EP0973902A1 - Methodes et compositions de concentration de radio-isotopes - Google Patents

Methodes et compositions de concentration de radio-isotopes

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Publication number
EP0973902A1
EP0973902A1 EP98920832A EP98920832A EP0973902A1 EP 0973902 A1 EP0973902 A1 EP 0973902A1 EP 98920832 A EP98920832 A EP 98920832A EP 98920832 A EP98920832 A EP 98920832A EP 0973902 A1 EP0973902 A1 EP 0973902A1
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Prior art keywords
gene
vector
cells
radioisotope
tumor
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EP98920832A
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German (de)
English (en)
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Robert B. Mandell
Charles J. Link, Jr.
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Human Gene Therapy Research Institute
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Human Gene Therapy Research Institute
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1203—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules in a form not provided for by groups A61K51/1206 - A61K51/1296, e.g. cells, cell fragments, viruses, virus capsides, ghosts, red blood cells, viral vectors
    • 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
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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)
    • 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
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/028—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a herpesvirus

Definitions

  • the present invention pertains to combination radiotherapy for imaging a destruction of cells and more specifically to pharmaceutical compositions, and methods of treatment by genetic engineering designed to concentrate radioisotopes in tumor cell destruction, non-neoplastic tissue destruction such as keloid scar tissue, adipose tissue, cardiac hypertrophy, etc., or for radioimaging of transduced cells either ex vivo or in vivo, as well as those additional cells subject to a bystander effect.
  • One alternative strategy entails the artificial creation of differences between normal and neoplastic cells through prophylactic use of gene insertion techniques. In other words, manufacturing biochemical differences which can be exploited to systematically and specifically target neoplastic cells for destruction.
  • This invention involves combination genetic engineering of cells and radiation treatment to inhibit proliferation and kill neoplastic cells for ex vivo protocols such as bone marrow purging or in vivo for treatment of tumors.
  • Gene insertion protocols are used to artificially manufacture biochemical differences in target tumor cells which are then exploited to specifically render the cells susceptible to the effects of radiation, by causing the cells to take- up and accumulate radioisotopes leading to tumor cell death.
  • an object of the present invention is to provide therapeutic materials and procedures for visualizing or treating tumors and for non neoplastic cell destruction as well, using, for example, radioisotopes such as
  • Another object of the invention is to kill tumor using gene therapy to engineer biochemical differences in tumor cells which are exploited to confer radiation therapy sensitivity.
  • Another object of the invention is to genetically engineer tumor cells to contain a gene which causes radioisotope concentration in that cell, which upon exposure to radioisotopes causes accumulation of the same within the cell and targeted cell death.
  • Another object of the invention is to genetically engineer cells for radioisotope concentration to provide for radioimaging of tumors or of transformed cells in vivo.
  • a method of concentrating radioisotopes in cells for diagnostic as well as therapeutic methods using radiopharmaceuticals include radioimaging of transduced cells and radiation therapy for destruction of tumor and non-neoplastic cells.
  • the method comprises transducing cells in vivo, ex vivo, or in vitro with a nucleic acid (DNA or RNA) sequence encoding an agent which is capable of providing for the uptake by transformed cells of radionuclides causing their accumulation in transformed cells as well as those cells subject to a bystander effect, upon expression of the nucleic acid sequence encoding the agent and subsequent radiation therapy.
  • DNA or RNA nucleic acid
  • the agent encoded by the nucleic acid is a radioisotope concentrator or transporter which targets and enhances the effects as well as lethality of administered radioactive elements whether for diagnostic radioimaging or for radiation therapy.
  • Figure 1 is a graph depicting an iodine uptake experiment for several murine and human tumor cell lines as described in the Examples section. As can be seen, the cells which contain the sodium iodide symporter experienced considerably higher iodine uptake than control cells which were the FRTL cells with ice or cells with no vector.
  • Figures 2(a)-2(d) are graphs depicting saturation kinetics of 125 I accumulation. Saturation kinetics of 125 I uptake is linear as a function of the external [r] as determined by Lineweaver-Burk plot. (Lineweaver H et al, "The determination of enzyme dissociation constants", J Am Chem Soc, 1934; 56:658-660). The calculated kM was 31.3 ⁇ M and 34.7 ⁇ M iodide in 2(A) A375 melanoma and 2(C) IGROV ovarian cancer cells, respectively. These values are similar to that previously reported for rat thyroid FRTL-5 cells.
  • Figure 3 is a graph depicting in vitro survival of transduced and non- transduced tumor cells upon exposure to lO ⁇ Ci/ml 131 I after 6 hours. Percent survival is based on counts of cell colonies still proliferating after treatment. This demonstrates that tumors expressing the NIS can be selectively killed using standard, well-established protocols for 131 I-mediated thyroid ablation.
  • Figure 4 is a graph depicting tumor cell lines after 12 hours of exposure to 133nM Na 131 I (lO ⁇ Ci/ml) 30 ⁇ M Nal.
  • Figure 5 is a graph depicting percent survival of BNL.1ME cells to 6 hour 131 I 8 days post exposure.
  • Figure 6 is a Plasmid map of the Herpes Simplex Viral pHE700 vector.
  • FIG. 7 is a diagram depicting the proposed mechanism of the effects of photochemical modification of pHE-tk vector containing tandem repeats of the Hstk gene expression unit. In permissive cells transduced with unmodified vector, both DNA replication and gene expression occur (top).
  • interstrand DNA crosslinks inhibit viral replication (Hanson CV, "Photochemical inactivation of viruses with psoralens: an overview", Blood Cells, 1992; 18:7-25), but permit transgene expression from unaffected transcription units.
  • Figure 8 is a graph depicting Hstk gene activity expressed from PUVA modified vectors in IGROV ovarian carcinoma cells.
  • the graph depicts IGROV cell proliferation after exposure to pHE-tk vector, measured by 3 H-thymidine incorporation.
  • Vectors were treated with TMP (0 or 1 ⁇ g/ml) and differing lengths of UVA exposure (0 to 7.5 KJ/m 2 ).
  • Tumor cells were plated at IO 4 cells/well and proliferation assays were done as previously described.
  • Link, CJ, et al. "Preliminary in vitro efficacy and toxicities studies of the herpes simplex thymidine kinase gene system for the treatment of breast cancer", Hybridoma.
  • Figure 9 is a graph depicting the theoretical effect of increasing tumor mass diameter on dose transmission. Increasing the radius of the tumor cell mass results in less of the ⁇ -particles being transmitted from the mass.
  • Figure 10 is a plasmid map of the Herpes Simplex Virus pHE850 vector.
  • Amp R ampicillin resistant; "a”, packaging signal; HSV-tk promoter, HSV-1 thymidine kinase promoter; hyg+, hygromycin resistance; MCS, multi-cloning site; ⁇ EBNA-1, modified EBV nuclear antigen gene; ori P, EBV unique latent replication origin; ori S, replication origin; GFP, green fluorescent protein.
  • the field of nuclear medicine has long used radiopharmaceuticals for diagnostic (radioimaging) as well as therapeutic (radiation therapy for destruction of both neoplastic and non neoplastic cells).
  • Radionucleics used in nuclear medicine are all synthetic and are either generator produced, reactor produced, or cyclotron produced.
  • the half life of the radionuclide is critical as it must be long enough for imaging or for destruction of target tissue yet must also have an acceptable clearance pattern so that effective life is long enough for imaging.
  • a radiopharmaceutical's effective half life should equal 1.5 times the duration of the diagnostic procedure.
  • Iodine 121, 131, and 125 all have acceptable half lives compared to their clearance pattern so that the effective life is long enough for imaging.
  • Availability is also a critical parameter as several of the radionuclides are rare.
  • Target to nontarget ratio is also essential.
  • An additional critical aspect of treatment by radiopharmaceuticals includes target to non-target ratio. If a ratio is not high enough, 5:1 minimum per planar imaging, and 2:1 for single photon emission computed tomography (SPECT) imaging, a non- diagnostic scan can result making it difficult or impossible to distinguish pathology from background.
  • SPECT single photon emission computed tomography
  • the results of a very low target to non-target ratio can be a non-diagnostic scan resulting in unnecessary radiation dose, a delay in the diagnosis and the ultimate need to repeat the procedure.
  • the target to non-target ratio where important in diagnostic radiopharmaceutical use, it is absolutely essential for therapeutic procedures.
  • a low target to non-target ratio can result in inadequate treatment of the primary disease and delivery of a potentially lethal radiation dose to bone marrow or other radiosensitive tissues.
  • Several groups of nuclides are currently used as radiopharmaceuticals.
  • the first group is comprised of the positive emitting isotopes carbon-11, nitrogen-13, oxygen-15 and fluorine- 18 all of which are cyclotron produced. These have very short half lives and the first three limit their use to facility at or near the cyclotron site. While 18 F is possible to transport, practicalities make it virtually impossible.
  • the second group includes the gamma emitters cobalt-57 67 Ga, m In, 123 I, and 201 T1. These are also cyclotron produced, have long half lives and are easily transportable. 201 TI is of particular interest. The majority of photons collected by the camera for image formation are the low energy mercury-201 x- rays since the percent abundance of the 135 and 167-keV gamma rays is low.
  • the third group includes gallium-68, krypton-81m, rubidium-82, "mTc and nsmjN a n 0 f which are generator produced radionuclides.
  • mTc because of its ideal imaging energy and physical half life as well as its ability to bind so many compounds.
  • the final group includes 133 Xe molybdenum 99, and 131 I all of which are by-products of the fission of uranium-235. These isotopes are produced in great quantity in nuclear reactors and are regarded by the nuclear power industry as waste products.
  • the limiting factor in design of appropriate radiopharmaceuticals is simply the physiological function of the target organ and is wholly dependent on organ processes such as antigen antibody reactions, physical trapping, receptor cite binding, removal of intentionally damaged cells from circulation, and transport of a chemical species across the cell membrane and into a cell by a normally operative metabolic process.
  • the organ may be modified to actively transport the radiopharmaceutical of the invention thereby allowing radionuclides with attractive features for both radioimaging and radionuclide therapy such as 131 I, 201 T1, and 99m TC, to be used with any organ by active transport through the thyroid iodide symporter gene.
  • Radioiodine has an excellent half life profile, 125 I has a half Ufe of sixty days, 131 I has a half life of 8.08 days. It is abundantly available and has a good target to nontarget ratio. It's use, however, has been limited to thyroid carcinoma due to its active transport in the thyroid. Thyroid uptake of radioiodide is by active transport.
  • the first step involves trapping of the iodide, it then undergoes intermediate synthesis involving a thyroglobuUn intermediate (organification) and is ultimately converted into T 3 and T 4 by the process of coupUng.
  • Initial locaUzation following intravenous injection is in the thyroid stomach teratoids and chloroidplexus.
  • the iodide is stored in the thyroid as thyroxines with a Tbiol of approximately 3 weeks or cleared through the kidneys.
  • T1 Another radionuclide with good parameters is 201 T1 which is also actively taken in through the Na + /K + pump.
  • Myocardial perfusion imaging is routinely formed with 2 o ⁇ Tl in the form of ThalUum (Tl 1+ ). This involves utilization of a normally operative metabolic pathway for handUng potassium since the Tl 1+ is a potassium analogue and is therefore handled efficiently by the well documented ATPase driven sodium potassium pump mechanism.
  • Initial locaUzation of the Tl 1+ following intravenous injection is in the heart, liver and muscle. Ultimately it is recycled so that very little is cleared to the kidneys. The whole body Tbiol is approximately ten days.
  • Imaging tumors of neuroendocrine origin also falls under the category of active transport although metabolic incorporation is perhaps a better description.
  • the 123 I or 131 ImBG injected is similar in structure to guanethidine.
  • the precursor of epinephrin so that these tumors which include pheochromocytomas, neuroblastomas, paragangliomas, carcinoid type tumors medullary hyperplasia attempt to use it as a substrate for synthesis of hormones.
  • the material thus accumulates within them and the accumulated tracer activity simply increases in the tumor as a function of time.
  • Radioiodine uptake studies are disclosed in Hee- Myung Park, Chapter 59, "The Thyroid Gland", Nuclear Medicine, Vol. 1; Robert E. Henkin, 1996, Mosby-Year Book Inc., the disclosure of which is incorporated herein by reference.
  • Technetium-99 Pertechnetate is also trapped by the same mechanism as iodide and can be used to estimate thyroid functional status as well.
  • Advantages of use of this radiopharmaceutical include much lower radiation absorbed doses and shorter duration for the test.
  • Applicants invention solves the problem of normal tissue toxicity due to nonspecific action.
  • the invention comprising gene-activated radionuclide accumulation, reduces normal tissue compUcation to negligible levels.
  • Another combination therapy comprises use of compounds which are selectively metaboUzed in Herpes transformed cells to radiation sensitizers. It involves use of Herpes Simplex Virus thymidine kinase gene expression " to phosphorylate radiation sensitizers that are otherwise inactive in mammalian cells. Upon administration of pyrimidine nucleoside analogs, tumor cells which have been transformed with the HSV-tk gene metabolize these compounds to an active radiation sensitizer and render those cells more susceptible to radiation therapy. This protocol is described at length in PCT Publication No. WO 96/26743, published September 6, 1996 to Link et al., the disclosure of which is expressly incorporated by reference.
  • the invention herein describes a methodology whereby radioisotopes are selectively accumulated in transformed cells for use in radioimaging or radiotherapy.
  • target cells are transformed with a nucleic acid which is a radioisotope concentrator and upon expression of the nucleic acid sequence encoding the agent and subsequent radiation therapy the radioisotopes are accumulated in target cells.
  • the agent is a radioisotope concentrator which causes uptake by transformed cells of radioisotopes to direct ionizing radiation to these cells and to thereby enhance the effects of radiation in these ceUs.
  • radioisotope concentrator gene shall be interpreted to include any nucleic acid sequence the expression of which causes the cell to import, either actively or passively, a radiopharmaceutical including precursors or conjugated derivatives thereof.
  • radioisotope concentrator gene useful for the present invention includes the sodium-iodide symporter gene, which causes uptake of iodine and upon exposure to radioactive iodine by the thyroid.
  • Radioisotope concentrator gene useful for the present invention includes the (Na + -K + ) activated ATPase which is responsible for uptake of the radioisotope ThalUum. See, Henkin et al., "Nuclear Medicine", 1996 by Mosby Year Book, Inc. Vol. II, Chapter 93 "Thallium-201 Chloride and Technetium- 99m Sestamibi in Tumor Imaging” incorporated herein by reference. ThaUium-201, ( 201 T1) binds 2 sites on the ATPase enzyme and is accumulated by ceUs. Its uptake is inhibited by ouabain, digitalis, and the diuretic furosemide because the agents block the sodium potasium pump.
  • 99m Tc-sestamibi is another agent taken up by cells through the Na+-K+ pump and can be used in radioimaging of transformed ceUs expressing the Na + -K + ATPase.
  • Any such radioisotope concentrator gene can be used in the methods of the invention and one of skill in the art can identify numerous variations of gene/radioisotope combinations which are intended to be within the scope of this invention.
  • the gene encoding Na + -K + ATPase can easily be located by one of skill in the art by searching data bases such as Genbank, or by developing primers and using PCR amplification using analogous sequences from other species according to methods known in the art, and as disclosed in, for example, Ausubel FM et al, (eds.) "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, New York, 1989. Recently, the cloning and characterization of the thyroid iodide transporter gene from the rat has been reported and is herein incorporated by reference. Dai, G., Levy, O.
  • NIS Na+/1- symporter
  • the NIS protein has 12 putative membrane spanning domains. This molecular pump functions as a NaH-/l- symporter and allows for the initial step of thyroid hormone synthesis. This protein therefore allows for the detection of l 125 uptake in thyroid tissue and for the ability to ablate hyperactive thyroid tissue or to treat well differentiated thyroid cancers with 131 I. This interesting and unique property of thyroid tissue allows for very focused radiotherapy with minimal, if any, systemic side effects from therapy except the potential need for thyroid supplements. Additional information on NIS gene and its activity, as well as methods for detecting iodine accumulation are disclosed in "Iodide Transport in a Continuous Line of Cultured Cells From Rat Thyroid". Weiss, S.J. et al., Endocrinology, Vol. 114(4):1090-1098 herein incorporated by reference.
  • the logical choice for thyroid scanning agent is an iodine tracer, namely, radioiodine.
  • radioiodine tracer There are several radioiodines available: 123 I, 131 I, and 125 I. However, 125 I is no longer used for in vivo thyroid studies because of its low energy (28 keV) and very long half-Ufe (60 days). For imaging the thyroid morphology, 9 °- m Tc pertechnetate is also satisfactory in most cases.
  • agents include thalUum-201, galhum-67, 131 I or 123 I metaiodobenzylguanidine (MIBG), " m Tc(V) dimercaptosuccinic acid (DMSA), fluorine-18 fluorodeoxyglucose (FDG), and " m Tc sestamibi. These secondary imaging agents are used mainly to image thyroid neoplasms and their metastases.
  • Radioimaging procedures using radioiodine have long been known in the art and are disclosed in detail in Nuclear Medicine, Vol. I and II, Mosby Press, 1996. The disclosure of which is incorporated by reference. Iodine in its various isotopic forms has also been used to radiolabel monoclonal antibodies for imaging and for therapy.
  • Classic radioiodination methods involve covalent attachment of radioiodine to tyrosine residues in proteins. The process involves mild oxidation of iodine at slightly alkaUne pH using oxidizing agents such as chloramine-T, idogen, or lactoperoxidase.
  • Indirect iodination procedures via the Bolton-hunter reagent involves radioiodinating a secondary molecule, typically an active ester which then reacts with N-terminal and lysine amino groups of monoclonal antibodies. Disclosure of radioiodination of monoclonal antibodies is disclosed at A. Michael Zimmer, "New approaches to radiolabeUng monoclonal antibodies", Chapter 40, Nuclear Medicine, Vol. 1, pp. 511-515, Henkin, Robert E (ed.) 1996, Mosby-Year Book Inc., supra.
  • the NIS gene has made radioactive iodine therapy a standard treatment for thyroid cancer.
  • a majority of adenocarcinomas are removed surgically and radioiodine has been used as an injunctive therapy for management of thyroid adenocarcinoma for more than twenty years.
  • the efficacy of radioiodine therapy is directly related to tumor uptake and retention due to the presence of the sodium iodide symporter gene which is expressed in thyroid cells. Efficient uptake and response to radioiodine is observed in tumors that are differentiated in cell type such as papillary or follicular whereas undifferentiated tumors and medullary carcinomas rarely concentrate radioiodine.
  • Effected tumor uptake is approximately 0.5% of the radioiodine dose per gram with a biologic half life of approximately 4 days. From administration of 5.6 Gbq (equivalent to 150 mCi of 131 I) a tumor may receive as much as 25 cGy or five times the absorbed dose that can be dehvered by a course of external radiation therapy. Moreover the dose is delivered to every functional metastases regardless of size or location in the body and tumor tissue will receive several hundred times the radiation exposure received by the rest of the body. Doses of radioiodine effective for treatment for uptake and killing of tumor cells are accomplished by the administration of 1.9 to 5.6 Gbq (50-150 mCi of 131 I.
  • Radio imaging procedures are used to identify the presence of iodine and to track its accumulation in tumor ceUs.
  • These bovine TSH thyroid stimulating hormone
  • TSH thyroid stimulating hormone
  • the dose of radioiodine varies between 3.7 to 7.4 Gbq (100-200 mCi).
  • the standard imperical dose is 150-200 mCi and methods of administration of radioiodine tracking its accumulation and managing side effects are known in the art and are described in the following publications which are incorporated in their entirety by reference. Cancer Treatment, 4th Ed., Charles M. Haskel, MD, FACP, W.B. Saunders Co., 1995. "Radiation to the thyroid can induce thyroid neoplasms both benign and malignant”; Nuclear Medicine Communications. (1993) 14:736-735. "Radionuclides and Therapy of Thyroid Cancer", O'Doherty, M.J.
  • any peptide or protein which causes the accumulation of radionuclides, whether radioisotope or beam therapy can be used in the method of invention.
  • the methods of the invention can be used for radioimaging of transformed cells. This can include diagnostic protocols such as radioimaging of tumors or for ex-vivo protocols as a marker to track the expression by transformed ceUs of incorporated genes, one of which includes a radioisotope concentrator gene.
  • the methods can also be used to target various compounds to transformed cells via conjugation of pharmaceuticals or other agents to a radioisotope which is selectively taken up by transformed cells.
  • the methods include radiotherapy for destruction of neoplastic cells such as tumor cells as well as non neoplastic cells to accumulate radiopharmaceuticals and destroy transformed cells.
  • the radiotherapy of the invention may also be combined with other traditional radio-sensitizers to further concentrate the effects.
  • traditional drugs which have been reported to sensitize cells to therapeutic radiation include those in U.S. Patent No. 4,628,047 reports use of diltiazem (chemical name: d-3-aceotxy-cis-2, 3-dihydro-5-[2-(dimethylamino) ethyl[-2-(p- methoxyphenyl]-l,5-benzo-thiazepin-4(5H)-l) to enhance the sensitivity of a variety of types of cancer cells toward cytotoxic agents such as doxorubicin.
  • the radiation sensitizer precursor or conjugate thereof can be combined with a pharmaceutically acceptable carrier such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives.
  • a suitable liquid vehicle or excipient such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives.
  • suitable liquid vehicles and excipients are conventional and are commerciaUy available. Illustrative thereof are distilled water, physiological saUne, aqueous solutions of dextrose and the like. Traditionally IV therapy is preferred.
  • the pharmaceutical compositions of this invention may contain suitable excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • active ingredients may be administered by a variety of specialized delivery drug techniques which are known to those of skill in the art, such as portable infusion pumps.
  • the method comprises transducing cells in vivo or in vitro with a nucleic acid (DNA or RNA) sequence encoding a radioisotope concentrates gene which is capable of providing for the accumulation of administered radionuclides upon expression of the nucleic acid sequence encoding the agent.
  • Radioisotopes useful for the invention include any which can be paired with a concentrator gene for cellular uptake. Potential radioisotopes include but are not limited to the radio nuclide metals 18 ⁇ 5 RE, 188 RE, ⁇ Cu, ⁇ "Cu, "yttrium, ⁇ > 9 Pd, 212 Bi, 203 PB, 212 Pb, 211 At, 97 Ru, weRh, 198 Au, 19 Ag, and i "I.
  • Any radioisotope concentrator gene the nucleotide sequence of which is known or is ascertainable by one of skiU in the art can be used.
  • One of skill in the art can search for such genes in publically accessible databases such as Genbank to identify other such genes which act similarly.
  • the radioisotope concentrator gene is an iodide symporter gene, Na/k+ ATPase, or calcium transporter.
  • radiotherapy is administered to the cells.
  • the radioisotope is administered according to known protocols disclosed and incorporated herein in an amount effective to visuaUze transformed cells or to destroy the growth of the transduced tumor cells. These dosages are standard and are known in the art as described supra.
  • the invention may allow for lower dosages of the radio element with equal effectiveness due to the concentration of the elements in transformed cells.
  • the radioisotope may be administered to the host or to the in vivo cells in an amount known in the art as radiotherapy has long been employed and tolerance as well as toxicity data is well known.
  • the radioisotopes are administered systemically, such as, for example, by intravenous administration, by parenteral administration, by intraperitoneal administration, or by intramuscular administration, or any other administration protocol acceptable for the in vivo or ex vivo protocol.
  • producer cells or other expression media including the radioisotope concentrator gene when administered to cells, metabolic cooperation, a "bystander effect", will result, i.e., tumor cells which are not transduced with the nucleic acid sequence encoding the radioisotope concentrator gene may also be selectively visualized or killed upon administration of radiation therapy.
  • the methods of the invention should have a potent bystander effect since 131 I energy can travel 7 mm from the point of particle decay in tissues. This fact has been repeatedly viewed as strong supportive evidence for using anti-tumor antibodies radiolabeled with 131 I as the therapeutic agents. Divgi, C.R., "Status of Monoclonal Antibodies for Diagnosis and Therapy of Cancer", Oncology 10:939-953 (1996).
  • Bystander killing is a key element of this novel approach because it implies that gene delivery does not have to target 100% of the tumor cells in a recurrent lesion. This is essential because current gene delivery methods that are being tested in the Phase I or II trials are fairly inefficient in humans.
  • 131 I in humans is well characterized in terms of dose and monitoring.
  • the administration of 5.6 Gbq of 131 I usually results in a radiation dose equivalent to 25,000 cGy to a thyroid tumor.
  • the invention in its simplest embodiment includes a polynucleotide sequence which provides for the expression of a radioisotope concentrator gene in transformed ceUs.
  • the polynucleotide sequence is an expression construct which includes a gene encoding a radioisotope concentrator element and certain regulatory elements operatively linked to the gene.
  • One such element is a promoter which when operatively linked to the gene can provide for the transcription and ultimate expression of the gene in cells.
  • the promoter can be constitutive or inducible.
  • promoters which are active in mammalian cells are known in the art and the selection of a suitable promoter is an expedient easily optimized by those of skill in the art and contemplated herein.
  • suitable promoters are disclosed in Maniantis, "Molecular Cloning", Cold Spring Harbor Press, 1989.
  • the promoter is a viral promoter that is located within a viral vector for gene delivery which is operatively linked to a multiple cloning site for insertion of the radioisotope concentrator gene.
  • the expression construct also typically includes a termination or polyadenylation sequence operatively linked to the radioisotope concentrator gene.
  • the construct of the invention may be further comprised within a vector or gene transfer vehicle as described hereinafter.
  • Inducible promoters can be used with the methods of the invention in radioimaging to visuaUze activity of the promoter in activating the radioisotope concentrator gene and any other genes operatively linked to the same promoter as in, for example, a diagnostic turbidoscan to identify whether a particular promoter is active in a particular cell.
  • tyrosine hydroxylase promoter could be used to define tyrosine hydroxylase activity in part of the brain. This could provide valuable prognostic or diagnostic information for Parkinson's disease.
  • the promoter is a tumor specific promoter which can provide for selective expression of the gene in not only transformed cells, but transformed tumor cells.
  • the polynucleotide sequences of the invention can be used diagnostically or therapeutically. Transformation of tumor cells with a NIS gene or its equivalent operatively Unked to a tumor specific promoter such as those disclosed in Example 7 can be used to image tumors in which regulated expression of the NIS gene occurs and 125 I is taken up.
  • the vectors of the invention can be tied to cell specific promoters or inducible promoters and with radioimaging based upon radioiodine uptake can give very specific information about the state of a cell.
  • Two methods of gene delivery have been shown successful in other experiments are preferred.
  • the first method is retroviral (RV) delivery, Moolten, F.L., "Tumor Chemosensitivity Conferred by Inserted Herpes Thymidine Kinase Genes: Paradigm for a Prospective Cancer Control Strategy", Cancer Res. 46:5276-5281 (1986); Link, C.J., Kolb, E.
  • the invention comprises in one embodiment a novel retroviral vector comprising a nucleic acid sequence which encodes a radioisotope concentrator protein such as NIS.
  • the second and more novel method of gene delivery is an amplicon system based on HSV-1.
  • This gene delivery vector was developed by applicants as described herein. It has been found that at an MOI of 3-10 plaque forming units (pfu/ml) of applicants pHE vector transduces >95% of tumor cells in vitro. HSV vectors will also allow for high efficiency gene transfer in vivo into rodent tumors. Boviatsis, E.J., et al., "Long-Term Survival of Rats Harboring Brain Neoplasms Treated With Ganciclovir and a Herpes Simplex Virus Vector That Retains an Intact Thymidine Kinase Gene", Cancer Res., 54:5745-5751 (1994). In these preferred experimental models, gene transfer was efficient enough to induce complete remission and long term survival in some animals.
  • a packaging cell line is transduced with a retroviral vector, such as those hereinabove described, which includes the Na + /I- symporter gene.
  • the transduced packaging cells are administered in vivo or ex vivo to the tumor in an acceptable pharmaceutical carrier and in an amount effective to inhibit, prevent, or destroy the growth of the tumor.
  • the producer cells Upon administration of the producer cells to the tumor, the producer cells generate viral particles including a gene encoding the Na+/I- symporter. Such viral particles transduce the adjacent tumor cells.
  • the human or animal host organism is then given the appropriate corresponding radioisotope.
  • the radioisotope is 131 I.
  • the radioisotopes are selectively concentrated in transformed cells.
  • a "bystander effect" may also occur, whereby non- transduced tumor cells also may be killed as well.
  • the method of the present invention for direct in vivo therapy utilizing retroviruses is particularly useful when the targeted tumor is in or surrounded by a tissue made up of cells which are relatively quiescent mitotically, such as liver, skin, bone, muscle, bladder, prostate, kidney, adrenal, pancreas, heart, blood vessel and thyroid tissue, among others.
  • a tissue made up of cells which are relatively quiescent mitotically such as liver, skin, bone, muscle, bladder, prostate, kidney, adrenal, pancreas, heart, blood vessel and thyroid tissue, among others.
  • the inventive approach also should be useful against tumors located in the subarachnoid space, in the peritoneum, and in the pleural cavity.
  • tumors in organs the loss of which, in whole or part, is generally well-tolerated are preferred targets of a treatment according to the present invention such as the liver, for example.
  • Targeting of the vectors to tumor sites for the methods of the invention can be accomplished through a number of protocols.
  • the vectors can be mechanically injected directly at the target site. This can be accomplished with plasmid DNA, retroviral vectors (MMKV or HIV based) Herpes Simplex Viral Vectors (either amplicons or near whole virus vectors), adenoviral vectors, Sandai virus vectors, or DNA polylysine antibody complexes. Direction can also occur through selective uptake of foreign material (phagocytosis or macrophage).
  • Another method includes viral targets on dividing tumor cells such as the epo receptor or use an antibody fused to viral envelopes. As discussed earlier tissue or tumor specific promoters could also be used as disclosed in Example 7.
  • Direct injection of the producer cells minimizes undesirable propagation of the virus in the body, especially when replication-competent retroviral vectors are used. Because most cells of the body express receptors for amphotropic retroviral vectors, any vector particle which escapes from the local environment of the tumor should immediately bind to another cell. Most cells are not in cycle, however, and therefore will not integrate the genes carried by the vector and will not express any genes which it contains. Thus, the proportion of potential target cells which are in cycle at the time of exposure will be small, and systemic toxic effects on normal tissues will be minimized.
  • Tumors which may be treated in accordance with the present invention include malignant and non-malignant tumors.
  • Malignant (including primary and metastatic) tumors which may be treated include, but are not Umited to, those occurring in the adrenal glands; bladder; bone; breast; cervix; endocrine glands (including thyroid glands, the pituitary gland, and the pancreas); colon; rectum; heart; hematopoietic tissue; kidney; liver; lung; muscle; nervous system; brain; eye; oral cavity; pharynx; larynx; ovaries; penis; prostate; skin (including melanoma); testicles; thymus; and uterus.
  • tumors examples include apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), plasmacytoma, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma, synovioma, adenofibro
  • the nucleic acid sequence which encodes the radioisotope concentrator agent is contained in an appropriate expression vehicle which transduces the tumor cells.
  • an appropriate expression vehicle which transduces the tumor cells.
  • Certain proprietary expression vehicles have been discussed supra, however, and appropriate gene transfer vehicle may be employed by the method of the invention.
  • the invention also includes these novel expression vehicles as well as their use in radiotherapy.
  • Such expression vectors include, but are not Umited to, eukaryotic vectors, prokaryotic vectors (such as, for example, bacterial vectors), and viral vectors.
  • the expression vector is a viral vector.
  • Viral vectors which may be employed include, but are not Umited to, retroviral vectors, adenovirus vectors, and adeno-associated virus vectors.
  • a packaging cell line is transduced with a viral vector containing the nucleic acid sequence encoding the agent or factor which provides for the inhibition, prevention, or destruction of the tumor cells upon expression of the nucleic acid sequence encoding the agent to form a producer cell line including the viral vector.
  • the producer cells then are administered to the tumor, whereby the producer cells generate viral particles capable of transducing the tumor cells.
  • the viral vector is a retroviral or adenoviral vector.
  • retroviral vectors which may be employed include, but are not Umited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus.
  • Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic ceUs. Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Most often, the structural genes (i.e., gag, pol, and env), are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal.
  • Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.
  • a packaging-defective helper virus is necessary to provide the structural genes of a retrovirus, which have been deleted from the vector itself.
  • the retroviral vector may be one of a series of vectors described in Bender, et al., J. Virol. 61:1639-1649 (1987), based on the N2 vector (Armentano, et al., J. Virol., 61:1647-1650) containing a series of deletions and substitutions to reduce to an absolute minimum the homology between the vector and packaging systems. These changes have also reduced the likelihood that viral proteins would be expressed. In the first of these vectors, LNL-XHC, there was altered, by site-directed mutagenesis, the natural ATG start codon of gag to TAG, thereby eliminating unintended protein synthesis from that point.
  • MoMuLV Moloney murine leukemia virus
  • pPr80S a S another glycosylated protein
  • MoMuSV Moloney murine sarcoma virus
  • the vector LNL6 was made, which incorporated both the altered ATG of LNL-XHC and the 5' portion of MoMuSV.
  • the 5' structure of the LN vector series thus eliminates the possibility of expression of retroviral reading frames, with the subsequent production of viral antigens in genetically transduced target cells.
  • Miller has eliminated extra env sequences immediately preceding the 3' LTR in the LN vector (Miner, et al., Biotechni ⁇ ues, 7:980-990, 1989).
  • the paramount need that must be satisfied by any gene transfer system for its application to gene therapy is safety. Safety is derived from the combination of vector genome structure together with the packaging system that is utilized for production of the infectious vector. Miller, et al.
  • the retroviral vector may be a Moloney Murine Leukemia Virus of the LN series of vectors, such as those hereinabove mentioned, and described further in Bender, et al. (1987) and MiUer, et al. (1989).
  • Such vectors have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon.
  • the term "mutated” as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or fragment or truncations thereof, are not expressed.
  • the retroviral vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs.
  • Preferred cloning sites are selected from the group consisting of NotI, SnaBI, Sail, and Xhol.
  • the retroviral vector includes each of these cloning sites.
  • a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of NotI, SnaBI, Sail, and Xhol located on the retroviral vector.
  • the shuttle cloning vector also includes at least one desired gene which is capable of being transferred from the shuttle cloning vector to the retroviral vector.
  • the shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.
  • the shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems.
  • the shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria.
  • the shuttle cloning vector may be derived from plasmids such as pBR322; pUC 18; etc.
  • the vector includes one or more promoters. Suitable promoters which may be employed include, but are not Umited to, the retroviral LTR; the SV40 promoter; and the human cy tome galo virus (CMV) promoter described in Miller, et al., Biotechni ⁇ ues, 7:(9):980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not Umited to, the histone, pol III, and ⁇ -actin promoters).
  • CMV human cy tome galo virus
  • viral promoters which may be employed include, but are not Umited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • the vector then is employed to transduce a packaging cell line to form a producer cell Une. Examples of packaging cells which may be transfected include, but are not Umited to the PE501, PA317, ⁇ 2, ⁇ -AM, PA12, T19-14X, VT-19-17-H2, ⁇ CRE, ⁇ CRIP, GP+E-86, GP+envAM12, and DAN cell Unes.
  • the vector containing the nucleic acid sequence encoding the agent which is capable of providing for the inhibition, prevention, or destruction of the growth of the tumor cells upon expression of the nucleic acid sequence encoding the agent may transduce the packaging cells through any means known in the art. Such means include, but are not Umited to, electroporation, the use. of liposomes, and CaPO-j precipitation.
  • the producer cells then are administered directly to or adjacent to the tumor in an amount effective to inhibit, prevent, or destroy the growth of the tumor upon subsequent radiation therapy. In general, the producer cells are administered in an amount tolerated by the patient, it is desirable to inject as many producer cells as possible. The exact amount of producer cells to be administered is dependent upon various factors, including but not Umited to, the type of the tumor and the size of the tumor.
  • the producer ceUs are administered directly to or adjacent to the tumor by injection.
  • the producer cells are administered in combination with a pharmaceutically acceptable carrier suitable for administration to a patient.
  • the carrier may be a liquid carrier such as, for example, a saline solution.
  • the producer cells Upon administration of the producer cells to the tumor, the producer cells generate viral particles. The viral particles then transduce the surrounding tumor cells. Because tumor ceUs, and in particular cancerous tumor cells, in general are actively replicating ceUs, the retroviral particle would be integrated into and expressed preferentially or exclusively in the tumor cells as opposed to normal cells.
  • the invention comprises a viral vector which commonly infects humans and packaging cell Une which is human based.
  • viral vectors derived from viruses which commonly infect humans such as
  • Herpes Virus, Epstein Barr Virus may be used which do not express an active ⁇ -galactosyl envelope.
  • Herpes simplex virus type-1 (HSV-1) has been demonstrated as a potential useful gene delivery vector system for gene therapy, Glorioso, J.C., "Development of Herpes Simplex Virus Vectors for Gene Transfer to the Central Nervous System. Gene Therapeutics: Methods and Applications of Direct Gene Transfer", Jon A. Wolff, Editor, 1994 Birkhauser Boston, 281-302; Kennedy, P.G., "The Use of Herpes Simplex Virus Vectors for Gene Therapy in Neurological Diseases", Q J Med, Nov. 1993, 86(ll):697-702; Latchman, D.S., "Herpes Simplex Virus Vectors for Gene Therapy", Mol Biotechnol, Oct. 1994, 2(2):179-95.
  • HSV-1 Herpes simplex virus type-1
  • HSV-1 vectors have been used for transfer of genes to muscle.
  • Huard, J. "Herpes Simplex Virus Type 1 Vector Mediated Gene Transfer to Muscle", Gene Therapy, 1995, 2, 385-392; and brain, KapUtt, M.G., "Preproenkephalin Promoter Yields Re ion- Specific and Long- Term Expression in Adult Brain After Direct In Vivo Gene Transfer Via a Defective Herpes Simplex Viral Vector", Proc Natl Acad Sci USA, Sep 13, 1994, 91(19):8979-83, and have been used for murine brain tumor treatment, Boviatsis, E.J., “Long-Term Survival of Rats Harboring Brain Neoplasms Treated With Ganciclovir and a Herpes Simplex Virus Vector That Retains an Intact Thymidine Kinase Gene", Cancer Res, Nov 15, 1994, 54(22):5745-51; Mineta, T., "Treatment of Malignant Gliomas Using Ganci
  • Helper virus dependent mini-viral vectors have been developed for easier operation and their capacity for larger insertion (up to 140 kb), Geller,
  • HSV amplicons contain large deletions of the HSV genome to provide space for insertion of exogenous DNA. Typically they comprise the HSV-1 packaging site, the HSV-1 "ori S" repUcation site and the IE 4/5 promoter sequence. These virions are dependent on a helper virus for propagation. Primarily two types of mutant helper viruses have been developed to minimize recombination. Other complementary HSV helper virus systems are contemplated herein and are within the scope of those of skill in the art. One such system which has been developed is a temperature-sensitive mutant. An HSV temperature-sensitive (TS) mutant has been developed with a TS mutation in the IE3 gene. Davison et al, 1984, J. Gen. Virol., 65:859-863.
  • TS temperature-sensitive
  • helper virus system is a deletion mutant with the majority of the IE3 gene simply deleted. These do not revert to wild type. Therefore HSV-1 vectors packaged using a deletion mutant as helper virus is the most preferred helper virus of the invention. See for example Patterson et al., 1990, J. Gen. Virol., 71:1775-1783.
  • Other replication incompetent helper viruses can be used and one of skiU in the art will appreciate that other mutations in the IE genes or other genes which result in a replication incompetent helper virus which will provide the appropriate replication and expression functions and which are coordinated with the helper cell Une and vector are contemplated within this invention.
  • Any ceU Une can be used for this step so long as it is capable of expressing the IE3 or replication dependent gene, or obtaining a helper cell line which has already been transformed and is commercially available.
  • Any cell line can be used by introducing pHE and the plasmid containing the IE3 gene simultaneously.
  • the vector is delivered to the helper cell line by electroporation, calcium phosphate DNA transfection or any other suitable method.
  • Any cell Une can be used by introducing pHE and the plasmid containing the IE3 gene simultaneously.
  • the cells are next infected with a helper virus IE3 deletion mutant or other corresponding deletion mutant which is replication incompetent.
  • the IE3 gene or other such gene in the helper cell Une complements the helper virus resulting in a productive HSV-1 infection and the resulting virus stock consists of HSV-1 particles containing either vector DNA or helper virus DNA, all of which are replication incompetent. Further information about helper cell lines and the methodology is disclosed in Geller et al., PNAS, 87:8950-8954, November 1990, "An Efficient Deletion Mutant Packaging System for Defective Herpes Simplex Virus Vectors: Potential Applications to Human Gene Therapy and Neuronal Physiology".
  • the invention comprises a HSV mini vector which combines a replication incompetent HSV amplicon with other viral sequences such as those from Epstein-Barr virus, human papillomavirus, or bovine papillomavirus type 1 which aUow the vector to be maintained in the cell in episomal form achieving a 10 times greater titer, and a very large DNA insert capacity.
  • helper virus- dependent mini-viral vector comprising: (a) the HSV-1 "a" sequence for the package/cleavage signal and an "ori S" repUcation origin for the replication packaging of the plasmid (in response to signals to replicate and package from the helper virus); (b) an Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) gene and an EBV latent origin of replication (ori P) which allow the vector to be maintained in episomal form within the nucleus for replication without integration to the host genome and for even replication into each of two dividing cells; preferably (c) genes from prokaryotic cells for propagation of the vector in E.
  • EBV Epstein-Barr virus
  • EBNA-1 Epstein-Barr virus
  • ori P EBV latent origin of replication
  • the vector may also comprise prokaryotic genes that provide for a second selectable marker such as the genes for positive Hygromycin selection.
  • the packaging function of mini-vector DNA into Herpes simplex viral capsids is provided by a helper virus and a helper cell Une.
  • the HSV vector can be engineered to produce a helper free viral vector as in Mann et al., "Construction of a Retro- Virus Packaging Mutant and its Use to Produce Helper-Free Defective Retrovirus", 33 Sal., p. 153-159, May 1983, Journal of Virology, September 1989, pp.
  • Breast cancer that locally recurs after radiation therapy and that is refractory to chemotherapy represents a difficult clinical challenge. Patients often cannot tolerate further external beam radiotherapy without being subjected to prohibitive toxicity. Likewise, further chemotherapy can provide significant toxicity without great benefit, especially after combined modality therapy. In this setting, an ideal therapy would provide more local and effective treatment for better palliation of these significant lesions without substantial or Ufe threatening side effects.
  • a number of laboratories are investigating molecular approaches that are based on targeted gene transfer, Han, X., Kasahara, N. & Kan, Y.W., "Ligand-Directed Retroviral Targeting of Human Breast Cancer Cells", Proc. Natl.
  • Retroviral or Herpes Viral vectors for effective transfer a functional thyroid symporter gene to breast cancer cells in vitro.
  • the rat FRTL-5 thyroid-derived cell line was used as a source for RNA that was purified using the Rneasy kit (Qiagen) to isolate the thyroid iodide transporter gene as described in Dai et al., Supra.
  • the RNA was then treated by a RT-PCR method to generate cDNA (CLONETECH). These were then used as templates for PCR reactions to amplify the transporter.
  • the primers for PCR amplification were a 5' 31mer that started at position #90 of the reported sequence and 3' 31mer starting at position #1950.
  • the NIS gene was then cloned into the LXSN retroviral backbone that that contains a multi-cloning site and the neo r gene under SV40 promoter control: Link C.J., Kolb E, Muldoon R., "PreUminary in vitro efficacy and toxicities studies of the herpes simplex thymidine kinase gene system for the treatment of breast cancer", Hybridoma, 1995, 14:143-147; Link CJ et al., "A phase I trial of in vivo gene therapy with the Herpes simplex thymidine kinase/ganciclovir system for the treatment of refractory or recurrent ovarian cancer", Human Gene Ther, 1996, 7:1161-1179.
  • the vector also contains modifications to minimize breakouts of replication competent retrovirus. Miller, A.D. & Buttimore, C, "Redesign of Retrovirus Packaging Cell Lines to Avoid Recombination Leading to Helper Virus Production", Molec. Cell Biol, 6:2895-2902 (1986).
  • the vector has been well described elsewhere and the inventors have previously used LXSN for in vitro transfer and killing of breast cancer cells using the HS-tk gene and ganciclovir approach.
  • Link C.J., Kolb E, Muldoon R. "Preliminary in vitro efficacy and toxicities studies of the herpes simplex thymidine kinase gene system for the treatment of breast cancer", Hybridoma, 1995, 14:143-147.
  • the resulting vector was named LNISN.
  • iodide uptake was initiated by adding 30 ⁇ Ci/ml of Nal 131 and 30 ⁇ M Nal.
  • the survival rate of tumor cells transduced with LNISN to cells with no vector (no NIS gene) was compared. Cells were exposed for 6 hours to the solution containing 131 I. ( Figure 3). Next, cells were trypsin digested and then plated at cell numbers between 1.25xl0 2 and lxlO 4 cells per plate in quadruplicate for each data point. After 7 to 10 days the cells were fixed and stained. Macroscopic colonies containing ⁇ 50 cells were counted. Survival measurements were corrected for plating efficiency. Results are shown in Figures 3-5.
  • the vector LTKOSN contains the Hstk gene transcribed from the viral long terminal repeat (LTR) and a bacterial neomycin resistance (neo r ) gene transcribed from an internal SV40 (simian virus 40) early promoter (LTR- HStk-SV-neo r -LTR) in the LXSN backbone.
  • Animals received injections of 2xl0 4 MC38 tumor cells suspended in 1 ml Hank's balanced salt solution (HBSS) into the peritoneal cavity on day 1. On day 6 the animals received the treatment cells suspended in 1 ml HBSS. Five days later the animals were treated with a 14 day course of GCV. All cones showed some degree of activity compared to animals injected with tumor alone.
  • LTKOSN.2 (clone 2), however, showed better activity than clones 9, 10, or 12. No evidence of toxicity was observed in the surviving animals. Animals that died during the experiment had death caused by progressive intraperitoneal carcinomatosis. This experiment establishes the efficacy of treating intraperitoneal adenocarcinoma with injections of LTKOSN.2 VPC followed by GCV treatment.
  • VPC have not been detected in these mice after 14 days. Two days later, the cells were used in a clonogenic assay to determine the percentage of MC 38 cells which had been transduced in vivo by the LTKOSN.2 vector. MC 38 NV cells (negative control) and MC 38 cells from the LTKOSN/2 VPC treated mice were seeded in triplicate in tissue culture dishes at selected densities. The next day 1 mg/ml G418 was added to 3 of 6 dishes in each set. Nine days later the cells were fixed with methanol: acetic acid and stained with crystal violet. Three separate clonogenic assays were set up from 2 animals (Table 2).
  • EXAMPLE 2 Anti-tumor therapy with Herpes simplex viral vectors.
  • HSV-based vectors One of two broad categories of HSV-based vectors are amplicons.
  • Plasmids with HSV-1 lytic replication origins (ori S) and HSV-1 terminal packaging signal sequences can be amplified and packaged into infectious HSV-1 virions in the presence of helper virus.
  • This plasmid system permits easier cloning and carries genomic information between prokaryotic and eukaryotic cells as a shuttle vector.
  • Kwong AD et al. "The Herpes simplex virus amplicon. Efficient expression of a chimeric chicken ovalbumin gene amplified within defective virus genomes", Virology, 1985; 142:421-425).
  • the amplicon systems retain the merits of HSV-1 vectors, but viral stocks tend to have lower titers and production is time consuming.
  • Spaete RR et al. "The herpes simplex virus amplicon: A new eucaryotic defective-virus cloning-amplifying vector", Cell, 1982; 30:295-304).
  • HSV vectors as anti-cancer therapeutics.
  • the cytopathic effects of modified HSV viruses and vectors can be used to excellent advantage against tumor cells.
  • Martuza and coworkers first used thymidine kinase (Hstk) negative HSV to treat human gliomas in nude mice.
  • Hstk thymidine kinase
  • Martuza RL et al. "Experimental therapy of human glioma by means of a genetically engineered virus mutant", Science, 1991; 252:854-856).
  • These initial modified HSV vectors were replication competent.
  • HSV vectors defective in other genes such as ribonucleotide reductase transcription factors ICP4 or ICPO, ⁇ ⁇ 34.5 gene.
  • ribonucleotide reductase transcription factors ICP4 or ICPO ribonucleotide reductase transcription factors
  • Plasmids containing a HSV-1 lytic replication origin (ori S) and a HSV-1 terminal packaging signal sequences can be amplified and packaged into infectious HSV-1 virions in the presence of transacting helper virus.
  • HSV-1 ori S contains HSV-1 ori S and packaging sequences that permit vector replication and packaging into HSV-1 virions.
  • HSV amplicons that also contain Epstein-Barr virus (EBV) sequences to maintain the plasmid as an episome in the transfected cell nucleus.
  • EBV Epstein-Barr virus
  • Westphal EM et al. "A novel infectious mini-HSV for high efficiency gene transfer into human cancer cells", Cancer Gene Ther, 1995; 2:324
  • EBV has been demonstrated to contain a unique latent replication origin (ori. P) which directs viral self-replication and maintenance in cells without entering the lytic cycle.
  • Epstein-Barr virus nuclear antigen 1 encodes a DNA binding transactivator for ori P.
  • the combination of the HSV amplicon with the EBV sequences improves the ease of use of the HSV amplicon system.
  • Our replication incompetent pHE vectors maintain wide tropism for delivering transgene(s) into both dividing and quiescent cells with high efficiency both in vitro and in vivo.
  • This improved vector could be produced at high titer and could package and carry a vector with a 21 kb DNA insert.
  • Episomal maintenance and amplicon vector packaging The maintenance of pHE vector as an episome was demonstrated by transfection of pHE700-lac into E5 cells and selection with hygromycin.
  • the pHE700-lac vector contains the bacterial LacZ gene cloned into the multi-cloning site of pHE700 ( Figure 6). By day 16 of drug selection, almost all cells expressed ⁇ - galactosidase. To generate viral stocks, these selected E5 cells containing pHE700-lac plasmid were infected with dl20 helper virus (kindly provided by N. DeLuca, University of Pittsburgh). The resulting supernatants contain both the pHE700-lac vector and helper virus.
  • the multiplicity of infection (MOI) of the helper virus added was between 0.01 to 0.1 to induce viral vector production within 24-36 hours.
  • the average titer obtained was 2xl0 6 bfu/ml with a ratio of pHE700-lac vector (bfu) to dl20 helper virus (pfu) of 1:10.
  • the pHE700-lac containing supernatants were used to transduce human target cells in vitro.
  • the ⁇ -galactosidase gene expression was evaluated after infection with pHE700-lac vector (3-10 MOI) in various cultured human cells, including VA13 normal fibroblasts. All cells were fixed and stained with X-gal two days after infection. The expression continued for approximately 2 weeks with a peak expression occurring 48-72 hours after transduction.
  • HSV amplicon vector cytotoxicity The dl20 HSV helper virus is necessary to package this HSV amphcon vector (DeLuca NA et al., "Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate -early regulatory protein ICP4", J Virol, 1985; 56:558-570; DeLuca NA et al., "Activities of herpes simplex virus type 1(HSV-
  • ICP4 genes specifying nonsense peptides", Nucleic Acids Res, 1987; 15:4491-
  • Helper virus dl20 has deletions of both IE3 gene loci to prevent viral replication in normal cells, but permits replication in the E5 helper cell line expressing the IE3 gene (DeLuca NA et al., "Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4", J Virol, 1985; 56:558-570; DeLuca NA et al., "Activities of herpes simplex virus type l(HSV-l) ICP4 genes specifying nonsense peptides", Nucleic Acids Res. 1987; 15:4491-4511).
  • This helper virus causes substantial cytotoxicity to infected normal cells in vitro (data not shown).
  • a cell proliferation assay that measured cellular DNA synthesis was used as previously described.
  • the pHE-tk vector was generated by inserting the Hstk gene into the pHE vector multi-cloning site under CMV promoter control ( Figure 6).
  • CeUs expressing Hstk can phosphorylate ganciclovir (GCV) to a toxic form that induces cell death.
  • crosslinked DNA retains measurable transgene expression while prohibiting replication of any single vector.
  • photochemical modification should prevent any lytic viral 5 replication that might occur due to the rescue of recombinant wild-type virus both in vitro or in vivo.
  • PUVA treated vectors are ideal candidates for in vivo anti-tumor strategies using therapeutic genes such as the NIS gene.
  • VECTORS GENE TRANSFER TO OVARIAN CANCER CELLS IN VITRO AND INCREASED CELL DESTRUCTION BY 13 *I
  • the dose from accumulated 131 I is affected by cell density, radius and the stopping power of the beta energy from the decay of 131 I.
  • the next step in the project is to test a HSV amplicon vector to transfer a functional NIS gene to tumor spheroids in culture.
  • HSV vector containing both the Green Fluorescent Protein and NIS gene.
  • a key part of evaluating and developing any gene therapy strategy is to accurately measure gene transfer efficiency. This data is essential in order to determine the level of vector transduction required , to obtain a therapeutic level of the transgene in vivo.
  • the second series of HSV vectors that will be cloned will not contain G418 drug selectable markers (neo r )- Our vectors will express a variant of the green fluorescent protein (GFP). This codon modified GFP gene also contains a serine 65 to threonine mutation to greatly enhance the fluorophore activity.
  • This marker allows the detection of in vitro and in vivo gene transfer by the direct observation of living tissues or frozen sections (without the need for fixation).
  • the marker allows gene transfer to be observed on an individual cell basis.
  • Excellent GFP gene expression and translation is visible in living tumor cell, transduced by a pHE700 vector.
  • pHE850 a new HSV amplicon vector has been constructed, pHE850, that contains a red-shifted, codon optimized GFP gene and a separate multicloning site ( Figure 10).
  • This new vector will be used for transfer of the NIS gene into tumor cells, since it also permits rapid determinations of gene transfer efficiency by observing GFP co-expression
  • NIS gene Cloning of the NIS gene into the pHE8NIS amplicon vector
  • the NIS gene open reading frame will be cloned into the multi-cloning site of the pHE850 vector backbone ( Figure 10).
  • This vector contains a large multicloning site that aUows for direct subcloning from pREP eukaryotic expression plasmids.
  • the NIS gene was previously subcloned into pREP7 flanked by Kpn I and Xho I sites and these will be used to subclone into the multi-cloning site of pHE850.
  • the amplicon undergoes rolling circle replication in E5 cells containing an IE 3 gene that permits trans- complementation of dl20 (IE3 gene defective) helper virus. In the presence of helper virus, the amplicon replicates until a viral DNA of 152 kb long is obtained and then packaged.
  • helper virus the amplicon replicates until a viral DNA of 152 kb long is obtained and then packaged.
  • the closed pHE8NIS plasmid will be transfected into E5 cells expressing the repUcation permissive IE3 gene with Lipofection per the manufacture's protocol (GibcoBRL).
  • transfected cells will be placed in selection with Hygromycin B (ICN Biomedical Inc., Aurora, Ohio). Hygromycin resistant colonies will be trypsin digested and cells plated onto dishes. After stable selection for two weeks in hygromycin, the population of transfected cells will be examined for GFP expression. If positive for fluorescence, the cells will then be transduced with dl20 helper virus.
  • 3 x IO 6 hygromycin resistant cells containing pHE8NIS will be plated on a 10-cm dish. After cells grow to confluence, 0.01 to 0.1 MOI of dl20 virus in 1 ml of Opti-MEM (Gibco, BRL) will be added for 2 hours at 37°C to allow infection. The virus solutions are then removed and fresh medium is added for an additional 24 to 36 hours. After 48-72 hours cells wiU be lysed. Supernates obtained, after centrifugation to remove debris, will be tested for green forming units (gfu/ml) to define supernate vector titer.
  • Opti-MEM Gibco, BRL
  • PUVA treatment of pHE8NIS vector supernates For PUVA treatment, TMP (Trioxsalen, Sigma) in various concentrations (0 to 5 ⁇ g/ml) will be added to virus stocks for 30 minutes at 37°C with differing lengths of UVA exposure (0-7.5 kJ/m 2 ).
  • the UVA source will be a Spectroline Model XX-15A bulb (Westbury, New York).
  • the virus replication ability will be measured 3 days after incubation by staining permissive E5 cells with crystal violet. GFP will be visualized by fluorescence microscopy.
  • the vector will first be transiently transfected into GP+E86 ecotropic packaging cells and then the resulting supernates wiU be used to transduce PA317 cells growing in log phase. The cells will be cloned by Umiting dilution and 20 clones will be titered for activity. Several of the highest titer LNISN VPC will be grown to large scale and used for in vivo tests.
  • Test efficacy of pretransduced tumor model for response with 131 I In order to insure that the planned 131 I treatment dose of 0.3 mCi is effective, an initial small experiment will be conducted with 131 I.
  • SK-OV-3 tumor cells will be transduced with the LNISN vector and an individual clone with stable transgene expression will be obtained.
  • Athymic nude mice (nu/nu) will be injected subcutaneously with lOxlO 6 SK-OV-3 tumor cells into the anterior abdominal wall. This cell number usually results in a solid 3-4 mm tumor mass within 5-7 days (data not shown).
  • the cells administered will be transduced ex vivo before implantation according to Table 3.
  • Group D will determine if tumors cells expressing the NIS gene combined with cells not expressing the NIS gene (one to one ratio) can be destroyed by bystander radiation killing after 131 I administration. Fourteen days later, animals will receive a single intravenous injection of 0.3 mCi of 131 I (Kasuga Y et al., "The effect of xenotransplantation of human thyroid tissue following radioactive iodine-induced thyroid ablation on thyroid function in the nude mouse", Clin Invest Med, 1991; 14:277-281; Walinder G, "Determination of the 131 1 dose to the mouse thyroid", Acta Radio Ther Phys Biol, 1971; 558-578). Responses will be determined by tumor measurements recorded biweekly (mm3).
  • mice Five positive control mice (group A) will be injected with lOxlO 6 SK-OV-3 cells pretransduced with LNISN vector. Eight mice each in groups B, C and D will be injected into the abdominal cavity with lOxlO 6 SK- OV-3 tumor cells followed by treatment with either (B) LNChRG VPC (transfers GFP gene, but no NIS gene), (C) HBSS only or (D) LNISN VPC. Three days after the injection of treatment cells, 5 mice in each group will be injected intravenously of 0.3 mCi of 131 I. Responses will be determined by biweekly observations of the animals for cachexia of ascites. Animals from group B through D not treated with 131 I will be evaluated for neo r expression by G418 selection by the same method used for Table 2. This aim should establish in vivo efficacy of the isotope concentrator concept.
  • HSV vectors offer both of these properties and will be the gene transfer vehicle for our anti-tumor approach with NIS gene therapy. These vectors are extremely attractive since they efficiently transduce nondividing cells (Go stage) and have large carrying capacity.
  • Plasmids containing a HSV-1 lytic replication origin (ori S) and a HSV-1 terminal packaging signal sequences can be amplified and packaged into infectious HSV-1 virions in the presence of transacting helper virus.
  • herpes simplex virus amplicon A new eucaryotic defective-virus cloning-amplifying vector", Cell, 1982; 30:295-304; Kwong AD et al, "Herpes simplex virus amplicon: Effect of size on replication of constructed defective genomes containing eukaryotic DNA sequences", J Virol.
  • mice The injection of 10 x IO 6 cells on day 1 will result in multiple intraperitoneal tumors in all animals by day 7-10 after inoculation (Link, unpubhshed observations). Three groups of 5 mice each will receive direct intraperitoneal injections of 1 ml of HBSS containing 1 x IO 7 , 1 x IO 8 or 1 x IO 9 gfu/ml of pHE8NIS vector. Analysis for GFP expression will be performed 2 to 5 days after injection.
  • mice will be obtained (Harpan-Sprague). Animals will be fed sterile mouse food (Teklad) and H2O ad libitum. Sani-chip and microisolator mouse cages (Lab products) will be used. Cages will be kept in our fill service rodent faciUty. Mice in groups A through E (Table 5) will be injected intraperitoneally with 10xl0 G SK-OV-3 cells on day 1.
  • Control groups will be (A) HBSS only and (B) pHE-tk vector (control for HSV vector cytotoxicity).
  • Groups (C), (D) and (E) will be injected with three dose levels of pHE8NIS vector (Table 5).
  • Mice in group F will receive SK-OV-3 cells pretransduced with LNISN retroviral vector and will serve as positive controls for tumor killing by 131 I.
  • Three days after the injection of treatment cells five animals from each group will receive an intravenous injection of 0.3 mCi of 131 I. Animals will be observed daily for evidence of toxicity or infection after vector administration.
  • mice surviving at 60 days will be harvested. A separate portion of this tumor will be sent for histopathology staining for cellular infiltrates present in the tumors.
  • SK-OV-3 ceUs Day 0 Tumor implantation into the peritoneal cavity
  • Day 5 Vector administered into the peritoneal cavity
  • Day 8 131 I administered (0.3 mCi)
  • Allogeneic BMT (bone marrow transplant) cures leukemia by means of myeloablation induced by the preparative regimen and by transfer in the bone marrow aUograft of immunocompetent donor cells that exert an anti-leukemic effect called Graft-versus-Leukemia (GvL).
  • GvL Graft-versus-Leukemia
  • T-cell depletion increases leukemia relapse in AML and ALL because of the loss of GvHD.
  • GvL effect independent of GvHD
  • Tumor Specific Promoters Useful for the Invention include but are not hmited to the following:
  • HrMA4 alpha muscle-specific actin ⁇ promoter ⁇ [Halocynthia
  • 0gb:SUSMSP130A S.purpuratus cell surface glycoprotein (mspl30) gene, 5' flank and
  • 0gb:SUSMSP130B S.purpuratus cell surface glycoprotein (mspl30) mRNA, 5' end. R95062574-95068440-/gopherlib/data/db/.genbank-92/gbinv.seq gopher.nih.gov 70
  • 0gb:MDU32208 Monodelphis domestica ubiquitin C-terminal hydrolase (PGP9.5) gene
  • 0gb:A15840 Promoter region proteinase gene from pSKIII.

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Abstract

La présente invention concerne la médecine nucléaire d'association faisant intervenir des agents radio-pharmaceutiques destinés à l'imagerie diagnostique et à la thérapie des tumeurs. Plus spécifiquement, l'invention concerne des compositions pharmaceutiques et des méthodes de traitement, de diagnostic et de radio-imagerie par génie génétique conçues pour accumuler des radio-isotopes dans des cellules. L'association du génie génétique pour conférer une accumulation de radio-isotope par des cellules avec l'introduction de radio-isotopes présente de nombreuses utilisations et notamment la radio-imagerie in vitro ou in vivo afin de localiser ou d'identifier l'expression de gènes, la radiothérapie destinée à tuer des cellules néoplasique et non néoplasiques, et des applications diagnostiques. L'invention concerne également des constructions génétiques, des vecteurs, des cellules transformées et des méthodes.
EP98920832A 1997-04-10 1998-04-09 Methodes et compositions de concentration de radio-isotopes Withdrawn EP0973902A1 (fr)

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EP1900749A1 (fr) 2006-09-12 2008-03-19 Institut National De La Sante Et De La Recherche Medicale (Inserm) Acide nucléique pour l'expression d'un polynucléotide d'interêt dans des cellules cancéreuses de mammiferès
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US9951117B2 (en) 2010-09-02 2018-04-24 Mayo Foundation For Medical Education And Research Vesicular stomatitis viruses
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