WO1996016179A1 - Therapie genique enzymatique catalysant la conversion extracellulaire d'un precurseur - Google Patents

Therapie genique enzymatique catalysant la conversion extracellulaire d'un precurseur Download PDF

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WO1996016179A1
WO1996016179A1 PCT/GB1995/002716 GB9502716W WO9616179A1 WO 1996016179 A1 WO1996016179 A1 WO 1996016179A1 GB 9502716 W GB9502716 W GB 9502716W WO 9616179 A1 WO9616179 A1 WO 9616179A1
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enzyme
prodrug
chimaera
cell
targetted
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PCT/GB1995/002716
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Inderjit Kumar Dev
John Tomlin Moore
Carol-Ann Ohmstede
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The Wellcome Foundation Limited
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Priority to JP8516671A priority Critical patent/JPH10509326A/ja
Priority to AU38773/95A priority patent/AU695375B2/en
Priority to EP95937956A priority patent/EP0792366A1/fr
Publication of WO1996016179A1 publication Critical patent/WO1996016179A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4813—Exopeptidases (3.4.11. to 3.4.19)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/84—Penicillin amidase (3.5.1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • 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.
  • antitumour agents which have differing degrees of efficacy.
  • Standard clinically useful agents include adriamycin, actinomycin D, methotrexate, 5-fluorouracil, cis-platinum, vincristine and vinblastine.
  • 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.
  • 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.
  • 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.
  • HIV tat protein 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.
  • ADPPT antibody directed enzyme prodrug therapy
  • 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.
  • 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.
  • the delivered chemotherapeutic is a cytotoxic agent
  • 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.
  • 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
  • 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.
  • 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.
  • 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 7, 1434-1441 (1993)). Examples of such tumour-associated proteases include urokinase, gelatinase (e.g. 72kD) and stromolysin.
  • 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.
  • MMP matrix metalloproteases
  • Well characterised members of the family include interstitial collagenase, neutrophil collagenase, stromelysin-1, stromelysin-2, matrilysin, gelatinase A and gelatinase B.
  • 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.
  • 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.
  • 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.
  • 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-Ile, 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-Le
  • '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 A1 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 ⁇ -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.
  • 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, hydrolas ⁇ , oxido-reductase, isomerase, lyase, or ligase.
  • the directed mutagenesis will generate a novel substrate specificity but preserve the class of catalytic activity involved.
  • 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).
  • CPA mammalian carboxypeptidase A
  • 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.
  • 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.
  • the mutant enzyme is mutant human pancreatic carboxypeptidase A1 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,
  • 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 A1 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 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 turn 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.
  • 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.
  • 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, antifolates, 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.
  • cytotoxic compounds such as nitrogen mustard agents, antifolates, nucleoside analogs, the vinca alkaloids, the anthracyclines, the mitomycin
  • doxorubicin 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.
  • 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.
  • 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.
  • 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 ⁇ -actin, glyceraldehyde-S-phosphate and tubulin.
  • 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.
  • 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.
  • AFP transcription in adult liver often increases dramatically in heptacellular carcinoma (HCC).
  • HCC heptacellular carcinoma
  • 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.
  • 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.
  • 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)).
  • 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.
  • ALB albumin
  • AFP alphafetoprotein
  • CEA carcinoembryonic antigen
  • TRS for ALB or AFP are used to direct liver specific or hepatoma specific expression respectively.
  • 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.
  • TRS target-cell specific TRS
  • 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.
  • 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.
  • 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.
  • 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.
  • TRSs derived from liver-specific gene promoters such as ALB or AFP
  • 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.
  • 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 hepatitis B virus
  • HBV primarily infects hepatocytes via specific receptor mediated binding.
  • the HBV proteins encoded by the pre-S1 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.
  • the TRS need not be target cell specific and TRSs derived from genes such as ⁇ -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.
  • TRSs derived from genes such as ⁇ -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 deam
  • 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.
  • retroviral shuttle vectors which are known in the art (see for example Mol. and Cell Biol. 6, 2895-2902 (1986)).
  • 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.
  • 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.
  • 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.
  • LTR long terminal repeat
  • 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.
  • the molecular chimaera is placed in opposite transcriptional orientation to the 5' retroviral LTR.
  • 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).
  • NEO aminoglycoside-3-phosphotransferase type II
  • 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)).
  • retroviral shuttle vectors 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)).
  • retroviral shuttle vector of the present invention are SIN vectors.
  • 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.
  • 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.
  • 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.
  • helper virus LTR regulatory sequences 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.
  • helper virus structural genes i.e. gag, pol and env
  • helper virus structural genes 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.
  • 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.
  • a liquid medium suitable for use as vehicles to introduce the infective virion into the patient 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.
  • 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.
  • 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.
  • 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.
  • 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-1-yl-3-cyclobutyl-L-phenylalamine) by culture medium from cells expressing secreted 268G-HCPAI;
  • FIG 4 shows cellular location of ⁇ -lactamase activity in mammalian cells transfected with a secretory ⁇ -lactamase construct
  • FIG 5 shows growth curves of E. coli DH5 ⁇ and DH5 ⁇ transformed with pCMV-pga1 or pCMV-pga2.
  • FIG 6 shows a schematic representation of pCMV-pga clones and E. coli pga genomic sequence.
  • a Hind III-Xba I fragment which contained the entire coding sequence of human 268G-HCPAl was inserted into the eucaryotic expression vector, pcDNAINeo (Invitrogen, Inc.) also restricted with Hind III and Xba I.
  • the resulting plasmid construct designated 268G-HCPA1 was transfected into HT1080 cells using the cationic lipid mixture, Lipofectin TM , (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.
  • 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 37oC 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
  • 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-1-yl-3-cyclobutyl-L-phenylalanine; 20 ⁇ M was added to the cells and aliquots were removed at 1h, 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.
  • Hind III - Sal I cDNA fragment encoding the rat preprocarboxypeptidase A1 (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 [32 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.
  • 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 ⁇ - lactamase coding region.
  • the reverse primer contains an Xba I restriction site (TCTAGA) adjacent to the stop codon (TAA) of the ⁇ -lactamase coding region.
  • 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 ⁇ M 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 ⁇ -lactamase insert in this vector places the ⁇ -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 ⁇ - lactamase.
  • 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 ⁇ 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.
  • PADAC Calbiochem, Corp.
  • a 500 ⁇ M PADAC stock was made in water, filtered through a 0.22 ⁇ m filter, and added to media to give a final concentration of 20 ⁇ M. Decreases in absorbance at 570 nm were measured using the auto-rate assay of a Kontron UVNis spectrophotometer.
  • 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 ⁇ - lactamase followed by HPLC analysis to determine the rate of prodrug conversion.
  • ⁇ - Lactamase efficiently activates both 5798W93 and 1614W94 with a k cat /K M (specificity constant) of 272 and 67 sec -1 mM -1 , respectively.
  • 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).
  • methotrexate and its prodrug 5798W93 were equally toxic (Table 1). This experiment implies that the delivery of the ⁇ - lactamase gene to tumor cells will make them sensitive to cephalosporin prodrugs.
  • Prodrug therapy (1614W94 (50 mg/kg; i.p., qd x 5) or 5-FC (500 mg/kg; i.p., qd ⁇ 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 ⁇ 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.
  • mice and mice treated with 5-FU (30 mg/kg i.p., qd ⁇ 5) died from tumour by 30 days.
  • CMV-BL/1614W94 treatment increased survival to 60%
  • CMV-CD/5-FC treatment also increased the survival to 40% (Table 4).
  • Penicillin G amidase and the related penicillin V amidase activate these cytotoxic drugs by removing a phenylacetyl group from these nontoxic drugs (Senter,
  • GDEPT gene directed enzyme prodrug therapy
  • 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)).
  • 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.
  • 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.
  • 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 DH5 ⁇ cells (Molecular Cloning, Sambrook et al, Cold Spring Harbor Press (1989)).
  • pCMV-pga1 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 405 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)).
  • RNA Analysis - - A549 human lung adenocarcinoma cells were cotransfected as described above with pCMV-pga1 and pCMV-BL, a construct identical to pCMV-pga1 with the exception of replacing the penicillin G amidase coding region with the coding region of TEM ⁇ -lactamase.
  • RNA was prepared as described in Current Protocols in Molecular Biology (Section 4.2.4).
  • RNA 10 ⁇ g was slot blotted onto Hybond-N filters (Amersham, Arlington Heights, IL), cross-linked by UV irradiation, and probed with [ 32 P]-labeled DNA representing either plasmid pCMV-pga1 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 [ 32 P]-labeled probe was quantitated using a phoshorimager (Molecular Dynamics) using ImageQuant software.
  • Penicillin G amidase 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.
  • RPLC RPLC-binding protein
  • k cat and K m 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 cat /K m ) of 8.3 sec -1 mM -1 .
  • pCMV-pga1 and pCMV-pga2 Two eukaryotic expression vectors, containing the E. coli penicillin G amidase coding region, designated pCMV-pga1 and pCMV-pga2 were constructed. Purification of pCMV-pga1 was problematic because the plasmid caused lysis of its bacterial host. When E. coli DH5 ⁇ transformed with pCMV-pga1 was grown at 37°C, the recombinant strain displayed the same growth kinetics as the parental DH5 ⁇ strain until reaching an OD 600 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-pga1 was grown on the surface of agar plates.
  • the bacterial lysis induced by pCMV-pga1 was due to production of active penicillin G amidase (data not shown).
  • pCMV-pga1 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.
  • pCMV-pga2 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).
  • 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-pga1 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.
  • transfection with control constructs in combination with the prodrug did not result in any significant reductions in viable cell number.
  • 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% .
  • 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.
  • A549 cells were transiently cotransfected with pCMV-pga1 and pCMV-BL, an expression vector which expresses the TEM /3-lactamase gene.
  • penicillin G amidase mRNA was significantly lower than the ⁇ -lactamase levels (data not shown).
  • the low levels of expression penicillin G amidase may be in part accounted for by low steady-state levels of mRNA.
  • 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.
  • 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).

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Abstract

L'invention porte sur une chimère moléculaire s'utilisant avec un précurseur et comprenant une séquence d'ADN régulatrice de transcription susceptible d'être activée dans une cellule cible de mammifère et une séquence de codage d'ADN fonctionnellement liée à la séquence d'ADN régulatrice de transcription et codant pour un peptide signal de sécrétion et pour une enzyme hétérologue, de manière à ce que lors de l'expression de ladite séquence de codage dans la cellule cible, l'enzyme hétérologue puisse traverser la membrane plasmique de la cellule et catalyser la conversion extracellulaire du précurseur pour produire un agent cytotoxique ou cytostatique. Si la chimère moléculaire est utilisée dans une thérapie antigénique ou antivirale par les précurseurs d'enzymes, le fait que l'enzyme soit sécrétée de manière à ce que la conversion du précurseur en agent cytotoxique ou cytostatique soit extracellulaire accroît la capacité de destruction des cellules voisines.
PCT/GB1995/002716 1994-11-18 1995-11-20 Therapie genique enzymatique catalysant la conversion extracellulaire d'un precurseur WO1996016179A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997019180A2 (fr) * 1995-11-20 1997-05-29 Glaxo Group Limited Vecteur constitue d'une sequence d'adn de regulation de transcription liee a une sequence d'adn codant pour une beta-lactamase pour therapie a base d'un promedicament enzymatique
WO1999033870A2 (fr) * 1997-12-31 1999-07-08 Incyte Pharmaceuticals, Inc. Proteines humaines regulatrices
US5928888A (en) * 1996-09-26 1999-07-27 Aurora Biosciences Corporation Methods and compositions for sensitive and rapid, functional identification of genomic polynucleotides and secondary screening capabilities
US6025340A (en) * 1994-07-27 2000-02-15 Cancer Research Campaign Technology Limited Surface expression of enzyme in gene directed prodrug therapy
WO2001093826A1 (fr) * 2000-06-06 2001-12-13 Sumitomo Pharmaceuticals Company, Limited Preparations polymere synthetiques biocompatibles
US6339070B1 (en) 1997-05-10 2002-01-15 Zeneca Limited Gene construct encoding a heterologous prodrug-activating enzyme and a cell targeting moiety
US6410328B1 (en) 1998-02-03 2002-06-25 Protiva Biotherapeutics Inc. Sensitizing cells to compounds using lipid-mediated gene and compound delivery
WO2002052020A2 (fr) * 2000-12-26 2002-07-04 Bayer Aktiengesellschaft Regulation de carboxypeptidase a humaine
US6451571B1 (en) 1994-05-02 2002-09-17 University Of Washington Thymidine kinase mutants
WO2005040387A1 (fr) * 2003-10-28 2005-05-06 Friesland Brands B.V. Production et/ou administration intestinale specifique de site de substances actives

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6451571B1 (en) 1994-05-02 2002-09-17 University Of Washington Thymidine kinase mutants
US6025340A (en) * 1994-07-27 2000-02-15 Cancer Research Campaign Technology Limited Surface expression of enzyme in gene directed prodrug therapy
WO1997019180A3 (fr) * 1995-11-20 1997-08-28 Glaxo Group Ltd Vecteur constitue d'une sequence d'adn de regulation de transcription liee a une sequence d'adn codant pour une beta-lactamase pour therapie a base d'un promedicament enzymatique
WO1997019180A2 (fr) * 1995-11-20 1997-05-29 Glaxo Group Limited Vecteur constitue d'une sequence d'adn de regulation de transcription liee a une sequence d'adn codant pour une beta-lactamase pour therapie a base d'un promedicament enzymatique
US5928888A (en) * 1996-09-26 1999-07-27 Aurora Biosciences Corporation Methods and compositions for sensitive and rapid, functional identification of genomic polynucleotides and secondary screening capabilities
US6339070B1 (en) 1997-05-10 2002-01-15 Zeneca Limited Gene construct encoding a heterologous prodrug-activating enzyme and a cell targeting moiety
WO1999033870A2 (fr) * 1997-12-31 1999-07-08 Incyte Pharmaceuticals, Inc. Proteines humaines regulatrices
WO1999033870A3 (fr) * 1997-12-31 1999-10-21 Incyte Pharma Inc Proteines humaines regulatrices
US6410328B1 (en) 1998-02-03 2002-06-25 Protiva Biotherapeutics Inc. Sensitizing cells to compounds using lipid-mediated gene and compound delivery
WO2001093826A1 (fr) * 2000-06-06 2001-12-13 Sumitomo Pharmaceuticals Company, Limited Preparations polymere synthetiques biocompatibles
WO2002052020A2 (fr) * 2000-12-26 2002-07-04 Bayer Aktiengesellschaft Regulation de carboxypeptidase a humaine
WO2002052020A3 (fr) * 2000-12-26 2003-09-04 Bayer Ag Regulation de carboxypeptidase a humaine
WO2005040387A1 (fr) * 2003-10-28 2005-05-06 Friesland Brands B.V. Production et/ou administration intestinale specifique de site de substances actives

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CA2205593A1 (fr) 1996-05-30
JPH10509326A (ja) 1998-09-14
EP0792366A1 (fr) 1997-09-03
AU695375B2 (en) 1998-08-13
GB9423367D0 (en) 1995-01-11
ZA959846B (en) 1997-05-20

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