WO2008095168A2 - Compositions et procédés apparentés à un vecteur adénoviral recombinant qui cible les récepteurs de l'il13 - Google Patents

Compositions et procédés apparentés à un vecteur adénoviral recombinant qui cible les récepteurs de l'il13 Download PDF

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WO2008095168A2
WO2008095168A2 PCT/US2008/052803 US2008052803W WO2008095168A2 WO 2008095168 A2 WO2008095168 A2 WO 2008095168A2 US 2008052803 W US2008052803 W US 2008052803W WO 2008095168 A2 WO2008095168 A2 WO 2008095168A2
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adenovirus
cell
segment
cells
cancer
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Llya V. Ulasoy
Maciej S. Lesniak
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University Of Chicago
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10345—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/852—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from cytokines; from lymphokines; from interferons

Definitions

  • the invention generally relates to the field of oncology and therapeutic adenoviruses. More particularly, it concerns compositions and methods of treating cancer using targeted adenoviruses.
  • Ad5 Ad5's primary receptor, or CAR
  • an RGD motif retargets the adenovirus to integrins ( ⁇ v ⁇ 3 , ⁇ v ⁇ s) which are over-expressed on the cell surface of gliomas (Koizumi et al, 2001; Mizuguchi et al, 2001 ; Lamfers et al, 2002; Fueyo et al, 2003; Tyler et al, 2006).
  • Another ligand which has shown enhanced gene transfer to glioma is polylysine motif incorporation into the carboxyl terminus of the knob domain, which has been shown to target the angiogenic heparan sulfate proteoglycans that are over- expressed in blood vessel endothelial cells of glioma (Staba et al, 2000).
  • Other fiber modifications involve the incorporation of specific ligands which possess high affinities for growth factor receptors (Dmitri ev et al, 2000; Gaden et al, 2004; Perera et al, 2005).
  • the adenoviral fiber protein may contain a T4 phage fibritin shaft (designated LU- 13 adenovirus).
  • a fibritin protein is provided as SEQ ID NO:9.
  • the fibritin shaft may include a 100, 150, 200, 250, 300, 350, 400, 450 or 487 amino acid segment of a fibritin polypeptide, including all values, integers and ranges there between.
  • a fibritin polypeptide or segment is at least, at most, or about 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical in amino acid sequence to all or a segment of SEQ ID NO:9, including all values and ranges there between.
  • Embodiments of the invention include modified adenoviral fiber proteins that bind IL13 ⁇ 2 receptors.
  • the fiber protein comprises an IL-13 ⁇ 2 receptor binding segment.
  • the fiber protein comprises in an amino terminal to carboxy terminal direction an adenoviral tail segment (typically comprising the first 30 amino acids of an adenoviral fiber protein), a shaft segment, and an IL-13 ⁇ 2 receptor binding segment.
  • the shaft segment can be a heterologous shaft segment, for example a fibritin shaft segment, or an adenoviral shaft segment.
  • the IL-13 ⁇ 2 receptor binding segment can comprise an amino acid segment of an IL- 13 polypeptide.
  • an IL- 13 polypeptide has a segment that is at least, at most, or about 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical in amino acid sequence to SEQ ID NO: 2 or SEQ ID NO:4, including all values and ranges there between.
  • the IL-13 ⁇ 2 receptor binding segment comprises at least 25, 30, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 114 contiguous amino acids of SEQ ID NO:2 or SEQ ID NO:4, including all values and ranges there between.
  • the IL-13 ⁇ 2 receptor binding segment can comprise the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
  • an IL- 13 polypeptide having a segment that is at least, at most, or about 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical in amino acid sequence to SEQ ID NO: 2 or SEQ ID NO:4 can comprise a lysine at amino acid 13, an aspartic acid at amino acid 66, an aspartic acid at amino acid 69, an arginine at amino acid 105, a lysine at amino acid 109 or various combinations thereof (Madhankumar et al, 2004, which is incorporated herein by reference in its entirety).
  • the adenoviral tail segment is an Ad5 tail segment.
  • the adenoviral tail segment is at least, or at most, or about the first 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids of an adenoviral fiber protein, an example of which can be found in GenBank accession number AP_000226.
  • an adenoviral fiber protein of the invention can be operatively coupled to an adenoviral particle, e.g., by its tail domain or other covalent or non-covalent attachment.
  • Other embodiments of the invention include polynucleotides encoding the adenoviral fiber protein of the invention.
  • the fiber protein comprises in an amino terminal to carboxy terminal direction an adenoviral tail segment, a shaft segment, and an IL-13 oc2 receptor binding segment.
  • Still other embodiments of the invention include a recombinant viral genome comprising a nucleic acid segment encoding an adenoviral fiber protein of the invention.
  • the fiber protein comprises in an amino terminal to carboxy terminal direction an adenoviral tail segment, a shaft segment, and an IL- 13 ⁇ 2 receptor binding segment.
  • Embodiments of the invention include an adenoviral particle comprising a nucleic acid segment encoding an adenoviral fiber protein of the invention.
  • the fiber protein comprises in an amino terminal to carboxy terminal direction an adenoviral tail segment, a shaft segment, and an IL-13 ⁇ 2 receptor binding segment.
  • the adenovirus is a replication competent or replication defective adenovirus.
  • the adenovirus replicates in a cancer cell (i.e., it is an oncolytic adenovirus) and in certain embodiments selectively (replication in the cancer cell is greater than replication in other non-cancer cells of a subject) or specifically (replication is primarily in the cancer cell with little detection (i.e., not significantly above background) of replication in other non-cancer cells of a subject) in a cancer cell.
  • a cancer cell includes, but is not limited to a neuronal, a glial, a bladder, a blood, a bone, a bone marrow, a brain, a breast, a colon, an esophageal, a gastrointestinal, a gum, a head, a kidney, a liver, a lung, a nasopharyngeal, a neck, an ovarian, a pancreatic, a prostate, a skin, a stomach, a testis, a tongue, or a uterus cancer cell.
  • the cancer or cancer cell is an epithelial ovarian cancer, Kaposi's sarcoma, head and neck cancer, or renal cell carcinoma.
  • the cancer is neuronal cancer or a glioma.
  • the adenovirus preferentially replicates in primary and/or metastatic cancer cells.
  • a further embodiment of the invention includes methods of infecting an IL-13 ⁇ 2 receptor expressing cell comprising contacting the IL-13 ⁇ 2 receptor expressing cell with an adenovirus vector having a fiber protein comprising an IL- 13 ⁇ 2 receptor binding region.
  • Still further embodiments of the invention include methods of treating cancer in a patient comprising contacting a cancer cell with an adenovirus having a recombinant fiber protein that binds an IL-13 ⁇ 2 receptor.
  • the cancer cell or subject will be contacted or administered an amount of adenovirus sufficient to result in a therapeutic effect, e.g., slowing of cancer cell growth, cessation of cell growth, or lysis of cancer cells, where some or all the cancer cells contacted undergo these processes.
  • a cancer cell can include, but is not limited to a neuronal, a glial, a bladder, a blood, a bone, a bone marrow, a brain, a breast, a colon, an esophageal, a gastrointestinal, a gum, a head, a kidney, a liver, a lung, a nasopharyngeal, a neck, an ovarian, a pancreatic, a prostate, a skin, a stomach, a testis, a tongue, or a uterus cancer cell.
  • the cancer or cancer cell is an epithelial ovarian cancer, Kaposi's sarcoma, head and neck cancer, or renal cell carcinoma.
  • the cancer is neuronal cancer or a glioma.
  • the adenovirus preferentially replicates in primary and/or metastatic cancer cells.
  • the method can further comprise determining whether the cancer cell expresses an IL- 13 ⁇ 2 receptor.
  • IL13 ⁇ 2 receptor expression can be assessed by either nucleic acid detection or protein detection.
  • IL-13 ⁇ 2 receptor expression can be determined by using an IL-13 ⁇ 2 receptor antibody.
  • the adenovirus can be directly injected into a tumor or administered intravenously, intraperitoneally or by other well know routes described in detail below.
  • the adenovirus may comprise a heterologous expression cassette.
  • the heterologous expression cassette can encode a therapeutic protein or comprise a therapeutic polynucleotide.
  • the heterologous expression encodes a reporter polypeptide.
  • the heterologous expression cassette encodes an enzyme to convert a pro-drug into an active chemotherapy drug.
  • a therapeutic polynucleotide can encode a tumor suppressor or apoptosis inducing protein.
  • Embodiments of the invention may also include administering to the patient or subject a second therapy, wherein the second therapy is chemotherapy, immunotherapy, surgery, radiotherapy, or immunosuppressive agents.
  • the second therapy is typically administered in amount sufficient to result in a therapeutic result.
  • chemotherapy can comprise an alkylating agent, mitotic inhibitor, antibiotic, or antimetabolite.
  • the chemotherapy comprises CPT-11, temozolomide, or a platin compound.
  • the methods can also include radiotherapy, for example X-ray irradiation, UV-irradiation, ⁇ -irradiation, proton beam radiation, or microwaves.
  • At least, at most or about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 , to 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10", 10 12 , 10 13 , 10 14 , or 10 15 viral particles are administered to the patient, including all values and ranges there between.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • FIGs. 1A-1B Schematic representations of an example of an IL- 13R targeting moiety (LU-13) (FIG. IA) and an example of a recombinant viral genome encoding an IL- 13R targeting moiety (FIG. IB).
  • FIG. 2 Validation of protein expression via Western blot analysis.
  • HEK293 cells (2 x 10 5 ) were plated the day before infection. Cells were then infected with LU- 13 or AdWT vectors at 1000 vp/cell. Controls were also mock infected. After 1 hr adsorption, media containing remnant viral particles was replaced with cell growing media and incubated for 48 hrs. Cells were then rinsed with PBS, trypsinized, sonicated and 50 ⁇ g/lane of cell lysates were electrophoresed into sodium dodecyl sulfate-polyacrylamide gel and subsequently transferred to a PVDF membrane.
  • the blots were probed with anti-human adenovirus fiber 5 antibodies (4D2) or anti-human IL- 13 proteins and then with a peroxidase-conjugated goat anti- mouse or goat anti-rabbit secondary antibody. Membranes were developed with enhanced-chemiluminescence reagent.
  • the gel is representative of at least two independent experiments from infections made at different time points. 4D2 antibodies cross-react with AdWT and LU- 13 chimera fibers.
  • AdWT fiber monomer size is a 65 kDa; LU- 13 monomer size is approximately 43 kDa. Lysates were boiled (+) or left unboiled (-) prior to electrophoresis to reveal monomelic (Fi) and trimeric (F 3 ) fibers.
  • FIG. 3 TRITC-ILl 3 staining of paraformaldehyde-f ⁇ xed human glioma cells.
  • Primary glioma cells were prepared and infected as described below. Cells were analyzed at x63 magnification by laser scanning confocal microcopy. A diffusely punctate distribution of LU-13 and ILl 3 ⁇ 2 is seen after 1 hour binding of LU- 13 vector to the target cell surfaces.
  • An overlay of the fluorescence channels yield a yellow fluorescence where Cy5-IL13 and TRITC-ILl 3 ⁇ 2 co-localize.
  • AdWT did not show a co-localization signal. Indirect fluorescence shows that LU- 13 virus binds to target glioma membrane through IL13 ⁇ 2 receptor.
  • FIGs. 4A-4B 3D image of ILl 3 -ILl 3 ⁇ 2 receptor interaction.
  • An immunofluorescence image was taken from LU- 13 infected cells.
  • Twenty-three optical 0.5 micron sections (FIG. 4A) were used to reconstitute the 3D image (FIG. 4B) using Leica 3D software.
  • FIGs. 5A-5B Transduction of human glioma cell lines (FIG. 5A) and primary cultures (FIG. 5B) by AdWT and LU-13 luciferase expressing vectors. Kings, U373MG, and U87MG were infected with AdWT and LU- 13 vectors in 24 well plates. After a 24 hr adsorption period, virus-containing media was replaced with cell growing media supplemented with 10% FBS. Twenty four hours later transgene expression was determined as relative luciferase units calculated per mg proteins. Each value was obtained from two independent experiments in triplicates. LU- 13 vector demonstrates significantly higher level of transgene expression in both passaged and primary tumor cells (p ⁇ 0.05).
  • FIGs. 6A-6B Role of ILl 3 ⁇ 2 receptor in transduction of glioma cells.
  • FIG. 6A Kings and U87MG were incubated with 1000 MOI per cell with LU- 13 or AdWT for 1 hr at +4°C. After adsorption, unbounded virus was removed, cells were rinsed with PBS three times, trypsinized, and total DNA was isolated and quantitated as described below. The results represent the mean (E4 copy number per ng DNA) ⁇ SD of duplicate data from 2 independent experiments.
  • FIG. 6B Inhibition of LU- 13 infectivity with anti-IL13 monoclonal antibodies was measured based on decreasing levels of transduction before and after incubation with antibodies.
  • the luciferase levels measured are expressed as % inhibition of infection, which represents a ratio of luciferase levels in lysed cells blocked with antibody to luciferase levels in untreated cells.
  • the experiment was performed twice in quadruplicates. Mean ⁇ the SD are shown. Statistically significant differences between treated and untreated cells are indicated; p ⁇ 0.05.
  • FIGs. 7A-7B IL- 13 -mediated adenoviral gene expression in U87MG- glioma established xenografts (FIG. 7A).
  • expression of reporter gene product (luciferase) was determined forty- eight hours later for each mouse per each group.
  • LU- 13 exhibited superior activity over AdWT.
  • FIG. 7B To support luciferase data, 5-mm slices were stained for hexon expression (FIG. 7B).
  • LU- 13 exhibited greater level of hexon expression vs. AdWt (x 40 magnifications).
  • IL13 ⁇ 2R appears to be a good target for cancer therapy, particularly glioma therapy.
  • IL13 ⁇ 2R is expressed on malignant glioma and not on normal, healthy astrocytes (Debinski et al, 1999a; Debinski et al, 1999b; Oshima and Puri, 2001; Kioi et al, 2004).
  • the receptor is not normally expressed by any other non-pathologic organ, tissue or cell, with the exception of testis.
  • the expression of this receptor can be associated with glial transformation and increased tumor grade (Debinski et al, 2000).
  • the natural ligand for IL13 ⁇ 2R is IL-13, a Th2 cytokine which regulates inflammatory and immune responses (de Vries et al, 1993; Arima et al, 2002).
  • IL-13 shares many functions with IL-4, a finding likely related to the fact that the receptor for IL- 13 consists of IL13 ⁇ Rl chain which requires heterodimerization with the IL4R ⁇ chain in order to bind IL- 13 with high affinity.
  • IL- 13 and IL-4 compete for the shared physiological signaling receptor IL13/IL4R present in normal cells (Debinski et al, 1996; Rolling et al, 1996).
  • the IL13 ⁇ 2R chain is non-signaling, IL-4 independent, and capable of binding IL- 13 with high affinity (Debinski et al, 1996; Rolling et al, 1996).
  • Preliminary clinical trials utilizing IL-13-Pseudomonas exotoxin via convection enhanced delivery for malignant brain tumors have been highly promising and a phase III clinical trial has just been completed.
  • an adenovirus was created which targets IL13 ⁇ 2R via means of a IL-13 ⁇ 2R binding fiber protein.
  • the IL-13 ⁇ 2R binding fiber protein is constructed by substituting the shaft 5/knob 5 of Ad5 with a fiber- fibritin-IL-13 element.
  • the IL- 13 targeting adenovirus, termed LU-13 was then tested in vitro and in vivo against malignant glioma, which is used as model for cells with upregulated or abnormal expression of IL-13R ⁇ 2R. Examples of methods of producing chimeric fiber proteins can be found in U.S. Patents 7,094,398, 7,045,348, 6,929,946, 6,905,678, 5,770,442, and 5,756,086, each of which is incorporated herein by reference in its entirety.
  • IL13 ⁇ 2R is an ideal target for glioma therapy as the receptor is selectively expressed on tumor cells.
  • the expression of IL13 ⁇ 2R is associated with increased tumor grade.
  • Embodiments of the invention include construction of an Ad vector with IL13 ⁇ 2R targeting potential.
  • the Ad vector is constructed by replacement of the fiber protein in Ad capsid with a chimeric molecule containing a heterologous trimerization motif and a receptor-binding ligand, IL-13. This approach has resulted in the generation of a genetically modified Ad5 targeted to IL13 ⁇ 2R.
  • In vitro and in vivo gene transfer studies employing this novel vector have demonstrated its capacity to efficiently deliver a transgene payload to passaged and primary glioma cells in a receptor-specific manner.
  • Human adenovirus normally infects human cells, which are quiescent (nondividing) or dividing cells (normal or cancer cells). Upon introduction of this virus into a human cell (viral infection), the adenovirus DNA is immediately transcribed by the synthesis of ElA adenoviral protein. The CR2 region of ElA protein interacts specifically with Rb protein and leads to release of E2F, forcing cell entry into S-phase (the DNA Synthesis phase) of the cell cycle and maintaining the cell in the dividing cycle. This series of events effectively commandeers the host cell exclusively for the purpose of expressing virally encoded proteins. Active production of adenoviral particles depends on this ability to drive cells into an active mode of replication, a critical feature of therapeutic or oncolytic viruses.
  • an adenovirus may be a replicating virus, conditionally replicating virus, or a replication defective virus.
  • mutations in critical sequences of the viral genome render the adenovirus unable to bind to and inactivate tumor suppressor proteins.
  • These modified adenoviruses are able to replicate exclusively in cells lacking a functional target tumor suppressor gene (tumor cells only, i.e., conditionally replicating virus).
  • an adenovirus may be used as an adenovirus expression vector.
  • Adenovirus expression vector is meant to include those vectors containing adenovirus sequences sufficient to (a) support packaging of the adenoviral DNA and (b) to express a heterologous polynucleotide that has been cloned therein. The insertion position of a polynucleotide encoding a heterologous polypeptide of interest within the adenovirus sequences is not critical to the invention.
  • the polynucleotide encoding the polypeptide of interest may be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al, (1986) or other regions that are not essential for viral replication in the target cell.
  • Traditional methods for the generation of adenoviral particles are co-transfection followed by subsequent in vivo recombination of a shuttle plasmid and an adenoviral helper plasmid into, for example, either 293 or 911 cells (Introgene, The Netherlands).
  • a helper cell may be required for viral replication.
  • helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells.
  • the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, for example Vero cells or other monkey embryonic mesenchymal or epithelial cells.
  • One such cell line is 293 cells.
  • the adenovirus may be replication- competent in cells expressing an IL-13 ⁇ 2R on their cell surface.
  • adenoviral plaques are isolated from the agarose overlaid cells and the viral particles are expanded for analysis. For detailed protocols the skilled artisan is referred to Graham and Prevac, 1991 and the like.
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • Adenovirus type 5 is the typical starting material for use in the present invention.
  • Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructs employing adenovirus as a vector.
  • Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers ⁇ e.g., 10 9 -10 ⁇ plaque-forming units (pfu) per ml), and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome.
  • Modifications of adenovirus described herein may be made to improve the ability of the adenovirus to treat cancer.
  • the present invention also includes any modification of oncolytic adenovirus that improves the ability of the adenovirus to treat neoplastic cells. Included are modifications to oncolytic adenovirus genome in order to enhance the ability of the adenovirus to infect and replicate in cancer cells by altering the receptor binding molecules.
  • the initial step for successful infection is binding of adenovirus to its target cell, a process mediated through the fiber protein.
  • the fiber protein has a trimeric structure (Stouten et al, 1992) with different lengths depending on the virus serotype (Signas et al 1985; Kidd et al 1993).
  • the fiber protein can be divided into three domains (see, e.g., Green et al. 1983).
  • the conserved N-terminus contains the sequences responsible for association with the penton base as well as a nuclear localization signal ⁇ i.e., the tail).
  • a rod-like shaft of variable length contains repeats of a 15 amino acid beta structure, with the number of repeats ranging from 6 in Ad3 to 22 in Ad5.
  • a conserved stretch of amino acids which includes the sequence TLWT marks the boundary between the repeating units of beta structure in the shaft and the globular head domain.
  • the C-terminal head domain ranges in size from 157 amino acid residues for the short fiber of Ad41 to 193 residues for AdI l and Ad34.
  • the fiber spike is a homotrimer and it is thought that the C-terminus is responsible for trimerization of the fiber homotrimer and there are 12 spikes per virion which are attached via association with the penton base complex.
  • the first 30 N- terminal amino acids ⁇ i.e., the tail are involved in anchoring of the fiber to the penton base (Chroboczek et al, 1995), especially the conserved FNPVYP (SEQ ID NO:5) region in the tail (Arnberg et al, 1997).
  • the tail segment of SEQ ID NO:8 is from amino acid 1 to 46, the shaft from amino acid 47-403, and the knob segment from amino acid 404-581.
  • the C-terminus, or knob is responsible for initial interaction with the cellular adenovirus receptor.
  • adenoviral fiber protein or segment of an adenoviral protein can be used.
  • adenovirus fiber protein examples include those described in GenBank as of the filing date of this application, for example GenBank accession numbers AP_000190 [gi56160507], AP_000135 [gi56160464], AP_000226 [gi56160559], NP_852715 [gi31540466], AP_000157 [gi56160488], AAP92365 [gi32967051], AP_000601 [gi56160945], AP_000564 [gi56160902], AP_000527 [gi56160869], YP_068048 [gi51527294], AAS66940 [gi45504820], AAA42490 [gi303964], AAP31232 [gi32127332], AAP31231 [gi32127330], AAP31230 [gi32127328], AAP31226 [gi32127320], AAP31223 [gi32127314], AAP31
  • SEQ ID NO: 8 is an example of an adenoviral type 5 fiber protein.
  • the carboxy terminal 150, 175, 180, 185, 190, 200 amino acids, including all integers there between, are designated the knob domain and, typically, amino acid 1 to the start of the knob domain are designated the shaft.
  • the first 30, 40, or 50 amino acids, including all values or ranges there between, are termed the tail.
  • the adenoviral shaft typically includes the first 20, 30, 35, 40, 50, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 175, 200, 250, 300, 350, 400, 401, 402, 403, 404 more amino acids of an adenoviral fiber protein.
  • the present invention provides a method and means by which adenoviruses can be constructed with a modified tropism.
  • the present invention further provides methods for the generation of chimeric adenoviruses that can be targeted to specific cell types in vitro as well as in vivo.
  • the present invention further provides a method and means by which such an adenovirus can be used as a protein or nucleic acid delivery vehicle to a specific cell type or tissue.
  • chimeric fiber protein refers to an adenovirus fiber protein comprising a non- native amino acid sequence, in addition to or in place of a portion of a native fiber amino acid sequence.
  • the non-native amino acid sequence may be from an adenoviral fiber protein of a different serotype.
  • the non-native amino acid sequence may be any suitable length (e.g., 3 to about 200 amino acids).
  • An exemplary "chimeric fiber protein" has a shaft segment engineered to provide for trimerization and a head segment comprising a ligand that binds a component of a cell to be targeted.
  • adenovirus receptor CAR
  • One mechanism for altering the tropism of an adenovirus is to include a peptide or protein motif that associates with or binds to cells to be treated, growth inhibited, or destroyed.
  • Various peptide motifs may be added to the fiber knob of adenovirus.
  • IL- 13 ⁇ 2R ligand or binding elements are directly or indirectly coupled to an adenovirus fiber protein on the surface of an adenovirus. A motif can be inserted into the HI loop of the adenovirus fiber protein.
  • Modifying the capsid allows CAR-independent target cell infection. This allows higher replication, more efficient infection, and increased lysis of targeted cells (Suzuki et al, 2001, incorporated herein by reference).
  • Peptide sequences that bind receptors preferentially expressed on target cells such as cancer cells and more particularly glioma cells, such as IL-13 ⁇ 2R may also be added. These specific receptors found exclusively or preferentially on the surface of cells may be used as a target for adenoviral binding and infection.
  • the adenoviral fiber is a chimeric molecule comprising an adenovirus association segment, a shaft segment, and a targeting segment. [0047] Lack of expression in normal cells and achievable targeting using peptides and antibodies make these targeting systems suitable for the development of targeted adenoviruses with high therapeutic indices.
  • aspects of the invention include adenovirus and adenoviral nucleic acids that have been modified to express therapeutic nucleic acids (heterologous nucleic acids) or encode detectable and/or therapeutic polypeptides.
  • the heterologous nucleic acid is a therapeutic, or therapeutic nucleic acid.
  • a "therapeutic nucleic acid” is a nucleic acid which can be administered to a subject for the purpose of treating or preventing a disease.
  • a therapeutic nucleic acid can be a nucleic acid administered to a subject for treatment or prevention of cancer.
  • therapeutic nucleic acids encoding a therapeutic polypeptide include, but are not limited to Rb, CFTR, pi 6, p21, p27, p57, p73, C- CAM, APC, CTS-I, zacl, scFV ras, DCC, NF-I, NF-2, WT-I, MEN-I, MEN-II, BRCAl, VHL, MMACl, FCC, MCC, BRCA2, IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, IL-8, IL-9, IL-10, IL-I l IL-12, GM-CSF, G-CSF, thymidine kinase, mda7, fus, interferon ⁇ , interferon ⁇ , interferon ⁇ , ADP, p53, ABLI, BLCl, BLC6, CBFAl, CBL, CSFIR,
  • the therapeutic nucleic acid is a tumor suppressor gene.
  • a "tumor suppressor gene” is a gene or coding segment that, when present in a cell, reduces the tumorigenicity, malignancy, or hyperproliferative phenotype of the cell. This definition includes both the full length nucleic acid sequence of the tumor suppressor gene, as well as non-full length sequences of any length derived from the full length sequences.
  • Nucleic acids encoding tumor suppressor genes include tumor suppressor genes, and nucleic acids or encoding segments derived therefrom (e.g., cDNAs, cRNAs, mRNAs, and subsequences thereof encoding active fragments of the respective tumor suppressor amino acid sequences), as well as vectors comprising these sequences. It being further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.
  • tumor suppressor nucleic acids within this definition include, but are not limited to APC, CYLD, HIN-I, KRAS2b, pi 6, pi 9, p21, p27, p27mt, p53, p57, p73, PTEN, Rb, Uteroglobin, Skp2, BRCA-I, BRCA-2, CHK2, CDKN2A, DCC, DPC4, MADR2/JV18, MENl, MEN2, MTSl, NFl, NF2, VHL, WRN, WTl, CFTR, C-CAM, CTS-I, zacl, scFV, MMACl, FCC, MCC, Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYALl), Luca-2 (HYAL2), 123F2 (RASSFl), 101F6, Gene 21 (NPRL2), or a gene encoding a SEM A3 polypeptide and FUS
  • tumor suppressor genes are described in a database of tumor suppressor genes available on the world wide web at cise.ufl.edu/ ⁇ yyl/HTML- TSGDB/Homepage.html. This database is herein specifically incorporated by reference (as of the filing date of this application) into this and all other sections of the present application.
  • One of ordinary skill in the art would be familiar with tumor suppressor genes that can be applied in the present invention.
  • the therapeutic gene is a gene that induces apoptosis (i.e., a pro-apoptotic gene).
  • a "pro-apoptotic gene amino acid sequence” refers to a polypeptide that, when present in a cell, induces or promotes apoptosis.
  • the present invention contemplates inclusion of any pro- apoptotic gene known to those of ordinary skill in the art, particularly those active in cells expressing IL-13 ⁇ 2R.
  • pro-apoptotic genes include CD95, caspase-3, Bax, Bag-1, CRADD, TSSC3, bax, hid, Bak, MKP-7, PERP, bad, bcl-2, MSTl, bbc3, Sax, BIK, BID, and mda7.
  • pro-apoptotic genes include CD95, caspase-3, Bax, Bag-1, CRADD, TSSC3, bax, hid, Bak, MKP-7, PERP, bad, bcl-2, MSTl, bbc3, Sax, BIK, BID, and mda7.
  • the therapeutic gene can also be a gene encoding a cytokine.
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators and includes a polypeptide that maintains some or all of the function of a cytokine. This definition includes full-length as well as non-full length sequences of any length derived from the full length sequences. It being further understood, as discussed above, that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell. Examples of such cytokines are lymphokines, monokines, growth factors and traditional polypeptide hormones.
  • cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor- ⁇ and - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF- ⁇ ; platelet-growth factor; transforming growth factors (TGFs) such as TGF- ⁇ and TGF- ⁇ ; insulin-like growth factor-I and -II; erythropoietin (
  • genes encoding enzymes include, but are not limited to, ACP desaturase, an ACP hydroxylase, an ADP-glucose pyrophorylase, an ATPase, an alcohol dehydrogenase, an amylase, an amyloglucosidase, a catalase, a cellulase, a cyclooxygenase, a decarboxylase, a dextrinase, an esterase, a DNA polymerase, an RNA polymerase, a hyaluron synthase, a galactosidase, a glucanase, a glucose oxidase, a GTPase, a helicase, a hemicellulase, a hyaluronidase, an integrase, an invertase, an isomerase, a kinase, a lac
  • therapeutic genes include the gene encoding carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha- 1 antitrypsin, glucose-6-phosphatase, low-density-lipoprotein receptor, porphobilinogen deaminase, factor VIII, factor IX, cystathione beta.- synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, g
  • Therapeutic genes also include genes encoding hormones. Examples include, but are not limited to, genes encoding growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, leptin, adrenocorticotropin, angiotensin I, angiotensin II, ⁇ -endorphin, ⁇ -melanocyte stimulating hormone, cholecystokinin, endothelin I, galanin, gastric inhibitory peptide, glucagon, insulin, lipotropins, neurophysins, somatostatin, calcitonin, calcitonin gene related peptide, ⁇ -calcitonin gene related peptide, hypercalcemia of malignancy factor, parathyroid hormone-related protein, parathyroid hormone-related protein, glucagon-like peptide, pancreastatin, pancreatic peptide, peptide
  • the term "therapeutic gene” includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or may be adapted to express nucleic acids, proteins, polypeptides, domains, peptides, fusion proteins, and mutants or variant thereof.
  • the nucleic acid molecule encoding a therapeutic gene may comprise a contiguous nucleic acid sequence of about 5 to about 12000 or more nucleotides, nucleosides, or base pairs.
  • nucleic acids of the invention need not encode a protein.
  • nucleic acids of the invention can express inhibitory, catalytic, and/or therapeutic RNAs or DNAs.
  • RNA interference also referred to as "RNA-mediated interference" or RNAi
  • dsRNA double-stranded RNA
  • dsRNA double-stranded RNA
  • dsRNA has been shown to silence genes in a wide range of systems, including plants, protozoans, fungi, C. elegans, Trypanasoma, Drosophila, and mammals (Grishok et al, 2000; Sharp et al, 1999; Sharp and Zamore, 2000; Elbashir et al, 2001). It is generally accepted that RNAi acts post-transcriptionally, targeting RNA transcripts for degradation. It appears that both nuclear and cytoplasmic RNA can be targeted (Bosher and Labouesse, 2000).
  • siRNAs small interfering RNAs
  • miRNAs microRNAs
  • siRNAs small interfering RNAs
  • miRNAs microRNAs
  • siRNAs direct target mRNA cleavage (Elbashir et al, 2001)
  • miRNAs block target mRNA translation (Lee et al, 1993; Reinhart et al, 2000; Brennecke et al, 2003; Xu et al, 2003).
  • Many known miRNA sequences and their position in genomes or chromosomes can be found on the World Wide Web at sanger.ac.uk.
  • siRNAs are designed so that they are specific and effective in suppressing the expression of the genes of interest. Methods of selecting the target sequences, i.e., those sequences present in the gene or genes of interest to which the siRNAs will guide the degradative machinery, are directed to avoiding sequences that may interfere with the siRNA's guide function while including sequences that are specific to the gene or genes. Typically, siRNA target sequences of about 21 to 23 nucleotides in length are most effective. This length reflects the lengths of digestion products resulting from the processing of much longer RNAs (Montgomery et al, 1998).
  • RNA for use in siRNA may be chemically or enzymatically synthesized. Both of these texts are incorporated herein in their entirety by reference.
  • the enzymatic synthesis contemplated in these references is by a cellular RNA polymerase or a bacteriophage RNA polymerase ⁇ e.g. , T3, T7, SP6) via the use and production of an expression construct as is known in the art. For example, see U.S. Patent 5,795,715.
  • the contemplated constructs provide templates that produce RNAs that contain nucleotide sequences identical to a portion of the target gene.
  • the length of identical sequences provided by these references is at least 25 bases, and may be as many as 400 or more bases in length.
  • An important aspect of this reference is that the authors contemplate digesting longer dsRNAs to 21-25mer lengths with the endogenous nuclease complex that converts long dsRNAs to siRNAs in vivo.
  • U.S. Patent 5,795,715 reports the simultaneous transcription of two complementary DNA sequence strands in a single reaction mixture, wherein the two transcripts are immediately hybridized.
  • the templates used are particularly of between 40 and 100 base pairs, and which is equipped at each end with a promoter sequence.
  • U.S. Patent App. 20050203047 which is incorporated herein by reference, describes a method of modulating gene expression through RNA interference by incorporating a siRNA or miRNA sequence into a transfer RNA (tRNA) encoding sequence.
  • the tRNA containing the siRNA or miRNA sequence may be incorporated into a nucleic acid expression construct so that this sequence is spliced from the expressed tRNA.
  • the siRNA or miRNA sequence may be positioned within an intron associated with an unprocessed tRNA transcript, or may be positioned at either end of the tRNA transcript.
  • a therapeutic nucleic acid can be an antisense nucleic acid.
  • Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a particular embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
  • complementary or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region ⁇ e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
  • genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
  • a genomic clone will need to be used.
  • the cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
  • a therapeutic nucleic acid is a ribozyme.
  • proteins traditionally have been used for catalysis of nucleic acids another class of macromolecules has emerged as useful in this endeavor.
  • Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cook, 1987; Gerlach et al, 1987; Forster and Symons, 1987).
  • ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cook et al, 1981; Michel and Westhof, 1990; Reinhold- Hurek and Shub, 1992).
  • This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
  • IGS internal guide sequence
  • Ribozyme catalysis has primarily been observed as part of sequence- specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cook et al, 1981).
  • U.S. Patent 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
  • sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991; Sarver et al, 1990).
  • ribozymes elicited genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme.
  • Amino acid sequence variants of the targeting elements may be substitutional, insertional or deletion variants.
  • Deletion variants lack one or more residues of the native protein.
  • Insertional mutants typically involve the addition of material at a nonterminal point in the polypeptide. This may include the insertion of a targeting element, a polymerization element, an immunoreactive epitope or simply a single residue. Terminal additions, called fusion proteins, are discussed below.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage or modified binding properties, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • biologically functional equivalent in certain aspects binding to IL-13 ⁇ 2 receptor (e.g., SEQ ID NO:2 or SEQ ID NO:4) or cell surface receptors of adenovirus (e.g., SEQ ID NO:8)) is well understood in the art and is further defined in detail herein.
  • sequences that have between about 60%, 70%, 80% to about 80%, 85%, 90%, 95%, 97%, 99%; or between about 81% and about 90%; or even between about 91% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of a particular polypeptide (e.g., SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:8 or SEQ ID NO:9) over 10, 20, 30, 40, 50, 100, 114, 150, 200, 250, 300, 350 400, 500 or more amino acids of a polypeptide segment, including all ranges and values there between, provided the biological activity (e.g., binding) of the polypeptide is maintained.
  • a particular polypeptide e.g., SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:8 or SEQ ID NO:9
  • amino acids of a particular polypeptide e.g., SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:8 or SEQ ID NO
  • nucleotide or protein sequences refer 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, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms or by visual inspection.
  • amino acid and nucleic acid sequences may include additional residues or nucleotides, such as additional N- or C-terminal amino acids or 5' or 3' nucleic acid sequences.
  • additional residues or nucleotides such as additional N- or C-terminal amino acids or 5' or 3' nucleic acid sequences.
  • terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, e.g., introns.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, nucleic acids, antibodies, antigens, and the like.
  • amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • the present invention concerns polynucleotides that are capable of expressing a protein, polypeptide, or peptide discussed above, such as an engineered adenoviral fiber protein and/or a therapeutic nucleic acid. Included within the term nucleic acid are polynucleotides, DNA segments, and recombinant vectors. Recombinant vectors may include plasmids, cosmids, phage, viruses, adenoviral genomes and the like. In certain embodiments recombinant adenoviruses are contemplated.
  • polypeptides of the invention may be altered or modified to produce a polypeptide, e.g., a fiber protein, that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450
  • a polynucleotide may comprise one or more therapeutic nucleic acids that typically are operatively coupled to various regulatory sequences.
  • the term "gene” is used for simplicity to refer to a nucleic acid encoding a protein, polypeptide, or peptide-encoding unit.
  • this functional term includes genomic sequences, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants.
  • isolated substantially away from other coding sequences means that the nucleic acid of interest, for example the polynucleotide encoding a wild-type, a polymorphic, or a mutant therapeutic polypeptide, forms part of the coding region of the nucleic acid segment, and that the nucleic acid segment does not contain large portions of naturally-occurring DNA.
  • this refers to the nucleic acid segment as originally isolated, and does not exclude nucleic acid sequence, polynucleotide or coding regions later added to the segment by human manipulation.
  • nucleic acid segments used in the present invention may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • Adenoviruses of the present invention can be constructed using methods known in the art and described herein. Expression requires that appropriate signals be provided which include various regulatory elements, such as enhancers/promoters that may be derived from both viral and mammalian sources that drive host cell expression of the genes of interest. Elements designed to optimize messenger RNA stability and translatability in host cells are contemplated.
  • a heterologous nucleic acid is under transcriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, that is required to initiate the specific transcription of a gene.
  • the phrase "under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • a promoter is a heterologous promoter, in which the promoter is associated with a nucleic acid that is not associated with its natural location.
  • the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.
  • a human cell it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either a human or viral promoter.
  • the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of the coding sequence of interest.
  • CMV cytomegalovirus
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the nucleic acid transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals.
  • a terminator is also contemplated as an element of the expression cassette. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • a nucleic acid of the invention may also include sequences related to a selectable marker.
  • the markers listed below can be inserted as a heterologous sequence in the adenovirus genome.
  • the cells contain a nucleic acid construct of the present invention; a cell may be identified in vitro or in vivo by including a marker in the nucleic acid construct. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector.
  • a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed.
  • Immunologic markers also may be employed.
  • the selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a polypeptide of interest. Further examples of selectable markers are well known to one of skill in the art.
  • IRES elements are used to create multigene, or polycistronic, messages.
  • IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988).
  • IRES elements from two members of the picornavirus family polio and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991).
  • IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages.
  • each open reading frame is accessible to ribosomes for efficient translation.
  • Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
  • An example of such a construct is described in U.S. Patent 5,665,567, which is herein incorporated by reference.
  • Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single vector and a single selectable marker.
  • Embodiments of the invention include adenovirus having a ligand for an IL- 13 receptor on the adenoviral surface.
  • Western blotting and sequence analysis can be used to confirm proper trimerization and/or ligand presentation on the adenoviral surface.
  • fluorescent microscopy can be used to co-localize an adenovirus and its targeted receptor.
  • confocal microscopic analysis of primary glioma suspensions incubated with viral recombinants showed that LU- 13 co- localized to the ILl 3 ⁇ 2 receptor.
  • Transduction of a target cell can be studied by using a reporter gene that can be detected once expressed in a cell, e.g., luciferase transduction assays.
  • luciferase assays using viral recombinants have been conducted on both primary and passaged glioma cell cultures and demonstrate that LU- 13 exhibited at least 10-fold enhanced gene transduction compared to AdWT. Moreover, the recombinant virus preferentially replicated in glioma cells, as documented by increased E4 copy number.
  • In vitro competition assays performed with anti-human IL- 13 mAb confirmed significant attenuation of LU- 13 transduction. These results were further confirmed in vivo, where LU-13 showed 300-fold increase in transgene expression.
  • we describe here the development of novel and targeted adenoviral vectors that binds ILl 3 ⁇ 2 receptor which is over-expressed on brain tumor cells. Our findings confirm the ability of LU-13 to bind IL13 ⁇ 2R and increase transgene expression, making it an attractive gene therapy vector for the treatment of malignant glioma in a clinical setting.
  • the present invention involves the treatment of hyperproliferative cells. It is contemplated that a wide variety of cells may be treated using the methods and compositions of the invention, including gliomas, sarcomas, lung, ovary, breast, cervix, pancreas, stomach, colon, skin, larynx, bladder, prostate, and/or brain metastases, as well as pre-cancerous cells, metaplasias, dysplasias, or hyperplasias.
  • glioma refers to a tumor or cancer cell originating in the neuroglia of the brain or spinal cord.
  • Gliomas are derived from the glial cell types such as astrocytes and oligodendrocytes, thus gliomas include astrocytomas and oligodendrogliomas, as well as anaplastic gliomas, glioblastomas, and ependymomas.
  • Astrocytomas and ependymomas can occur in all areas of the brain and spinal cord in both children and adults.
  • Oligodendrogliomas typically occur in the cerebral hemispheres of adults. Gliomas account for 75% of brain tumors in pediatrics and 45% of brain tumors in adults.
  • the remaining percentages of brain tumors are meningiomas, pineal region tumors, choroid plexus tumors, neuroepithelial tumors, embryonal tumors, peripheral neuroblastic tumors, tumors of cranial nerves, tumors of the hemopoietic system, germ cell tumors, and tumors of the sellar region.
  • Various embodiments of the present invention deal with the treatment of disease states comprised of cells that express IL- 13 receptors, in some aspects IL- 13 ⁇ 2 receptors.
  • the present invention is directed at the treatment of diseases, including but not limited to gliomas, sarcomas, tumors of lung, ovary, cervix, pancreas, stomach, colon, skin, larynx, breast, prostate and metastases thereof.
  • the term "therapeutic benefit” refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of his/her condition, which includes treatment of pre-cancer, cancer, and hyperproliferative diseases.
  • a list of non-exhaustive examples of this includes extension of the subject's life by any period of time, decrease or delay in the neoplastic development of the disease, decrease in hyperproliferation, reduction in tumor growth, delay of metastases, reduction in cancer cell or tumor cell proliferation rate, and a decrease in pain to the subject that can be attributed to the subject's condition.
  • adenoviral delivery to in vivo and ex vivo situations.
  • viral vectors one generally will prepare a viral vector stock. Depending on the kind of virus and the titer attainable, one will deliver 1 to 100, 10 to 50, 100-1000, or up to 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , or 1 x 10 12 or more adenoviral or infectious particles to a patient in a pharmaceutically acceptable composition as discussed below.
  • Various routes are contemplated for various tumor types. Where discrete tumor mass, or solid tumor, may be identified, a variety of direct, local, and regional approaches may be taken.
  • the tumor may be directly injected with the adenovirus.
  • a tumor bed may be treated prior to, during or after resection and/or other treatment(s).
  • Following resection or other treatment(s) one generally will deliver the adenovirus by a catheter having access to the tumor or the residual tumor site following surgery.
  • One may utilize the tumor vasculature to introduce the vector into the tumor by injecting a supporting vein or artery.
  • a more distal blood supply route also may be utilized.
  • the method of treating cancer includes treatment of a tumor as well as treatment of the region near or around the tumor.
  • residual tumor site indicates an area that is adjacent to a tumor. This area may include body cavities in which the tumor lies, as well as cells and tissue that are next to the tumor.
  • compositions in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • the active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route.
  • the routes of administration will vary, naturally, with the location and nature of the lesion, and include, e.g., intradermal, transdermal, parenteral, intracranial, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration and formulation. Certain embodiments include intracranial or intravenous administration. Administration may be by injection or infusion, see Kruse et al. (1994), specifically incorporated by reference, for methods of performing intracranial administration.
  • compositions would normally be administered as pharmaceutically acceptable compositions.
  • An effective amount of the therapeutic agent is determined based on the intended goal, for example, elimination of tumor cells.
  • unit dose refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses, discussed above, in association with its administration, i.e., the appropriate route and treatment regimen.
  • the quantity to be administered both according to number of treatments and unit dose, depends on the subject to be treated, the state of the subject and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.
  • the engineered viruses of the present invention may be administered directly into animals, or alternatively, administered to cells that are subsequently administered to animals.
  • in vitro administration refers to manipulations performed on cells removed from an animal, including, but not limited to, cells in culture.
  • ex vivo administration refers to cells that have been manipulated in vitro, and are subsequently administered to a living animal.
  • the term in vivo administration includes all manipulations performed on cells within a subject.
  • the compositions may be administered either in vitro, ex vivo, or in vivo.
  • An example of in vivo administration includes direct injection of tumors with the instant compositions by intracranial administration.
  • an adenoviral vector or nucleic acid can be introduced into a host cell and the host cell, producing adenoviral products, is then administered to a subject or patient.
  • the host cell can be a stem cell, e.g., a mesenchymal stem cell.
  • the host cell can be a heterologous or autologous cell.
  • adenoviral replication can be selective or substantially restricted to the host cell. Examples of such methods are found in Pereboeva et al. (2003), Nakamizo et al. (2004), Nakamizo et al. (2005), Komarova et al. (2006), and Stoff-Khalili et al.
  • a host cell can be used as an intermediate carrier for an adenovirus vector, particularly an adenovirus that selectively replicates in the host cell.
  • the host cell may migrate to the therapeutic target and therefore direct administration of such a cell may or may not be administered into or locally to the target site, e.g. , a tumor.
  • Intratumoral injection or injection into the tumor vasculature is specifically contemplated for discrete, solid, accessible tumors including tumor exposed during surgery. Local, regional or systemic administration also may be appropriate.
  • the injection volume will be 1 to 3 cc, preferably 3 cc.
  • the injection volume will be 4 to 10 cc, preferably 5 cc.
  • Multiple injections delivered as single dose comprise about 0.1 to about 0.5 ml volumes, preferable 0.2 ml.
  • the viral particles may be administering by multiple injections, spaced at approximately 1 cm intervals.
  • the present invention may be used preoperatively to render an inoperable tumor subject to resection.
  • the present invention may be used at the time of surgery, and/or thereafter, to treat residual or metastatic disease.
  • a resected tumor bed may be injected or perfused with a formulation comprising the adenovirus.
  • the perfusion may be continued post-resection, for example, by leaving a catheter implanted at the site of the surgery. Periodic post-surgical treatment is also envisioned.
  • Continuous administration preferably via syringe or catheterization, also may be applied where appropriate, for example, where a tumor is excised and the tumor bed is treated to eliminate residual, microscopic disease.
  • Such continuous perfusion may take place for a period from about 1-2 hr, to about 2-6 hr, to about 6-12 hr, to about 12-24 hr, to about 1-2 days, to about 1-2 wk or longer following the initiation of treatment.
  • the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs. It is further contemplated that limb perfusion may be used to administer therapeutic compositions of the present invention.
  • Treatment regimens may vary as well, and often depend on tumor type, tumor location, disease progression, and health and age of the patient. Obviously, certain types of tumor will require more aggressive treatment, while at the same time, certain patients cannot tolerate more taxing protocols. The clinician will be best suited to make such decisions based on the known efficacy and toxicity (if any) of the therapeutic formulations.
  • the adenovirus may be administered parenterally or intraperitoneally, or in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations may also be emulsified.
  • a typical composition for such purpose comprises a pharmaceutically acceptable carrier.
  • the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline.
  • Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride or Ringer's dextrose.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases.
  • the pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well known parameters.
  • the route is topical, the form may be a cream, ointment, or salve.
  • an adenovirus or a nucleic acid encoding an adenovirus may be delivered to cells using liposome or immunoliposome delivery.
  • the adenovirus or nucleic acid encoding an adenovirus may be entrapped in a liposome or lipid formulation.
  • Liposomes may be targeted to a cell by attaching a targeting moiety, such as the IL-13 ⁇ 2R binding element, or an antibody to the liposome that bind specifically to the cell surface.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium.
  • Tumor cell resistance to various therapies represents a major problem in clinical oncology.
  • One goal of current cancer research is to find ways to improve the efficacy of chemo- and radiotherapy, as well as other conventional cancer therapies.
  • One way is by combining such traditional therapies with adenovirus therapy.
  • Traditional therapy to treat cancers may include removal of all or part of the affected organ, external beam irradiation, xenon arc and argon laser photocoagulation, cryotherapy, immunotherapy and/or chemotherapy.
  • the choice of treatment is dependent on multiple factors, such as, (1) multifocal or unifocal disease, (2) site and size of the tumor, (3) metastasis of the disease, (4) age of the patient or (5) histopathologic findings (The Genetic Basis of Human Cancer, 1998).
  • adenoviral therapy could be used in conjunction with anti-cancer agents, including chemo- or radiotherapeutic intervention, as well as radiodiagnostic techniques. It also may prove effective to combine adenovirus therapy with immunotherapy.
  • a "target" cell contacted with an effective amount of adenovirus and optionally at least one other agent may kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce a hyperproliferative phenotype of target cells.
  • These compositions would be provided in a combined amount effective to kill or inhibit proliferation of the target cell.
  • This process may involve contacting the cells with an adenovirus and an agent(s) or factor(s) at the same or different times. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, wherein one composition includes an adenovirus and the other includes a second agent.
  • Adenoviral therapy may also be combined with immunosuppression.
  • the immunosuppression may be performed as described in WO 96/12406, which is incorporated herein by reference.
  • immunosuppressive agents include cyclosporine, FK506, cyclophosphamide, and methotrexate.
  • an adenovirus treatment may precede or follow the second agent or treatment by intervals ranging from minutes to weeks. In embodiments where the second agent and adenovirus are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the times of each delivery, such that the second agent and adenovirus would still be able to exert a combined effect on the cell.
  • both agents are delivered to a cell in a combined amount sufficient to affect the cell.
  • Agents or factors suitable for use in a combined therapy are any anti- angiogenic agent and/or any chemical compound or treatment method with anticancer activity; therefore, the term "anticancer agent” that is used throughout this application refers to an agent with anticancer activity.
  • These compounds or methods include alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA antimetabolites, DNA antimetabolites, antimitotic agents, as well as DNA damaging agents, which induce DNA damage when applied to a cell.
  • Examples of chemotherapy drugs and pro-drugs include, CPTI l, temozolomide, platin compounds and pro-drugs such as 5-fluorocytosine (5-FC).
  • Examples of alkylating agents include, BCNU ⁇ e.g., glialdel wafers), chloroambucil, cis-platinum, cyclodisone, flurodopan, methyl CCNU, piperazinedione, teroxirone.
  • Topoisomerase I inhibitors encompass compounds such as camptothecin and camptothecin derivatives, as well as morpholinodoxorubicin.
  • RNA/DNA antimetabolites include L-alanosine, 5-fluoraouracil, aminopterin derivatives, methotrexate, and pyrazofurin; while the DNA antimetabolite group encompasses, for example, ara-C, guanozole, hydroxyurea, thiopurine.
  • Typical antimitotic agents are colchicine, rhizoxin, taxol, and vinblastine sulfate.
  • agents and factors include radiation and other forms of energy that induce DNA damage that include ⁇ -irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, sound and the like.
  • Chemotherapeutic agents contemplated to be of use include, e.g., adriamycin, bleomycin, 5-fluorouracil (5-FU), etoposide (VP- 16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), podophyllotoxin, verapamil, and even hydrogen peroxide.
  • the invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide.
  • anti-angiogenesis agents include, but are not limited to, retinoid acid and derivatives thereof, 2-methoxyestradiol, ANGIOSTATIN R protein, ENDOSTATIN R protein, suramin, squalamine, tissue inhibitor of metalloproteinase-I, tissue inhibitor of metalloproteinase-2, plasminogen activator inhibitor- 1, plasminogen activator inhibitor-2, cartilage-derived inhibitor, paclitaxel, platelet factor 4, protamine sulphate (clupeine), sulphated chitin derivatives (prepared from queen crab shells), sulphated polysaccharide peptidoglycan complex (sp-pg), staurosporine, modulators of matrix metabolism, including for example, proline analogs ((l-azetidine-2-carboxylic acid (LACA), cishydroxyproline, d, 1-3,4- dehydroproline, thiaproline], ⁇ , ⁇ -dipyridyl,
  • anti-angiogenesis agents include antibodies, preferably monoclonal antibodies against these angiogenic growth factors: bFGF, aFGF, FGF-5, VEGF isoforms, VEGF-C, HGF/SF and Ang-l/Ang-2 (Ferrara and Alitalo, 1999).
  • Calbiochem (San Diego, Ca) carries a variety of angiogenesis inhibitors including (catalog number/product name) 658553/AG 1433; 129876/Amiloride, Hydrochloride; 164602/Amino ⁇ eptidase N Inhibitor; 175580/Angiogenesis Inhibitor; 175602/Angiogenin (108-123); 175610/Angiogenin Inhibitor; 176600/ Angiopoietin-2, His*Tag®, Human, Recombinant, Mouse, Biotin Conjugate; 176705/Angiostatin Kl -3, Human; 176706/ Angiostatin Kl -5, Human; 176700/Angiostatin® Protein, Human; 178278/Apigenin; 189400/ Aurintricarboxylic Acid; 199500/Benzopurpurin B; 211875/Captopril; 218775/Castanospermine, Castanospermum australe; 251400
  • Immunotherapy may be used as part of a combined therapy, in conjunction with adenovirus therapy.
  • the tumor cell must bear some marker that is amenable to targeting for immunotherapy, i.e., is not present on the majority of other cells or the combination of markers is not present on a majority of other cells (IL- 13 ⁇ 2R plus a second surface target).
  • IL- 13 ⁇ 2R IL- 13 ⁇ 2R plus a second surface target.
  • Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention.
  • Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase ( ⁇ 97), g ⁇ 68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pl55.
  • adenovirus therapies with chemo- and radiotherapies
  • combination with other gene therapies will be advantageous.
  • targeting of an adenovirus in combination with providing a therapeutic nucleic acid that is therapeutic upon transcription from the vector and/or upon translation by the target or neighboring cell(s) see genomic modification section above for more details.
  • the therapies described above may be implemented in combination with all types of surgery. Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. These types of surgery may be used in conjunction with other therapies, such as adenovirus therapies.
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed.
  • Tumor resection refers to physical removal or destruction of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection, systemic administration, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
  • the time between such treatment types may be about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or about 24 hours apart; about 1, 2, 3, 4, 5, 6, or 7 days apart; about 1, 2, 3, 4, or 5 weeks apart; and about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months apart, or more.
  • the target cell expresses IL-13 ⁇ 2R, particularly on its surface, which may be assessed using standard immunological and gene expression assessment techniques know in the art.
  • the cell may be administered compositions of the invention in vitro, in vivo, or ex vivo.
  • the cancer cell may be in a patient.
  • the patient may have a solid tumor.
  • embodiments may further involve performing surgery on the patient, such as by resecting all or part of the tumor.
  • Viral compositions may be administered to the patient before, after, or at the same time as surgery.
  • patients may also be administered directly, endoscopically, intratracheally, intratumorally, intravenously, intralesionally, intramuscularly, intraperitoneally, regionally, percutaneously, topically, intrarterially, intravesically, or subcutaneously.
  • Viral compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times, and they may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months.
  • the cancer cell that is administered viral compositions may be a neuronal, glial, bladder, blood, bone, bone marrow, brain, spinal, breast, colorectal, esophageal, gastrointestine, head, kidney, liver, lung, nasopharyngeal, neck, ovary, pancreas, prostate, skin, stomach, testicular, tongue, or uterus cell.
  • Cancers that may be evaluated and/or treated by methods and compositions of the invention include cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm malignant; carcinoma; carcinoma undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma
  • Animal models may be used as a screen for tumor suppressive effects of adenoviruses.
  • orthotopic animal models will be used so as to closely mimic the particular disease type being targeted and to provide the most relevant results.
  • One type of orthotopic model involves the development of an animal model for the analysis of microscopic residual cancer cell(s) and microscopic seeding of body cavities.
  • 1 x 10 7 cells are inoculated into a nude mouse.
  • the number of cells will be dependent upon various factors, such as the size of the animal, the site and technique of administration, the replicative capacity of the tumor cells themselves, the time intended for tumor growth, the potential anti-tumor therapeutic to be tested, and the like.
  • establishing an optimal model system for any particular type of tumor may require a certain adjustment in the number of cells administered, this in no way represents an undue amount of experimentation. For example, this can be accomplished by conducting preliminary studies in which differing numbers of cells are delivered to the animal and the cell growth is monitored.
  • Antibodies can be used to detect IL-13 binding proteins (e.g., IL-13 ⁇ 2R).
  • one or more antibodies may be produced that are immunoreactive with multiple antigens or epitopes. These antibodies may be used in various diagnostic or therapeutic applications, described herein below.
  • the term "antibody” is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting. Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Harlow and Lane (1988), incorporated herein by reference).
  • Monoclonal antibodies are recognized to have certain advantages (e.g., reproducibility and large-scale production).
  • the invention thus provides for monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and even chicken origin. Due to the ease of preparation and ready availability of reagents, murine monoclonal antibodies may be preferred.
  • humanized antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof.
  • MAbs monoclonal antibodies
  • polyclonal antibodies are well known in the art.
  • MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference. It is also contemplated that a molecular cloning approach may be used to generate monoclonals.
  • monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer, or by expression of full-length gene or of gene fragments in E. coli.
  • Adenoviral gene expression in a population of cells can be determined by western blot analysis using antibodies as probes to adenoviral hexon protein or other adenoviral encoded proteins. The level of protein detected indicates whether viral protein expression is occurring in the cell.
  • the present invention concerns immunodetection methods for binding, purifying, removing, quantifying and/or otherwise generally detecting biological components such as protein(s), polypeptide(s) or peptide(s) involved in adenoviral replication.
  • Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few.
  • the biological sample analyzed may be any sample that is suspected of containing a cancer cell or an adenoviral infected cell, such as, for example, a tissue section or specimen, a homogenized tissue extract, a cell, an organelle, separated and/or purified forms of any of the above compositions, or even any biological fluid that comes into contact with the cell or tissue, including blood and/or serum, although tissue samples or extracts are preferred.
  • the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches, such as ELISA, immunohistochemical (IHC) staining, western blotting, dot blotting, FACS analyses, and/or the like. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological and enzymatic tags.
  • a label or marker such as any radioactive, fluorescent, biological and enzymatic tags.
  • U.S. Patents concerning the use of such labels include 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference.
  • a secondary binding ligand such as a second antibody and/or a biotin/avidin ligand binding arrangement, as is known in the art.
  • K28 is a HEK293 derivate which stably expresses the adenovirus type 5 fiber protein from a selective zeocin antibiotic cassette containing vector (Invitrogen). K28 cells were provided by Dr. D.T Curiel (University of Alabam, Birmingham, AL, USA). Human glioma cells U87MG and U373MG were purchased from American Type Culture Collection (ATCC) and maintained in DMEM containing 10% FBS (HyClone). Kings cells were purchased from Japanese Tumor Tissue Bank (Tokyo, Japan) and cultured under standard conditions in RPMI media containing 10% FBS.
  • the human IL13 cDNA (Genebank accession: NM 002188) was amplified from a plasmid provided by Dr. W. Debinski (Wake Forest University, NC, USA) using ILl 3F 5'- GCCGGATCCGCTATGCATCCG-3' (SEQ ID NO:6) and SwaRev 5'- GATATTTAAATTTTATCAGTTGAACTGTCCCTCGCG-3'(SEQ ID NO:7).
  • EcoRI-Avrll-cut fragment of pxk.FFM13 was fused in Escherichia coli BJ5183 by homologous recombination with Swa-I linearized pVK700 adenoviral backbone vector (DHGT, UAB) to generate a packagable adenoviral genome.
  • the LU- 13 adenoviral vector was produced by transfecting Pad-digested LU- 13 into K28 cells with LipofectamineTM 2000 (#11668-019, Invitrogen Corp) reagent.
  • Adenoviruses were amplified in HEK293 cells and purified by cesium chloride gradient ultracentrifugation. Titers were calculated using previously described methods (Maizel et al, 1968a, Maizel et al, 1968b; Mittereder et al, 1996).
  • Membranes were incubated with mouse anti-adenovirus fiber 5 (4D2, 1:5000 dilution, Abeam, Cambridge, Ma, USA) Ab or rabbit polyclonal serum against human IL- 13 protein (sc-1292, 1 :500 dilution; Santa Cruz Biotechnology, Ca, USA). After one hour incubation, membranes were incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit antibodies. All secondary antibodies were purchased from Abeam (Cambridge, Ma, USA). Protein expression was visualized using an enhanced SuperSignal West Pico Chemiluminescent Substrate (#34077, Pierce, Rockford, IL, USA).
  • Transduction assay in glioma and non-glioma cells Cultured human glioma cells were harvested and resuspended in 10% FBS-DMEM medium. After centrifugation, 5x10 4 cells were seeded in 24- well tissue culture plates. Twenty- four hours later, cells were infected in triplicates with replication-deficient viruses reAd or LU- 13 at 1000 MOI with overnight adsorption at 37 0 C. Following adsorption, the media was replaced with complete culture media. Twenty four hours post- adsorptions, the cells were lysed in Lysis Reagent (Promega, Madison, WI) for detection of luciferase expression.
  • Lysis Reagent Promega, Madison, WI
  • Vector inhibition assay - Kings, U373MG, and U87MG cells were preincubated for 30 minutes at 4°C with transduction media (2% DMEM). Then, the cells were incubated with 50 ⁇ g/ml of anti-human IL- 13Ab (clone C- 19) for 1 hr at 37°C. After this incubation, cells were incubated with either AdWT or LU-13 virions (1000 MOI per cell) for 2 hr on ice. After adsorption, cells were washed and further incubated for 48 hrs in growth media until transgene expression was determined.
  • Adenovirus cell binding assays - 0.5 x 10 6 of U87MG, U373MG, and Kings human glioma cells were grown to 60% confluence and seeded onto 6-well plates each with 6 ml of F- 12 Dulbecco's modified eagle medium. The next day, media was aspirated and the cells were infected at 1000 MOI/cell and incubated at 37 0 C in humidified atmosphere for one hour.
  • Antigen staining of IL13 ⁇ 2 receptor was achieved with mouse anti- human IL13 ⁇ 2 antibodies (clone B-D13, Cell Sciences, Canton, MA, USA) and secondary Cy5 -conjugated, rabbit anti-mouse antibodies (AP160S; Chemicon International). Bound virus was detected by rabbit anti-IL13 ligand antibodies (sc- 1292; Santa Cruz Biotechnology, Ca, USA) and TRITC-conjugated goat anti-rabbit IgG (AP307R; Chemicon International). Negative controls: mock-infected cells showed negative staining for TRITC-conjugated goat anti-rabbit IgG antibodies.
  • DAPI (Sigma) staining of nuclei (434-503 nm light) was visualized using Leica-sp5 laser-scanning confocal microscopy. During the processing of captured pictures, Cy- 5 developed signal (650-750-nm light) was converted into green channel to obtain co- localization signal with TRITC-signal (570-620-nm light). To obtain merged figures, single-channel images were digitally captured and overlayed using Leica software (LAS AF, version 1.6.0. build 869; Microsytems).
  • a replication-deficient vector was created, LU- 13, containing several features, some of which are depicted in FIG. 1.
  • the tail domain of Ad5 was not deleted.
  • the presence of type 5 tail region facilitates incorporation of the chimera protein into the virion particle; in this particular case, only the shaft and knob fiber domains were replaced.
  • the inventors substituted the T4 phage's fibritin structural protein that is fused to the human IL- 13 cytokine. This transcription unit was verified by sequencing and restriction analysis of recombinant plasmid pxk.FFIL13 (data not shown).
  • Protein expression of the LU- 13 chimera was detected after infection of HEK293 with 100 MOI. As shown in FIG. 2, 4d2 antibodies recognize the trimer and monomer form of the wild-type fiber as well as the LU- 13 chimera. Using antibody to the COOH terminus of ILl 3 protein, incorporation of IL- 13 into fibritin chimera was confirmed. Boiled/unboiled conditions used in western blotting analysis confirmed trimerization of fiber- fibritin-IL 13 chimera.
  • FIG. 4A shows the 3D images of ILl 3 -stained cells that were generated by combining 23 optical sections. This image confirms that ligand-receptor interaction occur on the surface of glioma cells. Taken together, these results prove that the ILl 3 ligand incorporated into LU- 13 viral virions interacts with ILl 3 ⁇ 2 receptor.
  • LU-13 exhibits enhanced transduction activity in glioma cells.
  • glioma cells lines were infected with reAd or LU-13. Cells were infected with 1000 MOI/cell followed by a 24 hr adsorption period. Luciferase activity was measured after a second 24 hr incubation with a fresh portion of media. As shown in FIG. 5A, transduction of all three human glioma cells was significantly higher with LU-13 virus than control vector.

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Abstract

L'invention concerne des vecteurs adénoviraux modifiés ciblant de façon sélective les vecteurs adénoviraux de l'IL13a2R ainsi que les cellules, les tissus ou les organes contenant ceux-ci.
PCT/US2008/052803 2007-02-01 2008-02-01 Compositions et procédés apparentés à un vecteur adénoviral recombinant qui cible les récepteurs de l'il13 WO2008095168A2 (fr)

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US11077156B2 (en) 2013-03-14 2021-08-03 Salk Institute For Biological Studies Oncolytic adenovirus compositions
US11130968B2 (en) 2016-02-23 2021-09-28 Salk Institute For Biological Studies High throughput assay for measuring adenovirus replication kinetics
US11401529B2 (en) 2016-02-23 2022-08-02 Salk Institute For Biological Studies Exogenous gene expression in recombinant adenovirus for minimal impact on viral kinetics
US11813337B2 (en) 2016-12-12 2023-11-14 Salk Institute For Biological Studies Tumor-targeting synthetic adenoviruses and uses thereof

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US11130968B2 (en) 2016-02-23 2021-09-28 Salk Institute For Biological Studies High throughput assay for measuring adenovirus replication kinetics
US11401529B2 (en) 2016-02-23 2022-08-02 Salk Institute For Biological Studies Exogenous gene expression in recombinant adenovirus for minimal impact on viral kinetics
US11813337B2 (en) 2016-12-12 2023-11-14 Salk Institute For Biological Studies Tumor-targeting synthetic adenoviruses and uses thereof

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