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WO1997019183A2 - Transcription specifique pour le tissu d'une sequence d'adn codant une enzyme heterologue connue pour etre utilisee dans une therapie a base d'un promedicament enzymatique pour traiter le cancer du poumon - Google Patents

Transcription specifique pour le tissu d'une sequence d'adn codant une enzyme heterologue connue pour etre utilisee dans une therapie a base d'un promedicament enzymatique pour traiter le cancer du poumon Download PDF

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Publication number
WO1997019183A2
WO1997019183A2 PCT/GB1996/002846 GB9602846W WO9719183A2 WO 1997019183 A2 WO1997019183 A2 WO 1997019183A2 GB 9602846 W GB9602846 W GB 9602846W WO 9719183 A2 WO9719183 A2 WO 9719183A2
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Prior art keywords
prodrug
dna sequence
lung cancer
transcriptional regulatory
lactamase
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PCT/GB1996/002846
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English (en)
Inventor
Inderjit Dev
John Tomlin Moore
Phiroze Behram Sethna
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Glaxo Group Limited
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Application filed by Glaxo Group Limited filed Critical Glaxo Group Limited
Priority to AU77004/96A priority Critical patent/AU7700496A/en
Publication of WO1997019183A2 publication Critical patent/WO1997019183A2/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/552—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being an antibiotic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/67—Enzyme prodrug therapy, e.g. gene directed enzyme drug therapy [GDEPT] or VDEPT
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4721—Lipocortins
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    • C07K14/57572—Gastrin releasing peptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/785—Alveolar surfactant peptides; Pulmonary surfactant peptides
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • 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
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    • A—HUMAN NECESSITIES
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention relates to enzyme prodrug therapy and, in particular, to the application of this form of therapy to lung cancer.
  • Lung cancer is a major type of cancer in the countries of Western Europe and North America For example, it is the most common lethal cancer in the United States It was estimated that 172,000 people would develop lung cancer in the U.S. in 1994 and about 153,000 people would die of it . The number of deaths from lung cancer is steadily increasing. The prevalence of lung cancer in developing countries is relatively low but is expected to increase sharply with the fast spreading of tobacco smoking. It is estimated that by the year 2000, deaths related to lung cancer will increase worldwide to about 2 million, mainly as a result of an increase in cigarette smoking by young adults.
  • SCLC small cell lung carcinoma
  • NSCLC non-small cell lung carcinoma
  • SCLC For SCLC, combination chemotherapy forms the cornerstone of therapy Because of its relatively rapid growth rate, and its tendency to metastasize, SCLC can rarely be treated surgically. In the absence of treatment, median survival in SCLC is only a few months After aggressive and toxic combination chemotherapy, only 10% of patients with SCLC will be alive at 2 years after diagnosis and 5% at 5 years.
  • GDEPT or VDEPT Gene or virus directed enzyme prodrug therapy
  • GDEPT or VDEPT is potentially less toxic and more efficient as a therapy for cancer than existing therapies
  • GDEPT or VDEPT involves the use of a gene encoding an enzyme that is capable of converting a relatively nontoxic prodrug to its active, e g cytotoxic, form.
  • WO-A-90 07936 proposes 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 ncin 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 ncin 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, and 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.
  • Transcriptional regulatory sequences specifically mentioned are albumin, alfafetoprotein, carcino-embryonic antigen, tyrosine hydroxylase, choline acetyl transferase, neuron-specific enolase, glial fibro acidic protein, insulin, gamma glutamyltranspeptidase, dopa decarboxylase, HER2/neu and N-myc oncogenes.
  • prodrug/enzyme combinations disclosed are purine or pyrimidine analogs/VZV tk, FC/cytosine deaminase, phenoxyacetamide derivatives of adriamycin and melphalen/penicillin V amidase, phosphate salt of etoposide, adriamycin or mrtomycin C /alkaline phosphatase, para-N-bis-(2-CI-ethyl) aminobenzylglutamic acid/carboxypeptidase G2.
  • the present invention provides the use of a molecular chimaera for the manufacture of a medicament for use with a prodrug in the therapy of lung cancer, the molecular chimaera comprising a transcriptional regulatory DNA sequence derived from a gene encoding a lung-associated protein or a neuroendocrine marker protein and, operatively linked to the transcriptional regulatory DNA sequence, DNA sequence encoding a heterologous enzyme capable of catalysing the conversion of the prodrug into an agent toxic to a lung cancer cell.
  • the present invention provides a molecular chimaera for use in therapy of lung cancer with a prodrug, the molecular chimaera comprising a transcriptional regulatory DNA sequence derived from a gene encoding a lung-associated protein or a neuroendocrine marker protein and, operatively linked to the transcriptional regulatory DNA sequence, DNA sequence encoding a heterologous enzyme capable of catalysing the conversion of the prodrug into an agent toxic to a lung cancer cell.
  • the molecular chimaera of the present invention may be made utilising standard recombinant DNA techniques.
  • Lung is not the site for dose limiting toxicity of most anticancer agents because airway epithelial cells are well differentiated and non-dividing. Therefore, lung specific activation of prodrugs improves the selectivity of these agents. Also, the promoter elements for lung-specific genes can be used to target selectively the lung metastatic disease localized in other tissues. The majority of small cell lung carcinomas and about 30% of non-small cell lung cancer are of neuroendocrine origin.
  • Neuroendocrine (NE) tumors usually produce multiple markers for NE differentiation such as creatine kinase, neuron-specific enolase, L-dopa decarboxylase, chromogranin A, neural cell adhesion molecule, Leu-7, gastrin releasing peptide, synaptophysin, calcitonin, serotinin insulinoma-associated peptide and ACTH (a hormone produced from a precursor protein called proopiomelanocortin (POMC)).
  • POMC proopiomelanocortin
  • NE tumor cells selectively express the genes for most of these markers because the transcriptional regulatory sequence (TRS) elements of these genes are functional only in NE tumors and in a small nest of neurons, endocrine and ganglion cells of the central and peripheral nervous systems Therefore, the TRS elements for NE marker genes are highly specific for cancers of neuroendocrine origin.
  • TRS transcriptional regulatory sequence
  • TRS elements have been isolated and characterised for a number of NE marker genes.
  • the proopiomelanocortin (POMC) gene codes for the precursor of multiple peptide hormones, including ACTH, and is normally expressed only in the anterior pituitary
  • the sequence of the human POMC gene including 680 base pairs preceeding the transcriptional initiation site, have been determined (Takahashi et al., Nuci Acids Res., 11, 6847-6858 (1983)).
  • the chromogranin A (CgA) gene codes for an acidic glycoprotein which is involved in hormone packaging and secretion in neuroendocrine cells
  • the sequence of the human CgA gene including 250 base pairs preceeding the transcriptional initiation site have been determined (Moutand et al, J Biol. Chem., 269, 6918-6926 (1994)).
  • GRP Gastrin-releasing peptide
  • pulmonary surfactant which is composed of a mixture of lipids and surfactant proteins specifically expressed in the respiratory epithelium Their main function involves reduction in surface tension in the alveolar space and hence prevention of alveolar collapse.
  • surfactant proteins A, B, C, and D each interacting with the lipid component differently (Weaver and Whrtsett, Biochem J., 273, 249-264 (1991)).
  • SP-B which direct lung specific transcription, has been identified as a 259 bp fragment (Bohinski et al., J. Bioi Chem., 268, 11160-11166 (1993) and Tami et al., DNA, 8, 75-86 (1989)).
  • specificity of expression of the heterologous enzyme for lung cancer cells and hence selective conversion of the prodrug to the active cytotoxic form is achieved by the use of the TRS denved from a gene encoding a lung-associated or NE-marker protein.
  • TRS denved from a gene encoding a lung-associated or NE-marker protein is used in association with this lung specific TRS.
  • heterologous enzyme means any enzyme not present naturally in the targetted lung cancer 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 being recombinant DNA technology.
  • Suitable enzymes for use according to the present invention include any having 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 glutamtc acid into be ⁇ zoic 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-B-amidase which converts a prodrug which is a phenylacetamide denvative of doxorubicin or melphalan into its corresponding toxic metabolite.
  • Another preferred enzyme for use according to the present invention is ⁇ -lactamase which has particular advantages in terms of the range of toxic agents which can be presented in the form of prodrugs capable of conversion to the active agent by means of the enzyme.
  • any toxic agent can be converted to such a prodrug by conjugation with another compound through a bond capable of being cleaved by ⁇ -lactamase.
  • conjugates are formed between the toxic agent and a cephalosporin.
  • conjugates of 5-fluorouracil, methotrexate and adriamycin linked in each case to a cephalosporin see WO-A-94 01 137 and EP-A-0 382 411) and cephalosporin mustards (see EP-A-0 484 870).
  • cephalosporin/toxic agent conjugate shows markedly reduced toxicrty but can be converted to the active form by ⁇ -lactamase thus making it suitable for use as a prodrug in GDEPT.
  • Other toxic agents can be linked to cephalosporins in a similar way.
  • Prodrugs for use according to the present invention may thus be based on any compound showing a suitable chemotherapeutic effect
  • Preferred cytotoxic compounds include nitrogen mustard agents, antifolates, nucleoside analogs, the vinca alkaloids, the anthracyclines, the mrtomycins, 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 tnchothecenes and the cotchicines.
  • the molecular chimaera is selectively expressed in a target lung cancer cell population.
  • 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 is achieved by inclusion of the target-cell specific TRS (promoter with or without enhancer) as described above but may also be enhanced by 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.
  • 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
  • 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 molecufar biology techniques well known in the art. Retroviral shuttle vectors have the parental endogenous retroviral genes (e.g.
  • 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.
  • 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.
  • 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)).
  • 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)).
  • the retroviral shuttle vector of the present invention are
  • 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 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 genome of the packaging cell, 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 lung cancer.
  • 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 earner therefor.
  • Pharmaceutical acceptable earners 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 earner 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.
  • 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 descnbed compnsing mixing an artificial infective virion containing a molecular chimaera with a pharmaceutically acceptable earner.
  • 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.
  • a protein encoded by a molecular chimaera of the present invention is also included within the scope 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 dose of prodrug will advantageously be in the range of 0 1 to 250mg per kilogram body weight of recipient per day, preferably 0 1 to 100mg per kilogram bodyweight.
  • FIG 1 shows the results of use of the POMC promoter relative to the CMV promoter in small cell lung tumours.
  • FIG 2 shows the results of use of the CgA promoter relative to the CMV promoter in lung tumours.
  • FIG 3 shows the results of expression of the human uteroglobin promoter in different human tumor lines.
  • FIG 4 shows expression of the surfactant protein-B promoter in different human tumour lines.
  • FIG 5 shows cellular location of ⁇ -lactamase activity in mammalian cells transfected with ⁇ -lactamses constructs.
  • a 785 base pair sequence was amplified via PCR from human fibroblast genomic DNA (Clontech, Palo Alto, CA) using the following two primers: JM30;
  • JM30 represents POMC sequences ending at -680 of the sequence defined by
  • JM31 represents POMC sequences ending at +105 in the region containing the 5-prime untranslated portion of the POMC mRNA
  • the PCR reaction was carried out for 25 cycles using standard conditions and using Vent polymerase (New England Biolabs, Inc.). PCR thermal cycling conditions were 95°C, 1 min, 65°C, 3 min, 70°C, 2 min, 92°C, 1 min; 72°C, 2 min; 25 cycies then 75°C, 10 min
  • This PCR product was gel-purified using the Glass-Max kit (Life Technologies, Inc.) and subsequently used for a second PCR reaction using JM30 in combination with an internal primer.
  • the second primer consisted of the following sequence: JM32:
  • JM32 represents POMC sequences ending at +22 in the region representing the 5'-untranslated portion of the POMC mRNA. PCR was earned out as above except that thermal cycling conditions were 95°C, 1 min; 92°C, 1 min; 70°C, 1 min; 72°C, 2 mins;
  • the sequence of the human POMC gene including 680 base pairs preceeding the transcriptional initiation site, have been determined (Takahashi et al., supra).
  • the 680 base pair control region of the POMC promoter was fused to the ⁇ -lactamase coding region in order to utilize ⁇ -lactamase as a reporter of promoter strength.
  • Transient transfeetions were used to evaluate the expression of different promoters. Transfeetions were earned 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 ⁇ 15mm tissue culture plates containing approximately 3 ⁇ 10 5 to 1 ⁇ 10 6 cells were employed.
  • the POMC- ⁇ -lactamase construct was transfected into seven cell lines ( Figure 1). The relative strength of the promoter was quantitated by comparing the magnitude of expression of the POMC- ⁇ -lactamase construct to expression observed in parallel transfeetions using a CMV promoter- ⁇ -lactamase construct. The POMC promoter displayed apparent selectivity towards small cell carcinoma lines. EXAMPLE 4
  • the sequence of the human CgA gene including 250 base pairs preceeding the transcriptional initiation site have been determined (Mouland et al., supra).
  • the strength and specificity of the promoter were evaluated as described for POMC, using the ⁇ -lactamase gene as a reporter. This promoter was found to be active in all lung lines tested ( Figure 2).
  • Expression of CgA promoter was 0.4% of CMV in a colon line (WiDr) and was 1% of CMV in an ileocoecal (HCT-8) line.
  • the Uteroglobin Promoter A 465 base pair element of the 5'-flanking region of the human uteroglobin promoter was isolated. The element was placed in front of a ⁇ -lactamase reporter gene so that the reporter was under the transcriptional control of the uteroglobin promoter. Transient transfeetions using lipofectamine were carried out using the CMV promoter as a control as described above. Data obtained so far ( Figure 3) suggests that expression of the uteroglobin promoter is substantially restricted to non-small cell lung cancer lines.
  • Pulmonary surfactant is composed of a mixture of lipids and surfactant proteins These surfactant proteins are specifically expressed in the respiratory epithelium Their main function involves reduction in surface tension in the alveolar space and hence prevent alveolar collapse. There are four surfactant proteins, A, B, C, and D each interacting with the lipid component differently (Weaver and Whitsett, supra).
  • the regulatory element for SP-B which direct lung specific transcription, has been identified as a 259 bp fragment (Bohinski et al., and Tami et al., supra) This promoter sequence was generated using overlapping oligonucleotides in a PCR based strategy. The promoter element was tested for directing transcnption of a ⁇ -lactamase reporter, in vitro, in various tumor lines and the results are shown in Figure 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 ⁇ -lactamase coding region.
  • the reverse primer contains an Xba l 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, USA).
  • 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 ID NO 8 along with the amino acid sequence of inserted secretory ⁇ - lactamase.
  • This forward primer consists of a Hind III restriction site (AAGCTT), a concensus site for optimal traslation efficiency (GCCACC) in vertebrates (Kozak, 1991 supra) and an ATG initiator codon immediately adjacent to the sequence representing the mature amino-terminus of TEM ⁇ -lactamase (Sutcliffe, 1978 supra).
  • AAGCTT Hind III restriction site
  • GCCACC concensus site for optimal traslation efficiency
  • the resulting PCR product would contain a deleted signal peptide and a new initiator methionine codon adjacent to the mature coding region of ⁇ -lactamase.
  • This PCR reaction was carried out using PCR conditions identical to those described for pCMV-BL, except that JM30 was substituted for JM1.
  • the approximately 700 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 l, repurified by gel electrophoresis, and ligated into the multiple cloning site of the pRc/CMV vector (InVitrogen, Inc., San Diego, CA, USA) as described above for pCMV-BL.
  • the sequence of the construct (designated pCMV- ⁇ BL) is shown in SEQ ID NO 10 along with the ammo acid sequence of inserted intracellular ⁇ -lactamase.
  • a membrane-bound form of ⁇ -lactamase would be useful in prodrug therapies since the enzyme is active and does not diffuse from the site of expression and since the external activation of prodrug guarantees bystander effects of the activated drug.
  • This chimeric enzyme may also have potential as a potent immunostimulatory moiecule since the membrane location of the protein may enhance its presentation on MHC Class II molecules.
  • a membrane-spanning domain was appended to the carboxy-terminus of the secretory ⁇ -lactamase coding region contained in pCMV-BL
  • the membrane sequence was derived from the human C mu IgM heavy chain gene (Dorai, Nucl. Acids Res., 17, 6412 (1989)). This was done by fusing a 300 base pair sequence representing the human IgM membrane-spanning domain (from plasmid IgM/TM/PCRII which contains exons M1 and M2 separated by a single intervening sequence) in-frame to the carboxy-terminus of the secretory ⁇ -lactamase gene.
  • the first step in this process was to delete the termination codon in the ⁇ -lactamase sequence contained in pCMV-BL. This was done by PCR amplification of the insert using the forward primer JM1 (see above) in combination with the reverse primer MEM1.
  • MEM 1 consists of the sequence:
  • MEM1 contains sequence representing the carboxy-terminus of secretory ⁇ -lactamase excepting the translation termination signal (TAA) which is replaced by an Xba I restriction site.
  • TAA translation termination signal
  • Xba I hexameric Xba I sequence is in-frame with the coding region of ⁇ -lactamase and represents a Ser-Arg amino acid sequence.
  • This PCR product was amplified as described above, gel-purified, and cloned into the Hind III and Xba I sites of pRc-CMV. This plasmid was designated pCMV-MEM1.
  • MEM2 consists of the sequence:
  • MEM3 consists of the sequence:
  • MEM2 represents the amino-terminus of the IgM trans-membrane domain (beginning at nucleotide 489; GenBank Accession #X14939) flanked by an Xba I restriction site (TCTAGA).
  • MEM3 represents the carboxy-terminus of the trans-membrane domain (ending at nucleotide 815; GenBank Accession #X14939) flanked by an Apa I restriction site (GGGCCC). These oligos were used to carry out PCR as described above and the approximately 300 base-pair product was restriction digested, gel-purified, and cloned into the Xba I and Apa I sites of pCMV-MEM1.
  • Transfeetions were earned 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 ⁇ 15mm tissue culture plates containing approximately 3 ⁇ 10 5 to 1 ⁇ 10 6 cells were employed.
  • transfected cells were resuspended in 50 mM Tris-CI (pH 7.4), 0 1 mM EDTA containing PMSF and leupeptm, 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.
  • ⁇ -lactamase enzyme activity was measured using PADAC (-Calbiochem, Corp.) which serves as a chromogenic substrate of ⁇ -lactamase activity (Schindler and Huber, Enzyme Inhibitors, Brodbede, Ed., pp 169-176, Verlag Chemie, Weinheim (1980))
  • 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 UV ⁇ /is spectrophotometer.
  • Transient transfeetions of human lung adenocarcinoma with pCMV-BLIgM were carried out.
  • ⁇ -Lactamase activity was detected only if the assay media was in contact with the cells, indicating that the enzyme must be membrane-bound located on the exterior face of the membrane.
  • Activity was not detected using the same method when a stable cell line expressing the intracellular form of ⁇ -lactamase was used as a control, indicating that the substrate does not penetrate cells.
  • stable lines were generated for use in immunohistochemistry experiments.
  • large-scale transfeetions in A549 cells were performed. Since pCMV-BL, pCMV- ⁇ BL, and pCMV-BLIgM contain the neomycin R gene, stable lines could be selected after passaging the lines in media containing the antibiotic, G418.
  • 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-foid more toxic than the fluorouracil prodrug 1614W94 (Table 1).
  • A549 cells which contained stable integrated copie(s) of the secretory ⁇ -lactamase gene (A549-BL) were tested, 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 ⁇ 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.
  • 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)

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Abstract

L'invention concerne l'utilisation de chimères moléculaires avec un promédicament dans la thérapie du cancer du poumon, ces chimères moléculaires comprenant une séquence d'ADN de régulation de transcription dérivée d'un gène codant une protéine associée aux poumons ou une protéine de marqueur de neuroendocrine et, reliée à ladite séquence d'ADN de régulation de transcription, une séquence d'ADN codant une enzyme hétérologue capable de catalyser la conversion du promédicament en un agent toxique pour la cellule cancéreuse du poumon. L'utilisation de promoteurs spécifiques pour le poumon dans un contexte de GDEPT ou VDEPT, permet de réaliser un ciblage spécifique des cellules cancéreuses du poumon.
PCT/GB1996/002846 1995-11-20 1996-11-19 Transcription specifique pour le tissu d'une sequence d'adn codant une enzyme heterologue connue pour etre utilisee dans une therapie a base d'un promedicament enzymatique pour traiter le cancer du poumon WO1997019183A2 (fr)

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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
WO1999039741A2 (fr) 1998-02-03 1999-08-12 Inex Pharmaceuticals Corporation Administration systemique de particules lipidiques du plasmide stables dans le serum en cancerotherapie
US5955604A (en) * 1996-10-15 1999-09-21 The Regents Of The University Of California Substrates for β-lactamase and uses thereof
WO2000020608A1 (fr) * 1998-10-02 2000-04-13 Genotherapeutics, Inc. Procede de traitement du cancer de la prostate au moyen d'un vecteur d'expression adenoviral codant pour une enzyme de promedicament
US6291162B1 (en) * 1995-03-20 2001-09-18 The Regents Of The University Of California Cytosolic forms of beta-lactamase and uses thereof
US6410328B1 (en) 1998-02-03 2002-06-25 Protiva Biotherapeutics Inc. Sensitizing cells to compounds using lipid-mediated gene and compound delivery
US7541044B2 (en) 2004-01-09 2009-06-02 Oxford Biomedica (Uk) Limited Administration of 5T4 antigen and immune response of cells expressing 5T4 and CEA antigens
US7575916B2 (en) 2002-02-13 2009-08-18 Oxford Biomedica (Uk) Limited Nucleic acid molecule encoding a MHC class I peptide epitope from the human 5T4 tumor-associated antigen and vector comprising the nucleic acid molecule
US7601698B2 (en) 1998-11-18 2009-10-13 Oxford Biomedica (Uk) Limited Polypeptide
JP2010517945A (ja) * 2007-02-05 2010-05-27 パンバイオ・リミテツド 均一インビトロｆｅｃアッセイ及び成分
US7910109B2 (en) 2002-02-13 2011-03-22 Oxford Biomedica (Uk) Ltd. MHC class II epitopes of 5T4 antigen

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GB9709421D0 (en) 1997-05-10 1997-07-02 Zeneca Ltd Chemical compounds
CA2324286A1 (fr) * 1998-04-01 1999-10-07 Metamorphix, Inc. Sequences regulatrices du facteur 9 de differenciation de croissance et utilisations associees
US7227013B1 (en) 1999-03-31 2007-06-05 Metamorphix, Inc. Growth differentiation factor-9 regulatory sequences and uses therefor
GB9910077D0 (en) 1999-05-01 1999-06-30 Univ Manchester Chemical compounds

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GB8919607D0 (en) * 1989-08-30 1989-10-11 Wellcome Found Novel entities for cancer therapy
GB9423367D0 (en) * 1994-11-18 1995-01-11 Wellcome Found Enzyme prodrug therapy

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* Cited by examiner, † Cited by third party
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US6291162B1 (en) * 1995-03-20 2001-09-18 The Regents Of The University Of California Cytosolic forms of beta-lactamase and uses thereof
US6472205B1 (en) 1995-03-20 2002-10-29 The Regents Of The University Of California Cytosolic forms for β-lactamase and uses thereof
US7157575B2 (en) 1995-03-20 2007-01-02 The Regents Of The University Of California Substrates for β-lactamase and uses thereof
US8071761B2 (en) 1995-03-20 2011-12-06 The Regents Of The University Of California Substrates for beta-lactamase and uses thereof
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
US5955604A (en) * 1996-10-15 1999-09-21 The Regents Of The University Of California Substrates for β-lactamase and uses thereof
WO1999039741A2 (fr) 1998-02-03 1999-08-12 Inex Pharmaceuticals Corporation Administration systemique de particules lipidiques du plasmide stables dans le serum en cancerotherapie
US6410328B1 (en) 1998-02-03 2002-06-25 Protiva Biotherapeutics Inc. Sensitizing cells to compounds using lipid-mediated gene and compound delivery
WO2000020608A1 (fr) * 1998-10-02 2000-04-13 Genotherapeutics, Inc. Procede de traitement du cancer de la prostate au moyen d'un vecteur d'expression adenoviral codant pour une enzyme de promedicament
US7666669B2 (en) 1998-11-18 2010-02-23 Oxford Biomedica (Uk) Limited Polypeptide
US7601698B2 (en) 1998-11-18 2009-10-13 Oxford Biomedica (Uk) Limited Polypeptide
US7615612B2 (en) 1998-11-18 2009-11-10 Oxford Biomedica (Uk) Limited Polypeptide
US7575916B2 (en) 2002-02-13 2009-08-18 Oxford Biomedica (Uk) Limited Nucleic acid molecule encoding a MHC class I peptide epitope from the human 5T4 tumor-associated antigen and vector comprising the nucleic acid molecule
US7888115B2 (en) 2002-02-13 2011-02-15 Oxford Biomedica (Uk) Limited MHC class I peptide epitopes from the human 5T4 tumor-associated antigen
US7910109B2 (en) 2002-02-13 2011-03-22 Oxford Biomedica (Uk) Ltd. MHC class II epitopes of 5T4 antigen
US7541044B2 (en) 2004-01-09 2009-06-02 Oxford Biomedica (Uk) Limited Administration of 5T4 antigen and immune response of cells expressing 5T4 and CEA antigens
JP2010517945A (ja) * 2007-02-05 2010-05-27 パンバイオ・リミテツド 均一インビトロｆｅｃアッセイ及び成分

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