ENZYME GENE THERAPY CATALYSING PRODRUG EXTRACELLULAR CONVERSION

Abstract: The invention relates to a molecular chimaera for use with a prodrug, the molecular chimaera comprising a transcriptional regulatory DNA sequence capable of being activated in a targetted mammalian cell and a DNA coding sequence operatively linked to the transcriptional regulatory DNA sequence and encoding a secretion signal peptide and a heterologous enzyme, such that on expression of said coding sequence in the targetted cell, the heterologous enzyme is capable of passing through the cell plasma membrane and is capable of catalysing extracellular conversion of the prodrug into a cytotoxic or cytostatic agent. When the molecular chimaera is used in gene or virus directed enzyme prodrug therapy the fact that enzyme is secreted so that conversion of the prodrug to the cytotoxic or cytostatic agent is extracellular increases neighbouring cell kill.

Description: ENZYME GENE THERAPY CATALYSING PRODRUG EXTRACELLULAR CONVERSION The present invention relates to a novel targetted enzyme prodrug therapy. Targetted enzyme prodrug therapies provide a method for restricting the activity of a chemotherapeutic agent to a particular target site. This is desirable when the systemic presence of the chemotherapeutic agent produce unwanted side effects. Although applicable to any therapeutic regime for which a targetted approach is advisable, the technique is clearly particularly applicable to the treatment of cancer where therapeutic regimes have previously involved the systemic introduction of highly cytotoxic compounds which exert their effect in a non-selective manner on both healthy and tumourogenic cells. Research in the area of cancer chemotherapy has produced a variety of antitumour agents which have differing degrees of efficacy. Standard clinically useful agents include adriamycin, actinomycin D, methotrexate, 5-fluorouracil, cis-platinum, vincristine and vinblastine. However, these presently available antitumour agents are known to have various disadvantages such as toxicity to healthy cells and resistance of certain tumour types. Other forms of therapy such as surgery, are known. However it is clear that novel approaches and entities for cancer therapies are required if significant progress in the clinical management of this disease is to be achieved. Targetted enzyme prodrug therapies may offer significant improvements in cancer therapy, either alone or in combination with existing treatment regimes. One such therapy relates to the use of molecular chimaeras, which encode a heterologous enzyme and, which are delivered to targetted cells. Intracellular expression of the enzyme allows catalysis of a subsequently administered prodrug to its active cytotoxic or cytostatic form. The therapy is known as gene or virus directed enzyme prodrug therapy (GDEPT or VDEPT). WO-A-9007936 describes a treatment for an infection or a hyperproliferative disorder which is characterised by the presence, in the affected cells, of a trans-acting factor capable of regulating gene expression by inserting into the cells a polynucleotide construct having a cis- acting regulatory sequence which is regulated by the trans-acting factor and an effector gene which renders said cell susceptible to protection or destruction. For example, the cis-acting region may be homologous to the HIV tar region, and the effector gene may encode ricin A or HSV-1 thymidine kinase. Upon infection with HIV, the HIV tat protein activates the tar region, and induces transcription and expression of ricin A, resulting in cell death, or of HSV-1 tk, resulting in cell death upon treatment with dideoxynucleoside agents such as acyclovir and gancyclovir. EP-A-0 334 301 describes methods for the delivery of vectors using recombinant retrovirus wherein the vector construct directs the expression of a protein that activates a compound with little or no cytotoxicity into a toxic product in the presence of a pathogenic agent, thereby effecting localised therapy to the pathogenic agent. EP-A-0 415 731 describes molecular chimaeras for use with prodrugs, comprising transcriptional regulatory DNA sequences capable of being selectively activated in a mammalian cell, a DNA sequence operatively linked to the transcriptional regulatory DNA sequence and encoding a heterologous enzyme capable of catalysing the conversion of the prodrug into an agent toxic to the cell. Existing GDEPT and VDEPT regimes involve the intracellular expression of the heterologous enzyme and intracellular catalysis of the prodrug. This limits the efficiency of such therapy as in, for example tumour therapy using retroviral mediated targetting, only between 1 and 10% of cells of the solid tumour may be infected by retrovirus. Hence therapy is limited to those 1 to 10% of infected cells and the neighbouring 90-99% of cells remain untreated. Clearly in a hyperproliferative condition it is desirable to maximise cell kill and so the ability to kill uninfected neighbouring cells is extremely important. An alternative strategy for targetted enzyme prodrug therapy which may provide a limited 'neighbouring cell kill' is antibody directed enzyme prodrug therapy (ADEPT) which involves linkage of the heterologous enzyme to a targetting antibody which directs and attaches extracellularly the enzyme to a target cell population where extracellular conversion of a prodrug may occur. Active drug may then diffuse and exert its effect in neighbouring cells. However this approach, described for example in EP-A-0 382 411 and WO-A- 88/07378, may result in an immune reaction to the heterologous enzyme/ targetting antibody complex and due to the large size of the antibody/enzyme complex, delivers a limited amount of enzyme to the target site which may be insufficient to provide a significant therapeutic effect. Additionally GDEPT and VDEPT provides some degree of restriction of the resultant chemotherapeutic activity to a target cell population by either the use of retroviruses which preferentially infect dividing cells, or the presence of transcriptional regulatory sequences whose expression is restricted to certain cell- or cancer-types e.g. liver-specific promoters for use in the treatment of hepatocellular carcinoma. However, even this level of restriction only targets the therapeutic effect to a particular cell-type, i.e. dividing cells, liver cells, etc., whether they be cancerous or healthy. When the delivered chemotherapeutic is a cytotoxic agent, it is clearly desirable that the restriction on the cells being targetted be pathology- specific and that no significant expression of the chemotherapeutic agent occurs in non- pathological healthy cells. One object of the present invention is to substantially meet this desirable requirement by use of a system that is designed to exert a therapeutic effect preferentially in cells with the pathological disorder. The present invention provides a molecular chimaera for use with a prodrug, the molecular chimaera comprising a transcriptional regulatory DNA sequence capable of being activated in a targetted mammalian cell and a DNA coding sequence operatively linked to the transcriptional regulatory DNA sequence and encoding a signal peptide and a heterologous enzyme, such that on expression of said coding sequence in the targetted cell, the heterologous enzyme is capable of passing through the cell plasma membrane and is capable of catalysing extracellular conversion of the prodrug into a cytotoxic or cytostatic agent. Thus according to the invention, molecular chimaeras are introduced into target cells where ) expression of the coding sequence which they contain under control of the transcriptional regulatory sequence produces an enzyme having a signal peptide so that the enzyme is exported through the cell membrane. Administered prodrug is catalysed to active compound extracellularly by means of the enzyme and diffuses into neighbouring cells, where it exerts its therapeutic effects According to one embodiment of the invention the heterologous enzyme is obtained from an enzyme precursor by cleavage at a proteolytic cleavage site by a pathology associated protease extracellularly to the targetted mammalian cell. Enzyme precursors which require to be proteolytically cleaved into a mature enzyme before being capable of expressing significant catalytic activity occur naturally in the form of proenzymes or preproenzymes. Alternatively they may be generated artificially using recombinant DNA technology to produce an enzyme translated with an inhibitory peptide linked genetically or by recombinant DNA to the enzyme via a protease sensitive amino acid or amino acid sequence such that the catalytic activity of the enzyme is inhibited until removal of the inhibiting peptide via cleavage at a proteolytic cleavage site by a pathology- associated protease. All these relatively inert enzymes whether naturally-occurring or artificially created, are enzyme precursors of use in the present invention and may be referred to herein as proenzymes. In preferred embodiments of the present invention the enzyme precursor comprises the enzyme and a secretion signal sequence which facilitates the extracellular transport of the enzyme during which process the signal sequence is cleaved to produce extracellular mature enzyme. A pathology-associated protease is any protease which is either expressed exclusively in association with a cell population as a consequence of a particular cellular pathology or one which is expressed at elevated levels correlating to a particular cellular pathology. Elevated levels of proteases are characteristic of a range of tumour types and have been clearly implicated in the tumourogenic and particularly the metastatic process (FASEB ] "___, 1434-1441 (1993)). Examples of such tumour-associated proteases include urokinase, gelatinase (e.g. 72kD) and stromolysin. According to the invention such pathology-associated proteases provide a mechanism for elevating the fidelity of targetted enzyme prodrug therapy by exerting control over the post- translational modification of the enzyme such that unless and until the enzyme is cleaved by a pathology-associated protease, it is unable to express its catalytic activity at all or at least to a sufficient degree to obtain therapeutic effect. Such catalytic activity is necessary to convert the prodrug to a cytotoxic or cytostatic compound. Therefore the toxic agent is preferentially, if not exclusively, generated in association with target cells which are expressing a pathology-associated protease as a consequence of inflammation, viral infection, tumourogenesis, etc. In a cancer patient the metastatic process often produces secondary tumours and it is these secondary tumours which frequently are the eventual cause of death. Studies in vivo have demonstrated that the pharmacological inhibition of the activity of the secreted matrix metalloproteases results in prevention of metastasis (Reich, et al Cancer Res 40 3307-3312 (1988)). The matrix metalloproteases (MMP) are a family of zinc metallo-enzymes with well characterised structural and catalytic properties. These enzymes are all secreted as pro¬ enzymes that undergo proteolytic cleavage of an amino-terminal domain during activation and are further defined by sequence similarities, inhibition by metal chelators, and inhibition by tissue inhibitors of metalloproteases. Well characterised members of the family include interstitial collagenase, neutrophil collagenase, stromelysin-1, stromelysin-2, matrilysin, gelatinase A and gelatinase B. Studies have clearly demonstrated a positive correlation between MMP expression, invasive behaviour, and metastatic potential. Studies of MMP expression in human tumours suggest that these proteases are an important component of the invasive phenotype of many tumours, including breast, prostrate, colon, lung, ovarian, and thyroid cancers. Hence particularly preferred heterologous enzymes precursors for use according to the particular embodiment of the present invention include those possessing a protease cleavage site capable of cleavage by a matrix metalloprotease as described herein. Such protease cleavage sequences are well known to those in the art and may either be present naturally in a suitable proenzyme of a heterologous enzyme suitable for use according to the embodiment of the invention or may be engineered into such a proenzyme using recombinant DNA technology. Tumours particularly suitable for treatment according to the invention include those expressing such matrix-metalloproteases. Particularly preferred are pro-enzymes which can be cleaved to mature enzyme by collagenase, including those possessing dipeptide protease cleavage sites comprising one of Gly-Leu, Gly-Val, Gly-Ile, Ala-Leu, Ala-Met, His-Leu, Phe-Leu, Pro-Val, Ala-Val, Pro-He, Gln-Phe, Phe-Val, Val-Leu, Met-Leu, Phe-Leu, Pro-Met, and Ala-Leu; those which can be cleaved by stromelysin including those with sites comprising Glu-Val, His-Phe, Asn-Phe, His-Ile, Ala-Glu, Pro-Met, Ala-Leu, Arg-Ser, Gly-Phe, Gly-Leu, Phe-Tyr and Gly-Phe and those which can be cleaved by gelatinases/type IV B collagenases including those with sites comprising Gly-Val, Gly-Leu, Gly-Gly, Gly-Glx, Gly-Phe, Gly-Asn, Gly-Ser and Asn-Tyr. The term 'heterologous enzyme' as used herein means any enzyme not present naturally in the targetted mammalian cell. This comprises non-mammalian enzymes such as those derived from yeast or bacteria and mammalian enzymes including naturally occurring mutant mammalian enzymes or mutant mammalian enzymes which have been generated by recombinant DNA technology. Preferred enzymes for use according to the present invention include any capable of passing through the cell membrane and which have a catalytic activity appropriate to the conversion of a prodrug to a therapeutically active compound. Such enzymes include cytosine deaminase which converts the prodrug 5-fluorocytosine to toxic 5-fluorouracil, human carboxypeptidase Al which converts the prodrug para-N-bis(2-chloroethyl)-aminobenzoyl glutamic acid into benzoic acid mustard, the enzyme alkaline phophatase which converts the prodrugs etoposidephosphate, doxorubicin phosphate and mitomycin phosphate into the corresponding toxic dephosphorylated metabolite and the enzyme penicillin-V-amidase which converts a prodrug which is a phenylacetamide derivative of doxorubicin or melphalan into its corresponding toxic metabolite. Other preferred non-mammalian enzymes include β- lactamase which may be used with prodrugs disclosed in EP-A-0 404 070 and WO-A-94 01137. The most preferred enzyme for use in accordance with the present invention is a mutant mammalian enzyme as described in WO-A-95 13095. The mutant mammalian enzyme is preferably a wild-type mammalian enzyme with one or more mutations which generate novel substrate specificites. This mutation permits a subtle shift in the substrate specificity of the enzyme such that the appropriate prodrug may be catalysed while being refractory to catalysis by the corresponding endogenous wild-type enzyme. The essential characteristic of a mutant mammalian enzyme of the present invention is the presence of a mutant substrate-binding site irrespective of the amino acid sequences flanking this mutant substrate-binding site. Hence a chimaeric enzyme comprising a mutant substrate- binding site and flanking sequences derived from one or more different enzymes is included with the definitions of a mutant mammalian enzyme of the present invention. Such a chimaeric enzyme may be generated by recombinant DNA technology and/or protein engineering. Enzymes suitable for directed mutagenesis include any enzymes possessing a catalytic activity capable of converting a prodrug to a drug. Such catalytic activities include transf erase, hydrolasε, oxido-reductase, isomerase, lyase, or ligase. The directed mutagenesis will generate a novel substrate specificity but preserve the class of catalytic activity involved. For example a mutant isomerase of the present invention will possess a novel isomerase activity sufficiently different from the isomerase from which it was derived to ensure that prodrugs susceptible to activation by the mutant isomerase remain substantially stable in the presence of the isomerase from which the mutant enzyme was derived. This dramatic shift in activity may be achieved by the alteration of as little as one residue at the catalytic site and the alteration of the minimum number of residues necessary to obtain the required shift in activity ensures continuity of enzyme structure. A preferred mutant enzyme for use in the present invention is mutant mammalian carboxypeptidase A (CPA). This enzyme has the general activity of cleaving carboxy- terminal aromatic and aliphatic amino acid residues and has been characterised in a diversity of species and tissue-specific variants. Particularly preferred mutant carboxypeptidases include mutants derived from human pancreatic carboxypeptidase Al (Catasus et al Biochem. J. 287. 299-303, (1992)), human mast cell carboxypeptidase A (Reynolds et al J. Clin. Invest. 89 273-282, (1992)) and human pancreatic carboxypeptidase A2. It will be appreciated that the common characteristic of these carboxypeptidases is the presence of a CPA-like substrate-binding pocket and associated enzymatic activity irrespective of overall sequence or structure of the enzyme and that any enzyme possessing this CPA-like substrate- binding pocket is amenable to mutation to a mutant carboxypeptidase of the present invention. In a particularly preferred embodiment the mutant enzyme is mutant human pancreatic carboxypeptidase Al or A2 (CPA1 or CPA2) and yet more preferably, CPA1 or CPA2 • wherein amino acid substitutions are generated at one or more of residues 203, 210, 242, 244, 250, 253, 255, 267, 268, 269 and 305, of the amino acid sequences of the wild-type (w.t.) enzymes. Particularly preferred combinations of residue substitution include Gly at residues 250 and 268 when 253 and 255 are w.t.; Gly at 253 and 268 when 250 and 255 are w.t.; Gly at 250 and His at 268 when 253 and 255 are w.t.; Gly at 250 when 253, 255 and 268 are w.t.; Ala at 255 and His at 268 when 250 and 253 are w.t. and His at 268 when 250, 253 and 255 are w.t. The most preferred mutants are carboxypeptidase Al or A2 mutants comprising a single substitution; Gly at 268. Human pancreatic carboxypeptidase A is expressed as a preproenzyme which is processed to a proenzyme and subsequently to the mature enzyme. Mutant carboxypeptidases for use according to the present invention may be mutants of the preproenzyme, the proenzyme or of the mature enzyme but are preferably mutants of the mature enzyme. These mutants may be derived from either preproenzyme, the proenzyme or the mature enzyme. A mutant enzyme for use in the present invention may be generated from the DNA or RNA source of any enzyme possessing the previously discussed activities by methods well known in the art of molecular biology. The selection of the enzymatic activity and the primary sequence of a mutant enzyme for use in the present invention will clearly depend upon the nature of the relevant prodrug and, if a prodrug of the type which is activated by the removal of a prodrug moiety, on the precise structure of that moiety. The substrate-binding or active site of the mutant enzyme must, unlike the corresponding non-mutant enzyme, be capable of interacting with the prodrug in such a way that enzymatic catalysis is facilitated. The ability of the mutant enzyme to perform this activity will depend upon subtle alterations in the 3-dimensional structure of the active site, which is in tum dependent upon the primary amino acid sequence of this region of the protein. Alterations of the primary sequence of an enzyme by standard techniques of protein engineering will allow the generation of an appropriate mutant enzyme for use according to the invention with a corresponding prodrug. To be capable of secretion through the cell membrane, an enzyme should possess a signal sequence at the amino terminus either because it is a secreted enzyme with a naturally occurring signal sequence or because the chimaera expressing the enzyme has been engineered such that the expressed enzyme has an additional amino acid sequence which possesses the properties of a signal sequence. Such signal sequences are well known in the art and include those indicated or derivable from Nothwehr et al J. Biol. Chem. 265, 21797- 21803 (1990), Nothwehr and Gordon J. Biol. Chem. 265, 17202-17208 (1990) and Kohara et al FEBS Lett. 311, 226-230 (1992) and the references contained therein. Prodrugs for use according to the present invention may be any compound which upon catalysis by an enzyme suitable for use in the present invention, will generate a chemotherapeutic compound. Such prodrugs will generate chemotherapeutic compounds which are preferably anti-inflammatory, anti-viral or anti-cancer compounds, will more preferably be cytotoxic compounds such as nitrogen mustard agents, a tifolates, nucleoside analogs, the vinca alkaloids, the anthracyclines, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, the podophyophyllotoxins, the sulfonylureas (as described in EP-A-0 222,475) and low- molecular-weight toxins such as the trichothecenes and the colchicines. Particularly including doxorubicin, daunorubicin, aminopterin, methotrexate, taxol, methopterin, dichloromethotrexate, mitomycin C, porfirmoycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin, etoposide, melphalan, vinblastine, vincristine, desacetylvinblastine hydrazide, leurosidine, vindesine, leurosine, trichothecene and desacetylcolchicine. Particularly preferred prodrugs for use in the present invention, and particularly for use with the preferred carboxypeptidase mutant enzymes described herein are described in WO-A-95 13095. A mutant human enzyme as used herein shall be taken to be any human enzyme with a sequence differing by at least one amino acid from the amino acid sequence or sequences of that enzyme in the patient to which the therapy is applied. A "chemotherapeutic agent" which may also be referred to herein as a "drug" includes any molecule which has activity in human therapy. Such chemotherapeutic agents include but are not limited to cytostatic or cytotoxic compounds used in the therapy of cancers or viral infections. A "functionally inactive precursor" which may also be referred to herein as a "prodrug" includes any compound which may be converted into a chemotherapeutic agent under the action of an enzyme. Such functionally inactive precursors may typically be converted to a chemotherapeutic agent by the enzymatic cleavage of the functionally inactive precursor to yield a chemotherapeutic agent and a "prodrug moiety". Such conversion from functionally inactive precursor to chemotherapeutic agent may also occur by enzymatically mediated isomerisation. A functionally inactive precursor will generally not exhibit clinically significant levels of the therapeutic activity possessed by the chemotherapeutic agent into which it may be enzymatically converted for example as a result of having been chemically derivitized to decrease its normal pharmacological activity. The functionally inactive precursor of a cytotoxic chemotherapeutic agent will not itself exhibit clinically significant cytotoxicity and will be sufficiently stable in vivo such that during therapy, clinically significant levels of cytotoxicity are largely only generated at the site of conversion of functionally inactive precursor to chemotherapeutic agent i.e. at the site of the targetted mammalian cell. In the molecular chimaera according to the invention, the coding sequence is under the control of a transcriptional regulatory sequence (TRS) comprising at least a promoter and preferably an enhancer, each of which may either be capable of non-specific expression independent of the type of cell in which expression is occurring or may exhibit a selectivity of expression dependent upon the cellular environment. Preferred TRSs are non-specific, potent promoter/enhancer combinations such as cytomegalovirus promoter/enhancer, SV40 promoter/enhancer and retroviral long terminal repeat promoter/enhancer. Other preferred TRSs include those of 3-actin, glyceraldehyde-S-phosphate and tubulin. Also preferred are TRSs exhibiting cell-type dependent expression in which case the selection of the TRS, in particular the promoter and enhancer sequence, will depend on the targetted cell type. Examples include the albumin (ALB) and alpha-fetoprotein (AFP) TRS for normal hepatocytes and transformed hepatocytes respectively, the TRS for carcinoembryonic antigen (CEA) for use in transformed cells of the gastrointestinal tract, lung, breast and other tissues: the TRS for tyrosine hydroxylase, choline acetyl transferase or neuron specific enolase for use in neuroblastomas: the TRS for glial fibro acidic protein for use in glioblastomas and the TRS for insulin for use in tumours of the pancreas. Further examples include the TRS specific for gamma-glutamyltranspeptidase for use in certain liver tumours and dopa decarboxylase for use in certain tumours of the lung. In addition the TRS for certain oncogenes may be used as these are expressed predominantly in certain tumour types. These include the HER-2/neu oncogene TRS which is expressed in breast tumours and the TRS specific for the N-myc oncogene for neuroblastomas. The ALB and AFP genes exhibit extensive homology with regard to nucleic acid sequence, gene structure, amino acid sequence and protein secondary folding (for review see Ingram et al Proc. Natl. Acad. Sci. (USA) 78, 4694-4698 (1981)). These genes are independently but reciprocally expressed in ontogeny. In normal development ALB transcription is initiated shortly before birth and continues throughout adulthood. Transcriptional expression of ALB in the adult is confined to the liver. AFP is normally expressed in foetal liver, the visceral endoderm of the yolk sac and the foetal gastrointestinal tract, but declines to undetectable levels shortly after birth and is not significantly expressed in nonpathogenic or non- regenerating adult liver or in other normal adult tissues. However, AFP transcription in adult liver often increases dramatically in heptacellular carcinoma (HCC). In addition, transcription may also be elevated in non-seminomatous and fixed carcinoma of the testis: in endodermal sinus tumours in certain tertorcarcinomas and in certain gastrointestinal tumours. Liver-specific expression of AFP and ALB is the result of interactions of the regulatory sequences of their genes with trans-activating transcriptional factors found in nuclear extracts from liver. The AFP and ALB TRSs are preferred for generating hepatoma- specific or general liver-specific expression respectively of molecularly combined genes since the AFP and ALB genes are regulated at the transcriptional level and their mRNAs are among the most abundant polymerase II transcripts in the liver. Several mammalian ALB and AFP promoter and enhancer sequences have been identified (for review see Genes and Develop 1, 268-276 (1987); Science, 235, 53-58 (1987); J. Biol. Chem. 262, 4812-4818 (1987)). These sequences enable the selective and specific expression of genes in liver hepatocytes (normal and transformed) and hepatomas respectively. Similar to the regulatory structure of the ALB gene the regulatory elements of the AFP genes promote tissue-specific expression in certain liver pathologies, such as HCC (Mol. Cel. Biol. 6, 477-487 (1986); Science, 235, 53-58 (1987)). The regulatory elements of a mammalian AFP gene consist of a specific 5' promoter proximal region (located in some mammalian species between 85 and 52 bp 5' to the gene). This sequence is essential for transcription in hepatomas. In addition there are upstream (5') regulatory elements well defined for the murine AFP gene which behave as classical enhancers (Mol. Cell Biol. 6, 477-487 (1986); Science, 235, 53-58 (1987)). These upstream regulatory elements are designated elements I, II and III and are located between 1,000 to 7,600 bp 5' to the transcription initiation site for the AFP murine gene. These three enhancer domains are not functionally equivalent at generating tissue-specific expression of AFP. Elements I and II have the greatest capacity to direct liver-specific expression of AFP. It is important to note that the regulatory sequences of the alpha-fetoprotein gene advantageously contain the sequences not only for tissue-specific transcriptional activation but also for repression of expression in tissues which should not express AFP. In a similar fashion the regulatory regions of the human alpha- fetoprotein gene have been characterised (J. Biol. Chem. 262, 4812-4818 (1987)). A structural gene placed in the correct orientation 3' to the AFP regulatory sequences will enable that structural gene to be selectively expressed in fetal liver hepatomas, non- seminamatous carcinomas of the testis, certain teratocarcinomas, certain gastrointestinal tumours and other normal and pathological tissues which specifically express AFP. The promoter and enhancer sequences preferably are selected from the TRS for one of albumin (ALB), alphafetoprotein (AFP), carcinoembryonic antigen (CEA) (J. DNA Sequencing and Mapping, Vol 4, 185-196), tyrosine hydroxylase, choline acetyl transferase, neuron-specific enclase, glial fibro acid protein, insulin or gamaglutamytranspeptidase, dopadecarboxylase, HER-2/neu or N-myc oncogene or other suitable genes. Most preferably the TRS for ALB or AFP are used to direct liver specific or hepatoma specific expression respectively. In preferred embodiments of the present invention the molecular chimaera is selectively expressed in a target cell population. This may be taken to mean that the chimaera is expressed at a higher level in the target than in the non-target cell population and is preferably expressed predominantly or exclusively in that population. Selective expression may be achieved by inclusion of a target-cell specific TRS (promoter with or without enhancer) as described above or may be a product of the method of delivery of the chimaera to the target cell. Methods capable of providing target cell specific delivery of the chimaera, with subsequent stable integration and expression, include the techniques of calcium phosphate transfection, electroporation, microinjection, liposomal transfer, ballistic barrage or retroviral infection or infection using adenovirus or adeno-associated virus. For a review of this subject see Biotechniques Vol. 6 No. 7 (1988). Such selectivity may be obtained by a variety of such techniques. Physiologically localised delivery of the chimaera for the target cells will reduce the possibility of non-target cells expressing the chimaera. This may be achieved when for example using retroviral or liposome mediated delivery and would involve direct injection to a blood vessel known to supply the target cells. Selectivity may also be obtained using retroviral mediated chimaera delivery in the therapy of hyperproliferative disorders. Retroviruses only infect dividing cells and would therefore only introduce chimaeras to dividing cells. Liposome technology permits the delivery of the chimaera contained therein to be targetted to a particular cell type based on appropriate modifications made to the liposome coat structure. In a preferred embodiment according to the present invention, a number of such methods for obtaining selectivity will be combined to improve the fidelity of selective expression. For example in the treatment of hepatocellular carcinoma, the chimaera may comprise TRSs derived from liver-specific gene promoters such as ALB or AFP, and will be delivered in a retrovirus directly to the hepatic artery. Hence a three-fold level of specificity will be obtained, firstly by the localised delivery which ensures that effectively all the retroviruses infect liver cells, secondly by use of a retrovirus which only infects dividing carcinoma cells and thirdly by liver-specific expression of the TRSs. One particular method according to the present invention for obtaining selective expression of a molecular chimaera of the present invention delivered using a retrovirus is accomplished by promoting selective infection of liver cells. This technique involves the retroviral env gene present in the packaging cell line which defines the specificity for host infection. The env gene used in constructing the packaging cell line is modified to generate artificial infective virions that selectively infect hepatocytes. As an example a retroviral env gene introduced into the packaging cell may be modified in such a way that the artificial infective virion's envelope glycoprotein selectively infect hepatocytes via the specific receptor mediated binding utilised by the hepatitis B virus (HBV). HBV primarily infects hepatocytes via specific receptor mediated binding. The HBV proteins encoded by the pre-Sl and pre-S2 sequences play a major role in the attachment of HBV to hepatocytes (see Hepadna Viruses edited Robinson et al 189-203, 205-221 (1987)). The env gene of the packaging cell is modified to include the hepatocyte binding site of the large S HBV envelope protein. Such modifications of the env gene introduced into the packaging cell may be performed by standard molecular biology techniques well known in the art and will facilitate viral uptake in the target tissue. In the methods of obtaining selective activation which comprise target cell specific delivery systems, the TRS need not be target cell specific and TRSs derived from genes such as β- actin, glyceraldehyde-3-phosphate and cytomegalovirus (e.g. immediate early gene) (see Huber, et al Cancer Research, 53, 4619-4626 (1993)) and references therein) may be used. The molecular chimaera of the present invention may be made utilising standard recombinant DNA techniques. Thus the coding sequence and polyadenylation signal of for example the cytosine deaminate gene is placed in the proper 3' orientation to the ALB or AFP TRS. These molecular chimaeras enable the selective expression of cytosine deaminate in cells which normally express from ALB or AFP TRSs respectively. Accordingly there is also provided a method of constructing a molecular chimaera comprising operatively linking a DNA sequence comprising a TRS capable of being activated in a mammalian cell to a DNA sequence encoding an enzyme capable of passing through the cell membrane and capable of catalysing the conversion of the prodrug into a cytotoxic or cytostatic agent. The technique of retroviral infection of cells to integrate artificial genes employs retroviral shuttle vectors which are known in the art (see for example Mol. and Cell Biol. 6, 2895-2902 (1986)). Essentially retroviral shuttle vectors are generated using the DNA form of the retrovirus contained in a plasmid. These plasmids also contain sequences necessary for selection and growth in bacteria. Retroviral shuttle vectors are constructed using standard molecular biology techniques well known in the art. Retroviral shuttle vectors have the parental endogenous retroviral genes (e.g. gag pol and env) removed and the DNA sequence of interest inserted, such as the molecular chimaeras which have been described. They however contain appropriate retroviral regulatory sequences for viral encapsidation, proviral insertion into the target genome, message splicing, termination and polyadenylation. Retroviral shuttle vectors have been derived from the Moloney murine leukaemia virus (Mo- MLV) but it will be appreciated that other retroviruses can be used such as the closely related Moloney murine sarcoma virus. Certain DNA viruses may also prove to be useful as a delivery system. The bovine papilloma virus (BPV) replicates extrachromosomally so that delivery system based on BPV have the advantage that the delivered gene is maintained in a nonintegrated manner. Adenoviruses and adeno-associated viruses may also be used. Thus according to a further aspect of the present invention there is provided a retroviral shuttle vector containing a molecular chimaera as hereinbefore defined. The advantages of a retroviral-mediated gene transfer system are the high efficiency of the gene delivery to the targeted tissue sequence specific integration regarding the viral genome (at the 5' and 3' long terminal repeat (LTR) sequences) and little rearrangements of delivered DNA compared to other DNA delivery systems. Accordingly in a preferred embodiment of the present invention there is provided a retroviral shuttle vector comprising a DNA sequence comprising a 5' viral LTR sequence, a cis acting psi encapsidation sequence, a molecular chimaera as hereinbefore defined and a 3' viral LTR sequence. In a preferred embodiment and to help eliminate non-target-specific expression of the molecular chimaera, the molecular chimaera is placed in opposite transcriptional orientation to the 5' retroviral LTR. In addition a dominant selectable marker gene may also be included which is transcriptionally driven from the 5' LTR sequence. Such a dominant selectable marker gene may be the bacterial neomycin-resistance gene NEO (aminoglycoside- 3-phosphotransferase type II) which confers on eukaryotic cells resistance to the neomycin analogue G418 sulphate (Geneticin - trade mark). The NEO gene aids in the selection of packaging cells which contain these sequences. The retroviral vector used may be based on the Moloney murine leukaemia virus but it will be appreciated that other vectors may be used. Such vectors containing a NEO gene as a selectable marker have been described, for example, the N2 vector (Science, 230, 1395-1398 (1985)). A theoretical problem associated with retroviral shuttle vectors is the potential of retroviral long terminal repeat (LTR) regulatory sequences transcriptionally activating a cellular oncogene at the site of integration in the host genome. This problem may be diminished by creating SIN vectors. SIN vectors are self-inactivating vectors which contain a deletion comprising the promoter and enhancer regions in the retroviral LTR. The LTR sequences of SIN vectors do not transcriptionally activate 5 or 3 genomic sequences. The transcriptional inactivation of the viral LTR sequences diminishes insertional activation of adjacent target cell DNA sequences and also aids in the selected expression of the delivered molecular chimaera SIN vectors are created by removal of approximately 299 bp in the 3 viral LTR sequence (Biotechniques, 4, 504-512 (1986)). Thus preferably the retroviral shuttle vector of the present invention are SIN vectors. Since the parental retroviral gag, pol and env genes have been removed from these shuttle vectors a helper virus system may be utilised to provide the gag pol and env retroviral gene products trans to package or encapsidate the retroviral vector into an infective virion. This is accomplished by utilising specialised "packaging" cell lines which are capable of generating infectious synthetic virus yet are deficient in the ability to produce any detectable wild-type virus. In this way the artificial synthetic virus contains a chimaera of the present invention packaged into synthetic artificial infectious virions free of wild-type helper virus. This is based on the fact that the helper virus that is stably integrated into the packaging cell contains the viral structural genes but is lacking the psi site and cis acting regulatory sequence which must be contained in the viral genomic RNA molecule for it to be encapsidated into an infectious viral particle. Accordingly the present invention provides an infective virion comprising a retroviral shuttle vector as hereinbefore described said vector being encapsidated within viral proteins to create an artificial infective replication-defective retrovirus. In addition to removal of the psi site additional alterations can be made to the helper virus LTR regulatory sequences to ensure that the helper virus is not packaged in virions and is blocked at the level of reverse transcription and viral integration. Alternatively helper virus structural genes (i.e. gag, pol and env) may be individually and independently transferred into the packaging cell line. Since these viral structural genes are separated within the packaging cell's genome, there is little chance of covert recombinations generating wild-type virus. In a further aspect of the present invention there is provided a method for producing infective virions of the present invention by delivering the artificial retroviral shuttle vector comprising a molecular chimaera of the invention as hereinbefore described into a packaging cell line. The packaging cell line may have stably integrated within it a helper virus lacking a psi site and other regulatory sequence as hereinbefore described or alternatively the packaging cell line may be engineered so as to contain helper virus structural genes within its genome. The present invention further provides an infective virion as hereinbefore described for use in therapy particularly for use in the treatment of cancer and more particularly for use in the treatment of hepatocellular carcinoma, non-seminamatous carcinoma of the testis, certain teratocarcinomas and certain gastrointestinal tumours. The infective virion according to the invention may be formulated by techniques well known in the art and may be presented as a formulation with a pharmaceutically acceptable carrier therefor. Pharmaceutical acceptable carriers in this instance may comprise a liquid medium suitable for use as vehicles to introduce the infective virion into the patient. An example of such a carrier is saline. The infective virion may be a solution or suspension in such a vehicle. Stabilisers and antioxidants and or other excipients may also be present in such pharmaceutical formulations which may be administered to a mammal by any conventional method e.g. oral or parenteral routes. In particular the infective virion may be administered by intra-venous or intra-arterial infusion. In the case of treating HCC intra-hepatic arterial infusion may be advantageous. Accordingly the invention also provides pharmaceutical formulations comprising a molecular chimaera of the present invention contained within one of, an infective virion or a liposome or a packaging cell mix, in admixture with a pharmaceutically acceptable carrier, and pharmaceutical formulations comprising a molecular chimaera virion, vector, liposome or packaging cell mix of the present invention in admixture with a pharmaceutically acceptable carrier. Additionally the present invention provides methods of making pharmaceutical formulations as herein described comprising mixing an artificial infective virion containing a molecular chimaera with a pharmaceutically acceptable carrier. The invention also includes the use of any molecular chimaera, vector, virion, liposome or pharmaceutical formulation of the present invention in human therapy and in the manufacture of a medicament for use in the treatment of pathological states. The invention also includes methods of medical therapy comprising the use of any molecular chimaera, vector, virion, liposome or pharmaceutical formulation of the present invention. Also included within the scope of the present invention is a protein encoded by a molecular chimaera of the present invention and any combination of such a protein and a prodrug which can be catalysed by the enzyme component of that protein. The precise dosage to be administered to a patient will ultimately be dependent upon the discretion and professional judgement of the attendant physician and will be a product of the particular targetting mechanism chosen. References contained herein to the efficiency of targetting of retroviruses, liposome etc. may be used to determine appropriate dosage levels. The invention is illustrated by the following examples in which reference is made to the accompanying drawings. In the drawings: FIG 1 is a map of plasmid 268G-HCPAI which includes the human 286G-HCPAI in a suitable form for expression in HT1080 cells; FIG 2 shows inhibition of HT1080 cell growth in cells expressing secreted 268G-HCPAI with various prodrug treatments; FIG 3 shows production of methotrexate from prodrug B (N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl-L-glutan-l-yl-3-cyclobutyl-L-phenylalamine) by culture medium from cells expressing secreted 268G-HCPAI; FIG 4 shows cellular location of /3-lactamase activity in mammalian cells transfected with a secretory 3-lactamase construct; FIG 5 shows growth curves of E. coli DH5o and DH5α transformed with pCMV-pgal or pCMV-pga2. FIG 6 shows a schematic representation of pCMV-pga clones and E. coli pga genomic sequence. Example 1 (i) Engineering of HT1080 Cells To Express 268G-HCPA1. A Hind III-Xba I fragment which contained the entire coding sequence of human 268G- HCPAlwas inserted into the eucaryotic expression vector, pcDNAINeo (Invitrogen, Inc.) also restricted with Hind III and Xba I. The resulting plasmid construct designated 268G- HCPA1 (see Figure 1) was transfected into HT1080 cells using the cationic lipid mixture, Lipofectin™, (Gibco BRL, Inc.) and recombinant cells were selected in G418 (500 g/ml). Individual cell clones were obtained by the use of cloning rings and were characterized by the HCPA1 enzymatic activity produced in the cell culture medium. (ϋ) Inhibition of HT1080 Cell Growth in Cells Expressing Secreted 268G-HCPA1 with Prodrug Treatment. HT1080 cells, either control (pcDNA) or engineered to express 268G-HCPA1 (268G) were plated into 96-well plates in normal medium with 10 % human serum which was pretreated at pH 3.2 for 2 h at room temperature followed by neutralization. After 24 h the indicated drug was added at the specified concentration Plates were incubated at 37'C for three days and cell viability was determined using the MTT assay. The results are shown in Figure 2. (Hi) Production of Methotrexate from Prodrug B by culture Medium from Cells Expressing Secreted 268G-HCPA1. HT1080 cells as above were plated into dishes in the indicated medium and were incubated at 37'C for three days. Prodrug B (N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino) benzoyl)-L-glutam-l-yl-3-cyclobutyl-L-phenylalanine; 20μM) was added to the cells and aliquots were removed at lh, 3h and 20h for analysis of methotrexate production. The following cells were tested: pcDNA-control cells; 268G-cells expressing 268G-HCPA1; , 5%HS-cells plated in 5 % human serum; 5%HS-Acid-cells plated in 5% human serum pretreated in acid as above. Example 2 Sequencing of Human Carboxypeptidase Al cDNA A Hind III - Sal I cDNA fragment encoding the rat preprocarboxypeptidase Al (CPA1) (Gardell et al., Nature, 317, 551-555 (1985)) was isolated from pMP36 provided by Dr. M. Phillips (UCSF). The 1.2 kb Hind III - Sal I rat CPA1 cDNA was radiolabelled with p2 P]dCTP and Prime-it kit (Stratagene) and then used to screen a lambda gtll human pancreas cDNA library (Clontech) according to the method of Grunstein and Hogness (Proc. Natl. Acad. Sci. (USA), 72, 5016-5020 (1975)). Purified isolated plaques that hybridised to the rat CPA1 cDNA probe were obtained after three rounds of screening. Lambda DNA from purified plaques was prepared from overnight growth liquid cultures using Qiagen columns and according to the manufacturer's protocol (Qiagen). Human CPA1 cDAN inserts were liberated from lambda DNA by digestion with EcoRI and purification of the cDNA inserts by low-melting agarose gel electrophoresis. The EcoRI cDNA inserts were cloned into the EcoRI site of pGEM 7z(+) (Promega) and denoted as pHCPA plasmids. Following overnight growth in liquid culture, plasmid pHCPA DNA was prepared using Qiagen columns. DNA sequencing of isolated lambda plaques and of plasmids containing HCPA1 cDNA was performed with [ 3S S]dATP and the fmol sequencing system (Promega). Oligonucleotide primers that flanked the cloning site of lambda gtll or the multiple cloning sites in pGEM 7(+) and the TA vector (Invitrogen) were used initially to determine cDNA sequence. Oligonucleotide primers that correspond to regions of the determined sequence were synthesised which permitted the entire cDNA sequence of both strands of HCPA1 to be determined. The sequence is given in SEQ. ID. NOS. 1 and 2. Example 3 (i) Cloning of E. coli J-Lactamase for Human Cell Expression We have constructed unique DNA constructs containing the bacterial b-lactamase gene which, when delivered to human cells, result in expression of functional b-lactamase. The advantages of b-lactamase as a prodrug activating enzyme are 1) the enzyme is kinetically very efficient and 2) because of a unique activation mechanism, a prodrug of virtually any drug can be made as an efficient substrate for the enzyme. The implications of this to cancer therapy is that it permits the use of combination prodrug therapy to counter resistance phenomena as well as allows one to choose drugs appropriate to the tumor target. For example, to target lung cancer, prodrugs of methotrexate (5798W93) and 5-fluorouracil (1614W94) have been synthesized. (ϋ) Construction of Secretory 3-lactamase Constructs To create a DNA construct which would express secretory /3-lactamase in human cells, the coding region of TEM /3-lactamase (Sykes and Matthews, J. Antimicrob. Chemo., 2, 115- 157 (1976); Ambler and Scott Proc. Natl. Acad. Sci. USA, 75, 3732-3736 (1978)) was used. Since it exists in the periplasm of bacteria, the unmodified coding region of TEM β- lactamase contains a signal peptide (Sutcliffe, Proc. Natl. Acad. Sci. USA, 75, 3737-3741 (1978)). Sequences useful for the cloning and expression of this gene in a eukaryote were added to flanking sequence during PCR by including the sequences in the PCR primers. The sequence of the forward primer (JM1) was: 5'-TTGCATAAGCTTGCCACCATGAGTATTCAACATTTCCGTGTC (42-mer - SEQ ID NO 3). The sequence of the reverse primer (JM2) was: 5'-GATCTGTCTAGATTACCAATGCTTAATCAGTGAGGC (36-mer - SEQ ID NO 4). The forward primer contains a Hind III restriction site (AAGCTT) for subsequent cloning of the PCR product, and a sequence (GCCACC) which confers optimal translation effciency in vertebrates (Kozak, J. Cell Biol. 115, 887-903 (1991)) immediately 5-prime to the initiator methionine codon (ATG) of the /3-lactamase coding region. The reverse primer contains an Xba I restriction site (TCTAGA) adjacent to the stop codon (TAA) of the β- lactamase coding region. The PCR reaction was carried out for 25 cycles using standard conditions and using Vent DNA Polymerase (New England Biolabs, Inc., Beverly, MA, USA) in 4 mM MgSO 4 and 200 μM of each dNTP and 1 pmol/ l forward and reverse primers. PCR thermal cycling conditions were 95°C, 1 min; 60°C, 1 min; 75°C, 1 min, 25 cycles then 75°C, 5 min. The approximately 800 base pair PCR product was gel-purified using the Glass-Max kit (Life Technologies, Inc. , Gaithersburg, MD, USA). The purified PCR product was restriction digested with Hind III and Xba I, re-purified by gel electrophoresis, and ligated into the multiple cloning site of the pRc/CMV vector (InVitrogen, Inc., San Diego, CA). The orientation of the /3-lactamase insert in this vector places the /3-lactamase gene under the transcriptional regulation of the intermediate/early CMV promoter as well as followed a bovine growth hormone poly (A) addition signal. The sequence of the construct (designated pCMV-BL) is shown in SEQ IDS NOS 5 and 6 along with the amino acid sequence of inserted secretory /3-lactamase. (iii) Determination of Cellular Locations of Targeted -S-Lactamase Protein Confirmation of the predicted location of the /3-lactamase constructs was carried out using transient DNA transfection in a mammalian cell line. Transfection was carried out by liposome-mediated DNA delivery using lipofectamine (Life Technologies, Inc. , Gaithersburg, MD, USA). Experiments were performed according to manufacturer's instructions, varying the number of cells, amount of transfection reagent, and amount of DNA to determine optimum conditions. Typically, 60 x 15mm tissue culture plates containing approximately 3 x 10 s to 1 x 10 6 cells were employed. After transfection using pCMV-BL, transfected cells were resuspended in 50 mM Tris-Cl (pH 7.4), 0.1 mM EDTA containing PMSF and leupeptin, swollen on ice for 10 min, then lysed using a Dounce homogenizer. After centrifugation at 800 x g for 6 min, supernatant (cytosolic fraction) was recentrifuged at 30 psi for 20 minutes in a Beckman AirFuge. Pellets from both centrifugations (which include membranes and nuclei) were combined. Each fraction was assayed for activity using the chromogenic substrate PADAC, added to a final concentration of 20 mM (Calbiochem, Corp.). Absorbence at 570 nm was measured using the auto-rate assay of a Kontron Model 9310 spectrophotometer. To assess secreted /3-lactamase levels, PADAC assays were carried out on the cell-free media after transfections. /3-lactamase enzyme activity was measured using PADAC (Calbiochem, Corp.) which serves as a chromogenic substrate of 3-lactamase activity (Schindler and Huber, Enzyme Inhibitors, Brodbede, Ed., pp 169-176, Verlag Chemie, Weinheim (1980). A 500 μM PADAC stock was made in water, filtered through a 0.22 μ filter, and added to media to give a final concentration of 20 μM. Decreases in absorbance at 570 nm were measured using the auto-rate assay of a Kontron UV/Vis spectrophotometer. The data in Figure 4 show that at 48 hours after transfection with lipofectamine, large amounts of /3-lactamase are secreted from cells transfected with pCMV-BL. The cellular activity seen with this construct is presumably the enzyme contained in secretory granules in the process of being exported. Based on the magnitude of this activity, we estimate that the enzyme from the secretory /3-lactamase construct represents 5-10% of total cellular protein made per 24 hours per cell. (iv) /3-Lactamase Delivery to Cells Confers Sensitivity to Cephalosporin Prodrugs A. /3-lactamase efficiently activates 5798W93 and 1614W94 Prodrugs of methotrexate (5798W93) and 5-fluorouracil (1614W94) represent the parent drugs linked to cephalothin. The kinetic parameters of prodrug activation were measured by incubating various concentrations of prodrug with purified /3-lactamase followed by HPLC analysis to determine the rate of prodrug conversion. /3-Lactamase efficiently activates both 5798W93 and 1614W94 with a K K„ (specificity constant) of 272 and 67 sec"' mM ', respectively. B. Combination of the 3-Lactamase Gene with 5798W93 and 1614W94 Confers Toxicity We have evaluated the in-vitro toxicity of the /3-lactamase prodrugs in the presence and absence of the /3-lactamase gene. Cytotoxicity was quantitated by determining IC » s in treated A549 human lung adenocarcinoma cells using an SRB-based growth inhibition assay (Nair et al., J. Med. Chem. 32, 1277-1279 (1989)). In the absence of the /Mactamase gene, methotrexate was 10-fold more toxic than the methotrexate prodrug 5798W93, and fluorouracil was 20-fold more toxic than the fluorouracil prodrug 1614W94 (Table 1). When A549 cells which contained stable integrated copie(s) of the secretory /Mactamase gene (A549-BL) were tested, methotrexate and its prodrug 5798W93 were equally toxic (Table 1). This experiment implies that the delivery of the /8-lactamase gene to tumor cells will make them sensitive to cephalosporin prodrugs. The relatively small differential between the toxicity of methotrexate and 5-fluorouracil and their respective prodrugs in the absence of the /8-lactamase gene was unexpected. This is because, for both parent drugs, the mechanism of action is well understood and the chemical modification made by attaching cephalothin to these compounds should clearly detoxify the drugs. For example, transport of methotrexate into cells depends on availability of the terminal glutamate moiety which is blocked in 5798W93. Toxicity of 5 -fluorouracil depends on the availability of the Nl group since this group is necessary for glycosidic bond formation and concomitant nucleoside formation. The Nl group is blocked in 1614W94. It is clear that the observed toxicity of these prodrugs in-vitro reflects some degree of chemical instability of the prodrugs which could result in significant breakdown of the prodrugs during the 72-hour incubation utilized in the IC* determination. Support for this notion comes from two lines of evidence. The first is that no toxicity is observed when either prodrug is given to mice at a dose equivalent to an LD 1∞ for the parental drug. The lack of toxicity in these cases is explained by the relatively short half life of the drug in-vivo (t w «■* 20 minutes) in contrast to the exposure of cells to the prodrug for 72 hours in-vitro. The second line of evidence is shown by direct measurement of in-vitro toxicity by short- term assays (3 hour exposure of cells to prodrug). Using a sensitive assay for cell toxicity, a 6-[Η]-deoxyuridine based assay which measures inhibition of thy midy late synthase and DNA synthesis, we could measure toxicity over time as short as a three hour interval. During this shorter interval, the differential between prodrug and parent drug increased significantly (Table 2). These data are consistent with the idea that the prodrug toxicities reported in Table 1 result from chemical instability of the prodrugs over the long time-course (72 hours) of those experiments. (v) Antitumor Evaluation of Secretory /3-Lactamase in vivo Using Liposome- Mediated DNA Delivery Secretory /8-lactamase and cytosine deaminase DNA constructs were compared for antitumour effects in mice bearing subcutaneous (s.c.) A549 human lung adenocarcinoma tumours. Results are shown in Table 3. Plasmid DNA expression vectors encoding either cytosine deaminase (CD) or secretory /8-lactamase (BL) under the transcriptional control of the non¬ specific CMV promoter were encapsidated in cationic liposomes (25 g DNA; 25 nmol liposomes). Mice bearing A549 s.c. tumours were treated with five intratumoral injections of liposomal DNA. Prodrug therapy (1614W94 (50 mg/kg; i.p., qd x 5) or 5-FC (500 mg/kg; i.p., qd x 5) was initiated two days after DNA treatment. Inhibition of tumour growth was determined on day 47. Both CD and BL constructs resulted in similar antitumour activity in vivo. 1614W94 administration resulted in about 60% inhibition of tumour growth (Table 3). 5-FC administration resulted in about 70% inhibition of tumour growth, whereas DNA liposomes alone and 5-FU alone (25 mg/kg, i.p., qd x 5) resulted in only about 20% inhibition of tumour growth (Table 3). Thus, liposomal DNA/5-FU prodrug combinations resulted in s.c. tumour regressions. Secretory /3-lactamase and cytosine deaminase DNA constructs were also evaluated by intrathoracic (i.t.) injection of liposomal DNA into the pleural space of mice bearing tumors. Results are shown in Table 4. Mice bearing human large cell lung H460 i.t. tumours received DNA encoding either CD or BL under the transcriptional control of the CMV promoter. DNA was dosed by i.t. injection on days 6, 7, 12 and 13. Prodrugs for the respective enzyme were dosed on days 7-16 (5-FC, 500 mg/kg; 1614W94, 70 mg/kg; i.p., qd x 10). Animal survival was evaluated 30 days after tumour implantation. All nontreated mice and mice treated with 5-FU (30 mg/kg i.p., qd x 5) died from tumour by 30 days. CMV-BIJ1614W94 treatment increased survival to 60%, and CMV-CD/5-FC treatment also increased the survival to 40% (Table 4). Table 1 Cytotoxicity (SRB Assay) IC J0 A549 A549-BL Methotrexate 10 nM N.D. 1 μM, 3h 5798W93 100 nM N.D. 1 μM, 3h 5 -Fluorouracil 1.9 μM 1.4 μM 1 M, 5h 1614W94 40 μM 1.7 μM 1 μM, 5h Table 2 Cytotoxicity (6-[Η]-Deoxyuridine Assay) % Inhibition Δ542 A549-BL Methotrexate 82 ± 4 88 ± 6 1 μM, 3h 5798W93 3 ± 2 38 ± 3 1 μM, 3h 5-Fluorouracil 95 ± 8 91 ± 10 1 μM, 5h 1614W94 -2 ± 3 33 ± 6 1 μM, 5h Table 3 Antitumour Effects of Secretory /3-Lactamase and Cytosine Deaminase Genes in Mice Bearing Subcutaneous A549 Human Lung Adenocarcinoma Therapy Group Tunour Volume (mm 3 ) Percentage Inhibition (Relative to Control) Phosphate Buffered 1268 ± 212 0 Saline (No DNA) CMV-BL and 1614W94 511 ± 86 60 CMV-CD and 380 ± 237 70 5-Fluorocytosine 5-Fluorouracil alone 1021 ± 37 19 Table 4 Antitumour Effects of Secretory /3-Lactamase and Cytosine Deaminase Genes in Mice Bearing Intrathoracic H460 Human Large Cell Lung Tumours Therapy Group Mean Days of p Value Increased Life Survival Span (%) P h o s p h a t e 20 Buffered Saline (No DNA) CMV-CD + PBS 23 0.245 15 CMV-CD + 5- 27 0.009 40 Fluorocytosine C M V -BL + 32 0.012 60 1614W94 Example 4 (i) General Approach Acylation of an essential amino group of a wide variety of cytotoxic drugs leads to a significant loss of toxicity. Penicillin G amidase and the related penicillin V amidase activate these cytotoxic drugs by removing a phenylacetyl group from these nontoxic drugs (Senter, The FASEB Journal, 4, 188-193 (1990); Kerr et al, Cancer Immunol. Immunother., 31, 202-206 (1990); Bignami, et al, Cancer Research, 52, 5759-5764 (1992)). The goal is to pursue a gene directed enzyme prodrug therapy (GDEPT) approach in order to take advantage of the potential for long-term high levels of protein accumulation of enzyme at the tumor site using this approach. In order for the GDEPT approach to be successful using the penicillin G amidase gene, the gene must be expressed in human cells. In E. coli, expression of the penicillin G amidase gene is complex. The enzyme consists of two subunits with molecular weights of 24,000 daltons (a) and 65,000 daltons (b), produced by proteolytic cleavage of a 94,000 dalton precursor (Bock, et al, FEMS Microbil. Lett., 20, 141-144 (1983)) to remove an amino-terminal signal peptide (Oliver, et al. Gene 40, 9-14 (1985)). and a 54 amino acid endopeptide linking the two subunits (Schumacher et al, Nucl. Acids Res. 14, 5713-5727 (1986)). In E. coli, processing of the enzyme occurs during translocation of the enzyme to the periplasm. We show that the unmodified coding region representing this enzyme can be expressed, albeit at low levels, in human cells. (ϋ) Materials and Methods Preparation of genomic DNA from E. coli ATCC 11105 -- Bacterial genomic DNA was prepared using a modification of Current Protocols in Molecular Biology (Section 2.4.1). 50 ml of a confluent culture of ATCC 11105 (American Type Culture Collection, Rockville, MD, USA) grown in LB broth was pelleted and the supernatant removed. The pellet was resuspended in 2.2 ml of 10 mM Tris-Cl, pH 7.5, EDTA 1 mM, 0.5% SDS, and 0.1 mg/ml proteinase K and incubated for 1 hour at 37'C with occasional mixing. An equal volume of buffer-saturated phenol was added (Life Technologies. Inc., Gaithersburg, MD, USA), gently mixed by inversion, and subsequently centrifuged. The aqueous phase was removed using a large bore pipette and treated with phenol two more times as above. Ammonium acetate was added to a final concentration of 2M followed by the addition of 2 volumes of 100% ethanol at ambient temperature. Using a sealed Pasteur pipette, DNA was swirled around pipette and then transferred to new tube. The sample was centrifuged to remove excess liquid. The pellet was washed twice with 70% ethanol. After the second wash, DNA was resuspended in 300 μl of 10 mM Tris-Cl, pH 7.5, 1 mM EDTA. The DNA was sheared by passage through a 27-gauge needle followed by digestion of the DNA with DNAse-free RNAse from bovine pancreas (Boeringer Mannheim) at 5 micrograms/ml for 1 hour at 37°C. The DNA was then purified using the Promega DNA Clean Up Kit (Promega Corp.). The DNA was ethanol precipitated and resuspended in 10 mM Tris-Cl, pH 7.5, 1 mM EDTA and stored at 4°C. Isolation of the E. coli penicillin G amidase Coding Region -- The sequence of the E. coli penicillin G amidase gene from ATCC 11105 has been reported (Oh, et al, Gene, 56, 87-97 (1987), Schumacher et al, Nucl. Acids Res. 14, 5713-5727 (1986)). Oligonucleotide primers representing flanking regions of E. coli penicillin G amidase were synthesized (Oligo Therapeutics Inc. , Wilsonville, OR, USA) to produce a PCR product representing the E. coli penicillin G amidase sequence from nucleotides 199 (numbering from published sequence) to 2799 as well as attached flanking Hind III and Xba I restriction sites. An approximately 2.7 Kb PCR product was purified from an agarose gel using a Glass-Max kit (Life Technologies, Gaithersburg, MD, USA) and digested with Hind III and Xba I. The digested product was ligated to Hind III and Xba I digested pRc-CMV vector (InVitrogen, Inc.) and used to transform electrocompetent DH5α cells (Molecular Cloning, Sambrook et al, Cold Spring Harbor Press (1989)). Sequencing of the pCMV-pgal insert was performed using the fmol DNA Sequencing System (Promega Corp., Madison, WI, USA) ( Mead et al, Promega Notes, 16, (1988)). Two earlier reports on the sequence of E. coli penicillin G amidase differed at six positions (Oh et al (1987) and Schumacher et al (1986) supra). At all six positions, our sequence agreed completely with Schumacher et al., (1986) supra. Also, our penicillin G amidase sequence did not contain the 4 additional nucleotides reported by Valle et al. Gene, 50, 115-122 (1986)) in the ribosome binding site. Because constitutive expression of penicillin G amidase produced by pCMV-pgal was toxic during growth of the bacterial host carrying the modified penicillin G amidase gene, pCMV- pgal was further modified to give rise to pCMV-pga2 by removing all bacterial promoter sequences . One additional modification in this clone was the addition of a Kozak consensus (ACCGCC) sequence immediately 5' to the ATG start codon for possible enhanced translational efficiency in eukaryotic cells. Penicillin G Amidase Assays -- The catalytic activity of penicillin G amidase was routinely determined from the increase in A 403 during hydrolysis of 1 ml 0.4 mM 6-nitro- phenylacetamido-benzoic acid in 50 mM phosphate pH 7.5 (Kutzbach et al, Hoppe-Seyler's Z. Physiol. Chem., 354, 45-53 (1974)). In vitro Expression — Human cell lines A549 flung adenocarcinoma) and NCI-NCI-H441 (lung Clara-like) were obtained from ATCC (Rockville, MD, USA). Cells were transiently transfected via electroporation (manufacturer's instructions, Bio-Rad, Hercules, CA). Transfections using 6 mg of plasmid DNA were carried out in RPMI 16-40 media + glutamine (MediaTech, Washington, DC, USA) and 10 mM glucose and 0.1 mM DTT at 0.2 kilovolts and 960 microfarads in electroporation cuvettes containing a 0.4 cm gap (BioRad, Hercules, CA, USA). 24 hours after transfections, 4199W93 in 100% DMSO was added to a final concentration of 10 μM (DMSO concentration was 2% final). Viable cells were quantitated after 48 hours by counting the cells in the presence of trypan blue dye. RNA Analysis — A549 human lung adenocarcinoma cells were cotransfected as described above with pCMV-pgal and pCMV-BL, a construct identical to pCMV-pgal with the exception of replacing the penicillin G amidase coding region with the coding region of TEM /3-lactamase. RNA was prepared as described in Current Protocols in Molecular Biology (Section 4.2.4). 10 μg of purified RNA was slot blotted onto Hybond-N filters (Amersham, Arlington Heights, IL), cross-linked by UV irradiation, and probed with F ** P]-labeled DNA representing either plasmid pCMV-pgal or pCMV-BL. Plasmid DNAs were labeled by a random random primers DNA labeling system (Life Technologies, Gaithersburg, MD) following manufacturer's instructions. The relative amounts of RNA determined by hybridization of the ["Pj-labeled probe was quantitated using a phoshorimager (Molecular Dynamics) using ImageQuant software. (Abbreviations: E. coli, Escherichia coli; HSV, Herpes Simplex Virus; DTT, dithiothreitol; PCR, polymerase chain reaction.) (iii) Results Prodrugs; Cytotoxicity and Activation - Phenylacetylation of an essential amino group of a wide variety of cytotoxic drugs leads to a significant loss of cytotoxicity (Senter et al (1990) supra). We synthesized N-(2-phenylacetyl) daunomycin (4199W93) and N-(2- phenylacetyl) methotrexate and determined the cytotoxicity of these prodrugs in WiDr colon carcinoma cells relative to their respective parent drugs. The results are shown in Table 5. As expected phenylacetylation of daunomycin reduced the cytotoxicity of daunomycin and resulted in an increase of several orders of magnitude in its IC 50 value. In contrast, phenylacetylation of methotrexate increased its IC 50 value by only 5-fold (Table 5). In order to investigate the reasons for these differences we followed the generation of the parent drugs during these experiments by reverse phase liquid chromatography (RPLC). We observed that N-(2-phenylacetyl) daunomycin was very stable throughout the duration of the experiment as no daunomycin was detected in these cultures (data not shown). In contrast, a significant amount of methotrexate was generated in tissue cultures incubated with N-(2-phenylacetyl) methotrexate (data not shown). The half life for N-(2 -phenylacetyl) methotrexate in these experiments was about 50 hr. • Penicillin G amidase (PGA) can remove a phenylacetyl group from many different compounds (Senter et al (1990) supra). A purified preparation of PGA efficiently converted N-(2-phenylacetyl) daunomycin to daunomycin as followed by RPLC. However, we were unable to determine the detailed kinetics of activation due to the insolubility of the prodrug. We determined the kinetics of activation of N-(2-phenylacetyl) methotrexate by purified PGA using RPLC. The activation reaction was linear as a function of time and enzyme concentration when the substrate concentration was at saturation, and the enzyme followed normal Michaelis-Menton kinetics (data not shown). The values of k cti and Km, for PGA with N-(2-phenylacetyl) methotrexate as a substrate were determined and were 1.0 sec" 1 and 0.12 mM, respectively. This corresponded to a substrate specificity constant (k eu /K m ) of 8.3 sec 'mM 1 . Construction of Eucaryotic Expression Vectors - Two eukaryotic expression vectors, containing the E. coli penicillin G amidase coding region, designated pCMV-pgal and pCMV-pga2 were constructed. Purification of pCMV-pgal was problematic because the plasmid caused lysis of its bacterial host. When E. coli DH5a transformed with pCMV- pgal was grown at 37°C, the recombinant strain displayed the same growth kinetics as the parental DH5α strain until reaching an OD„, of 0.4, after which there followed rapid and apparently complete lysis (Figure 5). Bacterial lysis, as judged by the abnormal colony morphology, was also obvious when DH5a containing pCMV-pgal was grown on the surface of agar plates. The bacterial lysis induced by pCMV-pgal was due to production of active penicillin G amidase (data not shown). To facilitate plasmid production of pCMV-pga clones, the interfering expression of the penicillin G amidase gene in E. coli was blocked. To do this, pCMV-pgal was modified to give rise to pCMV-pga2 by removing all bacterial promoter sequences (Figure 6). One additional modification in this clone was the addition of a Kozak consensus sequence immediately 5' to the ATG start codon for possible enhanced translational efficiency in eukaryotic cells. Colonies harboring this plasmid had normal growth characteristics and produced large quantities of plasmid in plasmid purifications. When transiently transfected into A549 lung adenocarcinoma and NCI-H441 lung Clara-like cell lines, pCMV-pga2 conferred the same degree of sensitization to these cell lines to N-acetyl phenyl daunomycin as pCMV-pga2 (data not shown). Expression of PGA in Human Lung Tumor Cell Lines - We employed a cell-based assay to determine whether transient transfection of cultured cells with pCMV-pgal results in increased sensitivity of the cells to a relatively nontoxic and stable prodrug N-acetyl phenyl daunomycin (Table 6). Two human lung cancer cell lines, NCI-H441 and A549 were transiently transfected with pCMV-pgal. Twenty-four hours after the transfection, the prodrug was added at a final concentration of 10 μM. The results are shown in Table 6. Addition of prodrug alone had little effect on the cell morphology or on total number of viable A549 or H441 cells. In addition, transfection with control constructs in combination with the prodrug did not result in any significant reductions in viable cell number. Transfection of both NCI-H441 and A549 with pCMV-pgal plus prodrug resulted in a dramatic reduction in the number of viable cells after 48 hours and altered morphology and caused significant accumulation of cell debris. In contrast, transfection with control constructs in combination with the prodrug did not result in any significant reductions in viable cell number. By-Stander Tumor Cell Cytotoxicity and Low Levels of the Enzyme - When identical transfection conditions were assessed in A549 using a /3-galactosidase gene reporter, we found that only approximately 0.1 % of the total cells were transfected. Thus, the observed decrease in cell number in cells treated with the prodrug and pCMV-pgal must be due to transfer of a killing effect of the transfected cells to the non-transfected cells, presumably through diffusion of daunomycin catalytically produced by extracellular enzyme. However, the following observations suggest that the amount of extracellular enzyme activity produced is relatively low. The concentration of the prodrug used in above sensitivity experiments was about 200-fold more than a concentration of daunomycin which reduces the viability of WiDr by 100% . Thus, an equivalent dose of parent drug was not generated in the experiments described above, indicating that either activity of the produced enzyme or the levels of the enzyme are relatively low. The relatively low levels of enzyme produced could be due to several factors including, 1) instability of mRNA in human cells, 2) instability of the protein, 3) inefficiency in post- translational processing and secretion of the precursor. To investigate the factors involved in the low levels of penicllin G amidase produced in transfections, we examined the first step in expression and quantitated the levels of penicillin G amidase mRNA steady-state levels relative to another gene produced from the same expression vector utilizing the same expression regulatory signals, such as the CMV immediate/early promoter. In these experiments, A549 cells were transiently cotransfected with pCMV-pgal and pCMV-BL, an expression vector which expresses the TEM /3-lactamase gene. Total RNA was prepared from the cells, equal amounts were blotted onto a solid support, and each probed with either a penicillin G amidase or a TEM /3-lactamase [ 32 P] -labeled probed, respectively. We found that the steady-state levels of penicillin G amidase mRNA were significantly lower than the /8-lactamase levels (data not shown). Thus, the low levels of expression penicillin G amidase may be in part accounted for by low steady-state levels of mRNA. (iv) Discussion We have shown that the pCMV-pga constructs give rise to active penicillin G amidase when delivered to tumor cells. Since the unprocessed precursor protein of penicillin G amidase has been shown to be inactive (Burtscher et al, Brit. J. Biochem. 205, 77-83 (1992)), expression of functional enzyme from the gene representing the precursor in human cells implies that human cells are capable of removing part or all of the endopeptide sequence from the penicillin G amidase precursor protein. In E. coli, the penicillin G amidase precursor is processed as it is being translocated across the periplasmic membrane. It is possible that human cells have a homologous enzyme, or that the endopeptide is proteolytically sensitive and is removed by general protease activity present in the cell. We have also provided evidence that the enzyme produced by the pCMV-pga vectors is secreted from human cells. This is consistent with the fact that the enzyme contains a bacterial signal peptide, and that bacterial signal peptides have been shown to be functional in eukaryotic cells (Muller et al, J. Biol. Chem. 257, 11860-11863 (198?)). Extracellular prodrug activation by a secreted enzyme has the potential advantage of maximizing bystander effects, i.e. transfer of cytotoxic effects from transfected cells to neighboring cells. This characteristic is critical given the limitations of current gene transfer technology. Of course, this effect would be influenced by other factors such as diffusion of the enzyme away from the tumor site and stability of the enzyme in the extracellular space. The fact that penicillin G amidase is capable of activating multiple prodrugs allows the evaluation of multiple drugs for efficacy. Since each drug and prodrug will display its own characteristic pharmacokinetics and pharmacodynamics, each drug and drug combination must be explored under many conditions. Increased bystander effects could also conceivably be achieved through judicious choice of a prodrug(s). Table 5 IC 50 , μM Daunomycin 0.07 N-(2-Phenylacetyl) Daunomycin > 100 Methotrexate 0.02 N-(2-Phenylacetyl) Methotrexate 0.11 Table 6 % Cell Viability Cell Line Addition A549 H441 pCMV-pgal 94 96 4199W93 88 82 4199W93 + pCMV-pgal 30 35 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: The Wellcome Foundation Limited (B) STREET: Unicorn House (C) CITY: 160, Euston Road (D) STATE: London (E) COUNTRY: Great Britain (F) POSTAL CODE (ZIP): N 1 2BP (ii) TITLE OF INVENTION: Enzyme Prodrug Therapy (iii) NUMBER OF SEQUENCES: 6 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1260 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: CDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:!..1257 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATG CGG GGG TTG CTG GTG TTG AGT GTC CTG TTG GGG GCT GTC TTT GGC 48 Met Arg Gly Leu Leu Val Leu Ser Val Leu Leu Gly Ala Val Phe Gly 1 5 10 15 AAG GAG GAC TTT GTG GGG CAT CAG GTG CTC CGA ATC TCT GTA GCC GAT 96 Lye Glu Asp Phe Val Gly His Gin Val Leu Arg lie Ser Val Ala Asp 20 25 30 GAG GCC CAG GTA CAG AAG GTG AAG GAG CTG GAG GAC CTG GAG CAC CTG 144 Glu Ala Gin Val Gin Lye Val Lys Glu Leu Glu Asp Leu Glu Hie Leu 35 40 45 CAG CTG GAC TTC TGG CGG GGG CCT GCC CAC CCT GGC TCC CCC ATC GAC 192 Gin Leu Asp Phe Trp Arg Gly Pro Ala His Pro Gly Ser Pro lie Asp 50 55 60 GTC CGA GTG CCC TTC CCC AGC ATC CAG GCG GTC AAG ATC TTT CTG GAG 240 Val Arg Val Pro Phe Pro Ser lie Gin Ala Val Lys lie Phe Leu Glu 65 70 75 80 TCC CAC GGC ATC AGC TAT GAG ACC ATG ATC GAG GAC GTG CAG TCG CTG 288 Ser His Gly lie Ser Tyr Glu Thr Met lie Glu Asp Val Gin Ser Leu 85 90 95 CTG GAC GAG GAG CAG GAG CAG ATG TTC GCC TTC CGG TCC CGG GCG CGC 336 Leu Asp Glu Glu Gin Glu Gin Met Phe Ala Phe Arg Ser Arg Ala Arg 100 105 110 TCC ACC GAC ACT TTT AAC TAC GCC ACC TAC CAC ACC CTG GAG GAG ATC 384 Ser Thr Asp Thr Phe λsn Tyr Ala Thr Tyr His Thr Leu Glu Glu lie 115 120 125 TAT GAC TTC CTG GAC CTG CTG GTG GCG GAG AAC CCG CAC CTT GTC AGC 432 Tyr Asp Phe Leu Asp Leu Leu Val Ala Glu Asn Pro His Leu Val Ser 130 135 140 AAG ATC CAG ATT GGC AAC ACC TAT GAA GGG CGT CCC ATT TAT GTG CTG 480 Lys lie Gin lie Gly Asn Thr Tyr Glu Gly Arg Pro lie Tyr Val Leu 145 150 155 160 AAG TTC AGC ACG GGG GGC AGT AAG CGT CCA GCC ATC TGG ATC GAC ACG 528 Lys Phe Ser Thr Gly Gly Ser Lys Arg Pro Ala lie Trp lie Asp Thr 165 170 175 GGC ATC CAT TCC CGG GAG TGG GTC ACC CAG GCC AGT GGG GTC TGG TTT 576 Gly lie His Ser Arg Glu Trp Val Thr Gin Ala Ser Gly Val Trp Phe 180 185 190 GCA AAG AAG ATC ACT CAA GAC TAT GGG CAG GAT GCA GCT TTC ACC GCC 624 Ala Lys Lys He Thr Gin Asp Tyr Gly Gin Asp Ala Ala Phe Thr Ala 195 200 205 ATT CTC GAC ACC TTG GAC ATC TTC CTG GAG ATC GTC ACC AAC CCT GAT 672 He Leu Asp Thr Leu Asp He Phe Leu Glu He Val Thr Asn Pro Asp 210 215 220 GGC TTT GCC TTC ACG CAC AGC ACG AAT CGC ATG TGG CGC AAG ACT CGG 720 Gly Phe Ala Phe Thr His Ser Thr Asn Arg Met Trp Arg Lys Thr Arg 225 230 235 240 TCC CAC ACA GCA GGC TCC CTC TGT ATT GGC GTG GAC CCC AAC AGG AAC 768 Ser His Thr Ala Gly Ser Leu Cys He Gly Val Asp Pro Asn Arg Asn 245 250 255 TGG GAC GCT GGC TTT GGG TTG TCC GGA GCC AGC AGT AAC CCC TGC TCG 816 Trp Asp Ala Gly Phe Gly Leu Ser Gly Ala Ser Ser Asn Pro Cys Ser 260 265 270 GAG ACT TAC CAC GGC AAG TTT GCC AAT TCC GAA GTG GAG GTC AAG TCC 864 Glu Thr Tyr His Gly Lys Phe Ala Asn Ser Glu Val Glu Val Lys Ser 275 280 285 ATT GTA GAC TTT GTG AAG GAC CAT GGG AAC ATC AAG GCC TTC ATC TCC 912 He Val Asp Phe Val Lys Asp His Gly Asn He Lys Ala Phe He Ser 290 295 300 ATC CAC AGC TAC TCC CAG CTC CTC ATG TAT CCC TAT GGC TAC AAA ACA 960 He His Ser Tyr Ser Gin Leu Leu Met Tyr Pro Tyr Gly Tyr Lys Thr 305 310 315 320 GAA CCA GTC CCT GAC CAG GAT GAG CTG GAT CAG CTT TCC AAG GCT GCT 1008 Glu Pro Val Pro λap Gin Asp Glu Leu Asp Gin Leu Ser Lys Ala Ala 325 330 335 GTG ACA GCC CTG GCC TCT CTC TAC GGG ACC AAG TTC AAC TAT GGC AGC 1056 Val Thr Ala Leu Ala Ser Leu Tyr Gly Thr Lys Phe Asn Tyr Gly Ser 340 345 350 ATC ATC AAG GCA ATT TAT CAA GCC AGT GGA AGC ACT ATT GAC TGG ACC 1104 He He Lys Ala He Tyr Gin Ala Ser Gly Ser Thr He Asp Trp Thr 355 360 365 TAC AGC CAG GGC ATC AAG TAC TCC TTC ACC TTC GAG CTC CGG GAC ACT 1152 Tyr Ser Gin Gly He Lys Tyr Ser Phe Thr Phe Glu Leu Arg Asp Thr 370 375 380 GGG CGC TAT GGC TTC CTG CTG CCA GCC TCC CAG ATC ATC CCC ACA GCC 1200 Gly Arg Tyr Gly Phe Leu Leu Pro Ala Ser Gin He He Pro Thr Ala 385 390 395 400 AAG GAG ACG TGG CTG GCG CTT CTG- ACC ATC ATG GAG CAC ACC CTG AAT 1248 Lys Glu Thr Trp Leu Ala Leu Leu Thr He Met Glu His Thr Leu Asn 405 410 415 CAC CCC TAC TGA 1260 His Pro Tyr (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 419 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 : Met Arg Gly Leu Leu Val Leu Ser Val Leu Leu Gly Ala Val Phe Gly 1 5 10 15 Lye Glu Asp Phe Val Gly Hia Gin Val Leu Arg He Ser Val Ala Asp 20 25 30 Glu Ala Gin Val Gin Lya Val Lya Glu Leu Glu Asp Leu Glu His Leu 35 40 45 Gin Leu Asp Phe Trp Arg Gly Pro Ala Hia Pro Gly Ser Pro He Asp 50 55 60 Val Arg Val Pro Phe Pro Ser He Gin Ala Val Lys He Phe Leu Glu 65 70 75 80 Ser His Gly He Ser Tyr Glu Thr Met He Glu Asp Val Gin Ser Leu 85 90 95 Leu Asp Glu Glu Gin Glu Gin Met Phe Ala Phe Arg Ser Arg Ala Arg 100 105 110 Ser Thr Asp Thr Phe Asn Tyr Ala Thr Tyr His Thr Leu Glu Glu He 115 120 125 Tyr Asp Phe Leu Asp Leu Leu Val Ala Glu Asn Pro His Leu Val Ser 130 135 140 Lys He Gin He Gly Asn Thr Tyr Glu Gly Arg Pro He Tyr Val Leu 145 150 155 160 Lya Phe Ser Thr Gly Gly Ser Lys Arg Pro Ala He Trp He Aap Thr 165 170 175 Gly He His Ser Arg Glu Trp Val Thr Gin Ala Ser Gly Val Trp Phe 180 185 190 Ala Lya Lya He Thr Gin Aap Tyr Gly Gin Asp Ala Ala Phe Thr Ala 195 200 205 He Leu λap Thr Leu Asp He Phe Leu Glu He Val Thr Asn Pro Asp 210 215 220 Gly Phe Ala Phe Thr His Ser Thr Asn Arg Met Trp Arg Lys Thr Arg 225 230 235 240 Ser His Thr Ala Gly Ser Leu Cys He Gly Val Asp Pro Asn Arg Asn 245 250 255 Trp Asp Ala Gly Phe Gly Leu Ser Gly Ala Ser Ser Asn Pro Cya Ser 260 265 270 Glu Thr Tyr His Gly Lys Phe Ala Asn Ser Glu Val Glu Val Lys Ser 275 280 285 He Val Asp Phe Val Lys Asp His Gly Asn He Lys Ala Phe He Ser 290 295 300 He His Ser Tyr Ser Gin Leu Leu Met Tyr Pro Tyr Gly Tyr Lys Thr 305 310 315 320 Glu Pro Val Pro Asp Gin Asp Glu Leu Asp Gin Leu Ser Lys Ala Ala 325 330 335 Val Thr Ala Leu Ala Ser Leu Tyr Gly Thr Lys Phe Asn Tyr Gly Ser 340 345 350 He He Lys Ala He Tyr Gin Ala Ser Gly Ser Thr He Asp Trp Thr 355 360 365 Tyr Ser Gin Gly He Lys Tyr Ser Phe Thr Phe Glu Leu Arg Asp Thr 370 375 380 Gly Arg Tyr Gly Phe Leu Leu Pro Ala Ser Gin He He Pro Thr Ala 385 390 395 400 Lys Glu Thr Trp Leu Ala Leu Leu Thr He Met Glu His Thr Leu Asn 405 410 415 Hia Pro Tyr (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: CDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TTGCATλAGC TTGCCλCCλT GAGTATTCAA CATTTCCGTG TC 42 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GATCTGTCTA GATTλCCAAT GCTTAλTCλG TGAGGC 36 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (λ) LENGTH: 6229 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: CDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:693..1550 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CGATGTACGG GCCAGATATA CGCGTTGACA TTGATTATTG ACTAGTTATT AATAGTAATC 60 AATTACGGGG TCATTAGTTC ATAGCCCATA TATGGAGTTC CGCGTTACAT AACTTACGGT 120 AAATGGCCCG CCTGGCTGAC CGCCCAACGA CCCCCGCCCA TTGACGTCAA TAATGACGTλ 180 TGTTCCCλTA GTAACGCCAλ TλGGGλCTTT CCATTGACGT CAATGGGTGG ACTATTTACG 240 GTAλλCTGCC CACTTGGCλG TλCATCAAGT GTATCATATG CCAAGTACGC CCCCTATTGλ 300 CGTCAATGAC GGTAAATGGC CCGCCTCGCA TTATGCCCAG TACATGACCT TATGGGACTT 360 TCCTACTTGG CAGTACλTCT λCGTλTTAGT CATCGCTATT ACCATGGTGA TGCGGTTTTG 420 GCAGTACATC AλTGGGCGTG GλTλGCGGTT TGACTCACGG GGATTTCCAA GTCTCCACCC 480 CATTGACGTC λλTGGGλGTT TGTTTTGGCλ CCλλλλTCλλ CGGGACTTTC CAAAATGTCG 540 TλλCλλCTCC GCCCCATTGA CGCAλλTGGG CGGTλGGCGT GTACGGTGGG AGGTCTATAT 600 λλGCλGλGCT CTCTGGCTλλ CTλGλGλλCC CACTGCTTAA CTGGCTTATC GAAATTAATA 660 CGACTCλCTλ TAGGGAGACC AλGCTTGCCλ CC ATG AGT ATT CAA CAT TTC CGT 713 Met Ser He Gin Hia Phe Arg 420 425 GTC GCC CTT ATT CCC TTT TTT GCG GCA TTT TGC CTT CCT GTT TTT GCT 761 Val λla Leu He Pro Phe Phe Ala λla Phe Cys Leu Pro Val Phe λla 430 435 440 CλC CCλ Gλλ λCG CTG GTG Aλλ GTA AAA GAT GCT GAA GAT CAG TTG GGT 809 His Pro Glu Thr Leu Val Lys Val Lys λsp λla Glu λsp Gin Leu Gly 445 450 455 GCλ CGA GTG GGT TAC ATC GAA CTG GAT CTC AAC AGC GGT AAG ATC CTT 857 Ala Arg Val Gly Tyr He Glu Leu λsp Leu Asn Ser Gly Lys He Leu 460 465 470 GλG λGT TTT CGC CCC Gλλ Gλλ CGT TTT CCλ λTG λTG λGC λCT TTT λλλ 905 Glu Ser Phe λrg Pro Glu Glu λrg Phe Pro Met Met Ser Thr Phe Lys 475 480 485 490 GTT CTG CTA TGT GGC GCG GTA TTA TCC CGT ATT GλC GCC GGG CAA GλG 953 Val Leu Leu Cys Gly λla Val Leu Ser λrg He λsp λla Gly Gin Glu 495 500 505 Cλλ CTC GGT CGC CGC λTλ CλC TAT TCT CAG AAT GAC TTG GTT GAG TAC 1001 Gin Leu Gly λrg λrg He His Tyr Ser Gin λsn λsp Leu Val Glu Tyr 510 515 520 TCλ CCλ GTC λCλ GAA λλG CλT CTT λCG GλT GGC λTG λCλ GTλ λGλ Gλλ 1049 Ser Pro Val Thr Glu Lys Hia Leu Thr λap Gly Met Thr Val λrg Glu 525 530 535 TTλ TGC λGT GCT GCC λTλ λCC λTG λGT GλT AAC ACT GCG GCC AAC TTA 1097 Leu Cya Ser λla λla He Thr Met Ser λap λsn Thr λla λla λsn Leu 540 545 550 CTT CTG ACλ λCG λTC GGλ GGλ CCG λλG GλG CTλ λCC GCT TTT TTG CλC 1145 Leu Leu Thr Thr He Gly Gly Pro Lys Glu Leu Thr λla Phe Leu Hia 555 560 565 570 λλC λTG GGG GλT CλT GTλ λCT CGC CTT GλT CGT TGG GAA CCG GAG CTG 1193 λβn Met Gly λap Hia Val Thr λrg Leu λβp λrg Trp Glu Pro Glu Leu 575 580 585 AAT GAA GCC ATA CCA AAC GAC GAG CGT GAC λCC λCG λTG CCT GTλ GCλ 1241 λβn Glu λla He Pro λβn λβp Glu λrg λβp Thr Thr Met Pro Val λla 590 595 600 λTG GCλ λCλ λCG TTG CGC λλλ CTλ TTλ λCT GGC Gλλ CTλ CTT λCT CTλ 1289 Met λla Thr Thr Leu λrg Lye Leu Leu Thr Gly Glu Leu Leu Thr Leu 605 610 615 GCT TCC CGG Cλλ Cλλ TTλ λTλ GλC TGG λTG GλG GCG GAT Aλλ GTT GCλ 1337 λla Ser λrg Gin Gin Leu He λβp Trp Met Glu λla λsp Lys Val λla 620 625 630 GGλ CCλ CTT CTG CGC TCG GCC CTT CCG GCT GGC TGG TTT λTT GCT GλT 1385 Gly Pro Leu Leu λrg Ser λla Leu Pro λla Gly Trp Phe He λla λβp 635 640 645 650 λλλ TCT GGλ GCC GGT GλG CGT GGG TCT CGC GGT λTC λTT GCλ GCλ CTG 1433 Lye Ser Gly λla Gly Glu λrg Gly Ser λrg Gly He He λla λla Leu 655 660 665 GGG CCλ GλT GGT AAG CCC TCC CGT ATC GTA GTT λTC TλC λCG λCG GGG 1481 Gly Pro λβp Gly Lye Pro Ser λrg He Val Val He Tyr Thr Thr Gly 670 675 680 λGT CAG GCA ACT λTG GλT Gλλ CGA AAT λGλ CAG ATC GCT GAG λTλ GGT 1529 Ser Gin λla Thr Met λβp Glu λrg λβn λrg Gin He λla Glu He Gly 685 690 695 GCC TCλ CTG λTT λλG CλT TGG TλλTCTλGλG GGCCCTλTTC TλTλGTGTCλ 1580 λla Ser Leu He Lye Hie Trp 700 705 CCTλλλTGCT λGλGCTCGCT GλTCλGCCTC GλCTGTGCCT TCTλGTTGCC λGCCλTCTGT 1640 TGTTTGCCCC TCCCCCGTGC CTTCCTTGλC CCTGGλλGGT GCCλCTCCCλ CTGTCCTTTC 1700 CTλλTλλλλT GλGGλλλTTG CλTCGCλTTG TCTGAGTAGG TGTCλTTCTA TTCTGGGGGG 1760 TGGGGTGGGG CAGGλCλGCλ AGGGGGλGGλ TTGGGλλGλC AATAGCλGGC ATGCTGGGGλ 1820 TGCGGTGGGC TCTATGGAλC CAGCTGGGGC TCGλGGGGGG λTCCCCλCGC GCCCTGTλGC 1880 GGCGCλTTλλ GCGCGGCGGG TGTGGTGGTT λCGCGCλGCG TGλCCGCTλC λCTTGCCλGC 1940 GCCCTAGCGC CCGCTCCTTT CGCTTTCTTC CCTTCCTTTC TCGCCACGTT CGCCGGCTTT 2000 CCCCGTCAλG CTCTλλλTCG GGGCλTCCCT TTλGGGTTCC GATTTAGTGC TTTλCGGCλC 2060 CTCGλCCCCλ λλλλλCTTGλ TTλGGGTGAT GGTTCACGTA GTGGGCCATC GCCCTGATAG 2120 λCGGTTTTTC GCCTTTλCTG λGCλCTCTTT λλTλGTGGλC TCTTGTTCCA AλCTGGλλCλ 2180 λCλCTCλλCC CTλTCTCGGT CTλTTCTTTT GλTTTλTAAG ATTTCCλTCG CCATGTAλλλ 2240 GTGTTλCλλT TλGCλTTλλλ TTλCTTCTTT λTλTGCTλCT λTTCTTTTGG CTTCGTTCλC 2300 GGGGTGGGTλ CCGλGCTCGλ λTTCTGTGGA ATGTGTGTCA GTTAGGGTGT GGλλAGTCCC 2360 CλGGCTCCCC λGGCλGGCλG λλGTλTGCλλ λGCλTGCλTC TCAATTAGTC AGCλλCCλGG 2420 TGTGGAλλGT CCCCAGGCTC CCCAGCλGCC AGAAGTATGC λλλGCλTGCλ TCTCλλTTλG 2480 TCλGCλλCCλ TλGTCCCGCC CCTλλCTCCG CCCλTCCCGC CCCTλλCTCC GCCCAGTTCC 2540 GCCCλTTCTC CGCCCCλTGG CTGλCTλλTT TTTTTTλTTT λTGCλGλGGC CGλGGCCGCC 2600 TCGGCCTCTG λGCTλTTCCλ GλλGTλGTGλ GGλGGCTTTT TTGGλGGCCT λGGCTTTTGC 2660 λλλλλGCTCC CGGGλGCTTG GλTλTCCλTT TTCGGλTCTG λTCλλGλGλC λGGλTGλGGλ 2720 TCGTTTCGCλ TGλTTGλλCλ λGλTGGλTTG CλCGCλGGTT CTCCGGCCGC TTGGGTGGλG 2780 λGGCTλTTCG GCTλTGλCTG GGCλCλλCλG λCλλTCGGCT GCTCTGλTGC CGCCGTGTTC 2840 CGGCTGTCλG CGCλGGGGCG CCCGGTTCTT TTTGTCλλGA CCGACCTGTC CGGTGCCCTG 2900 λλTGλλCTGC AGGACGAGGC λGCGCGGCTλ TCGTGGCTGG CCλCGλCGGG CGTTCCTTGC 2960 GCAGCTGTGC TCGACGTTGT CACTGλλGCG GGAλGGGλCT GGCTGCTλTT GGGCGλλGTG 3020 CCGGGGCAGG λTCTCCTGTC λTCTCλCCTT GCTCCTGCCG λGλλλGTλTC CλTCλTGGCT 3080 GATGCAATGC GGCGGCTGCA TλCGCTTGλT CCGGCTACCT GCCCATTCGλ CCλCCλλGCG 3140 AλλCλTCGCλ TCGAGCGλGC ACGTACTCGG ATGGλλGCCG GTCTTGTCGA TCAGGλTGλT 3200 CTGGACGAAG AGCλTCλGGG GCTCGCGCCA GCCGλλCTGT TCGCCAGGCT CλλGGCGCGC 3260 ATGCCCGλCG GCGAGGATCT CGTCGTGACC CλTGGCGATG CCTGCTTGCC GAATATCATG 3320 GTGGAλλλTG GCCGCTTTTC TGGλTTCλTC GλCTGTGGCC GGCTGGGTGT GGCGGλCCGC 3380 TλTCAGGACA TAGCGTTGGC TλCCCGTGAT ATTGCTGAAG AGCTTGGCGG CGAATGGGCT 3440 GACCGCTTCC TCGTGCTTTλ CGGTλTCGCC GCTCCCGATT CGCAGCGCAT CGCCTTCTλT 3500 CGCCTTCTTG ACGAGTTCTT CTGAGCGGGλ CTCTGGGGTT CGAAATGACC GACCλλGCGλ 560 CGCCCλλCCT GCCλTCλCGλ GλTTTCGλTT CCλCCGCCGC CTTCTλTGλλ λGGTTGGGCT 3620 TCGGλλTCGT TTTCCGGGλC GCCGGCTGGλ TGλTCCTCCλ GCGCGGGGλT CTCλTGCTGG 3680 λGTTCTTCGC CCλCCCCλλC TTGTTTλTTG CλGCTTλTλλ TGGTTλCλλλ TλλλGCλλTλ 3740 GCλTCλCλλλ TTTCλCλλλT AλAGCλTTTT TTTCλCTGCλ TTCTλGTTGT GGTTTGTCCλ 3800 λλCTCλTCλA TGTATCTTλT CλTGTCTGGλ TCCCGTCGλC CTCGλGλGCT TGGCGTλλTC 3860 ATGGTCATAG CTGTTTCCTG TGTGAλλTTG TTλTCCGCTC λCλATTCCλC ACλλCλTλCG 3920 AGCCGGAAGC ATAAAGTGTA AλGCCTGGGG TGCCTAATGA GTGAGCTAλC TCACATTAAT 3980 TGCGTTGCGC TCACTGCCCG CTTTCCλGTC GGGλλλCCTG TCGTGCCλGC TGCλTTAATG 4040 AλTCGGCCλA CGCGCGGGGλ GλGGCGGTTT GCGTλTTGGG CGCTCTTCCG CTTCCTCGCT 4100 CλCTGλCTCG CTGCGCTCGG TCGTTCGGCT GCGGCGλGCG GTλTCλGCTC λCTCλAAGGC 4160 GGTAATACGG TTATCCλCλG AλTCλGGGGλ TAλCGCλGGλ AλGλλCλTGT GAGCλλλλGG 4220 CCAGCAAλλG GCCλGGλλCC GTA AAAGGC CGCGTTGCTG GCGTTTTTCC ATλGGCTCCG 4280 CCCCCCTGAC GAGCATCλCλ λλλATCGACG CTCAAGTCAG λGGTGGCGAλ λCCCGλCλGG 4340 λCTλTAAλGA TλCCλGGCGT TTCCCCCTGG λλGCTCCCTC GTGCGCTCTC CTGTTCCGλC 4400 CCTGCCGCTT λCCGGλTλCC TGTCCGCCTT TCTCCCTTCG GGλλGCGTGG CGCTTTCTCλ 4460 ATGCTCλCGC TGTλGGTλTC TCλGTTCGGT GTλGGTCGTT CGCTCCλλGC TGGGCTGTGT 4520 GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTλTCC GGTAACTATC GTCTTGAGTC 4580 CAλCCCGGTA AGACACGACT TATCGCCλCT GGCλGCλGCC ACTGGTλλCλ GGATTAGCAG 4640 λGCGλGGTAT GTλGGCGGTG CTλCλGλGTT CTTGλλGTGG TGGCCTλλCT λCGGCTλCλC 4700 TAGAAGGACA GTATTTGGTA TCTGCGCTCT GCTGλλGCCλ GTTACCTTCG GAAAAAGAGT 4760 TCGTAGCTCT TGλTCCCGCλ λλCλλλCCλC CCCTGGTAGC CGTGGTTTTT TTGTTTGCλλ 4820 GCAGCAGATT λCGCCCλGλλ λλλλλGGλTC TCAAGAAGAT CCTTTGATCT TTTCTACGGG 4880 GTCTGλCGCT CλGTGGλλCG AAAACTCACG TTAAGGGλTT TTGGTCλTGλ GATTATCAAA 4940 λλGGλTCTTC λCCTλGλTCC TTTTλλATTλ AλλλTGλλGT TTTA ATCλλ TCTAλλGTλT 5000 λTλTGλGTλλ λCTTGGTCTG λCλGTTλCCA ATGCTTλλTC λGTGλGGCλC CTλTCTCλGC 5060 GλTCTGTCTλ TTTCGTTCλT CCλTλGTTGC CTGλCTCCCC GTCGTGTλGλ TλλCTλCGλT 5120 λCGGGλGGGC TTλCCλTCTG GCCCCλGTGC TGCλλTGλTλ CCGCGλGλCC CλCGCTCλCC 5180 GGCTCCAGAT TTATCAGCAA TAλλCCλGCC AGCCGGAλGG GCCGAGCGCλ GλλGTGGTCC 5240 TGCλλCTTTλ TCCGCCTCCA TCCAGTCTλT TλλTTGTTGC CGGGλλGCTλ GλGTλλGTλG 5300 TTCGCCλGTT λλTλGTTTGC GCλλCGTTGT TGCCλTTGCT λCλGGCATCG TGGTGTCACG 5360 CTCGTCGTTT GGTλTGGCTT CλTTCλGCTC CGGTTCCCAA CGATCλλGGC GλGTTλCλTG 5420 λTCCCCCλTG TTGTGCλλAA AλGCGGTTλG CTCCTTCGGT CCTCCGλTCG TTGTCλGλλG 5480 TλAGTTGGCC GCλGTGTTλT CλCTCλTGGT TλTGGCλGCA CTGCATAλTT CTCTTλCTGT 5540 CλTGCCλTCC GTλ GλTGCT TTTCTGTGλC TGGTGλGTAC TCAλCCAAGT CATTCTGAGλ 5600 λTAGTGTATG CGGCGACCGλ GTTGCTCTTG CCCGGCGTCA ATλCGGGλTλ ATλCCGCGCC 5660 λCATλGCλGλ ACTTTAAAAG TGCTCATCλT TGGAλλλCGT TCTTCGGGGC GAλλλCTCTC 5720 AλGGλTCTTλ CCGCTGTTGλ GλTCCλGTTC GλTGTλλCCC λCTCGTGCλC CCλλCTGλTC 5780 TTCλGCλTCT TTTACTTTCλ CCAGCGTTTC TGGGTGλGCλ AλλλCλGGλλ GGCAλλλTGC 5840 CGCAAAAAAG GGAATAAGGG CGACλCGGλλ ATGTTGAATA CTCATλCTCT TCCTTTTTCA 5900 ATλTTλTTGλ λGCλTTTλTC λGGGTTλTTG TCTCλTGλGC GGλTλCλTλT TTGλλTGTλT 5960 TTλGλλλAAT λAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCTGλCGT 6020 CGACGGATCG GGAGATCTCC CGATCCCCTA TGGTCGACTC TCAGTACAAT CTGCTCTGAT 6080 GCCGCλTAGT TAAGCCAGTA TCTGCTCCCT GCTTGTGTGT- TGGAGGTCGC TGAGTAGTGC 6140 GCGAGCλλλλ TTTAλGCTλC AλCλλGGCλλ GGCTTGACCG λCλATTGCAT GAAGAATCTG 6200 CTTAGGGTTλ GGCGTTTTGC GCTGCTTCG 6229 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 286 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Met Ser He Gin His Phe Arg Val Ala Leu He Pro Phe Phe Ala λla 1 5 10 15 Phe Cys Leu Pro Val Phe λla Hie Pro Glu Thr Leu Val Lys Val Lys 20 25 30 λβp λla Glu λsp Gin Leu Gly λla λrg Val Gly Tyr He Glu Leu λsp 35 40 45 Leu λβn Ser Gly Lye He Leu Glu Ser Phe λrg Pro Glu Glu λrg Phe 50 55 60 Pro Met Met Ser Thr Phe Lye Val Leu Leu Cyβ Gly λla Val Leu Ser 65 70 75 80 λrg He λβp λla Gly Gin Glu Gin Leu Gly λrg λrg He Hie Tyr Ser 85 90 95 Gin λβn λβp Leu Val Glu Tyr Ser Pro Val Thr Glu Lye Hie Leu Thr 100 105 110 λβp Gly Met Thr Val λrg Glu Leu Cyβ Ser λla λla He Thr Met Ser 115 120 125 λβp λβn Thr λla λla λβn Leu Leu Leu Thr Thr He Gly Gly Pro Lye 130 135 140 Glu Leu Thr λla Phe Leu Hie λsn Met Gly λβp Hie Val Thr λrg Leu 145 150 155 160 λβp λrg Trp Glu Pro Glu Leu λβn Glu λla He Pro λβn λβp Glu λrg 165 170 175 λβp Thr Thr Met Pro Val λla Met Ala Thr Thr Leu Arg Lye Leu Leu 180 185 190 Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gin Gin Leu He Asp Trp 195 200 205 Met Glu λla λβp Lye Val λla Gly Pro Leu Leu λrg Ser λla Leu Pro 210 215 220 λla Gly Trp Phe He λla λβp Lye Ser Gly λla Gly Glu λrg Gly Ser 225 230 235 240 λrg Gly He He λla λla Leu Gly Pro λsp Gly Lys Pro Ser λrg He 245 250 255 Val Val He Tyr Thr Thr Gly Ser Gin λla Thr Met λsp Glu λrg λβn 260 265 270 λrg Gin He λla Glu He Gly λla Ser Leu He Lye Hie Trp 275 280 285

Claims: CLAIMS: 1. A molecular chimaera for use with a prodrug, the molecular chimaera comprising a transcriptional regulatory DNA sequence capable of being activated in a targetted mammalian S cell and a DNA coding sequence operatively linked to the transcriptional regulatory DNA sequence and encoding a secretion signal peptide and a heterologous enzyme, such that on expression of said coding sequence in the targetted cell, the heterologous enzyme is capable of passing through the cell plasma membrane and is capable of catalysing extracellular conversion of the prodrug into a cytotoxic or cytostatic agent. 2. A molecular chimaera according to Claim 1 wherein the heterologous enzyme is obtained from an enzyme precursor by cleavage at a proteolytic cleavage site by a pathology associated protease extracellular to the targetted mammalian cell. 3. A chimaera according to Claim 2 wherein the pathology associated protease is a tumour associated protease. 4. A chimaera according to Claim 2 or 3 wherein the proteolytic cleavage site has a sequence susceptible to cleavage by a tumour-associated matrix metalloprotease. 5. A chimaera according to any of Claims 1 to 4 wherein the secretion signal peptide is encoded by a DNA sequence engineered to that encoding the heterologous enzyme. 6. A chimaera according to any of Claims 1 to 5 wherein the heterologous enzyme is a mutant mammalian enzyme. 7. A chimaera according to any of Claims 1 to 5 wherein the heterologous enzyme is a non-mammalian enzyme. 8. A chimaera according to any of Claims 1 to 7 wherein the transcriptional regulatory DNA sequence is selectively activated in the targetted mammalian cell. 9. A chimaera according to Claim 8 wherein the transcriptional regulatory DNA sequence is selected from the transcriptional regulatory DNA sequences of the genes for albumin, alphafetoprotein, carcinoembryonic antigen, tyrosine hydroxylase, choline acetyl transf erase, neuron specific enolase, glial fibro acid protein, insulin, gammaglutamyltranspeptidase, dopa decarboxylase, HER2/neu, and N-myc. 10. A vector containing a chimaera as claimed in any of Claims 1 to 9. 11. A packaging cell line containing a vector as claimed in Claim 10. 12. An infective virion generated from a packaging cell line as claimed in Claim 11. 13. An infective virion according to Claim 12 which is a retrovirus, an adenovirus or an adeno-associated virus. 14. An infective virion for use with a prodrug, capable of selective infection of a targetted mammalian cell, said virion encapsidating a molecular chimaera comprising a transcriptional regulatory DNA sequence capable of being activated in a targetted mammalian cell and a DNA coding sequence operatively linked to the transcriptional regulatory DNA sequence and encoding a signal peptide and a heterologous enzyme, such that on expression of said coding sequence in the targetted cell, the heterologous enzyme is capable of passing through the cell plasma membrane and is capable of catalysing extracellular conversion of the prodrug into a cytotoxic or cytostatic agent. 15. An infective virion according to Claim 14 wherein the heterologous enzyme is obtained from an enzyme precursor by cleavage at a proteolytic cleavage site by a pathology associated protease extracellular to the targetted mammalian cell. 16. An infective virion according to Claim 14 or 15 wherein the signal peptide is encoded by a DNA sequence engineered to that encoding the heterologous enzyme. 17. An infective virion according to any of Claims 14 to 16 wherein the transcriptional regulatory DNA sequence is selected from the transcriptional regulatory DNA sequences of the genes for cytomegalovirus, glyceraldehyde-3-phosphate, S-actin or tubulin, albumin, alphafetoprotein, carcinoembryonic antigen, tyrosine hydroxylase, choline acetyl transferase, neuron specific enolase, glial fibro acid protein, insulin, gammaglutamyltranspeptidase, dopa decarboxylase, HER2/neu, and N-myc. 18. An infective virion according to any of Claims 14 to 17 being a retrovirus, an adenovirus or an adeno-associated virus. 19. A packaging cell line capable of producing an infective virion as claimed in any of Claims 14 to 18. 20. Use of an infective virion according to any of Claims 14 to 18 for the manufacture of a medicament for use in therapy wherein the therapy comprises the selective infection of a targetted mammalian cell using retroviral infection, adenoviral infection or adeno-associated viral infection, physically localised delivery or engineered viral coat proteins. 21. A pharmaceutical formulation comprising a molecular chimaera as claimed in any of Claims 1 to 9, a vector as claimed in Claim 10, a packaging cell line as claimed in either of claims 11 and 19 or an infective virion as claimed in any of Claims 12 to 18. 22. Use of a molecular chimaera according to any of Claims 1 to 9 for the manufacture of a medicament for use in therapy wherein the therapy comprises administering said molecular chimaera by a method selected from calcium phosphate transfection, electroporation, microinjection, liposomal transfer, ballistic barrage, retroviral infection and adeno or adeno-associated virus infection.

Inventors: DEV INDERJIT KUMAR, MOORE JOHN TOMLIN, OHMSTEDE CAROL-ANN, DEV, INDERJIT, KUMAR, MOORE, JOHN, TOMLIN, OHMSTEDE, CAROL-ANN
Applicants: DEV INDERJIT KUMAR, MOORE JOHN TOMLIN, OHMSTEDE CAROL ANN, WELLCOME FOUND, DEV, INDERJIT, KUMAR, MOORE, JOHN, TOMLIN, OHMSTEDE, CAROL-ANN, THE WELLCOME FOUNDATION LIMITED
Application year: 1995
Main classification: C12N15
IPC codes: A61K9, A61K35, A61K38, A61K45, A61K47, A61K48, A61P35, C12N5, C12N9, C12N15