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WO2007064971A2 - Composition de particules infectieuse comprenant un gene cytocide et methodes d'utilisation correspondantes - Google Patents

Composition de particules infectieuse comprenant un gene cytocide et methodes d'utilisation correspondantes Download PDF

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Publication number
WO2007064971A2
WO2007064971A2 PCT/US2006/046145 US2006046145W WO2007064971A2 WO 2007064971 A2 WO2007064971 A2 WO 2007064971A2 US 2006046145 W US2006046145 W US 2006046145W WO 2007064971 A2 WO2007064971 A2 WO 2007064971A2
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Prior art keywords
gene
capsid protein
papovavirus
infectious particle
pseudomonas exotoxin
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WO2007064971A3 (fr
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Chava Kimchi-Sarfaty
Michael M. Gottesman
Wilfred D. Vieira
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US Department of Health and Human Services
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US Department of Health and Human Services
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22023—Virus like particles [VLP]
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/22042—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule

Definitions

  • the present invention relates generally to a infectious particle composition comprising a papovavirus capsid protein and an exogenous cytocidal gene.
  • the present invention further relates to methods of treating disease and methods of use for the infectious particles.
  • Pseudomonas exotoxin A has been demonstrated to kill cancer cells.
  • Pseudomonas exotoxin A is a 66 kDa pathogenic protein secreted by Pseudomonas aeruginosa as a proenzyme, under the selective pressure of a low iron environment. This toxin assists bacteria in the invasion of animal tissues, including human tissues. As such, it enters cells via the low-density lipoprotein receptor-related protein (LRP).
  • LRP low-density lipoprotein receptor-related protein
  • PE is comprised of three major domains: domain Ia is the cell binding domain and domain Ib may serve to enhance toxin stability; domain II harbors the protease processing site and mediates translocation; and domain III has ADP-ribosylating activity, and therefore is responsible for the toxic activity of the protein. Chen et al, BiomedSci. 6:727-32, 1999.
  • Recombinant immunotoxins have been created in E. coli. Pastan, Cancer Immunol Immunother. 52:338-341, 2003.
  • the Fv portion of a monoclonal antibody (MAb) in a single chain form was fused directly to mutants of PE missing domain Ia.
  • MAb monoclonal antibody
  • the advantage of this method is that it combines the higher specificity of the antibody with the killing power of the toxin, hi order to achieve stability of the recombinant immunotoxin at 37°C, a recombinant immunotoxin molecule was designed in which the light and heavy chains of the Fv portion are held together by a disulfide bond.
  • the present invention relates to infectious particle compositions and pharmaceutical compositions thereof.
  • the infectious particle can comprise a papovavirus capsid protein and an exogenous cytocidal gene.
  • a method for inducing cell death is provided, and a method for treating disease, e.g., neoplastic disease or infectious disease, in a mammalian subject is provided.
  • the method for inducing cell death provides contacting a cell with the infectious particle comprising a papovavirus capsid protein and an exogenous cytocidal gene to induce cell death.
  • the method for treating neoplastic disease provides administering to the mammalian subject an infectious particle comprising a papovavirus capsid protein and an exogenous cytocidal gene, wherein the infectious particle directs the cytocidal gene to a neoplastic cell in the mammalian subject, in an amount effective to reduce or eliminate the neoplastic disease or to prevent its occurrence or recurrence.
  • a method of reducing side effects of chemotherapeutic treatment for neoplastic disease in a mammalian subject comprising administering to the mammalian subject an infectious particle comprising a papovavirus capsid protein and an exogenous toxin gene, and a chemotherapeutic agent, in an amount effective to reduce or eliminate the neoplastic disease or to prevent its occurrence or recurrence and reduce side effects of chemotherapeutic treatment.
  • An infectious particle comprising a papovavirus capsid protein and an exogenous cytocidal gene.
  • the exogenous cytocidal gene includes, but is not limited to, an exogenous bacterial toxin gene or an exogenous suicide gene.
  • the papovavirus capsid protein is SV40 capsid protein VPl, papilloma virus capsid protein Ll, or polyoma virus capsid protein VP 1.
  • the papovavirus capsid protein is S V40 capsid protein VPl, VP2, or VP3, or a combination thereof.
  • the SV40 capsid protein VPl, VP2, or VP3, or a combination thereof further comprises SV40 agno protein.
  • the papovavirus capsid protein is SV40 E capsid protein or polyoma E capsid protein.
  • the papovavirus capsid protein is papilloma virus capsid protein Ll or L2, or a combination thereof.
  • the papovavirus capsid protein is polyoma virus capsid protein VPl, VP2, or VP3, or a combination thereof.
  • the exogenous bacterial toxin gene is a Pseudomonas exotoxin gene PE38.
  • the toxin gene can further comprise a truncated Pseudomonas exotoxin gene PE38.
  • the truncated Pseudomonas exotoxin gene PE38 can be lacking domain Ia.
  • the truncated Pseudomonas exotoxin gene PE38further can further comprise an immunoglobulin Fv portion fused to the truncated Pseudomonas exotoxin gene PE38.
  • the toxin gene is a Pseudomonas exotoxin gene, a ricin gene, or a diphtheria toxin gene.
  • the exogenous suicide gene is an HSV thymidine kinase gene.
  • the infectious particle further comprises a targeting element.
  • the targeting element includes, but is not limited to, an immunotoxin, transforming growth factor, TGF- ⁇ , caspase, or caspase-6.
  • the infectious particle further comprises a fusion protein of the targeting element and the capsid protein.
  • a pharmaceutical composition which comprises, and a pharmaceutically acceptable carrier.
  • the pharmaceutical can further comprise a chemotherapeutic agent.
  • the chemotherapeutic agent is doxorubicin.
  • an infectious particle comprising a papovavirus capsid protein and an exogenous cytocidal gene wherein the papovavirus capsid protein is a semi-purified or pure papovavirus capsid protein.
  • the cytocidal gene is an exogenous bacterial toxin gene.
  • the cytocidal gene is an exogenous suicide gene.
  • the papovavirus capsid protein is SV40 capsid protein VPl, papilloma virus capsid protein Ll, or polyoma virus capsid protein VPl.
  • the papovavirus capsid protein is SV40 capsid protein VPl, VP2, or VP3, or a combination thereof.
  • the SV40 capsid protein VPl, VP2, or VP3, or a combination thereof further comprises SV40 agno protein.
  • the papovavirus capsid protein is SV40 E capsid protein or polyoma E capsid protein.
  • the papovavirus capsid protein is papilloma virus capsid protein Ll or L2, or a combination thereof.
  • the papovavirus capsid protein is polyoma virus capsid protein VPl, VP2, or VP3, or a combination thereof.
  • the exogenous bacterial toxin gene is a Pseudomonas exotoxin gene PE38.
  • the toxin gene can further comprise a truncated Pseudomonas exotoxin gene PE38.
  • the exogenous suicide gene is an HSV thymidine kinase gene.
  • a method for treating a disease comprising contacting cells with a cytocidal amount of an infectious particle comprising a papovavirus capsid protein and an exogenous cytocidal gene to induce cell death.
  • the cytocidal gene is an exogenous bacterial toxin gene.
  • the cytocidal gene is an exogenous suicide gene.
  • the papovavirus capsid protein is SV40 capsid protein VPl, papilloma virus capsid protein Ll, or polyoma virus capsid protein VPl .
  • the papovavirus capsid protein is SV40 capsid protein VPl, VP2, or VP3, or a combination thereof.
  • the SV40 capsid protein VPl, VP2, or VP3, or a combination thereof further comprises SV40 agno protein.
  • the papovavirus capsid protein is SV40 E capsid protein or polyoma E capsid protein.
  • the papovavirus capsid protein is papilloma virus caps ⁇ d'protein.X ' 1 ⁇ rT2, or a combination thereof.
  • the papovavirus capsid protein is polyoma virus capsid protein VPl, VP2, or VP3, or a combination thereof.
  • the exogenous bacterial toxin gene is aPseudomonas exotoxin gene PE38.
  • the toxin gene can further comprise a truncated Pseudomonas exotoxin gene PE38.
  • the truncated Pseudomonas exotoxin gene PE38 can be lacking domain Ia.
  • the toxin gene is a Pseudomonas exotoxin gene, a ricin gene, or a diphtheria toxin gene.
  • the exogenous suicide gene is an HSV thymidine kinase gene.
  • the method comprises contacting the cells with a cytocidal amount of a chemotherapeutic agent.
  • the chemotherapeutic agent is doxorubicin.
  • the method comprises an infectious particle comprising a fusion protein of a targeting element and the capsid protein.
  • the targeting element is an immunotoxin, transforming growth factor, TGF- ⁇ , caspase, or caspase-6.
  • a method for treating neoplastic disease in a mammalian subject comprising administering to the mammalian subject an infectious particle comprising a papovavirus capsid protein and an exogenous cytocidal gene, wherein the infectious particle directs the cytocidal gene to a neoplastic cell in the mammalian subject, in an amount effective to reduce or eliminate the neoplastic disease or to prevent its occurrence or recurrence.
  • the cytocidal gene is an exogenous bacterial toxin gene.
  • the cytocidal gene is an exogenous suicide gene.
  • the papovavirus capsid protein is SV40 capsid protein VPl, papilloma virus capsid protein Ll, or polyoma virus capsid protein VPl.
  • the papovavirus capsid protein is SV40 capsid protein VPl, VP2, or VP3, or a combination thereof.
  • the SV40 capsid protein VPl, VP2, or VP3, or a combination thereof further comprises SV40 agno protein.
  • the papovavirus capsid protein is SV40 E capsid protein or polyoma E capsid protein.
  • the papovavirus capsid protein is papilloma virus capsid protein Ll or L2, or a combination thereof.
  • the papovavirus capsid protein is polyoma virus capsid protein VPl, VP2, or VP3, or a combination thereof.
  • the exogenous bacterial toxin gene is a Pseudomonas exotoxin gene PE38.
  • the toxin gene can further comprise a truncated Pseudomonas exotoxin gene PE38.
  • the truncated Pseudomonas exotoxin gene PE38 can be lacking domain Ia.
  • the toxin gene is a. Pseudomonas exotoxin gene, a ricin gene, or a diphtheria toxin gene.
  • the exogenous suicide gene is an HSV thymidine kinase gene.
  • the method comprises an infectious particle comprising a fusion protein of a targeting element and the capsid protein.
  • the targeting element is an immunotoxin, transforming growth factor, TGF- ⁇ , caspase, or caspase-6.
  • the method comprises contacting the cells with a cytocidal amount of a chemotherapeutic agent.
  • the chemotherapeutic agent is doxorubicin.
  • the method further comprises administering the infectious particle to the mammalian subject by intraperitoneal injection.
  • the method comprises administering the infectious particle to the mammalian subject by intratumoral, intravenous, intraarterial, intraperitoneal, lymphatic, or intraocular injection.
  • the neoplastic disease includes, but is not limited to, solid tumor, hematological malignancy, adenocarcinoma, leukemia, lymphoma, lymphoblastoma, colorectal cancer, benign, or malignant breast cancer, uterine cancer, uterine leiomyomas, ovarian cancer, endometrial cancer, polycystic ovary syndrome, endometrial polyps, prostate cancer, prostatic hypertrophy, pituitary cancer, adenomyosis, meningioma, melanoma, bone cancer, multiple myeloma, CNS cancer, glioma, or astroblastoma.
  • the neoplastic disease is tumor cell metastasis in the mammal or the neoplastic disease state is adenocarcinoma metastasis in the mammal.
  • a method of reducing side effects of chemotherapeutic treatment for neoplastic disease in a mammalian subject comprising administering to the mammalian subject an infectious particle comprising a papovavirus capsid protein and an exogenous toxin gene, and a chemotherapeutic agent, in an amount effective to reduce or eliminate the neoplastic disease or to prevent its occurrence or recurrence and reduce side effects of chemotherapeutic treatment.
  • the chemotherapeutic agent is doxorubicin.
  • the toxin gene is Pseudomonas exotoxin gene PE38.
  • the toxin gene is a truncated Pseudomonas exotoxin gene PE38.
  • the truncated Pseudomonas exotoxin gene PE38 is lacking domain Ia.
  • the toxin gene includes, but is not limited to, a Pseudomonas exotoxin gene, ricin gene, or diphtheria toxin gene.
  • Figures Ia, Ib, Ic and Id show expression of PE38 packaged in vitro in four cell lines.
  • Figures 2a, 2b, 2c, 2d, 2e and 2f show tumor size and weight of PE38 packaged in vitro and control mice treated from the day of tumor challenge.
  • Figures 3a, 3b, 3c, 3d ,3e and 3f show tumor size and weight of PE38 packaged in vitro and control mice treated several days post tumor challenge via SC.
  • Figures 4a, 4b, 4c, 4d, 4e and 4f show tumor size and weight of PE38 packaged in vitro and control mice treated intraperitoneally several days post tumor challenge.
  • Figure 5 shows tissue fluorescence of EGFP packaged in vitro and control mice treated intraperitoneally several days post tumor challenge.
  • Figure 6 shows viability of KB-3-1 cells treated in tissue culture with PE38 packaged in vitro and doxorubicin.
  • Figures 7a and 7b show tumor size and weight of mice treated with a combination of PE38 packaged in vitro and doxorubicin via intraperitoneal injection.
  • An infectious particle comprising a papovavirus capsid protein and an exogenous cytocidal gene.
  • the present invention has demonstrated efficient delivery to various human cells of an infectious particle comprising a papovavirus capsid protein and a cytocidal gene product, such as an exogenous bacterial toxin gene, e.g., Pseudomonas exotoxin A (PE) 38 toxin, or an exogenous suicide gene, e.g., HSV thymidine kinase.
  • PE Pseudomonas exotoxin A
  • suicide gene e.g., HSV thymidine kinase.
  • the infectious particle comprising a papovavirus capsid protein and a cytocidal gene product can be used for treatment of neoplastic disease or for treatment of infectious disease.
  • the infectious particle can further include a chemotherapeutic agent, e.g., doxorubicin, delivered to a tumor cell in a mammalian subject, wherein the tumor cell is sensitized to the chemotherapeutic agent, while minimizing the side effects of the chemotherapeutic agent to the mammalian subject.
  • the infectious particle can be an SV40 capsid protein in combination with a cytocidal gene, e.g., PE38 toxin.
  • SV40 infectious particle delivery of PE38 toxin is effective in the treatment of human adenocarcinomas growing in mice either by direct injection into the tumor, intraperitoneally, or administered systemically.
  • a chemotherapeutic agent e.g., doxorubicin
  • the efficacy of doxorubicin chemotherapy was maintained while the side effects of chemotherapy were greatly reduced.
  • the infectious particle comprising a papovavirus capsid protein and a cytocidal gene product can be used for treatment of a disease in a mammalian subject, for example, neoplastic disease or infectious disease.
  • the infectious particle comprising a papovavirus capsid protein and a cytocidal gene product can be used for treatment of disease, including but not limited to: Graft vs.
  • Infectious particles comprising papovavirus capsid protein, for example, simian virus 40 (SV40) capsid protein, are an attractive vector for gene transfer to kill cancer cells.
  • the vectors can be prepared with nuclear extract of SF9 insect cells containing the main viral capsid protein of the SV40 wild-type virus, VPl.
  • the SV40 major capsid protein demonstrates high transduction efficiency and can be introduced into a wide variety of human, murine and monkey tissues.
  • In vitro packaging of DNA with SV40 capsid protein enables efficient delivery of plasmids with a length of up to 17.7 kb, with or without SV40 sequences. Moreover, it does not require any SV40 sequence, thus providing efficient gene delivery at the same level of safety when using nonviral vectors.
  • a truncated Pseudomonas exotoxin gene (PE38) was delivered into various human cells (HeLa, KB-3-1 human adenocarcinoma, human lymphoblastoids, and erythroleukemia cells) in vitro using pseudo virions. The number of viable cells was reduced significantly in the PE38 -transduced cells. Human KB adenocarcinomas growing in mice were treated with intratumoral injection of PE38 packaged in vitro. Tumor size was reduced significantly. Intraperitoneal treatments were as effective in reducing tumor size as intratumoral treatments.
  • mice In order to check the viability of the treated mice, three tests were carried out in mock- or PE38-treated mice: the weight of the mice was recorded every four days, various tissues were screened for pathology, and blood was analyzed. All parameters showed that the in vzYro-packaged vectors, given into tumor or intraperitoneally, caused no abnormalities in mice.
  • the combined treatment of doxorubicin with in vzYr ⁇ -packaged PE38 reduced tumor size only slightly more than each of the treatments separately. However, the combined treatment did not cause the weight loss seen with doxorubicin alone.
  • tibds invention is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell” includes a combination of two or more cells, and the like.
  • infectious particle refers to infectious, non-replicative viral particles assembled from viral capsid proteins and encapsidating a nucleic acid molecule, e.g., an exogenous cytocidal gene, an exogenous bacterial toxin gene, or an exogenous suicide gene.
  • Infectious pseudoviral particles can be derived from viruses such as papovavirus, which include, but are not limited to, SV40, polyoma, or papilloma, which have the ability to self-assemble using either the major viral capsid protein VPl, all capsid proteins VPl, VP2 and VP3, the E capsid protein, or a combination thereof.
  • the viral capsid proteins are recombinant proteins prepared in insect, bacteria or mammalian cells. The assembled capsid proteins maintain their ability to transduce a wide variety of cells through natural receptors that make these pseudovirions infectious particles.
  • Cytocidal gene refers to a gene or gene product that is toxic to a cell when presented at the cell surface or intracellularly via an infectious viral particle. Cytocidal genes include, but are not limited to, exogenous toxin genes, exogenous bacterial toxin genes, or exogenous suicide genes. Compositions and methods that incorporate an infectious particle comprising a papovavirus capsid protein and an exogenous cytocidal gene provide a therapeutic strategy for making cancer cells more vulnerable to chemotherapy. One approach has been to provide infectious particles comprising cytocidal genes with a chemotherapeutic agent.
  • the cytocidal gene can be a bacterial toxin gene, such as PE38 toxin, or a suicide gene, such as HSV thymidine kinase (tk) gene.
  • a further approach has been to link parts of genes expressed in cancer cells to suicide genes, e.g., suicide genes which are genes for enzymes not found in mammals that can convert a harmless substance into one that is toxic to the tumor.
  • suicide genes e.g., suicide genes which are genes for enzymes not found in mammals that can convert a harmless substance into one that is toxic to the tumor.
  • suicide genes e.g., suicide genes which are genes for enzymes not found in mammals that can convert a harmless substance into one that is toxic to the tumor.
  • tk thymidine kinase-mediated activation of ganciclovir to increase cyctocidal effects.
  • Infectious particles can be used to transfer the thymidine kinase (tk) gene to tumors or neoplastic cells in mice, and improves the ability of S V40 infectious particle-tk to render these tumors sensitive to the cytocidal effects of ganciclovir.
  • tk thymidine kinase
  • Cell death refers to a process which prevents a cell from carrying out its normal metabolic functions. Cell death can occur by either of two distinct mechanisms, necrosis or apoptosis (programmed cell death). In addition, certain chemical compounds and cells are said to be cytotoxic to the cell, that is, to cause its death. “Necrosis” refers to the pathological process which occurs when cells are exposed to a serious physical or chemical insult. Necrosis occurs when cells are exposed to extreme variance from physiological conditions (e.g., hypothermia, hypoxia) which may result in damage to the plasma membrane.
  • physiological conditions e.g., hypothermia, hypoxia
  • Infectious particles comprising a papovavirus capsid protein and an exogenous cytocidal gene can target to various tissues and to tumor cells in a mammalian subject. Targeting can occur by a number of different mechanisms. Surface targeting to cells by the infectious particle, e.g., papovavirus, SV40, papilloma, or polyoma infectious particles, occurs on both dividing and non-dividing target cells. Infectious particles comprising a papovavirus capsid protein and an exogenous cytocidal gene can target to tissues in a mammalian subject including, but not limited to, lung, heart, liver, spleen, kidney, and across the blood brain barrier to the brain and central nervous system.
  • Infectious particles comprising a papovavirus capsid protein and an exogenous cytocidal gene can target to tumor cells or neoplastic cells in a mammalian subject.
  • the tumor cells or neoplastic cells include, but are not limited to, solid tumor, hematological malignancy, adenocarcinoma, leukemia, lymphoma, lymphoblastoma, colorectal cancer, benign or malignant breast cancer, lung cancer, uterine cancer, uterine leiomyomas, ovarian cancer, endometrial cancer, polycystic ovary syndrome, endometrial polyps, prostate cancer, prostatic hypertrophy, bladder cancer, pituitary cancer, adenomyosis, meningioma, melanoma, bone cancer, multiple myeloma, brain cancer, CNS cancer, glioma, or astroblastoma.
  • Targeting of infectious particles can also occur by use of gene promoters that are specific to tumor cells, e.g., cancer-specific promoter sequences, for example, prostate- specific promoters.
  • Targeting of infectious particles can also occur by use of gene promoters that are specific to tumor cells, e.g., cancer-specific promoter sequences, for example, prostate- specific promoters.
  • Infectious particles that display targeting elements as a fusion protein with the papovavirus capsid protein can be targeted specifically to certain cancer cell types.
  • the fusion protein comprises the papovirus capsid protein including, but not limited to, SV40 capsid protein VPl, VP2, or VP3; papilloma virus capsid protein Ll or L2; polyoma virus capsid protein VPl, VP2, or VP3; SV40 agno protein, SV40 E capsid protein, or polyoma E capsid protein.
  • antibodies fused to papovavirus capsid protein can target the infectious particle to a cell-specific surface receptor.
  • Infectious particles with papovavirus capsid protein fused to an antibody or binding moiety to transferrin receptor can target to cells expressing transferrin receptor on the cell surface.
  • Infectious particles comprising papovavirus capsid protein can further provide a targeting elements, for example, TGF- ⁇ or cas ⁇ ase-6, to target to specific cancer cell types.
  • the infectious particles can be targeted to neoplastic cells, including but not limited to, bladder carcinoma, breast cancer, glioma, brain tumor, or lung tumor.
  • the targeting element can be provided in an expression vector in addition to the expression vector for the exogenous cytocidal gene, or the targeting element can be a fusion with the capsid protein to contact a cell surface target or an intracellular target.
  • Chimeric fusion proteins of capsid proteins and targeting elements are envisioned to direct infectious particles to target cells, e.g., tumor cells, internalize the infectious particles, and deliver the cytocidal gene product to the cell to initiate a cell death mechanism.
  • a method of treating neoplastic disease comprises administering an infectious particles comprising a papovavirus capsid protein and an exogenous cytocidal gene, wherein the disease state is a neoplastic disease, including, but not limited to, solid tumor, hematological malignancy, adenocarcinoma, leukemia, lymphoma, lymphoblastoma, colorectal cancer, benign or malignant breast cancer, uterine cancer, uterine leiomyomas, ovarian cancer, endometrial cancer, polycystic ovary syndrome, endometrial polyps, prostate cancer, prostatic hypertrophy, pituitary cancer, adenomyosis, meningioma, melanoma, bone cancer, multiple myeloma, CNS cancer, glioma, or astroblastoma.
  • the neoplastic disease is tumor cell metastasis in the ma
  • a method of reducing side effects of chemotherapeutic treatment for neoplastic disease in a mammalian subject comprises administering to the mammalian subject an infectious particle comprising a papovavirus capsid protein and an exogenous toxin gene, and a chemotherapeutic agent, in an amount effective to reduce or eliminate the neoplastic disease or to prevent its occurrence or recurrence and effective to reduce side effects of chemotherapeutic treatment.
  • the toxin gene can be a Pseudomonas exotoxin gene PE38 or a truncated Pseudomonas exotoxin gene PE38.
  • the truncated Pseudomonas exotoxin gene PE38 is lacking domain Ia.
  • the toxin gene can be a ricin gene, or diphtheria toxin gene.
  • the chemotherapeutic agent can be doxorubicin.
  • chemotherapeutic agents useful in a combined treatment with an infectious particle comprising a papovavirus capsid protein and an exogenous bacterial toxin gene include, but are not limited to, aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, duocarmycin, epoetin alpha, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole, leu
  • a preferred chemotherapeutic agent with which anti-activated integrin receptor can be combined is paclitaxel (TaxolTM).
  • paclitaxel TaxolTM
  • DTIC dacarbazine
  • Cell is used in its usual biological sense, of a component of a multicellular organism.
  • the cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats.
  • the cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
  • the cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing.
  • the cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.
  • the cell can be a human cell, for example, HeLa, KB-3-1 human adenocarcinoma, human lymphoblastoid cell, or human erythroleukemia cells.
  • mammalian cells containing one or more infectious particles comprising a papovavirus capsid protein and an exogenous cytocidal gene, an exogenous bacterial toxin gene, e.g., a Pseudomonas exotoxin gene PE38, or an exogenous suicide gene.
  • the exogenous bacterial toxin can independently be targeted to different sites, for example, solid tumor cells, adenocarcinoma cells, lymphoblastoid cells, or erythroleukemia cells.
  • RNA refers to a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide refers to a nucleotide with a hydroxyl group at the 2' position of a ⁇ -O- ribofuranose moiety.
  • the terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of the RNA or internally, for example at one or more nucleotides of the RNA.
  • Nucleotides in the RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
  • the nucleic acid molecules e.g., an exogenous cytocidal gene, an exogenous bacterial toxin gene, such as a Pseudomonas exotoxin gene PE38, or an exogenous suicide gene, or derivatives thereof, individually, or in combination or in conjunction with other drugs, can be used to for preventing or treating disease in a subject or organism.
  • an exogenous cytocidal gene e.g., an exogenous bacterial toxin gene, such as a Pseudomonas exotoxin gene PE38, or an exogenous suicide gene, or derivatives thereof, individually, or in combination or in conjunction with other drugs
  • an exogenous cytocidal gene e.g., an exogenous bacterial toxin gene, such as a Pseudomonas exotoxin gene PE38, or an exogenous suicide gene, or derivatives thereof, individually, or in combination or in conjunction with other drugs
  • the infectious particle comprising a papovavirus capsid protein and an exogenous cytocidal gene, an exogenous bacterial toxin gene, e.g., a. Pseudomonas exotoxin gene PE38 or an exogenous suicide gene, further provides the PE38 exotoxin gene or the HSV tk gene to be administered to a subject or to be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more chemotherapeutic drugs under conditions suitable for the treatment.
  • an exogenous bacterial toxin gene e.g., a. Pseudomonas exotoxin gene PE38 or an exogenous suicide gene
  • the infectious particle comprising a papovavirus capsid protein and an exogenous cytocidal gene, an exogenous bacterial toxin gene, e.g., a Pseudomonas exotoxin gene PE38, or an exogenous suicide gene
  • an exogenous bacterial toxin gene e.g., a Pseudomonas exotoxin gene PE38
  • an exogenous suicide gene can be used in combination with other known treatments to prevent or treat disease in a subject or organism.
  • the described molecules could be used in combination with one or more known compounds, treatments, or procedures to prevent or treat neoplastic disease in a subject or organism as are known in the art.
  • one or more infectious particles comprising a papovavirus capsid protein and an exogenous bacterial toxin gene, which allows expression of the exogenous bacterial toxin gene e.g., a Pseudomonas exotoxin gene PE38.
  • the infectious particle can contain sequence(s) encoding the bacterial toxin gene , a targeting element, or a fusion protein containing a targeting element and a bacterial toxin gene.
  • Non-limiting examples of such expression vectors include the PE38 gene construct under the CMV promoter, and the pEGFP-Cl construct (4.7Kb; Clontech, Palo Alto, CA). Hafkemeyer et al, Hum Gene Titer. 10:923-934, 1999.
  • “Expression cassette” and “expression vector” refer to nucleic acid constructs generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell.
  • the recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
  • the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.
  • expression cassette may be used interchangeably herein with "DNA construct” and its grammatical equivalents.
  • Vector and "cloning vector” refer to nucleic acid constructs designed to transfer nucleic acid sequences into cells.
  • Expression vector refers to a vector that has the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors is within the knowledge of those of skill in the art.
  • Plasmid refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in some eukaryotes or integrates into the host chromospmes.
  • “Expression” refers to the process by which a polypeptide is produced based on the nucleic acid sequence of the gene or the chemical synthetic peptide. The process includes both transcription and translation of the gene to produce polypeptide/protein.
  • Gene means the segment of DNA involved in producing a polypeptide chain that may or may not include regions preceding or following the coding region.
  • Nucleic acid molecule and “nucleic acid sequence” include sequences of any form of nucleic acid, including, but not limited to RNA, DNA and cDNA molecules. It will be understood that, as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding a given protein may be produced, in addition to mutant proteins.
  • Codon refers to a sequence of three nucleotides in a DNA or mRNA molecule that represents the instruction for incorporation of a specific amino acid into a polypeptide chain.
  • each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.
  • Treating" or “treatment” of neoplastic disease or infectious disease administering to a mammalian subject an infectious particle comprising a papovavirus capsid protein and an exogenous cytocidal gene, an exogenous bacterial toxin gene, e.g., a Pseudomonas exotoxin gene PE38, or an exogenous suicide gene includes preventing the onset of symptoms in a subject that may be at increased risk of disease or infection but does not yet experience or exhibit symptoms of disease or infection, inhibiting the symptoms of disease or infection (slowing or arresting its development), providing relief from the symptoms or side- effects of disease or infection (including palliative treatment), and relieving the symptoms of disease or infection (causing regression).
  • treating refers to any indicia of success in the treatment or amelioration or prevention of an neoplastic disease or infectious disease, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician.
  • treating includes the administration of the infectious particle compounds or agents to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with ocular disease.
  • therapeutic effect refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.
  • Dosage unit refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).
  • Disease-related genes and proteins are targets of a pharmaceutical composition which is an infectious particle comprising a papovavirus capsid protein and an exogenous cytocidal gene, an exogenous bacterial toxin gene, e.g., a Pseudomonas exotoxin gene PE38, or an exogenous suicide gene, in a mammalian subject in need of treatment for a neoplastic disease or an infectious disease.
  • the bacterial toxin gene can be a fusion protein of a Pseudomonas exotoxin gene PE38 and a targeting protein.
  • nucleic acids or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same ⁇ i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region) such as a nucleotide sequence encoding a protein of interest, such as an exogenous cytocidal gene, an exogenous bacterial toxin gene, e.g., a Pseudomonas exotoxin gene PE38, or an exogenous suicide gene, e.g., HSV TK gene, or an amino acid sequence of the exogenous cytocidal gene), when compared and aligned for maximum correspondence over a comparison window
  • exogenous cytocidal gene can be identified as having sequence identity (about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or higher identity over a specified region) to an exogenous cytocidal gene as described herein, such as an exogenous cytocidal gene, an exogenous bacterial toxin gene, e.g., a Pseudomonas exotoxin gene PE38, or an exogenous suicide gene, e.g., HSV TK gene.
  • sequences are then said to be “substantially identical.” This term also refers to, or can be applied to, the compliment of a test sequence.
  • sequences that have deletions and/or additions, as well as those that have substitutions can account for gaps and the like.
  • identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50- 100 amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman and Wunsch, J MoI. Biol.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et ah, supra).
  • These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • Polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -cafboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al, Molecular Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980).
  • Primary structure refers to the amino acid sequence of a particular peptide.
  • Secondary structure refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains.
  • Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include domains with enzymatic activity, e.g., a kinase domain. Typical domains are made up of sections of lesser organization such as stretches of jS-sheet and ⁇ -helices. "Tertiary structure” refers to the complete three dimensional structure of a polypeptide monomer. "Quaternary structure” refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.
  • a particular nucleic acid sequence also implicitly encompasses "splice variants.”
  • a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid.
  • "Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript can be spliced such that different (alternate) nucleic acid splice products encode different polypeptides.
  • Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read- through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition.
  • Recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • Stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-1O 0 C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 0 C, or, 5x SSC, 1% SDS, incubating at 65 0 C, with wash in 0.2x SSC, and 0.1% SDS at 65 0 C.
  • nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary "moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37 0 C, and a wash in IX SSC at 45 0 C. A positive hybridization is at least twice background.
  • Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., Ausubel et al, supra.
  • a temperature of about 36°C is typical for low stringency amplification, although annealing temperatures can vary between about 32°C and 48°C depending on primer length.
  • a temperature of about 62°C is typical, although high stringency annealing temperatures can range from about 50 0 C to about 65°C, depending on the primer length and specificity.
  • Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 9O 0 C - 95°C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72°C for 1 - 2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis ⁇ t al. PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N. Y. (1990).
  • Antibody refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.
  • “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • “Pharmaceutically acceptable salts and esters” means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed where acidic protons present in the compounds are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g. sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g. ethanolamine, diethanolamine, triethanolamine, tromethamine, N methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g.
  • esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the compounds, e.g. C 1-6 alkyl esters.
  • a pharmaceutically acceptable salt or ester can be a mono-acid-mono-salt or ester or a di-salt or ester; and similarly where there are more than two acidic groups present, some or all of such groups can be salified or esterified.
  • Compounds named in this invention can be present in unsalified or unesterified form, or in salified and/or esterified form, and the naming of such compounds is intended to include both the original (unsalified and unesterified) compound and its pharmaceutically acceptable salts and esters. Also, certain compounds named in this invention may be present in more than one stereoisomeric form, and the naming of such compounds is intended to include all single stereoisomers and all mixtures (whether racemic or otherwise) of such stereoisomers.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.
  • a “therapeutically effective amount” means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.
  • the terms “subject” or “patient” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Accordingly, the term “subject” or “patient” as used herein means any mammalian patient or subject to which the infectious particle compositions can be administered. In some embodiments of the present invention, the patient will be suffering from a condition that causes lowered resistance to disease, e.g., HIV.
  • a pharmaceutical composition comprising one or more infectious particles comprising a papovavirus capsid protein and an exogenous bacterial toxin gene, e.g., a Pseudomonas exotoxin gene PE38 to treat neoplastic disease according to the methods described herein
  • accepted screening methods are employed to determine the status of an existing disease or condition in a subject or risk factors associated with a targeted or suspected disease or condition.
  • Subject also refers to an organism, which is a donor or recipient of explanted cells or the cells themselves.
  • Subject also refers to an organism to which the infectious particle compositions can be administered.
  • a subject can be a mammal or mammalian cells, including a human or human cells.
  • Conscomitant administration of a known cancer therapeutic drug with a pharmaceutical composition means administration of the chemotherapeutic drug and the composition comprising an infectious particle comprising a papovavirus capsid protein and a cytocidal gene product, such as an exogenous bacterial toxin gene or an exogenous suicide gene, at such time that both the known chemotherapeutic drug and the composition will have a therapeutic effect.
  • Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the chemotherapeutic drug with respect to the administration of the infectious particle compound.
  • a person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention.
  • An infectious particle comprising a papovavirus capsid protein and a cytocidal gene product, such as an exogenous bacterial toxin gene or an exogenous suicide gene, can further comprise an antibody gene, e.g., a single chain Fv region, fused to the bacterial toxin gene, or carried within the infectious particle and expressed separately from a gene encoding an antibody.
  • An antibody-bacterial toxin gene fusion is useful to target the toxin protein to a neoplastic cell, tumor, or tumor vasculature.
  • Humanized antibody genes can be constructed by methods known to those of skill in the art.
  • either or both the heavy and light chain variable regions are produced by grafting the CDRs from the originating species into the hybrid framework regions.
  • Assembly of hybrid antibodies or hybrid antibody fragments having hybrid variable chain regions with regard to either of the above aspects can be accomplished using conventional methods known to those skilled in the art.
  • DNA sequences encoding the hybrid variable domains described herein i.e., frameworks based on the target species and CDRs from the originating species
  • the nucleic acid encoding CDR regions may also be isolated from the originating species antibodies using suitable restriction enzymes and ligated into the target species framework by ligating with suitable ligation enzymes.
  • suitable restriction enzymes e.g., restriction enzymes for ligating the target species framework
  • suitable ligation enzymes e.g., ligation enzymes for ligation the framework regions of the variable chains of the originating species antibody may be changed by site-directed mutagenesis.
  • hybrids are constructed from choices among multiple candidates corresponding to each framework region, there exist many combinations of sequences which are amenable to construction in accordance with the principles described herein. Accordingly, libraries of hybrids can be assembled having members with different combinations of individual framework regions. Such libraries can be electronic database collections of sequences or physical collections of hybrids.
  • oligonucleotides are designed to have overlapping regions so that they could anneal and be filled in by a polymerase, such as with " polyinerase cnain reactidn " (PCR). Multiple steps of overlap extension are performed in order to generate the VL and VH gene inserts. Those fragments are designed with regions of overlap with human constant domains so that they could be fused by overlap extension to produce full length light chains and Fd heavy chain fragments. The light and heavy Fd chain regions may be linked together by overlap extension to create a single Fab library insert to be cloned into a display vector.
  • PCR polyinerase cnain reactidn
  • the library may be assembled from overlapping oligonucleotides using a Ligase Chain Reaction (LCR) approach.
  • LCR Ligase Chain Reaction
  • variable genes can be cloned into a vector that contains, in-frame, the remaining portion of the necessary constant domain.
  • additional fragments that can be cloned include whole light chains, the Fd portion of heavy chains, or fragments that contain both light chain and heavy chain Fd coding sequence.
  • the antibody fragments used for humanization maybe single chain antibodies (scFv).
  • Any selection display system may be used in conjunction with a library according to the present disclosure.
  • Selection protocols for isolating desired members of large libraries are known in the art, as typified by phage display techniques.
  • Such systems in which diverse peptide sequences are displayed on the surface of filamentous bacteriophage have proven useful for creating libraries of antibody fragments (and the nucleotide sequences that encode them) for the in vitro selection and amplification of specific antibody fragments that bind a target antigen.
  • Scott et al Science 249:386, 1990.
  • the nucleotide sequences encoding the VH and V L regions are linked to gene fragments which encode leader signals that direct them to the periplasmic space of E.
  • phage-based display systems An advantage of phage-based display systems is that, because they are biological systems, selected library members can be amplified simply by growing the phage containing the selected library member in bacterial cells. Furthermore, since the nucleotide sequence that encode the polypeptide library member is contained on a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation is relatively straightforward.
  • Activators are agents that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize or up regulate the activity of disease-related genes, e.g., neoplastic disease or infectious disease.
  • Modulators include agents that, e.g., alter the interaction of disease-related genes with: proteins that bind activators or inhibitors, receptors, including proteins, peptides, lipids, carbohydrates, polysaccharides, or combinations of the above, e.g., lipoproteins, glycoproteins, and the like.
  • Modulators include genetically modified versions of naturally-occurring disease-related genes, e.g., with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • Such assays for inhibitors and activators include, e.g., applying putative modulator compounds to a cell expressing disease-related gene and then determining the functional effects on tumor metastasis, as described herein.
  • Samples or assays comprising a protein of interest that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) can be assigned a relative tumor cell growth activity value of 100%.
  • Inhibition of a disease-related gene is achieved when the neoplastic disease-related cell or infectious-related disease cell growth activity value relative to the control is about 80%, optionally 50% or 25-0%.
  • Activation of disease-related gene is achieved when the tumor cell growth activity value relative to the control is 110% ' , optionally 150%, optionally 200-500%, or 1000-3000% higher.
  • Modulate indicates that the expression of the target gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator.
  • modulate can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.
  • “Inhibit”, “down-regulate”, or “reduce” refers to that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the exogenous bacterial toxin or exogenous bacterial toxin gene.
  • inhibition, down-regulation or reduction with exogenous bacterial toxin molecule is below that level observed in the presence of an inactive or attenuated molecule.
  • inhibition, down-regulation, or reduction with exogenous bacterial toxin molecules is below that level observed in the presence of, for example, a control or placebo treatment.
  • inhibition, down-regulation, or reduction of gene expression with an infectious particle comprising a papovavirus capsid protein and an exogenous bacterial toxin gene e.g., a Pseudomonas exotoxin gene PE38
  • inhibition, down regulation, or reduction of gene expression is associated with inhibition of translation by the exogenous bacterial toxin gene, e.g., the Pseudomonas exotoxin gene PE38.
  • inhibition, down regulation, or reduction of gene expression is associated with inhibition of translation in the target cell or neoplastic cell.
  • Inhibitors “Inhibitors,” “activators,” and “modulators” of gene expression are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for disease related genes that are affected by exogenous bacterial toxin or exogenous bacterial toxin gene expression.
  • a composition comprising an infectious particle comprising a papovavirus capsid protein and a cytocidal gene product, such as an exogenous bacterial toxin gene or an exogenous suicide gene or in a method for killing cells in a mammalian subject or a method for treating neoplastic disease in a mammalian subject, comprising administering a bacterial toxin gene, e.g., a Pseudomonas exotoxin gene PE38, or administering a bacterial toxin gene, e.g., a Pseudomonas exotoxin gene PE38, in combination with a chemotherapeutic agent, is useful in the present compositions and methods.
  • a bacterial toxin gene e.g., a Pseudomonas exotoxin gene PE38
  • a bacterial toxin gene e.g., a Pseudomonas exotoxin gene PE38
  • compositions and methods can be administered to a human patient per se, in the form of a stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N-oxide or isomorphic crystalline form thereof, or in the form of a pharmaceutical composition where the compound is mixed with suitable carriers or excipient(s) in a therapeutically effective amount, for example, to treat neoplastic disease or infectious disease.
  • compositions for administering the infectious particle compositions (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18 th ed., 1990, incorporated herein by reference).
  • the pharmaceutical compositions generally comprise an infectious particle comprising a papovavirus capsid protein and a cytocidal gene product, such as an exogenous bacterial toxin gene or an exogenous suicide gene, in a form suitable for administration to a patient.
  • the pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • the particular label or detectable group used in the assay is not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the bacterial toxin gene, e.g., a Pseudomonas exotoxin gene PE38, used in the assay.
  • the detectable group can be any material having a detectable physical or chemical property.
  • detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels include, but are not limited to, magnetic beads ⁇ e.g.
  • DynabeadsTM fluorescent dyes ⁇ e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels ⁇ e.g., 3 H 3 14 C, 35 S, 125 1, 121 1, 112 In, "mTc), other imaging agents such as microbubbles (for ultrasound imaging), 18 F, 11 C, 15 O, (for Positron emission tomography), 99m TC, 111 In (for Single photon emission tomography), enzymes ⁇ e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic ⁇ e.g.
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the ait. As indicated above, a wide variety of labels maybe used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to an anti- ligand ⁇ e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a signal system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a number of ligands and anti-ligands can be used. Where a ligand has a natural anti- ligand, for example, biotin, thyroxine, and Cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an infectious particle composition.
  • the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like
  • Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
  • Means of detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter or photographic film as in autoradiography.
  • the label is a fluorescent label, it may be detected by exciting the fluoro chrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • CCDs charge coupled devices
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple calorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
  • agglutination assays can be used to detect the presence of the target antibodies.
  • antigen-coated particles are agglutinated by samples comprising the target antibodies.
  • none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
  • the bacterial toxin gene e.g. , a Pseudomonas exotoxin gene PE38
  • the bacterial toxin gene will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • compositions comprising one or a combination of an infectious particle comprising a papovavirus capsid protein and a cytocidal gene product, such as an exogenous bacterial toxin gene, e.g., a
  • compositions include a combination of multiple (e.g., two or more) infectious particles. In some compositions, each of the infectious particles of the composition binds to a distinct, pre-selected region of a target nucleic acid.
  • compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of a disease or condition (e.g., neoplastic disease or infectious disease) in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
  • a disease or condition e.g., neoplastic disease or infectious disease
  • compositions or medicants are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes in development of the disease.
  • An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose.
  • agents are usually administered in several dosages until a sufficient immune response has been achieved.
  • the effect of bacterial toxin gene e.g., a Pseudomonas exotoxin gene PE38, alone or in combination with a chemotherapeutic agent, on disease cells or on levels of a disease related polypeptide is monitored and repeated dosages are given if the levels of a disease related polypeptide starts to increase.
  • Effective doses of an infectious particle comprising a papovavirus capsid protein and a cytocidal gene product, such as an exogenous bacterial toxin gene, e.g., a Pseudomonas exotoxin gene PE38, or an exogenous suicide gene, alone or in combination with a chemotherapeutic agent, for the treatment of neoplastic disease or infectious disease vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
  • the patient is a human but nonhuman mammals including transgenic mammals can also be treated. Treatment dosages need to be titrated to optimize safety and efficacy.
  • an infectious particle comprising a papovavirus capsid protein and an exogenous bacterial toxin gene, e.g., a Pseudomonas exotoxin gene PE38
  • the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.
  • dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.
  • An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months.
  • two or more infectious particles with different binding specificities are administered simultaneously, in which case the dosage of each infectious particle composition administered falls within the ranges indicated.
  • Infectious particle composition is usually administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of infectious particles in the patient. In some methods, dosage is adjusted to achieve a plasma infectious particle concentration of 1-1000 ⁇ g/ml and in some methods 25-300 ⁇ g/ml. Alternatively, infectious particle composition can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the infectious particle composition in the patient. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic, hi prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time.
  • Doses for infectious particles or nucleic acids derived therefrom can range from about 10 ng to 1 g, 100 ng to 100 mg, 1 ⁇ g to 10 mg, or 30-300 ⁇ g DNA per patient. Doses for infectious particles vary from 10-100, or more, virions per dose.
  • An infectious particle comprising a papovavirus capsid protein and a cytocidal gene product, such as an exogenous bacterial toxin gene, e.g., aPseudomonas exotoxin gene PE38, or an exogenous suicide gene, alone or in combination with a chemotherapeutic agent, for the treatment of neoplastic disease, e.g., adenocarcinoma, can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic as inhalants for infectious particle preparations targeting neoplastic disease or infectious disease, and/or other therapeutic treatment.
  • a cytocidal gene product such as an exogenous bacterial toxin gene, e.g., aPseudomonas exotoxin gene PE38, or an exogenous suicide gene, alone or in combination with a chemotherapeutic
  • infectious particle agent The most typical route of administration of an infectious particle agent is intraperitoneal although other routes can be equally effective.
  • the next most common route is intravenous, intratumoral, subcutaneous, or intramuscular injection.
  • Intramuscular injection is most typically performed in the arm or leg muscles.
  • agents are injected directly into a particular tissue where a tumor is found, for example intracranial injection or convection enhanced delivery.
  • Intramuscular injection or intravenous infusion are preferred for administration of infectious particle composition.
  • particular therapeutic infectious particle composition are delivered directly into the cranium.
  • infectious particle composition are administered as a sustained release composition or device, such as a MedipadTM device.
  • Infectious particles can optionally be administered in combination with other agents that are at least partly effective in treating various diseases including various immune-related diseases.
  • both primary and metastatic, infectious particle can also be administered in conjunction with other agents that increase passage of the infectious particle across the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • An infectious particle comprising a papovavirus capsid protein and a cytocidal gene product, such as an exogenous bacterial toxin gene, e.g., a Pseudomonas exotoxin gene PE38or an exogenous suicide gene, alone or in combination with a chemotherapeutic agent, for the treatment of neoplastic disease or infectious disease, are often administered as pharmaceutical compositions comprising an active therapeutic agent, i.e., and a variety of other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15 th ed., Mack Publishing Company, Easton, Pa., 1980). The preferred form depends on the intended mode of administration and therapeutic application.
  • compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • diluents are selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • the compounds employed in the methods of the present invention may exist in prodrug form.
  • Prodrug is intended to include any covalently bonded carriers which release the active parent drug, for example, Pseudomonas exotoxin gene PE38 alone or in combination with a chemotherapeutic agent, or other formulas or compounds employed in the methods of the present invention in vivo when such prodrug is administered to a mammalian subject. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.) tiie compounds employed in the present methods may, if desired, be delivered in prodrug form. Thus, the present invention contemplates methods of delivering prodrugs.
  • Prodrugs of the compounds employed in the present invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound.
  • prodrugs include, for example, compounds described herein in which a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a mammalian subject, cleaves to form a free hydroxyl, free amino, or carboxylic acid, respectively.
  • Examples include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups; and alkyl, carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, propyl, is ⁇ -propyl, butyl, isobutyl, .sec-butyl, tert-bntyl, cyclopropyl, phenyl, benzyl, or phenethyl esters.
  • alkyl, carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, propyl, is ⁇ -propyl, butyl, isobutyl, .sec-butyl, tert-bntyl, cyclopropyl, phenyl, benzyl, or phenethyl esters.
  • Examples of prodrugs of Pseudomonas exotoxin gene PE38 alone or in combination with a chemotibierapeutic agent further include, but are not limited to, an amide derivative, thioamide derivative, carbamate derivative, thiocarbamate derivative, imide derivative, sulphonamide derivative, irm ' ne derivative, protonated imine derivative, isocyanate derivative, or isothiocyanate derivative of Pseudomonas exotoxin gene PE38 alone or in combination with a chemotherapeutic agent.
  • compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (i.e., adjuvants).
  • infectious particle compositions can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water oils, saline, glycerol, or ethanol.
  • a pharmaceutical carrier that can be a sterile liquid such as water oils, saline, glycerol, or ethanol.
  • auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions.
  • Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil.
  • glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • Infectious particle composition can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient.
  • An exemplary composition comprises infectious particles at 5 mg/mL, formulated in aqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted to pH 6.0 with HCl.
  • compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997.
  • the infectious particles can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.
  • binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.
  • Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%- 95% of active ingredient, preferably 25%-70%.
  • Topical application can result in transdermal or intradermal delivery.
  • administration can be facilitated by co-administration of the agent with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins. Glenn et ah, Nature 391:851, 1998.
  • Co-administration can be achieved by using the components as a mixture or as linked molecules obtained by chemical crosslinking or expression as a fusion protein.
  • transdermal delivery can be achieved using a skin patch or using transferosomes. Paul et ah, Eur. J. Immunol. 25:3521-24, 1995; Cevc et al., Biochem. Biophys. Acta 1368:201-15, 1998.
  • compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • a therapeutically effective dose of an infectious particle comprising a papovavirus capsid protein and a cytocidal gene product such as an exogenous bacterial toxin gene, e.g., a. Pseudomonas exotoxin gene PE38, or an exogenous suicide gene alone or in combination with a chemotherapeutic agent, described herein will provide therapeutic benefit without causing substantial toxicity.
  • a cytocidal gene product such as an exogenous bacterial toxin gene, e.g., a. Pseudomonas exotoxin gene PE38, or an exogenous suicide gene alone or in combination with a chemotherapeutic agent, described herein
  • Toxicity of the proteins described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 50 (the dose lethal to 50% of the population) or the LD 100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human.
  • the dosage of the proteins described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et ah, 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1,
  • kits comprising the compositions ⁇ e.g., an infectious particle comprising a papovavirus capsid protein and a cytocidal gene product, such as an exogenous bacterial toxin gene, e.g., a Pseudomonas exotoxin gene PE38 or an exogenous suicide gene, alone or in combination with a chemotherapeutic agent) and instructions for use.
  • the kit can further contain a least one additional reagent, or one or more additional infectious particle compositions (e.g., an infectious particle having a complementary activity which binds to a region of the target gene distinct from the first infectious particle).
  • Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
  • Figures Ia, Ib, Ic and Id show expression of PE38 packaged in vitro in four cell lines.
  • Cells were transduced with full reaction of PE38 plasmid DNA packaged in vitro (square/light magenta), and the number of cells was compared to mock transduced cells (diamond/ dark blue). Cells were counted every other day in order to monitor their viability.
  • Cell lines were: HeLa cells (a), KB-3-1 cells (b), K562 human erythroleukemia cells (c), and .45 human lymphoblastoid cells (d).
  • Figure 2 panels A-C clearly shows a major growth in tumor size 30 days after inoculation when PBS was used for treatment, while only very small tumors appeared in PE38 -treated mice. Tumor size and growth rate obtained when using a control consisting of empty SV40 pseudovirions were similar to those using PBS treatment.
  • the weight of the mice was measured every 4-6 days (Figure 2, panels D-F): both PE38-treated mice (Figure 2, panel D) and control-treated mice (Figure 2, panel E) gained weight.
  • Figure 2, panel F demonstrates the average weight of the groups shown in panels A and B with standard deviations. One of the mice died on day 25.
  • FIG. 1 Three of the PE38-treated mice (# 342, 343, 345) were followed more than a year and half after the 30 days of PE38-m vitro packaged treatment. No tumors appeared and the mice continued to grow and gain weight. The results of pathology tests were the same for tissues from PE38-treated mice as for control, PBS- treated animals. Blood tests for both groups were within the normal range. " [0124] Figures 2a72b, 2c, 2d, 2e and 2f show tumor size and weight of PE38 packaged in vitro and control mice treated from the day of tumor challenge. Mice were injected with 2X10 6 KB-3-1 cells along with the treatment. Tumor size as well as mouse weight were followed for 30 days.
  • IVP-PE with 200 or 300 ⁇ l IP 4-5 times a week
  • IVP-EGFP 200 or 300 ⁇ l IP 4-5 times a week a The amounts varied from experiment to experiment and are specified in the Results section.
  • SC subcutaneous
  • IP intraperitoneal
  • the amount is based on 1% packaging efficiency estimation d, Combined treatment
  • Figures 3a, 3b, 3c, 3d, 3e and 3f show tumor size and weight of PE38 packaged in vitro and control mice treated several days post tumor challenge via SC. Treatment was followed six days after mice were injected with 2 X 10 6 KB-3-1 cells. Tumor size as well as mouse weight were followed for 41 days.
  • Figure 3 (a-c) demonstrates tumor size in mm 3 of empty vector-treated mice (panel a) or IVP-PE38 (panel b) (five mice in each group, each line represents one mouse), and panel c is the average of each five mice (PVP-PE is shown in diamond symbol/ dark blue, and control-treated mice in square symbol/ light magenta) with standard deviation.
  • Figure 3 (d-f) demonstrates the weight in grams for these groups in this order (panels d, e, and f respectively).
  • IP Intraperitoneal
  • mice [0127] 2x10 6 KB-3-1 cells were injected subcutaneously into mice. Three to seven days after inoculation, five to ten mice were injected intraperitoneally with 200 ⁇ l PBS 4-5 times a week (Table 1, line X), and five to ten mice were injected intraperitoneally with 200 ⁇ l IVP- PE38 in vitro vectors intraperitoneally (Table 1, line 8). Weight and tumor size were measured every 4-5 days.
  • mice treated with PBS and mice treated with EGFP Five tissues were examined from mice treated with PBS and mice treated with EGFP: kidney, heart, liver, spleen and tumor. All tissues from GFP -treated mice expressed high fluorescence as compared to tissues from PBS-treated mice ( Figure 5), except kidney tissue, which expressed similar high background fluorescence in both PBS and EGFP mice. The difference in fluorescence intensity between tumors of mice treated with EGFP to those treated with PBS is the most obvious. When the delivery of PE38 by SV40 started later than 7 days after inoculation, and tumor size exceeded 150 mm 3 on average, it did not significantly decrease tumor size. Moreover, pathology and blood tests were done on animals that received IP treatment, and all results were within normal ranges.
  • Figures 4a, 4b, 4c, 4d, 4e and 4f show tumor size and weight of PE38 packaged in vitro and control mice treated intraperitoneally several days post tumor challenge. Treatment was followed three days after mice were inoculated with 2 X 10 6 KB-3-1 cells. Tumor size as well as mouse weight were followed for 34 days.
  • Figure 5 shows tissue fluorescence of EGFP packaged in vitro and control mice treated intraperitoneally several days post tumor challenge. Treatment was followed three days after mice were inoculated with 2 X 10 6 KB-3-1 cells. Tumor size as well as mouse weight were followed for 30 days.
  • Figure 5 shows five different tissues (from the top) kidney, heart, liver, spleen and tumor of IVP-EGFP (left) and PBS-treated mice (right). Figures were taken at 400X magnification.
  • the following four controls were added to the experiment: cells only, cells transduced with in vz ⁇ ro-packaged PE38, cells treated with 6 ng/ml doxorubicin only, and cells treated with 12 ng/ml doxorubicin only. Cell counts were performed every 2-3 days. The viability of cells in the combined treatment was higher than in doxorubicin alone, but lower than tihe viability of cells transduced with PE38 ( Figure 6). The experiment was repeated three times with similar results.
  • Figure 6 shows viability of KB-3-1 cells treated in tissue culture with PE38 packaged in vitro and doxorubicin. 10 5 KB-3-1 cells were transduced with PE38 plasmid DNA packaged in vitro (square symbol/ light magenta line), and the number of cells was compared to mock transduced cells (diamond symbol/ dark blue line).
  • Figures 7a and 7b show tumor size and weight of mice treated with a combination of PE38 packaged in vitro and doxorubicin via IP. Treatment was followed three days after inoculation of mice with 2 X 10 6 KB-3-1 cells. Tumor size as well as the weight of the mice were followed for 56 days.
  • Figure 7a demonstrates average tumor size in mm of IVP- PE-treated mice (diamond symbol/ dark blue line), doxorubicin-treated mice (square symbol/ light magenta line), arid the combined treatment of IVP-PE38 with 5 mg/kg doxorubicin-treated mice (triangle symbol/ green line) (five mice in each group).
  • PE Pseudomonas exotoxin
  • Pastan Cancer Immunol Immunother. 52:338-341, 2003; Fitzgerald and Pastan, Ann N Y Acad Sd. 685:740-745, 1993; Chaudhary et al., J Biol Chem. 265:16306-16310, 1990; FitzGerald et al, Int J Med Microbiol. 293:577-582, 2004; Kreitman et al, Blood. 83:426-434, 1994; Kreitman et al, Proc Natl Acad Sd USA.
  • mice did not lose weight and serological parameters were very similar to those of control-injected mice, hi the present study, intraperitoneal injection of an infectious particle comprising a papovavirus capsid protein and an exogenous bacterial toxin gene into nude mice with adenocarcinomas also results in significant reduction of tumor size.
  • This delivery mode has not previously been used with SV40 vectors. Animals receiving the treatment did not show any abnormal pathological manifestations as compared to control treated animals.
  • the Pseudomonas exotoxin delivered in vitro using the infectious particle comprising a papovavirus capsid protein and an exogenous bacterial toxin gene very efficiently eradicated various types of cell lines, adherent cells (KB-3-1 cells and HeLa cells) and cells in suspension (K562, human erythroleukemia cells, and .45 human lymphoblastoid cells).
  • adherent cells KB-3-1 cells and HeLa cells
  • cells in suspension K562, human erythroleukemia cells, and .45 human lymphoblastoid cells.
  • the efficiency of delivery was very similar between the different cell lines, which indicates that the PE protein has a similar toxicity in cells of various origins.
  • the plasmid DNA contains the PE protein C-terminus domain 3, the catalytic domain, which is responsible for the inactivation of eukarytic elongation factor 2 (eEF2) by catalyzing the ADP-ribosylation of this target protein once in the cytoplasm, thus inducing cell death.
  • eEF2 eukarytic elongation factor 2
  • Bacterial toxins which have been targeted to cancer cells include Pseudomonas exotoxin, ricin, and diphtheria toxin. These can be designed to form recombinant fusion toxins, and delivered by injection of the fused protein or by gene delivery using adenoviral, retroviral and liposome delivery systems. Vallera et al, Hum Gene Ther. 14:1787-1798, 2003; Jain, Expert Opin Biol Ther. 1:291-300, 2001. Therefore, the majority of the research on lethal gene delivery has been done on fused-PE proteins. TGF- ⁇ fused with PE 5 for example, was delivered to bladder carcinoma. Theuer et al., J Urol. 149:1626-1632, 1993.
  • IL13-PE a chimeric protein comprised of human IL- 13 and a truncated version of an exotoxin from Pseudomonas
  • IL13-PE selectively targeted human pulmonary fibroblasts grown from IIP SLBs, and had a minimal effect on fibroblasts grown from biopsies from normal patients.
  • GFP expressing cells in tumors from IP treated mice It is hypothesized that the SV40 pseudovirions could circulate in the blood, but probably because of the proliferation of blood vessels in the tumors they are absorbed by the tumors. Probably the PE38 is absorbed even more than EGFP by the tumor, because otherwise, tissue abnormalities in the PE-treated mice would have been seen.
  • mice do not exhibit any adverse effects from prolonged 3-5 times a week in vivo administration of the pseudovirions during tumor treatment, which indicates that the capsid proteins are also harmless.
  • SV40 in vitro packaging delivery system One of the main advantages of the SV40 in vitro packaging delivery system is its high efficiency. However, following a detailed examination of the pathway of the SV40 pseudovirus particles in human lymphoblastoid cells (which were tested among the four cell lines for cell viability), it is suggested that the low expression provided by the delivery system might be also due to the DNA trapped in the cytoplasm. Moreover, expression levels of toxin in cancer cells must be very low since even one molecule in the cytosol may lead to cell death, and these levels could not be quantified. Therefore, the combination of a delivery system that is highly efficient, although with a low-level of expression, and a very potent toxin results in high levels of cell death.
  • a combined treatment of doxorubicin and infectious particles comprising in vitro packaged PE38 to cultured cells resulted in a major arrest of cell proliferation, but cell numbers were higher than when each of the treatments were applied separately.
  • doxorubicin acts on DNA synthesis
  • PE inhibits protein synthesis and induces caspase-dependent programmed cell death. This partially antagonistic effect could result from the need for protein synthesis as part of the doxorubicin killing process.
  • Cogan and Koch J Med Chem. 47:5690- 5699, 2004.
  • the present study demonstrated successful gene delivery of Pseudomonas exotoxin using SV40 pseudovirions in mice that actually continue to gain weight.
  • Treating the same adenocarcinoma tumors using chemotherapy resulted in less tumor growth, but in significant weight loss.
  • the combined treatment in vivo resulted in only slightly less tumor growth than when each of the treatments was applied separately. However, the mice did not lose weight.
  • GFP was expressed in normal tissues to a lesser degree, which might suggest that PE targets normal cells as well. It is possible that through this pathway, the pathway of toxicity of doxorubicin is changed which reduces its cytotoxic activity.
  • the present study establishes a new method of delivery of a lethal gene into cells in vitro and in vivo.
  • the present study demonstrates efficient delivery of the truncated Pseudomonas exotoxin gene to dramatically reduce the size of human adenocarcinomas using SV40 pseudovirions. Delivery is very effective either by direct injection into the tumor or intraperitoneally. The method presented here could improve chemotherapy by reducing tumor size with less-pronounced side effects.
  • the nuclei were then extracted by buffering the cell culture in 20 mM Hepes pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, ImM DTT and a protease inhibitor cocktail tablet (Roche). DTT, PMSF and the protease inhibitor cocktail were added to the buffer immediately before use.
  • DTT, PMSF and the protease inhibitor cocktail were added to the buffer immediately before use.
  • Nuclear extract concentration was measured using BCA Protein Assay protocol (Pierce, Rockford, Illinois). The nuclear extract was kept for more than a year at -2O 0 C.
  • KB-3-1 cell line derived from human HeLa epidermal carcinoma cells, and the HeLa cells were maintained in DMEM media (Dulbecco's modified Eagle medium, Invitrogen, Carlsbad, CA) with 10% fetal bovine serum, 50 ⁇ g /ml penicillin, 50 ⁇ g/ml streptomycin and 5 mM L-glutamine (all obtained from Invitrogen, Carlsbad, CA).
  • DMEM media Dulbecco's modified Eagle medium, Invitrogen, Carlsbad, CA
  • 10% fetal bovine serum 50 ⁇ g /ml penicillin, 50 ⁇ g/ml streptomycin and 5 mM L-glutamine (all obtained from Invitrogen, Carlsbad, CA).
  • Akiyama et al Somat Cell MoI Genet. 11:117-126, 1985. Cells were incubated at 37 0 C, in 5% CO 2 . Splitting of cells was done using 0.25% trypsin-EDTA (
  • a reaction of in vzYro-packaged SV40 vectors carrying the PE38 plasmid DNA or empty SV40 vectors without DNA as a control were suspended in 340 ⁇ L of DMEM, supplemented with 5 mM L-glutamine, 50 ⁇ g /ml penicillin and 50 ⁇ g /ml streptomycin. Cells were transduced and placed on a rotary shaker at 30 rpm for 2.5 hours (at 37°C in 5% CO 2 ). Post-infection, the dishes were supplemented with 4 ml of growth medium. Every two days the cells were counted and replated in new T-25 flasks. Fresh media was added to bring the total to 5 ml.
  • mice were supplied by the National Cancer Institute's Fredrick Animal Facility and housed under standard conditions based on the guidelines of the NIH Office of Animal Care and Use. The conditions in mouse cages were uniform concerning feed and water.
  • all of the mice grew subcutaneous adenocarcinomas.
  • mice From zero to fifteen days subsequent to cell injection, mice were divided into equal groups, so that each group was comprised of 5-10 mice.
  • the treatments are shown in Table 1. The animals were regularly monitored for changes in weight and tumor size.
  • Palpable tumors were measured with calipers, and the rumor diameter in two orthogonal dimensions was measured every four days over a period of several months. Tumor size (in mm 3 ) was calculated for each mouse by multiplying the two measured dimensions together and then multiplying by the smaller dimension. Mice were inspected every day for the condition of their health and behavior. Mouse mortality in both experimental and control cages was similar. Tumors were allowed to grow until they reached approximately 2 cm in diameter. At this endpoint, the mice were euthanized according to Standard Operating Procedures.
  • Serum analyses were performed within 2 hours of blood collection for albumin, amylase, blood urea nitrogen, calcium, glucose, inorganic phosphorus, total protein, alkaline phosphatase, ⁇ -glutamyl transferase, lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, and total bilirubin.
  • GFP expression some tissues were harvested, and frozen, and frozen slides were screened in Leica fluorescent microscope.
  • Serological studies, including liver function tests, were performed on both control and treated mice. These experiments were conducted under approved NCI animal protocol LCB-004.

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Abstract

La présente invention concerne une composition de particules infectieuses comprenant une protéine capside du papovavirus et un gène cytocide exogène. Cette invention concerne également des méthodes thérapeutiques ainsi que des méthodes permettant d'utiliser les particules infectieuses.
PCT/US2006/046145 2005-12-01 2006-12-01 Composition de particules infectieuse comprenant un gene cytocide et methodes d'utilisation correspondantes Ceased WO2007064971A2 (fr)

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US60/741,831 2005-12-01

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WO2007064971A2 true WO2007064971A2 (fr) 2007-06-07
WO2007064971A3 WO2007064971A3 (fr) 2007-11-15

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2036980A1 (fr) * 2007-09-14 2009-03-18 Gruber, Jens Dérégulation de l'expression génétique à l'aide de particules semblables à un virus chargées d'acide nucléique

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69734829D1 (de) * 1996-07-17 2006-01-12 Us Health Infektiöse pseudovirale papillomavirus partikel
WO1999027123A2 (fr) * 1997-11-26 1999-06-03 Board Of Regents, The University Of Texas System Vecteurs viraux sv40 modifies

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2036980A1 (fr) * 2007-09-14 2009-03-18 Gruber, Jens Dérégulation de l'expression génétique à l'aide de particules semblables à un virus chargées d'acide nucléique
WO2009036933A3 (fr) * 2007-09-14 2009-08-13 Jens Gruber Régulation à la baisse de l'expression génique à l'aide de particules pseudovirales chargées d'acide nucléique
JP2010538625A (ja) * 2007-09-14 2010-12-16 グルベル，ジェンス 核酸担持ウイルス様粒子を用いた遺伝子発現の下方制御
US8729038B2 (en) 2007-09-14 2014-05-20 Jens Gruber Down regulation of the gene expression by means of nucleic acid-loaded virus-like particles
US9951329B2 (en) 2007-09-14 2018-04-24 Gabriele Jansen Down regulation of the gene expression by means of nucleic acid-loaded virus-like particles

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