreductase according to any of the first to fifth aspects of the invention conjugated to a targeting moiety. The targeting moiety can suitably comprise an antibody specific for a target cell. Alternatively, the targeting moiety is a moiety preferentially 15 accumulated by or taken up by a target cell. A further example of delivery of the enzyme of the invention is achieved in a gene therapy-based approach for targeting cancer cells, as described in WO 95/12678. As described by Knox R.J. et al, the basis of this further 20 prodrug therapy is delivery of a drug susceptibility gene into target, usually tumour or cancer, cells. The gene encodes a nitroreductase that catalyses the conversion of a prodrug into a cytotoxic derivative. The nitroreductase itself is not toxic and cytotoxicity used to treat the tumour cells arises after administration of a prodrug which is converted into the cytotoxic form. A 25 bystander effect may be observed as cytotoxic drug may diffuse into neighbouring cells. Thus, in this gene-based therapy, the nitroreductase is expressed inside a cell, in contrast to other delivery systems in which, for example, the 30 enzyme itself is delivered accompanied by a targeting moiety. Targeting of gene-based therapies may be achieved by providing a virus or WO 00/47725 PCT/GBOO/00431 -8 liposome with altered surface components so that the delivery vehicle is recognised by target cells. Typically, transcriptional elements are chosen so that the gene coding for the nitroreductase enzyme will be expressed in the target cells, and preferably substantially only in the target cells. A 5 number of viral-based vectors are suitable for this delivery. Retro-viral based vectors typically infect replicating cells. Adenoviral vectors and lentiviral-vectors are also believed to be suitable. This delivery technology has been demonstrated by Bridgewater et al (Eur 10 J Cancer 31a, 236-2370, 1995). A recombinant retrovirus encoding a nitroreductase was used to infect mammalian cells, it being observed that infected cells expressing the nitroreductase were killed by application of CB1 954. 15 Accordingly, a further aspect of the invention provides the use of a DNA sequence coding for a nitroreductase of the invention in manufacture of a medicament for prodrug therapy. The medicament may take the form of a viral vector, comprising a DNA 20 encoding the nitroreductase of the invention operatively coupled to a promoter for expression of the DNA. The medicament may take the form of a mini-gene comprising a DNA operatively linked to a promoter for expression of the DNA, the mini-gene being suitable for inclusion or incorporation into a targeting vehicle such as a microparticle. 25 Thus, an embodiment of the invention provides a viral vector comprising a nucleotide sequence encoding a nitroreductase according to any of aspects 1 to 5 of the invention, which nitroreductase converts a prodrug into a cytotoxic drug, and also a kit comprising the viral vector and the 30 prodrug, and also a method of treatment of tumours which comprises administering an effective amount of the viral vector together with an effective amount of the prodrug.

WO 00/47725 PCT/GBOO/00431 -9 The preparation and administration of these viral vectors may be substantially as described in WO 95/12678, the contents of which is incorporated herein by reference. The present invention relates to providing nitroreductase enzymes and genes and DNA coding therefore. 5 The uses of those enzymes and genes may be as set out in WO 95/12678. A nitroreductase can also be delivered by putting a gene of the invention into a bacteria that selectively colonises tumours, such as a clostridial (Lemmon et al, Gene Therapy, 1997, 4, 791-796) or Salmonella species. 10 A further aspect of the invention provides an isolated DNA encoding a nitroreductase according to any of the first to fifth aspects of the invention. The DNAs of this further aspect of the invention, and also the DNAs incorporated into vectors of the invention, preferably comprise a sequence 15 which is selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 22, 24, 26 or 28, together with fragments, derivatives and analogs thereof retaining nitroreductase activity according to one of the first to fifth aspects of the invention. The fragments, derivatives and analogs are suitably selected from sequences which retain at least 70% identity with the specific 20 embodiments of the invention, or preferably at least 90% identity and most preferably at least 95% identity. The enzymes of the invention can also be obtained by purification from cell extracts and may also be obtained by recombinant expression of DNA. A 25 still further aspect of the invention lies in a method of preparing a nitroreductase enzyme, comprising expressing a gene in a bacterial cell, wherein the gene codes for a nitroreductase enzyme of the invention. In an example of the invention described below in more detail, the gene 30 expressed is a Bacillus gene or is a gene obtained by substitution, deletion and/or addition of nucleotides in or to a Bacillus gene.

WO 00/47725 PCT/GBOO/00431 - 10 The invention also provides the use of a nitroreductase according to any of the aspects of the invention in manufacture of a medicament for anti tumour therapy, and the use of a compound comprising a nitroreductase according to any aspect of the invention conjugated to a targeting moiety 5 in manufacture of a medicament for anti-tumour therapy. The invention is now illustrated by the following specific examples and in the accompanying sequence listing in which: SEQ ID NO: 2 is a nitroreductase from B.amy/o/iquefaciens (coded 10 for by SEQ ID NO: 1) and designated "Bam YrwO"; SEQ ID NO: 4 is a nitroreductase from B.subti/is (coded for by SEQ ID NO: 3) and designated "Bs YwrO"; SEQ ID NO: 6 is a nitroreductase from B.subtilis (coded for by SEQ ID NO: 5) and designated "YrkL"; 15 SEQ ID NO: 8 is a nitroreductase from B.subti/is (coded for by SEQ ID NO: 7) and designated "YdeQ"; SEQ ID NO: 10 is a nitroreductase from B.subti/is (coded for by SEQ ID NO: 9) and designated "Ydgl"; SEQ ID NO: 12 is a nitroreductase from B.subti/is (coded for by SEQ 20 ID NO: 11) and designated "YodC"; SEQ ID NO: 14 is a nitroreductase from E.coli (coded for by SEQ ID NO: 13) and designated "YabF" SEQ ID NO: 16 is a nitroreductase from E.coli (coded for by SEQ ID NO: 15) and designated "YheR"; 25 SEQ ID NO: 17 is a nitroreductase from H.influenzae; SEQ ID NO: 18 is a nitroreductase from T.aquaticus; SEQ ID NO: 19 is a nitroreductase from Synechocystis sp PCC 6803; SEQ ID NO: 20 is a nitroreductase from A.fulgidus; SEQ ID NO: 21 is a nitroreductase from A.fulgidus. 30 SEQ ID NO: 23 is a nitroreductase from Camp ylobacterjejuni (coded for by SEQ ID NO: 22); SEQ ID NO: 25 is a nitroreductase from Porphyromonas gingiva/is WO 00/47725 PCT/GBOO/00431 - 11 (coded for by SEQ ID NO: 24); SEQ ID NO: 27 is a nitroreductase from Yersinia pestis (coded for by SEQ ID NO: 26); and SEQ ID NO: 29 is a nitroreductase from Helicobacter pylori (coded 5 for by SEQ ID NO: 28). The invention is also illustrated by reference to the accompanying Tables 1-4 and Figures 1 and 2, in which Figs 1 and 2 show sequence comparisons as set out in more detail in Example 8. 10 Example 1 A Nitroreductase Enzyme/Gene from Bacillus amylo/iquefaciens Briefly, extracts of Bacillus amyloliquefaciens were shown to possess 15 nitroreductase activity. To purify this activity, crude cell extracts were subjected to ammonium sulphate, fractionation and anion exchange chromatography. The purified material was subject to N-terminal amino acid sequence analysis and the information obtained used to cloned the gene via a PCR-based strategy. Following determination of its nucleotide 20 sequence the gene was overexpressed in E. co/i and the resultant recombinant protein purified and characterised see table 1. This analysis showed that the enzyme had properties which were distinct from that of E.coli NfnB. Thus the protein had a more favourable Km for 25 CB1 954 (1.5-fold lower than the E. coli B NfnB) and furthermore converted CB1954 into the 4HX form alone. It also differed from the E. co/i B NfnB in that the enzyme showed no activity against the prodrug SN23862. The isolated enzyme/gene represents a significant improvement over the 30 E. co/i NfnB enzyme with respect to its activity against the prodrug CB1 954 ie., it produces only the 4HX derivative and has an improved Km for CB1 954.

WO 00/47725 PCT/GB00/00431 - 12 A comparison of the amino acid sequence of the isolated enzyme revealed that it shared a very low level of homology to the rat DTD (c. 25%), but exhibited high homology (70% sequence identity) with the predicted product of a gene that has been discovered in the Bacillus subtilis genome 5 sequencing project, designated ywrO. On this basis, we have designated the cloned Bacillus amyloliquefaciens gene ywrO, and its encoded enzyme YwrO. YwrO BAM is a tetrameric flavoprotein (monomeric molecular mass 10 approximately 22.5 kDa by SDS-PAGE, native molecular mass approximately 90 kDa by gel filtration). Although it shares sequence homology with rat DTD it differs in its enzymic properties in that it can use only NADPH as cofactor (Km 40 pM). In common with DTD it can reduce CB1 954 but not SN23862, reduction of CB1 954 resulting in formation of 15 the 4HX product only (Km 617 pM, kca 8.2). It shows a high affinity for the quinone menadione (Km 3.4pM) and has azoreductase and flavin reductase activity (Km for FMN 53 iM, Km for FAD 209 pM). In more detail, N-terminal amino acid sequencing of the purified Bacillus 20 amyloliquefaciens nitroreductase enzyme resulted in the following sequence,Met-Lys-Val-Leu-Val-Leu-Ala-Val-His-Pro-Asp-Met-Glu-Asn-Ser Ala-Val-Asn. When this sequence was used to search available protein databases strong homology was noted with the predicted amino acid sequence of a hypothetical protein, YrkL, identified in the Bacillus subtilis_ 25 genome sequencing project. Significant homology was also evident with two proteins, YabF and YheR, identified during the course of the determination of the Escherichia co/i genome. These three hypothetical proteins shared weak homology with a number of mammalian quinone reductases and NAD(P)H-oxidoreductases, such as the rat DTD. 30 In view of this observation, a strategy was formulated whereby sequence homology between the identified bacterial proteins, together with the WO 00/47725 PCT/GBOO/00431 - 13 determined N-terminal amino acid sequence of the discovered Bacillus amyloliquefaciens enzyme, was used to amplify a region of the desired encoding gene from the Bacillus amyloliquefaciens genome. The one primer utilised in PCR was a degenerate oligonucleotide sequence which 5 corresponded to a DNA sequence capable of coding for the N-terminal octa-peptide Val-His-Pro-Asp-Met-Glu-Asn. It was composed of the following nucleotides, 5'-GTNCAYCCNGATATGGARAA-3', where Y indicates the presence of a T or C, R indicates the presence of A or G, and N indicates the presence of either T, C, G or A. The second primer was 10 based on the hypothetical sequence His-Gly-Trp-Ala-Tyr-Gly which was found to be entirely conserved between the hypothetical bacterial proteins YrkL (Bacillus sub ti/is) and YabF (E.co/i), and partially conserved in YheR (E. col). The degenerate oligonucleotide mixture synthesised corresponded to the antisense DNA coding strand, viz., 5'-CCRTANGCCCANCCRTG-3'. 15 E.coli YheR (90-95) Arg Gly Phe Ala Ser Gly E.coli YabF (84-89) His Gly Trp Ala Tyr Gly B. sub ti/is YrkL (85-90) His Gly Trp Ala Tyr Gly 20 The two primers were employed in PCR using chromosomal DNA isolated from Bacillus amyloliquefaciens and an amplified DNA fragment of the expected size (approximately 230 bp) obtained. This was cloned into plasmid pCR2.1TOPO (Invitrogen) and its nucleotide sequence determined. Translation of the sequence obtained demonstrated the presence of an 25 open reading frame which encoded a polypeptide which shared 66% sequence similarity with YrkL. To obtain the entire structural gene, an approach was employed based on inverse PCR. In essence, B. amy/oliquefaciens DNA was cleaved with the 30 restriction enzyme Styl and the fragments generated circularised through their subsequent incubation with DNA ligase. The ligated DNA was then used as the template for a PCR employing two divergent primers based on WO 00/47725 PCT/GB0O/00431 -14 the sequenced 220 bp fragment. These were BamNTR1 1 (5' GCTTATTGACCGCTGAG-3') and BamNTR1 4 (5'-GTACAGTGCGCCTCCGC 3'). A 2.9 kb fragment was generated, cloned into pCR2.1TOPO (Invitrogen) and the sequence of the insert determined. This allowed the 5 identification of the nucleotide sequence of the remaining parts of the B. amyloliquefaciens gene. Using this information, a contiguous copy of the entire structural gene was amplified from the B. amylo/iquefaciens chromosome using primers which encompassed the translational start codon (5'-GGTGTGATACATATGAAAGTATTG-3') and resided 3' to the 10 translational stop codon (5'-CGGGGATTCGAATTCTTTCTCAGG-3'). The primer at the 5'-end of the gene was designed such the sequence immediately 5' to the ATG start codon became CAT. This change created an Ndel restriction site (CATATG), thereby allowing the cloning of the gene into the equivalent site of the expression vector pMTL1015. This 15 manipulation facilitated the subsequent overexpression of the gene, as insertion of the gene at this point positions the start codon at an optimum distance from the vector borne ribosome binding site. The strategy employed to clone the BM YwrO gene could be similarly 20 employed to clone further genes encoding novel nitroreductases. This would involve purifying the desired enzyme activity from a cell lysate, and then determining the N-terminal sequence. The data obtained could then be used to design an oligonucleotide primer corresponding to the sense strand of the DNA encoding part or all of the determined amino acid 25 sequence. This primer could then be used, in conjunction with a second primer, to amplify part of the gene encoding the nitroreductase from the chromosome of the bacterial host using PCR. The second primer would correspond to the antisense strand of an internal portion of the targeted gene. Its design would be based on regions of homology which are 30 conserved amongst the type of nitroreductase family that is sought. Thus, in the case of the DTD-like family, the oligonucleotide would, for example be based on the conserved motif His-Gly-Trp-Ala-Tyr-Gly (ie., amino acid WO 00/47725 PCT/GBOO/00431 - 15 residues 85-90 in the BS YrkL protein). In the case of the NfnB-like family, the oligonucleotdie could be based on the motif Glu-Arg-Tyr-Val-Pro-Val Met (ie., amino acid residues 170-176 in the BS YodC protein). 5 Such amplified fragments could then be cloned and sequenced, and new primers designed based on this sequence to isolate the flanking regions of the gene by PCR. Once these have been cloned and sequenced, the entire, contiguous structural gene may be amplified using primers which extend beyond the 5' and 3' end of the translational start and stop codons. 10 Cloning of genes encoding novel nitroreductases may also be achieved without recourse to N-terminal sequencing of the enzyme, or even its purification. This would involve basing the sequence of both of the oligonucleotides used in the initial PCR reaction on amino acid sequence 15 motifs conserved amongst the two identified nitroreductase families. Thus, in the case of the NfnB-like family, a sense primer (eg., 5' ATTTCTAAAGAAGAGCTGACGGAA-3') based on the motif lle-Ser-Lys-Glu Glu-Leul-Thr-Glu (ie., amino acid residues 13 to 20 of BS YodC) could be employed with the an antisense primer (eg., 5' 20 CATTACCGGTACATAGCGTTC-3') based on the sequence motif Glu-Arg Tyr-Val-Pro-Val-Met (ie., amino acid residues 170 to 176). In the case of the DTD-family a sense primer (eg., 5'-CATCCGGATATGGAAAAT-3') based on the motif His-Pro-Asp-Met-Glu-Asn (ie., amino acid residues to 9 to 14 of BM YwrO) could be employed with the an antisense primer (eg., 25 5'-TCCATATGCCCATCCATA-3') based on the sequence motif Tyr-Gly Trp-Ala-Tyr-Gly (ie., amino acid residues 85 to 90). Once amplified, the rest of the gene could be isolated using the same procedure as outlined above. 30 Example 2 Bacillus subti/is Nitroreductases WO 00/47725 PCT/GB0O/00431 - 16 As indicated above in Example 1, comparative analysis of the B. subti/is genome sequence with the amino acid sequence of the isolated B. amylo/iquefaciens enzyme demonstrated the existence of an enzyme (YwrO) which shared 70% sequence identity. Unexpectedly, B.subti/is was 5 found to possess two homologues, YrkL and YdeQ, which share 54% and 51% sequence homology, respectively, with the B. amylo/iquefaciens enzyme. All three enzymes share no homology with the E.co/i B NfnB. They do, however, exhibit weak similarity (c. 25%) to the rat DT Diaphorase (DTD). Whilst these proteins share a low level of sequence 10 similarity to DTD, and other mammalian equivalents, they are characteristically smaller. This is because of the absence of an extensive internal protein domain at the N-terminus of the protein. Thus, the functional equivalent domain of the rat DTD between amino acid residues 51 to 82, are missing from the BM YwrO protein. In addition, the rat DTD 15 has an extra COOH-terminal domain. These bacterial enzymes are thus distinct from their mammalian equivalents. A further analysis of the B.subtilis genome, demonstrated that two homologues of the E.coliNfnB gene were present. Their encoded proteins 20 (Ydgl and YodC) share a barely detectable level of sequence conservation with EC NfnB, of around 20% sequence identity. Bacillus subti/is was thus found to carry at least 5 different enzymes with nitroreductase activity. These are split into two families, thus; 25 DTD-like - 3 members:- YwrO, YrkL, YdeQ NfnB-like - 2 members:- Ydgl, YodC Example 3 30 Recombinant Production of Nitroreductases from Bacillus subti/is The DNA encoding all 5 B.subtilis nitroreductase enzymes were cloned WO 00/47725 PCT/GBOO/00431 - 17 from genomic DNA using PCR and the resultant genes, following authentification by nucleotide sequencing, subcloned into a propriety CAMR expression vector (pMTL1015). The expression clones generated have been used to overproduce each of the 5 proteins and the enzymic 5 activity of each assessed in crude lysates. This analysis has demonstrated that whilst the B.subtilis YwrO shares similar properties to the B. amyloliquefaciens homologue (ie., converts CB1 954 to the 4HX derivative alone, but is inactive against SN23862), YrkL and YdeQ have no activity against either of the two prodrugs tested (CB1 954 or SN23862) but they 10 may be active against other prodrugs. Despite the extremely limited sequence similarity to EC NfnB, Ydgl and YodC are active against both CB1 954 and SN23862. They do, however, produce both the 2HX and 4HX derivatives of CB1 954. Their 15 characterisation has shown that they turn over CB1 954 at higher rates than EC NfnB (YodC kct 58, Ydgl ke,, 30.3 cf 6 for NfnB). Both show a high affinity for menadione and flavins, but they differ in that whereas Ydgl uses both NADH and NADPH, YodC shows a preference for the latter. The native molecular mass of YodC (approximately 90kDa) indicates that it is 20 tetrameric (molecular mass estimated from amino acid sequence and by SDS-PAGE being approximately 22 kDa) whereas Ydgl appears to be a dimer in the native state (molecular mass by gel filtration approximately 49 kDa). 25 These finding are further illustrated in Table 2. Example 4 Bacillus lautus & Bacillus pumilis nitroreductases 30 From 103 soil sample isolates tested, two strains (Bacillus pumi/is CP044 and Bacillus lautus CPO60) had been previously chosen as possessing extracts which showed the most rapid reduction of both CB1 954 and WO 00/47725 PCT/GBOO/00431 - 18 SN23862. Purification experiments demonstrated that the activity in both extracts was distributed across three distinct peaks. The presence of more than one enzyme activity is consistent with our discovery of multiple forms of proteins in Bacillus able to turnover prodrugs. Eventual purification of the 5 three enzymes of B. pumi/is CP044 revealed that no one candidate exhibited properties which were an improvement on the E.coli NfnB enzyme. In contrast, the proteins in peak 1 and peak 3 of the B./autus CPO60 were determined to offer advantage over NfnB. 10 Thus, whilst the enzyme in peak 1 did not produce the required 4HX derivative of CB1954, it exhibited a 4-fold lower Km with the prodrug SN23862. The enzyme of peak 3 was, however, deemed to be of greatest value as it converted CB1 954 solely into the 4HX derivative and had a Km approximately 4-fold lower than NfnB. Furthermore, it also had activity 15 against SN23862. In this respect it shares the properties of both the Bacillus DTD-like family (ie., it produces only the 4HX derivative) and the NfnB-like family (ie., it is active against SN23862) - these findings are illustrated in Table 3. 20 Example 5 N-terminal Sequencing of B. lautus Nitroreductase Electrophoretic separation of the peak 3 demonstrated that 4 protein bands were present which could account for the observed prodrug activity. All 25 four were subjected to N-terminal amino acid sequencing and the activity localised to the fourth protein band from which the nitroreductase may be purified. Example 6 30 Detection of Nitroreductase Activity in Thermophile Extracts As an alternative source novel enzymes, a preliminary screen of CAMRs WO 00/47725 PCT/GBOO/00431 - 19 thermophile collection was undertaken. Enzymes from this source may have the advantage of greater stability, and therefore longevity of action. Strains were selected on the basis either of sensitivity to CB1 954, or those which are resistant but which impart a yellow/golden coloration to agar 5 containing prodrug. Two of these strains (B. thermoflavus and B. /icheniformis) generated the cytotoxic 4HX form and were selected for further study. 10 Example 7 Identification Of Further Nitroreductase Enzymes Having identified the two families of nitroreductase in Bacillus, a search was undertaken of both finished and unfinished genomes for homologues, 15 using YwrO and YodC/NfnB. On the basis of this search homologues of YwrO were identified in the genomes of Yersiniapestis and Porphyromonas gingivalis, and homologues of NfnB in the genomes of Pyrococcus furiosus, Haemophilus influenza, Synechocystis PCC 6803, Camp y/obacter jejuni, Archaeglobus, Helicobacter pylori, Heliocbacter fulgidus and Thermus 20 aquaticus. In addition to the above, two E.coligenes were found to be homologues of rat DTD and YwrO, and were designated Yher and YabF. They were discovered to share the characteristic of YwrO in that they lack the internal 25 protein domain found in the rat DTD enzyme and functional mammalian homologues. (i) P.gingiva/is YwrO homologue P.gingivalis YwrO homologue is a dimeric flavoprotein with native molecular mass estimated by gel filtration at 40 kDa. Although it shares sequence homology with DTD and forms only the 4HX reduction product of CB1 954 WO 00/47725 PCT/GBOO/00431 - 20 (Km 1 200iM, kcat 3.2), it differs from DTD in that it is active with SN23862 and it can only use NADH as cofactor (cf DTD which can use either NADH or NADPH and is inactive with SN23862). It can reduce azodyes but it is inactive with menadione or flavins. (ii) C.jejuni NfnB homologue C.jejuni NfnB homologue produces only the 4HX reduction product of CB1 954 (Km 143 pM, kcat 11 .2) using NADPH as cofactor and it is also active with SN23862. It can use the quinone menadione as substrate as well as azodyes and the flavins FMN and FAD. (iii) Archaeoglobus fulgidus NfnB homologue Archaeoglobus fulgidus NfnB homologue is a dimeric flavoprotein of 42 kDa native molecular mass, producing the 4HX derivative of CB1 954 only (Km 690 pM, kcat 56.2) using NADPH as cofactor. It is also active with SN23862 and menadione (Km 9 pM), but does not decolourise azodyes and has only weak flavin reductase activity. (iv) H.influenzae and H.pylori NfnB homologues Both these enzymes are dimeric flavoproteins and form the 4HX reduction product of CB1 954 using NADPH in preference to NADH, but have no activity with azodyes. The former also lacks activity with the quinone menadione and flavins FMN or FAD. Both however have weak activity with SN23862 and may be active with other prodrugs. (v) Y.pestis nfnB homologue and Synechocystis YwrO homologue Both these proteins reduce CB1 954 but produce only the relatively non toxic 2HX derivative using NADPH as cofactor. They do however show WO 00/47725 PCT/GBOO/00431 - 21 activity with SN23862 and the former can also reduce azodyes. Example 8 Comparison of Nitroreductase Sequences We compared the amino acid sequences of nitoreductases according to the invention with each other and with known rat, human and E.coi sequences, and the results are illustrated in Figures 1 and 2. In Figure 1, rat, mouse and two human sequences make up the first four lanes for comparison purposes. It is evident that nitroreductases of the invention are lacking a sequence from positions 51-82 of the rat sequence. In Figure 2, sequences of nitroreductases of the invention are compared with the known E. coi sequence, which is designated nfmB in the second to-last lane. The invention thus provides nitroreductase enzymes, DNA and genes therefor and methods of obtaining such enzymes and of using the enzymes and DNA coding therefor in clinical applications. SUBSTITUTE SHEET (RULE 26) WO 00/47725 PCT/GBOO/00431 -22 ENZYME M.Wt CB1 954 SN23862 ACTIVITY (kDa) Product Km Km B. pumilis CP044 Peak 1 ND 4HX v. low ND Peak 2 ND 4HX >1000 ND Peak 3 ND 2/4HX 999 ND B.lautus CP060 Peak 1 35 2HX 211 325 Peak 2 42 41-IX >2000 none Peak 3 47 4HX 257 active Table 3: Fractionation of nitroreductase activity in cell extracts of Bacillus lautus and Bacillus pumilis STRAIN CB1954 SN23862 Product NADH NADPH NADH NADPH 1078 2/4HX 13.8 22.6 8.5 17.6 2122a 2/4HX 36.6 56.0 33.4 62.8 6012 b 4>2HX 15.2 37.8 8.2 35.2 6013 c 2HX 9.8 49.4 6.4 39.0 6031 d 2HX 11.9 42.1 8.2 33.8 6036 2HX 10.7 26.7 7.3 26.2 6044 2HX 4.0 21.3 4.5 9.9 Table 4: Characteristics of nitroreductase activity of thermophiles identified as being sensitive to CB1954 [Identified as Bacillus thermoflavus a, Bacillus licheniformis b, Bacillus licheniformis C, Bacillus alkophilus d] RTTRTTTTTTr RTTFFT IDTTN 7I9 WO 00/47725 PCT/GBOO/00431 - 23 ENZYME M.Wt CB1954 SN23862 ACTIVITY (kDa) Product Km Km B. pumilis CP044 Peak I ND 4HX v. low ND Peak 2 ND 4HX >1000 ND Peak 3 ND 2/4HX 999 ND B.lautus CP060 Peak 1 35 2HX 211 325 Peak 2 42 4HX >2000 none Peak 3 47 4HX 257 active Table 3: Fractionation of nitroreductase activity in cell extracts of Bacillus lautus and Bacillus pumilis STRAIN CB1954 SN23 862 Product NADH NADPH NADH NADPH 1078 2/4HX 13.8 22.6 8.5 17.6 2122a 2/4HX 36.6 56.0 33.4 62.8 6012 b 4>2HX 15.2 37.8 8.2 35.2 6013 c 2HX 9.8 49.4 6.4 39.0 6031 d 2HX 11.9 42.1 8.2 33.8 6036 2HX 10.7 26.7 7.3 26.2 6044 2HX 4.0 21.3 4.5 9.9 Table 4: Characteristics of nitroreductase activity of thermophiles identified as being sensitive to CB1954 [Identified as Bacillus thermoflavus a, Bacillus licheniformis b, Bacillus licheniformis C, Bacillus alkophilus d) SUBSTITUTE SJRT rUTT r

Claims (28)

1. A nitroreductase characterised in that it preferentially reduces CB1 954 to a cytotoxic 4-hydroxylamine (4HX) derivative instead of a non 5 cytotoxic 2-hydroxylamine derivative.

2. A nitroreductase according to Claim 1 further characterised in that it reduces CB1 954 to the 4HX derivative with a Km of less than 700 micromolar. 10

3. A nitroreductase according to Claim 1 or 2 further characterised in that it is NADPH dependant.

4. A nitroreductase according to any of Claims 1 to 3, further 15 characterised in that it reduces CB1 954 to a cytotoxic 4-hydroxylamine (4HX) derivative substantially without producing the non-cytotoxic 2 hydroxylamine derivative.

5. A nitroreductase according to any of Claims 1 to 4 which reduces 20 the prodrug to the toxic derivative with a Kcat of at least 8.

6. A nitroreductase according to any of Claims 1 to 5, which reduces CB1 954 or an analogue thereof to a toxic derivative, shares at least 50% sequence identity with the rat DTD sequence and does not contain a 25 domain that is the same as or corresponds to amino acids 51 to 82 of the rat DTD sequence.

7. A nitroreductase characterised in that it reduces a prodrug to a toxic derivative with a Km of less 700 micromolar, wherein the prodrug is 30 selected from CB1 954 and analogues thereof.

8. A nitroreductase according to Claim 7 which reduces the prodrug to SUBSTITUTE SHEET (RULE 26) WO 00/47725 PCT/GBOO/00431 - 25 the toxic derivative with a Km of less 300 micromolar.

9. A nitroreductase according to Claim 7 or 8 which reduces the prodrug to the toxic derivative with a Kcat of at least 8. 5

10. A nitroreductase according to Claim 9 which reduces the prodrug to the toxic derivative with a Kcat of at least 10.

11. A nitroreductase according to any of Claims 7 to 10, further 10 characterised in that it reduces CB1954 to a toxic derivative, it reduces SN23862 to a toxic derivative, it can use both NADH and NADPH as electron donor and in that it shares no more than 30% sequence identity with the E.coi NfnB sequence. 15

12. A nitroreductase according to any of Claims 7 to 11 further characterised in that it shares at least 50% sequence identity with the rat DTD sequence and in that it does not contain a domain that is the same as or corresponds to amino acids 51 to 82 of the rat DTD sequence. 20

13. A nitroreductase characterised in that it reduces a prodrug to a toxic derivative with a Kcat of at least 8.

14. A nitroreductase according to Claim 13, further characterised in that it reduces CB1 954 to a toxic derivative, it reduces SN23862 to a toxic 25 derivative, it can use both NADH and NADPH as electron donor and in that it shares no more than 30% sequence identity with the E.coi NfnB sequence.

15. A nitroreductase according to Claim 13 or 14, further characterised 30 in that it reduces CB1 954 or an analogue thereof to a toxic derivative, in that it shares at least 50% sequence identity with the rat DTD sequence and in that it does not contain a domain that is the same as or corresponds SUBSTITUTE SHEET (RULE 26) WO 00/47725 PCT/GB0O/00431 - 26 to amino acids 51 to 82 of the rat DTD sequence.

16. A nitroreductase characterised in that it reduces CB1 954 to a toxic derivative, it reduces SN23862 to a toxic derivative, it can use both NADH 5 and NADPH as electron donor and in that it shares no more than 30% sequence identity with the E.coli NfnB sequence.

17. A nitroreductase according to Claim 16, wherein the sequence identity is about 25% or less. 10

18. A nitroreductase characterised in that it reduces CB1954 or an analogue thereof to a toxic derivative, in that it shares at least 50% sequence identity with the rat DTD sequence and in that it does not contain a domain that is the same as or corresponds to amino acids 51 to 15 82 of the rat DTD sequence.

19. Use of a DNA sequence coding for a nitroreductase according to any preceding Claim in manufacture of a medicament for prodrug therapy.

20 20. A viral vector, comprising (a) a DNA encoding nitroreductase according to any of Claims 1 to 18 operatively coupled to (b) a promoter for expression of the DNA. 25

21. A mini-gene comprising (a) a DNA encoding nitroreductase according to any of Claims 1 to 18 operatively coupled to (b) a promoter for expression of the DNA. 30

22. A pharmaceutical composition comprising a nitroreductase according to any of Claims 1 to 18 in combination with a pharmaceutically acceptable carrier. SUBSTITUTE SHEET (RULE 26) WO 00/47725 PCT/GBOO/00431 -27

23. A pharmaceutical composition for use in a directed-enzyme prodrug therapy, comprising a pharmaceutically acceptable carrier and a compound for converting a prodrug into a drug, wherein a compound comprises a nitroreductase according to any of Claims 1 to 18 conjugated to a targeting 5 moiety.

24. A pharmaceutical composition according to Claim 23 wherein the targeting moiety comprises an antibody specific for a target cell. 10

25. A pharmaceutical composition according to Claim 23 wherein the targeting moiety is a moiety preferentially accumulated by or taking up by a target cell.

26. A method of preparing a nitroreductase, comprising expressing a 15 gene in a bacterial cell, wherein the gene codes for a nitroreductase according to any of Claims 1 to 18.

27. Use of a nitroreductase according to any of Claims 1-18 in manufacture of a medicament for anti-tumour therapy. 20

28. Use of a compound comprising a nitroreductase according to any of Claims 1 to 18 conjugated to a targeting moiety in manufacture of a medicament for anti-tumour therapy. SUBSTTITT ST-FT (RTTI2 71