US20060147420A1 - Oncolytic adenovirus armed with therapeutic genes - Google Patents

Oncolytic adenovirus armed with therapeutic genes Download PDF

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US20060147420A1
US20060147420A1 US11/077,513 US7751305A US2006147420A1 US 20060147420 A1 US20060147420 A1 US 20060147420A1 US 7751305 A US7751305 A US 7751305A US 2006147420 A1 US2006147420 A1 US 2006147420A1
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tumor
cells
adenovirus
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cell
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Juan Fueyo
Candelaria Gomez-Manzano
W.K. Yung
Charles Conrad
Frederick Lang
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University of Texas System
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University of Texas System
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Assigned to BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM reassignment BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FUEYO, JUAN, GOMEZ-MANZANO, CANDELARIA, CONRAD, CHARLES A., LANG, JR., FREDERICK F., YUNG, W.K. ALFRED
Publication of US20060147420A1 publication Critical patent/US20060147420A1/en
Priority to US12/370,232 priority patent/US9061055B2/en
Priority to US14/703,876 priority patent/US10080774B2/en
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    • C12Y305/04001—Cytosine deaminase (3.5.4.1)

Definitions

  • the invention generally relates to the field of oncology and oncolytic adenoviruses. More particularly, it concerns compositions and methods of treating cancer of the brain in a patient using oncolytic adenoviruses armed with therapeutic transgenes.
  • tumor suppressor genes as opposed to proto-oncogenes, is to antagonize cellular proliferation.
  • a tumor suppressor gene is inactivated, for example by point mutation or deletion, the cell's regulatory machinery for controlling growth is upset.
  • the studies of several laboratories have shown that the neoplastic tendencies of such mutated cells can be suppressed by the addition of a nucleic acid encoding a wild-type tumor suppressor polypepitde (a functional tumor suppressor) (Levine, 1995).
  • Brain tumors are metastases to the brain from a primary tumor outside of the central nervous system (CNS). Brain tumors derived from metastases are typically more common than primary tumors of the brain. The most common primary tumors that metastasize to the brain are lung, breast, melanoma, and kidney. These brain metastases are usually in multiple sites, but solitary metastases may also occur.
  • Gene therapy is a promising treatment for brain tumors including gliomas because conventional therapies typically fail and are toxic.
  • identification of genetic abnormalities contributing to malignancies is providing crucial molecular genetic information to aid in the design of gene therapies.
  • Genetic abnormalities indicated in the progression of tumors include the inactivation of tumor suppressor genes and the overexpression of numerous growth factors and oncogenes.
  • Tumor treatment may be accomplished by supplying a polynucleotide encoding a therapeutic polypeptide or other therapeutic that target the mutations and resultant aberrant physiologies of tumors. It is these mutations and aberrant physiology that distinguishes tumor cells from normal cells.
  • a tumor-selective virus would be a promising tool for gene therapy.
  • Targeting the Rb pathway has noted relevance for the treatment of gliomas because abnormalities of the p16/Rb/E2F pathway are present in most gliomas (Fueyo et al., 1998a; Gomez-Manzano et al., 1998). Targeting this pathway by replacement of lost tumor suppressor activity through the transfer of p16 and Rb genes has produced cytostatic effects (Fueyo et al., 1998a; Gomez-Manzano et al., 1998). Transfer of E2F-1 resulted in powerful anti-cancer effect since the exogenous wild-type E2F-1 induced apoptosis and inhibited tumor growth in vivo (Fueyo et al., 1998b).
  • Additional treatments include an adenovirus with therapeutic capabilities or with an ability to be tracked in vivo.
  • the present invention provides an oncolytic adenovirus capable of killing target cells, such as a tumor cells, with a greater efficiency.
  • the invention takes advantage of the discovery that an adenovirus encoding an E1A polypeptide unable to bind the tumor suppressor protein Rb may not replicate in or kill a cell that has a functional Rb pathway, but may replicate in and kill a cell that has a defective Rb pathway.
  • the oncolytic adenovirus is armed or encodes a therapeutic or diagnostic polypeptide. “Armed” is a term that indicates that the virus contains a heterologous nucleic acid sequence encoding a polypepitde of interest or a nucleic acid comprising a polynucleotide of interest.
  • the nucleic acid encoding a therapeutic polypeptide may encode angiopoietin 2 (Ang-2), humanized yeast cytosine deaminase polypeptide (hyCD) or a sodium-iodide symporter (NIS) polypeptide.
  • Ang-2 angiopoietin 2
  • hyCD humanized yeast cytosine deaminase polypeptide
  • NIS sodium-iodide symporter
  • the NIS polypepitde may be used in detecting the location of oncolytic adenovirus within a subject.
  • the adenovirus of the present invention can be delivered by a number routes including, but not limited to intracranial (into the skull cavity) or intravenous administration.
  • the tumor may be a primary tumor or it may be a tumor resulting from a metastasis to the skull or brain.
  • Embodiments of the invention include an oncolytic adenovirus and replication defective adenovirus, as well as wildtype adenoviruses.
  • Certain aspects of the invention include an oncolytic adenovirus comprising an E1A deletion, in particular where the E1A deletion is a deletion of nucleotides encoding amino acids 122 to 129 of the E1A protein (Delta 24) and/or Delta-24-300 adenovirus and an expression cassette encoding a therapeutic or diagnostic gene, including but not limted to an Ang-2 gene, a yeast cytosine deaminase gene, a humanized yeast cytosine deaminase gene, and/or a NIS gene.
  • the nucleic acid encoding the yeast cytosine deaminase polypeptide may be a humanized nucleic acid encoding a yeast cytosine deaminase polypeptide.
  • the humanized nucleic acid encoding the yeast cytosine deaminase comprises the nucleic acid sequence of SEQ ID NO:5.
  • An adenovirus of the invention may comprise additional modifications, such as a nucleic acid encoding a modified adenoviral fiber protein, which in certain aspects may comprise a heterologous peptide motif, which targets various proteins expressed on the surface of a cell, including but not limited to adhesion molecules and/or cell surface receptors, such as EGFR, EGFRvIII, Tie, and Tie2.
  • the heterologous peptide motif can be an RGD motif, an EGFR targeting motif, or a Tie2 targeting motif.
  • the targeting motif alters the tropism of the virus by providing a chimeric fiber protein that includes the particular targeting motif.
  • An adenovirus of the invention typically, will selectively replicate in a cell having a defective Rb pathway.
  • the defective Rb pathway may comprise a defective Rb protein or a defect in other proteins that make up the Rb pathway in a cell.
  • some embodiments of the invention may be used in conjunction with replication defective adenoviruses or other replication selective, replication competent adenoviruses, or combinations thereof.
  • methods of treating cancer in a patient include administering to a patient an effective amount of a composition comprising an oncolytic adenovirus, preferably Delta 24, comprising an expression cassette encoding therapeutic gene, including but not limited to an Ang-2 gene, a yeast cytosine deaminase gene, a humanized yeast cytosine deaminase and/or a NIS, and administering an effective amount of a pro-drug, wherein the pro-drug is metabolized to a cytotoxic drug by a polypeptide encoded by the yeast cytosine deaminase gene.
  • a composition comprising an oncolytic adenovirus, preferably Delta 24, comprising an expression cassette encoding therapeutic gene, including but not limited to an Ang-2 gene, a yeast cytosine deaminase gene, a humanized yeast cytosine deaminase and/or a NIS, and administering an effective amount of a pro-drug, wherein the pro-d
  • the nucleic acid encoding a yeast cytosine deaminase is preferably a humanized nucleic acid encoding a yeast cytosine deaminase.
  • the cancer to be treated may include one or more cells comprising a mutated Rb pathway.
  • the cancer may comprise one or more cells comprising a mutated Rb polypeptide.
  • the cell to be treated may be a tumor cell or a brain tumor cell, more particularly a glial cell or glial derived cell.
  • the methods of the invention may further comprise determining whether the cell or cells has a defect, e.g. a mutation, in a gene encoding a polypeptide in the Rb pathway, in a gene encoding Rb or both.
  • a defect may be caused by a deletion or a mutation in the nucleic acid encoding a gene, include coding and non-coding regions of the gene.
  • a cell is not killed if it does not comprise a mutated polypeptide in the Rb pathway.
  • the oncolytic adenovirus may be suitably dispersed in a pharmacologically acceptable formulation.
  • the composition may comprise a suitable buffer and may further comprise one or more lipids.
  • the composition may be administered through various routes including: intradermal, transdermal, parenteral, intracranial, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral routes of administration.
  • the compositions may be directly injected into a tumor. Furthermore, the administration of a composition may occur more than once and may be administered at least three times to the patient.
  • the methods of the invention may further comprise administering to the patient a second therapy, wherein the second therapy is anti-angiogenic therapy, chemotherapy, immunotherapy, surgery, radiotherapy, immunosuppresive agents, or gene therapy with a therapeutic polynucleotide.
  • the second therapy may be administered to the patient before, during, after or a combination thereof relative to the administration of the onclolytic adenovirus composition.
  • Chemotherapy includes, but is not limited to an alkylating agent, mitotic inhibitor, antibiotic, or antimetabolite.
  • the chemotherapy may comprise administration of CPT-11, temozolomide, or a platin compound.
  • Radiotherapy may include X-ray irradiation, UV-irradiation, ⁇ -irradiation, or microwaves.
  • the oncolytic adenovirus may be administered to the patient preferably in doses of approximately 10 3 to about 10 15 viral particles; more preferably about 10 5 to about 10 12 viral particleseven more preferably about 10 7 to about 10 10 viral particles.
  • methods include methods for treating a brain tumor in a patient comprising identifying a patient having a brain tumor; and contacting the tumor with an oncolytic adenovirus comprising an expression cassette, preferably an expression cassette comprising an Ang-2 gene, a yeast cytosine deaminase gene, and/or a NIS gene.
  • the oncolytic adenovirus may also comprise a targeting moiety.
  • the methods may include contacting the tumor with the adenovirus by injecting the adenovirus intracranially into the patient.
  • Embodiments of the invention include methods for treating a subject having a brain tumor by determining that a cell in the tumor has a mutation in the Rb pathway; administering intracranially to the patient an oncolytic adenovirus comprising an expression cassette, wherein the expression cassette comprises a therapeutic or diagnostic gene.
  • the methods may further include administering to the patient a pro-drug that is converted by cytosine deaminase to a therapeutic drug.
  • Methods of the invention also include treatment of a subject having a tumor that has metastasized to the brain by determining that a cell in the tumor has a mutation in the Rb pathway; administering intracranially to the patient an oncolytic adenovirus and a therapeutic gene such as, but not limited to Ang-2 gene, yeast cytosine deaminase gene, humanized yeast cytosine deaminase, and/or NIS gene.
  • the methods may further include administering to the patient a pro-drug that is converted by cytosine deaminase to a therapeutic drug, a therapeutic or an anti-angiogenic agent(s) (e.g., anti-sense VEGF or its equivalents).
  • Embodiments of the invention also include an adenovirus (e.g., Delta 24, Delta-24-300) comprising; and an expression cassette encoding a sodium-iodide symporter (NIS) gene.
  • the nucleic acid encoding NIS may comprise the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3.
  • the adenovirus may further comprise additional modifications, such as a nucleic acid encoding a modified adenoviral fiber protein, which may comprise a heterologous peptide motif, preferably the heterologous peptide motif is an RGD, vIII, or PEPHC1 motif.
  • the expression cassette may comprise a chimeric polynucleotide encoding Ang-2, hyCD, or NIS and a nucleic encoding a second peptide or polypeptide, wherein a sequence encoding a protease-cleavable amino acid linker is between the NIS coding sequence and the nucleic acid encoding the second peptide or polypeptide.
  • the protease-cleavable amino acid linker may be an auto-cleaving amino acid sequence.
  • the sequence encoding a protease-cleavable linker can be fused in-frame to the 3′ or 5′ end of the Ang-2, NIS and/or hyCD encoding polynucleotide.
  • the protease cleavable linker is cleaved by furin.
  • the protease-cleavable linker may be identical to a linker present in a cytoplasmic protein.
  • the expression cassette may comprises a chimeric polynucleotide comprising the Ang-2, NIS or hyCD encoding polynucleotide, or combination thereof, and a nucleic acid sequence encoding a second peptide or polypeptide, wherein the chimeric polynucleotide encode an internal ribosome entry site between NIS encoding polynucleotide and a second nucleic sequence encoding a second polypeptide.
  • FIG. 1 A block diagram adenovirus
  • the step of detecting can be performed quantitatively to determine the amount of transported diagnostic agent in the mammal.
  • the diagnostic agent may be iodine.
  • Iodine may be iodine radionuclides 131 I, 123 I, 124 I, or 125 I.
  • the diagnostic agent may also be technesium pertechnetate, rhenium perrhenate, radioactive iodine, or other diagnostic elements used in the art.
  • a nucleic acid encoding an oncolytic adenovirus comprises a chimeric polynucleotide comprising a nucleic acid sequence encoding NIS and a second transgene.
  • a second transgene may encode a second therapeutic polypeptide.
  • Embodiments of the invention include methods of treating cancer in a patient comprising administering to a patient an effective amount of a composition comprising an oncolytic adenovirus comprising an expression cassette encoding an Ang-2, NIS or hyCD polynucleotide; and administering an effective amount of a diagnostic agent, a therapeutic agent a therapy enhancing agent or an anti-angiogenic agent.
  • the diagnostic agent, the therapeutic agent or the therapy enhancing agent is transported into a cell by a NIS polypeptide encoded by the NIS polynucleotide.
  • the cancer may comprise one or more cells comprising a mutated polypeptide in the Rb pathway, a mutated Rb polypeptide or both.
  • the method may further comprise determining whether one or more cell has a mutation, resulting in a defective protein, in a gene encoding a polypeptide in the Rb pathway, in the gene encoding Rb, or both.
  • the method may comprise assaying Rb activity.
  • Rb activity may be assayed for using an anti-Rb antibody or by determining whether Rb in the cell inhibits E2F activation of transcription.
  • a cell may be a glial cell and/or a tumor cell.
  • the methods may include an adenovirus composition suitably dispersed in a pharmacologically acceptable formulation.
  • the composition may comprise a suitable buffer and/or a lipid.
  • Composition may be administered by a variety of routes including intradermal, transdermal, parenteral, intracranial, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral routes of administration.
  • the composition is directly injected into a tumor. Administration of a composition may occur more than once, twice, three times or more.
  • the method may further comprising administering to the patient a second therapy, wherein the second therapy is anti-angiogenic therapy, chemotherapy, immunotherapy, surgery, radiotherapy, immunosuppresive agents, or gene therapy with a therapeutic polynucleotide.
  • the second therapy may be administered to the patient before, during, after or a combination thereof relative to the administration of the onclolytic adenovirus composition.
  • Chemotherapy includes, but is not limited to an alkylating agent, mitotic inhibitor, antibiotic, or antimetabolite.
  • the chemotherapy may comprise administration of CPT-11, temozolomide, or a platin compound.
  • Radiotherapy may include X-ray irradiation, UV-irradiation, ⁇ -irradiation, or microwaves.
  • the oncolytic adenovirus may be administered to the patient preferably in doses of approximately 10 3 to about 10 15 viral particles; more preferably about 10 5 to about 10 12 viral particleseven more preferably about 10 7 to about 10 10 viral particles.
  • Embodiments discussed in the context of a methods and/or composition of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method may be applied to other methods of the invention as well.
  • FIG. 1 is a schematic representation of the ⁇ 24 adenovirus. Shown are the 2 Rb-binding regions of the E1A sequence (hatched boxes). The 24 nucleotides that have been deleted and the corresponding amino acid translation are indicated.
  • FIG. 2 shows the effects of ⁇ -24 in a mouse xenograft intracranial glioma tumor model.
  • FIG. 3 shows visualization of NIS expression with a transfected U87MG tumor implanted subcutaneously in NuNu mice flanks.
  • a 2-3 mm palpable tumor in the animal on the left is clearly visible after injection with 0.2 mCi of 99mTcO4 in 0.1 ml PBS.
  • the animal to the right did not have a palpable tumor.
  • the control animals below do not have hNIS expressing flank tumors.
  • FIG. 4 shows an MRI of nude mouse glioma xenograft model using U87MG cells and a correlated pathologic section (H&E).
  • the MRI panels from left to right show the noncontrast T 1 , and Gd-contrast T 1 , and T 2 sequences.
  • the increase in hematoxylin staining of the tumor can be appreciated in the pathologic sections immediately infereior to the Teflon screw.
  • FIGS. 5A-5C shows viral inclusion bodies (indicative of actively replicating adenovirus), FIG. 5A , staining for late transcribed viral genes (hexon protein), FIG. 5B and the typical “zonal” spread through a tumor, FIG. 5C U87MG xenograft infected with ⁇ 24-RGD incubated with E1A antibody.
  • FIG. 6 shows a western blot of the transgene expression of yeast CD in U87MG glioma cells using ⁇ 24 adenovirus: lane 1 U87MG cells unifected; lane 2 ⁇ 24-CD at 1 MOI and 24 h post-infection; lane 3 ⁇ 24-CD at 10 MOI and 24 h post-infection; lane 4 ⁇ 24-CD at 100 MOI and 24 h post-infection; lanes 5-7 are similar to lanes 2-4 except that they are 48 h post-infection instead of 24 h; lane 8 ⁇ 24 adenovirus.
  • FIG. 7 shows expresion of cytosine deaminase by ⁇ 24-hyCD relative to a stable expressing clone.
  • the X-axis demonstrates two cell lines at increasing MOI from 0.1 to 100 MOI and from 24 to 96 h.
  • the Y axis shows the increase of expression in logarithmic increase.
  • FIGS. 8A-8D shows a demonstration of animal whole-mount preparation and autoradiography imaging of NIS expression by 188 Re accumulation.
  • Breast tumor (13762F) bearing rat was injected with the eluent from W-188/Re-188 generator.
  • FIG. 9 shows the pharmacodynamic studies of animals injected with eluant from an W/Re-188 Rhenium generator at 100 ⁇ Ci.
  • the uptake of 188 ReO 4 corresponds to tissues that express NIS (thyroid and stomach). Urine and later fecal accumulation (at 24 hours) is also observed. Importantly, the organ tumor sites to be examined (brain and lung) do not demonstrate NIS-dependent active accumulation of radionuclides.
  • FIG. 10 shows accumulation of Tc 99 O 4 in a U251MG glioma cell line after transient transfections with the hNIS-containing plasmid (pcDNA3.1-Zeo). Comparing the accumulations of Tc 99 O 4 peripate is determined by cpm after incubation for 30 min with 2 ⁇ Ci. Cells were then washed with ice-cold Hanks balanced salt solution and Tc 99m O 4 released by adding absolute ethanol. Parental cell line was compared to the active clone in the first two lanes. Controls using the inhibitor to the hNIS pump (NaClO 4 ) against the parental cell line, vector control and finally positive clone simultaneousely inhibited by NaClO 4 .
  • NaClO 4 the inhibitor to the hNIS pump
  • FIG. 11 shows accumulations of Tc 99 O 4 in U251 glioma cell line infected with ⁇ 24-NIS.
  • FIGS. 12A-12F shows a specimen from a patient treated with a single injection of Ad-p53 as a dose of 3 ⁇ 10 10 vp in 1 ml (level 1).
  • FIG. 12A photograph of sugical specimen that was removed en bloc. The injection catheter is protruding from the surface of the tumor.
  • FIG. 12B formalin fixed tumor blocks. Specimen from A has been cur perpendicular to the catherter. The hole created by the catheter is evident.
  • FIG. 12C low power view (300 ⁇ ) of same speciment immunostained with antibody to p53 protein. The hole from the catheter is at the top of the photomicrogaph. Transfected tumor cells stain darkly and are distributed within 5 mm of the injection site.
  • FIG. 12D High power view (500 ⁇ ) of same section a C demonstrating transfected cells.
  • FIG. 12E View of adjacent section of that shown distrubution as p53 staining.
  • FIG. 12F low power (10 ⁇ ) view of cress-section of whole speciment. The catheter was within the central hole. Blue staining around hole shows distribution of exogenous p53.
  • FIGS. 13A-13B shows Generation of ⁇ 24-hyCD adenovirus.
  • FIG. 13A The humanized yeast cytosine deaminase sequence is depicted as the complete nucleotide sequence of yeast CD as well as the nucleotide substitutions (highlighted in bold) for optimized humanized codon preference. A Kozak sequence is placed immediately before the starting codon (italicized). Proximal HindIII and distal XbaI restriction sites are placed between parentheses.
  • FIG. 13B Schematic illustration of ⁇ 24-hyCD showing the 24-bp deletion in the E1A region (nucleotides and corresponding amino acid residues are shown) and the insertion of the modified cytosine deaminase (hyCD) expression mini-cassette in the deleted E3 region.
  • FIGS. 14A-14B shows analyses of the expression and enzymatic activity of hyCD.
  • FIG. 14A Western blot analyses of the expression of exogenous hyCD in U251MG cells. Expression was apparent by 24 h after infection in a dose-dependent manner. As expected, mock (M) or ⁇ 24-treatment at an pfu/cell of 100 did not result in the expression of CD. The expression level of actin is showed as a loading control.
  • FIG. 14B Thin layer chromatography analyses of cytosine deaminase. U251MG cells stably transfected with the hyCD encoding polynucleotide were treated with cytosine at the indicated times and assessed for hyCD activity. The migrated uracil spot was visualized with ultraviolet excitation at 260 ⁇ . Lanes 5 and 6 showed a dose-dependence positive result. Negative (cytosine) and positive (uracil) controls are shown in lanes 1 and 2, respectively.
  • FIG. 15 shows dose response curves of parental and hyCD stable-transfected U251MG cells treated with 5-FC, as assessed by cell viability assay. Note the shift to the left of the IC 50 curve for the hyCD stable-transfected U251MG cells in the presence of 5-FC. IC 50 values for both cultures are indicated.
  • FIGS. 16A-16C shows in vitro antiglioma effect of ⁇ 24-hyCD.
  • FIG. 16A Crystal violet analyses of the cytopathic effect of ⁇ 24-hyCD or ⁇ 24 in U251MG cells treated with either 5-FU or 5-FC. Each well represents a different time period of 5-FU or 5-FC treatment indicated in days.
  • FIG. 16B Quantification of the viability of U251MG cells by MTT assay, treated with ⁇ 24 (left) or ⁇ 24-hyCD (right) and 5-FC at the indicated doses. UVi, UV-inactivated adenoviral treatment, 5 MOI.
  • FIG. 16C Demonstration of ⁇ 24-hyCD-mediated bystander effect.
  • U25 1 MG cells were treated with ⁇ 24-hyCD or ⁇ 24 (at an pfu/cell of 10) 24 h after infection, and conditioned media was collected.
  • the conditioned media were inactivated by UV to ensure that no replication-competent virus was carried over.
  • Conditioned media at the indicated volumes were transferred to fresh U251MG cultures and incubated for 48 h. Viability was assessed by MTT assay.
  • FIG. 17 shows the treatment with ⁇ 24-hyCD+5-FC significantly improves survival of nude mice implanted with U87MG intracranial xenografts.
  • Data are represented as Kaplan-Meier survival curves from the day of U87MG intracranial implantation (day 0) after intratumoral injection (day 3) with a single dose of experimental viral groups or PBS alone controls.
  • 5-FC was administered on day 5 after treatment (E) or on day 15 after treatment (L).
  • E E
  • L day 15 after treatment
  • FIGS. 18A-18B show Tie2 expression in glioma cell lines: a) RT-PCR, and b) Western blotting analysis (membrane subfraction) of the expression of Tie2 in a panel of glioma cell lines. HUVEC cells and NIH3T3 cells were used as positive and negative cotnrols.
  • FIG. 18B shows Ang-2 downregulates VEGF secretion. Shown here is the secreted VEGF expressed as a percentage relative to that of the mock-infected cells (equal to 100%) (ELISA). Values shown as mean ⁇ SD (+, P ⁇ 0.005; *, P>0.5).
  • FIG. 19 shows Ang-2 reduced expression of HIF-1 ⁇ in glioma cells.
  • HIF-1 ⁇ Protein nuclear levels of HIF-1 ⁇ decreased after Ang-2 transfer, compared with control-treated cells in normoxia (21% O2) and hypoxia (1% O2) conditions in U-87 MG and D54 MG cells. Note that the expression of HIF-1 ⁇ , did not modify after Ang-2 transfer into U-251 MG cells, CMV, AdCMV; Ang-2, AdAng-2.
  • FIG. 20 shows Ang2 downmodulates HIF-DNA binding activity.
  • U-87 MG cells were treated with AdAng2 (Ang2), AdCMV-pA (CMV) or were mock-infected. Equal amounts of nuclear extracts were analyzed for HIF-DNA binding activity in a hypoxic or normoxic setting.
  • Competitive experiments were performed using wild-type (wt) or mutant (mut) oligonucleotides. Data are represented as the mean of three independent experiements (SD was less than 15%).
  • FIG. 21 shows Ang-2 inhibits Ang-1 mediated MEK/ERK phosphorylation of U-87 MG glioma cells.
  • HUVEC and U-87 MG cells were mock-, AdCMV- or AdAng-2-Infected. 24 hours later they were overnights serum-starved, and then stimulated with rhAng-2 for 10 min.
  • Cell lysates were collected and analyzed by Western blotting for expression of phospho- and total p42/p44 MAPK.
  • Ang-1 increased ERK 1/2 phosphorylation what was inhibited by the viral-transduced Ang-2.
  • FIG. 22 shows Transcriptional modulation of VEGF by Ang-2 is probably related to the downmodulation of HIF-1 ⁇ proteins levels (U-87 MG). Luciferase activity is expressed as relative to the VEGF-1.5 kb promoter activity in mock-treated cells (equal to 100%). The result shows that Ang-2 decreased the transcriptional activity of the two contstructs that contain the HIF-1 ⁇ binding site (2.6 and 1.5 kb promoters; Gomez-Manzano et al., 2003), however did not modified the activity of the 0.35-kb promoter, susceptivle to p53NHL regulation.
  • FIG. 23 is a presentation of the E1A region of wild-type (wt), Delta-24, Delta-300 and Delta-24-300 adenoviruses. Solid area: p300 binding area; hatched area: Rb Binding area.
  • FIG. 24 shows Anti-glioma effect of Delta-24-300, Delta-24, Delta-300 and wt-Ad in vitro. Cellse were infected at indicated MOI and viability was assessed ty Trypan blue exclusion. Data shown as the relative percentage of cells alive with UVi-infected cultures equal to 100%
  • FIG. 25 shows Anti-glioma effect of Delta-24-300, Delta-24, Delta-300 and wt-Ad in vitro.
  • Cell viability was assessed by crystal violet staining after viral infection with a range of MOI, 0.5-10 MOI, as indicated in top left panel.
  • M mock-infection; Uvi. UV-inactivated wt-Ad.
  • FIGS. 26A-26B show differential expression of Delta-24-300 viral proteins in glioma and normal human astrocyte (NHA) cultures.
  • FIG. 26A Western blot analysis of the E1A and fiber proteins in U-251 MG and NHA cell extracts 16 hours after infection with Delta-24, Delta-300 or Delta-24-300 at an MOI of 50.
  • FIG. 26B Actin expression is used as a loading control. Quantification of the fiber protein signal by densitometry following normalization to actin levels. Fiber levels from Delta-24-trated cultures are arbitrarily given the value of 100%.
  • FIGS. 27A-27B show analyses or adenoviral-induced E2F-1 promoter activity. Proliferating NHA were transfected with the E2F-1 reporter construct, and 1 h later the cells were treated with the indicated adenovirus at 5 MOI. Luciferase activity was determined 20 h after infection. Data is shown as relative means ⁇ SEM of normalized luciferase measurements (Ad300, equal to 100%).
  • FIG. 27B shows Viral replication analysis. Replicating NHA and U-87 MG giloma cell lines were infected with wt Ad, Delta-24, Delta-300 or Delta-24-300 at 1 MOI. Three days later viral titers were determined by TCID50 method. Viral titers from two independent experiments were normalized to wild-type adenovirus titers. Values in the chart represent the difference between the viral titers in U-87 MG cells and NHA.
  • FIG. 28 shows chimeric fiberprotein.
  • Tail N-terminal of AdS fiber protein (1-83aa); T4 febritin; bacteriophage T4 fibritin helican domain and fold on (233-487 aa);
  • Linker G4SG4SG4S linker;
  • Ligand PEPHC1 (HFLIIGFMRRALCGA (SEQ ID NO:19).
  • FIG. 29 shows infectivity assay of human mesenchymal stem cells. Light (left) and fluorescence (right) microscopy of the same fieldof human mesenchymal stem cells at the indicated hours after infection with a replication-deficient adenovirus carrying the GFP cDNA (Ad-GFP) or the tropism-modified Ad-GFP adenovirus (Ad-GFP-RGD) at 100 MOI.
  • Ad-GFP replication-deficient adenovirus carrying the GFP cDNA
  • Ad-GFP-RGD tropism-modified Ad-GFP adenovirus
  • FIG. 30 shows MRI studies nude mice bearing intracranial U-87 MG xenografts. Animals were imaged 7 and 14 days after cell implantation. Shown are T2 images (left) and gross morphologic features (right, H&E) of the brains of tumors treated with UV-inactivated Delta-24-300 or Delta-24-300. Note that Delta-24-300 treatment resulted in inhibition of the tumoral growth as seen simulteaneously in both pathological and imaging studies.
  • FIG. 31 shows specific binding of EGFRvIII-peptide (PEPCH1) to EGFRvIII-expressing cells. Briefly, the inventors performed membrane fluorescence staining of single-cell suspensions after incubating U-87 MG, U87.wtEGFR, and U-87. ⁇ EGFRvIII (Nishikawa et al., 1994) with the anti-EGFRvIII peptide (PEPCH1)-FITC (1 ⁇ M) (Campa et al., 2000) for 30 min.
  • PPCH1-FITC anti-EGFRvIII peptide
  • the cells were then washed twice with PBS and then analyzed for fluorescence with an EPICS XL-MCL flow cytometer (Beckman-Coulter Inc., Miami, Fla.) using a 488 nm argon laser for excitation. Fluorescence was tetected with a 520 nm band-pass filter, and all cytometric data was analyzed with the System II software (Beckman-Coulter Inc.). Data are shown as the percentages of FICT-positive cells (empty graph) compared to mock-treated cells (solid graphs)from a representative experiment.
  • FIG. 32 shows data are shown after normalization of the binding value, as FITC-positive U87. ⁇ EGFR cells equal to 100%.
  • FIG. 33 shows construction of pVK.Delta-24-vIII. Schema showing key information of the adenoviral vector pVK500C and accomplished homologous recombinations.
  • FIGS. 34A-34B shows analysis of the Delta-24-vIII genome structure.
  • FIG. 34A shows confirmation of the mutant-E1A region.
  • DNA isolated from Delta-24-vIII adenovirus was subjected to PCR assay utilizing pair of primers flanking the deletion in the E1A region and
  • FIG. 34B shows posteriorly subjected to restriction enzyme analysis using BstXI which cleaves tice in the E1A region amplified from Wild-type adenovirus, and one in the E1A from Delta-24. Two clones were asolited and tested (Clon#12 and #13). pVK500C and pVK.Delta24 were used as controls.
  • FIGS. 35A-35B Analysis of the Delta-24-vIII genome structure.
  • FIG. 35A shows confirmation of the chimeric fiber.
  • FIG. 35B shows DNA isolated from Delta-24-vIII adenovirus was subjected to PCR assay utilizing pair of primers as depicted below. Two clones were asolited and tested (Clons #12 and #13). pVK500C and pVK.Delta24 were used as controls.
  • Malignant tumors that are intrinsically resistant to conventional therapies are significant therapeutic challenges.
  • Such malignant tumors include, but are not limited to malignant gliomas and recurrent systemic solid tumors such as lung cancer.
  • Malignant gliomas are the most abundant primary brain tumors having an annual incidence of 6.4 cases per 100,000 (CBTRUS, 2002-2003). These neurologically devastating tumors are the most common subtype of primary brain tumors and are one of the deadliest human cancers.
  • GBM glioblastoma multiforme
  • median survival duration for patients ranges from 9 to 12 months, despite maximum treatment efforts (Hess et al., 1999).
  • a prototypic disease, malignant glioma is inherently resistant to current treatment regimens (Shapiro and Shapiro, 1998). In fact, in approximately 1 ⁇ 3 of patients with GBM the tumor will continue to grow despite treatment with radiation and chemotherapy. Median survival even with aggressive treatment including surgery, radiation, and chemotherapy is less than 1 year (Schiffer, 1998). Because few good treatment options are available for many of these refractory tumors, the exploration of novel and innovative therapeutic approaches is essential.
  • the ⁇ 24 adenovirus is derived from adenovirus type 5 (Ad-5) and contains a 24-base-pair deletion within the CR2 portion of the E1A gene. Significant antitumor effects of ⁇ 24 have been shown in cell culture systems and in malignant glioma xenograft models.
  • Oncolytic adenoviruses include conditionally replicating adenoviruses (CRADs), such as Delta 24, which have several properties that make them candidates for use as biotherapeutic agents.
  • CRADs conditionally replicating adenoviruses
  • Delta 24 have several properties that make them candidates for use as biotherapeutic agents.
  • One such property is the ability to replicate in a permissive cell or tissue, which amplifies the original input dose of the oncolytic virus and helps the agent spread to adjacent tumor cells providing a direct antitumor effect.
  • Embodiments of the present invention couple the oncolytic component of Delta 24 with a transgene expression approach to produce an armed Delta 24.
  • Armed Delta 24 adenoviruses may be used for producing or enhancing bystander effects within a tumor and/or producing or enhancing detection/imaging of an oncolytic adenovirus in a patient, or tumor associated tissue and/or cell. It is contemplated that the combination of oncolytic adenovirus with various transgene strategies will improve the therapeutic potential against a variety of refractory tumors, as well as provide for improved imaging capabilities.
  • an oncolytic adenovirus may be administered with a replication defective adenovirus, another oncolytic virus, a replication competent adenovirus, and/or a wildtype adenovirus. Each of which may be adminstered concurrently, before or after the other adenoviruses.
  • Embodiments of the invention include the Delta 24 adenovirus comprising an expression cassette containing a heterologous gene.
  • heterologous genes include therapeutic genes, pro-drug converting enzymes, cytosine deaminase (to convert 5-FC to 5-FU), a yeast cytosine deaminase, a humanized yeast cytosine deaminase, an image enhancing polypeptides, a sodium-iodide symporter, anti-sense or ihibitory VEGF, Bcl-2, Ang-2, or interferons alpha, beta or gamma.
  • a Delta 24 oncolytic adenoviral strategy is coupled with an Ang-2 transgene, sodium-iodide symporter (NIS) transgene, humanized yeast CD or a yeast CD transgene approach for augmenting bystander effects and/or obtaining imaging of the replicating virus within an in vivo tumor setting.
  • NIS sodium-iodide symporter
  • adenovirus is a 36 kb, linear, double-stranded DNA virus (Grunhaus and Horwitz, 1992).
  • Adenoviral infection of host cells results in adenoviral DNA being maintained episomally, which reduces the potential genotoxicity associated with integrating vectors.
  • adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.
  • Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.
  • NIS sodium Iodide Symporter
  • Embodiments of the invention include a ⁇ 24 adenovirus encoding an NIS polypeptide.
  • NIS is a transmembrane pump that was originally isolated from the thyroid gland and functions physiologically by concentrating iodide within thyroid tissue. NIS is also able to mediate the uptake and concentration of other diagnostic and therapeutically important radionuclides, such as Technicium-99m pertechnetate ( 99m TcO 4 ) and Rhenium-188 perrenate ( 188 ReO 4 ).
  • 99m TcO 4 Technicium-99m pertechnetate
  • Rhenium-188 perrenate 188 ReO 4
  • the mechanism mediating iodide uptake across the basloateral membrane of thyroid follicular cells has been elucidated by cloning and characterization of the sodium iodide symporter (NIS) (Smanik et al., 1996; Dai et al., 1996).
  • NIS sodium iodide symporter
  • the human NIS encoding polynucleotide contains an open reading frame of 1,928 nucleotides and encodes a 643-amino acid trans-membrane protein, which has an expected molecular weight of 69 kDa. This pump has been shown to be capable of concentrating multiple isotopes within the intracellular compartment, including 123 I, 125 I and 131 I.
  • NIS is an intrinsic membrane glycoprotein with 13 putative transmembrane domains which is responsible for the ability of cells of the thyroid gland to transport and sequester iodide.
  • An NIS of the present invention is comprised of a polypeptide having the activity of a sodium iodide symporter, including, but not limited to polypeptides of SEQ ID NO:2 and 4 that are encoded by the nucleic acid sequences of SEQ ID NO:1 and 3, for human and rat respectively.
  • NIS expression in thyroid tissues is dependent upon stimulation of the cells by pituitary-derived thyroid stimulating hormone (TSH) and can therefore be readily suppressed in this tissue by treatment with Thyroxine.
  • TSH pituitary-derived thyroid stimulating hormone
  • TSH-regulated NIS expression is specific for thyroid cells, whereas many other organs do not concentrate iodine due to lack of NIS expression.
  • Cloning and characterization of the human and rat NIS genes (SEQ ID NO:1 and SEQ ID NO:3 respectively; GenBank Accession numbers AC005796 and U60282 respectively) permits NIS encoding nucleic acid delivery into non-thyroid cells, thereby allowing these cells to trap and sequester radio-labeled iodine.
  • NIS functions well as a localization tag for several reasons.
  • the NIS is synthesized in the mammal, using the mammals own protein synthetic machinery, and thus is recognized as self, thereby avoiding a potential immune response.
  • the NIS is a useful localization tag according to the present invention as it should have no significant effect on the biological properties of the genetically modified cells. Given that the only known function of the NIS is to transport iodine across the cell membrane, it should not adversely affect endogenous cellular function (U.S. Pat. No. 6,586,411, which is incorporated herein by reference).
  • the therapeutic index of successful treatment of thyroid carcinomas and their metastases by 131 I is based on the expression of the NIS within thyrocytes (Levy et al., 1998a; Kohrle, 1999; Levy, 1998b). Additionally, treatment failure parallels the loss of expression of NIS. Because radioisotopes are widely established in diagnostic nuclear radiographic studies as well as being useful in treatment strategies, a NIS encoding polynucleotide is an attractive target for radioisotope-mediated cancer gene therapy (La Perle et al., 2002). Recently, many tumor lines, including glioma cell lines, have been genetically modified to express NIS using both viral and nonviral transfer vectors (Cho et al., 2000 and 2002).
  • this pump was sodium dependent and capable of transporting a wide, variety of anions (I ⁇ , CIO ⁇ , SCN ⁇ , SeCN ⁇ , NO 3 ⁇ , Br ⁇ , BF 4 ⁇ , IO 4 ⁇ ;, and BrO 3 ⁇ ), although, perchlorate (ClO 4 ) inhibited the NIS pump.
  • anions I ⁇ , CIO ⁇ , SCN ⁇ , SeCN ⁇ , NO 3 ⁇ , Br ⁇ , BF 4 ⁇ , IO 4 ⁇ ;, and BrO 3 ⁇
  • perchlorate ClO 4
  • compositions and methods described herein are designed to improve transgene expression by combining the power of an oncolytic virus with NIS expression for radionuclide accumulation.
  • Cytosine deaminase (CD, EC 3.5.4.1) catalyzes the hydrolysis of cytosine to uracil.
  • the enzyme which plays an important role in microbial pyrimidine metabolism (O'Donovan and Neuhard, 1970), has been isolated from several different microorganisms, but does not appear to be present in mammalian cells (Nishiyama et al., 1985).
  • CD from various organisms have been shown to differ significantly in terms of molecular weight, stability, and subunit composition.
  • CD from Salmonella typhimurium has been purified to homogeneity (by SDS-PAGE) and is composed of 4 subunits of 54 kilodaltons (kDa) each (West et al., 1982) while the enzyme from Escherichia coli has a molecular weight of 200 kDa and is composed of 35 and 46 kDa subunits (Katsuragi et al., 1986). Both of these enzymes are highly thermostable, and maintain high activity at 55° C.
  • Bakers' yeast Saccharomyces cerevisiae
  • CD previously obtained from Saccharomyces cerevisiae has a molecular weight of 34 kDa as determined by gel filtration (Ipata et al., 1971, 1978) and 32-33 kDa as determined by SDS-PAGE and amino acid analysis (Yergatian et al., 1977).
  • the CD enzyme that has been previously isolated from bakers' yeast therefore appears to be a monomeric protein.
  • a humanized yeast cytosine deaminase (hyCD) (SEQ ID NO:5 and 6) may be used minimize any immune reactions to the protein.
  • CD has been used therapeutically for the conversion of the pro-drug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU) (Katsuragi et al., 1987; Nishiyama et al., 1985; Senter et al., 1991).
  • bacterial sources of CD are impractical for such use, requiring large-scale cultivation in order to obtain adequate activity.
  • Yeast can be used as a source of CD to overcome these problems.
  • the thermal instability of the previous yeast-derived product requires that the enzyme be immobilized prior to its use (Katsuragi et al., 1987).
  • the isolation and purification of a thermally stable yeast CD provides an improved enzyme for use in anticancer therapy.
  • Such novel constructs increase the efficiency or usefulness of the enzyme in anticancer therapy (U.S. Pat. No. 5,545,548, which is incorporated herein by reference).
  • cytosine deaminase were expressed in the setting of a ⁇ 24 infection.
  • the ⁇ 24 backbone was modified using a yeast cytosine deaminase encoding polynucleotide cloned into the E3 region of ⁇ 24.
  • yeast cytosine deaminase encoding polynucleotide cloned into the E3 region of ⁇ 24.
  • the cytosine deaminase gene has superior catalytic properties compared with the bacterial form of the enzyme (Kievit, 1999). Extremely high specific activity of this enzyme has been detected when the oncolytic virus is allowed to infect human glioma tumors.
  • Assays have demonstrated a highly specific conversion of cytosine to uracil or 5-fluorocytosine (5-FC) to 5FU.
  • GDEPT gene-dependent enzyme/pro-drug therapy
  • Angiogenesis is critical for the development and maintenance of glioblastomas, the most malignant and common form of primary brain tumors.
  • Current evidence indicates that recruitment of tumor vessels from the normal surrounding tissue requires a delicate balance between the timing and level of expression of two major angiogenesis factors: angiopoietin 2 (Ang-2) and the vascular endothelial growth factor (VEGF).
  • Ang-2 is typically expressed at sites of vascular remodeling in the adult, notably in the female reproductive tract.
  • VEGF vascular endothelial growth factor
  • oncolysis with anti-angiogenesis may produce a synergistic effect since the anticancer mechanisms are different but complementary.
  • the success of the strategy is assisted by the relatively slow rhythm of oncolytic propagation. That allows time for an anti-angiogenic nucleic acid or polypeptide, sucy as, but not limted to an Ang-2 protein, to be produced, ultimately favoring delivery to the extracellular compartment. For that reason, the oncolytic adenovirus is used as an improved adenoviral vector to deliver high and continuous levels of Ang-2 to the tumor.
  • replication-competent adenoviruses that are currently being pursued is applying replication-competent systems as facilitators for the delivery of replication-deficient E1-deleted adenovirus vectors.
  • This is a useful approach because co-infection of a cancer cell with both a replication-deficient E1-deleted adenovirus and a replication-competent adenovirus results in the replication of both adenoviruses.
  • the replication-deficient vector uses the expression of the E1A protein by the replication-competent adenovirus to replicate. This system can thus generate and multiply the number of copies of exogenous protein.
  • the inventors will use this strategy to simultaneously deliver an anti-angiogenic nucleic acid or polypeptide, such as Ang-2 (in a replication competent, oncolytic or replication defective adenovirus) and antisense VEGF (in a replication competent, oncolytic or replication defective adenovirus) (Im et al., 1999) adenoviruses to human glioma xenografts.
  • an anti-angiogenic nucleic acid or polypeptide such as Ang-2 (in a replication competent, oncolytic or replication defective adenovirus) and antisense VEGF (in a replication competent, oncolytic or replication defective adenovirus) (Im et al., 1999) adenoviruses to human glioma xenografts.
  • Ang-2 in a replication competent, oncolytic or replication defective adenovirus
  • VEGF in a replication competent, oncolytic or replication defective adenovirus
  • Delta-24 based antiangiogenic agents may be compared to or used in conjunction with other antiangiogenesis agents that are or will be tested in clinical trials for patients with malignant gliomas.
  • the inventors are using cell lines with a greater clinical relevance, as LN229 (SNB19 as alternative). These cell lines exhibit an invasive phenotype when implanted intracranialy in animal models. Both cell lines express the Tie2 receptor and are tumorigenic in intracranial settings.
  • VEGF regulates Ang-2 in tumors there is not any evidence of a feedback signaling loop which will be required to coordinate and modulate the expression of the Ang-2 and VEGF molecules.
  • the specific time when the expression of Ang-2 is required during the different stages of tumor formation is also not known.
  • no treatments have been developed on the basis of dysregulation of the putative feedback loop and the consequent induction of an imbalance caused by the expression of Ang-2 together with the downregulation of VEGF.
  • the inventors put forth the idea that there is a regulatory signaling loop involving the coordinated and sequential expression of VEGF and Ang-2, and elucidating the underlying mechanisms can lead to developing a better model for the angiogenic process that occurs in human gliomas, as well development of improved compositions and methods or the treatment of such. Moreover, exploiting the interdependence between VEGF and Ang-2 could be used to develop more effective and rational anti-angiogenesis therapies for brain tumors than currently exist.
  • the inventors use oncolytic adenoviruses for the simultaneous targeting of Ang-2 or Ang-2 and VEGF (the inhibition or down regulation).
  • the inventors also plan on characterizing the interaction between Ang-2 and VEGF; and the role of Ang-2 expression in a dynamic tumor model of glioma angiogenesis.
  • the inventors will obtain efficacious preclinical evidence for supporting the translation of their studies to the clinic for single and/or combination anti-angiogenesis treatments for malignant gliomas.
  • Angiogenesis refers to vessel formation by remodeling the primary vascular network or by sprouting from existing vessels (reviewed in Yancopoulos et al., 2000).
  • the “angiogenesis switch” is “off” when the effect of pro-angiogenic molecules is balanced by the activity of anti-angiogenic molecules, and is “on” when the net balance between the molecules is tipped in favor of angiogenesis (reviewed in Carmeliet and Jain, 2000).
  • Angiogenesis has an essential role in the development and maintenance of solid tumors, including malignant gliomas.
  • GBM glioblastoma multiforme
  • VEGF vascular endothelial growth factor
  • Angiopoietin 2 Angiopoietin 2.
  • VEGF inhibitors may include VEGF-neutralizing chimeric proteins such as soluble VEGF receptors.
  • VEGF-receptor-IgG chimeric proteins may be VEGF-receptor-IgG chimeric proteins.
  • Another VEGF inhibitor contemplated for use in the present invention is antisense phosphorothio oligodeoxynucleotides (PS-ODNs).
  • anti-angiogenesis agents include, but are not limited to, retinoid acid and derivatives thereof, 2-methoxyestradiol, ANGIOSTATIN® protein, ENDOSTATIN® 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 ((1-azetidine-2-carboxylic acid (LACA), cishydroxyproline, d,1-3,4-dehydroproline, thiaproline], ⁇ , ⁇ -dipyridyl, ⁇ -amino
  • 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-1/Ang-2. (Ferrara and Alitalo (1999) Nature Medicine 5:1359-1364.
  • Calbiochem (San Diego, Calif.) carries a variety of angiogensis inhibitors including (catalog number/product name) 658553/AG 1433; 129876/Amiloride, Hydrochloride; 164602/Aminopeptidase N Inhibitor; 175580/Angiogenesis Inhibitor; 175602/Angiogenin (108-123); 175610/Angiogenin Inhibitor; 176600/Angiopoietin-2, His ⁇ Tag®, Human, Recombinant, Mouse, Biotin Conjugate; 176705/Angiostatin K1-3, Human; 176706/Angiostatin K1-5, Human; 176700/Angiostatin(& Protein, Human; 178278/Apigenin; 189400/Aurintricarboxylic Acid; 199500/Benzopurpurin B; 211875/Captopril; 218775/Castanospermine, Castanospermum australe; 25
  • angiopoietin-1 promotes remodeling and stabilization of VEGF-induced vessels (Suri et al.. 1996; Suri et al., 1998) through interactions between endothelial cells and surrounding pericytes and the extracellular matrix.
  • Ang-2 appears to be a natural antagonist of Ang-1, responsible for destabilizing mature vessels in the context of vessel regression or angiogenesis in a VEGF-dependent manner (Maisonpierre et al., 1997). Consequently, Ang-2 expression results in reversal of the maturation process mediated by Ang-1, leading to a disruption of interactions between endothelial cells, pericytes, and the extracellular matrix.
  • Ang-2-mediated effect is followed by sprouting and ingrowth of new vessels (i.e., neovascularization).
  • Ang-2-mediated destabilization leads to the regression of blood vessels (Holash et al., 1999).
  • the inventors envision a therapeutic strategy targeted to the tumor in which Ang-2 is induced in the setting of VEGF inhibition, a combination predicted to cause vascular collapse in the tumors.
  • the strong genetic validation has justified ongoing angiopoietin-directed drug development initiatives.
  • Tie2 in angiogenesis has been demonstrated in studies that have involved the deletion of this receptor. Specifically, Tie2 knockout mice die early in development because of immature blood vessels and lack of vessel development, (Sato et al., 1995). Ligands for the Tie2 receptor include Ang-1 and Ang-2 (Davis et al., 1996; Maisonpierre et al., 1997). Ang-1 phosphorylates Tie2 in cultured endothelial cells, whereas Ang-2 is a naturally occurring antagonist of Ang-1, competing with Ang-I for binding to Tie2 (Maisonpierre et al., 1997).
  • Tie2 is a vascular-specific receptor
  • preliminary evidence suggests that the expression of Tie2 is not limited to endothelial cells (Valable et al., 2003; Poncet et al., (2003).
  • cells of neuroectodermic origin express Tie2 (Valable et al., 2003). Therefore, the negative regulation of the Tie2 receptor and the growth proliferation transduction signal that its activation triggers can be used as an anti-proliferation target in cells other than endothelial cells that express Tie2, including cancer cells.
  • Tumor angiogenesis begins when tumor cells release molecules that send signals to the surrounding normal host tissue. Such signaling activates specific genes in the host tissue that, in turn, generate proteins that encourage the growth of new blood vessels. Among these molecules, two proteins appear to be the most important for sustaining tumor growth: VEGF and Ang-2. Imbalances in the coordinated timing and expression of these molecules lead to vessel regression. The confirmation of the Ang-2-mediated downmodulation of HIF is underscored by the observation that the overexpression of HIF-1 ⁇ has been associated with increased patient mortality in several cancer types. Moreover, in preclinical studies, inhibiting the activity of HIF-1 has marked effects on tumor growth. This approach may yield important mechanistic information about the abnormal regulation of angiogenesis in gliomas.
  • Characterizing an active Tie2 pathway in glioma cells will have an enormous scientific and clinical relevancy. This description together with the explanation of the overall role of Tie2 role in cell proliferation will define a novel target for glioma therapy, one that will permit therapies to be simultaneously directed against the glioma cell mass as well as the angiogenic vascular network. Furthermore, the data obtained from these studies will provide a rational basis for the development of a phase I/II clinical trial to assess the toxicity and efficacy of a triple treatment for malignant gliomas, one that will combine the overexpression of Ang-2 a decrease in the effect VEGF, and the production of oncolysis.
  • aspects of the invention include nucleic acids or genes that encode a detectable and/or therapeutic polypeptide.
  • the gene is a therapeutic, or therapeutic gene.
  • a “therapeutic gene” is a gene which can be administered to a subject for the purpose of treating or preventing a disease.
  • a therapeutic gene can be a gene administered to a subject for treatment or prevention of diabetes or cancer.
  • therapeutic genes include, but are not limited to, Rb, CFTR, p16, p21, p27, p57, p73, C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF, G-CSF, thymidine kinase, mda7, fus, interferon ⁇ , interferon ⁇ , interferon ⁇ , ADP, p53, ABLI, BLC1, BLC6, CBFA1, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS2, ETV6, FGR
  • the therapeutic gene is a tumor suppressor gene.
  • a tumor suppressor gene is a gene 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. 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-1, KRAS2b, p16, p19, p21, p27, p27mt, p53, p57, p73, PTEN, Rb, Uteroglobin, Skp2, BRCA-1, BRCA-2, CHK2, CDKN2A, DCC, DPC4, MADR2/JV18, MEN1, MEN2, MTS1, NF1, NF2, VHL, WRN, WT1, CFTR, C-CAM, CTS-1, zac1, scFV, MMAC1, FCC, MCC, Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), 101F6, Gene 21 (NPRL2), or a gene encoding a SEM A3 polypeptide and FUS1.
  • tumor suppressor genes are described in a database of tumor suppressor genes at www.cise.ufl.edu/ ⁇ yy1/HTML-TSGDB/Homepage.html. This database is herein specifically incorporated by reference into this and all other sections of the present application.
  • Nucleic acids encoding tumor suppressor genes include tumor suppressor genes, or nucleic acids 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.
  • cDNAs, cRNAs, mRNAs, and subsequences thereof encoding active fragments of the respective tumor suppressor amino acid sequences
  • vectors comprising these sequences.
  • 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.
  • Exemplary pro-apoptotic genes include CD95, caspase-3, Bax, Bag-1, CRADD, TSSC3, bax, hid, Bak, MKP-7, PERP, bad, bcl-2, MST1, bbc3, Sax, BIK, BID, and mda7.
  • pro-apoptotic gene amino acid sequence refers to a polypeptide that, when present in a cell, induces or promotes apoptosis.
  • Exemplary pro-apoptotic genes include CD95, caspase-3, Bax, Bag-1
  • 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.
  • a “cytokine” refers to a polypeptide that, when present in a cell, 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.
  • cytokines lymphokines, monokines, growth factors and traditional polypeptide hormones. Included among the cytokines are 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
  • therapeutic genes include genes encoding enzymes. Examples 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 lactase
  • therapeutic genes include the gene encoding carbamoyl synthetase I, omithine 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, gly
  • 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 YY,
  • the term “therapeutic gene” includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants.
  • 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.
  • 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 E1A adenoviral protein. The CR2 region of E1A 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 oncolytic viruses.
  • tumor cells provide a replicating environment that favors such activity. 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).
  • an E1A protein with a 24 base pair deletion in the CR2 region prevents the protein from binding to and inactivating Rb.
  • This attenuated E1A-mutant adenovirus is unable to replicate within normal quiescent cells that have a funtionally active Rb pathway.
  • tumor cells are permissive to viral replication, which in turn efficiently invade and lyse human glioma cells both in vitro and in vivo.
  • the oncolytic potential of ⁇ 24 is dramatic compared with other conditionally replication-deficient adenoviruses, such as Onyx-015.
  • the effects of ⁇ 24 in a mouse xenograft intracranial glioma tumor model are shown in FIG. 2 .
  • the curve representing RA55 carries the deletion in the E1B region as in Onyx-015.
  • the oncolytic adenovirus does not have the same degree of potency as ⁇ 24 at comparable doses used (in this case 1 ⁇ 10 8 pfu).
  • the negative control ⁇ 24 that is inactivated by ultraviolet exposure.
  • ⁇ 24 has been demonstrated in various human tumor cell lines and in animal xenograft models with known defects of the p16/Rb/E2F pathway. Permissive replication of ⁇ 24 in cell lines with p16/Rb/E2F defects is contrasted with the highly attenuated replication in normal astrocytes and normal quiescent fibroblasts. Additionally, the activity of this virus is attenuated when introduced into tumor cells in which Rb has been functionally restored through stable or transient transfection techniques.
  • gliomas do not metastasize, and therefore an efficient local approach should be enough to cure the disease.
  • every glioma harbors several populations of cells expressing different genetic abnormalities (Sidransky et al., 1992; Collins and James, 1993; Furnari et al., 1995; Kyritsis et al., 1996).
  • the spectrum of tumors sensitive to the transfer of a single gene to cancer cells may be limited.
  • replication competent adenoviruses can infect and destroy cancer cells that are arrested in G 0 . Since gliomas invariably include non-cycling cells, this property is important.
  • the p16-Rb pathway is abnormal in the majority of gliomas (Hamel et al., 1993; Henson et al., 1994; Hirvonen et al., 1994; Jen et al., 1994; Schmidt et al., 1994; Costello et al., 1996; Fueyo et al., 1996b; Kyritsis et al., 1996; Ueki et al., 1996; Costello et al., 1997), thus making the ⁇ 24 strategy appropriate for most of these tumors.
  • the loss of the retinoblastoma tumor suppressor gene function has been associated with the causes of various types of tumors and is not limited to treatment of gliomas.
  • an E1A mutation (e.g., a ⁇ 24 mutation in E1A) may be used in combination with mutations in the E1B region of the same adenovirus, thus producing a double mutant adenovirus.
  • an adenovirus may comprise a ⁇ 24 mutation and a deletion in the E1B region that prevents expression or function of the E1B55 kD protein.
  • the E1B55 kD protein has been shown to bind to and inactivate p53.
  • the E1B region mutation may include a deletion of adenovirus sequences from 2426 bp to 3328 bp of genebank accession number NC-001406, which is incorporated herein by reference.
  • an oncolytic 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 vector and (b) to express a 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 region that are not essential for viral replication in the target cell.
  • Traditional methods for the generation of adenoviral particles is co-transfection followed by subsequent in vivo recombination of a shuttle plasmid and an adenoviral helper plasmid into either 293 or 911 cells (Introgene, The Netherlands).
  • 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.
  • 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. As preferred helper cell line is 293.
  • the adenovirus is typically replication-competent in cells with a mutant Rb pathway. After transfection, 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.
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • Adenovirus type 5 is the preferred 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 constructions 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 11 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 oncolytic adenovirus described herein may be made to improve the ability of the oncolytic 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.
  • adenovirus receptor CAR
  • Various peptide motifs may be added to the fiber knob, for instance an RGD motif (RGD sequences mimic the normal ligands of cell surface integrins), Tat motif, poly-lysine motif, NGR motif, CTT motif, CNGRL motif, CPRECES motif or a strept-tag motif (Rouslahti and Rajotte, 2000).
  • RGD motif RGD sequences mimic the normal ligands of cell surface integrins
  • Tat motif poly-lysine motif
  • NGR motif NGR motif
  • CTT motif CNGRL motif
  • CPRECES motif CPRECES motif
  • strept-tag motif strept-tag motif
  • Peptide sequences that bind specific human glioma receptors such as EGFR or uPR may also be added.
  • Specific receptors found exclusively or preferentially on the surface of cancer cells may used as a target for adenoviral binding and infection, such as EGFRvIII.
  • EGFR Cell surface receptors are attractive candidates for the targeted therapy of cancer. Growth factors and their receptors play important roles in the regulation of cell division, development, and differentiation. Among those receptors, EGFR was the first to be identified as amplified and/or rearranged in malignant gliomas. EGFR gene amplification in gliomas is often accompanied by gene rearrangement, resulting in deletions of the coding region. The most common variant, de2-7 EGFR or EGFRvIII, is characterized by an in-frame deletion of 801-bp spanning exons 2-7 of the coding sequence. This truncation removes 267 amino acids from the extracellular domain, producing a unique junctional peptide, and renders EGFR unable to bind any known ligand.
  • EGFRvIII is expressed on the cell surface and contains a new tumor-specific protein sequence in its extracellular domain (Sugawa et al. 1990; Ekstrand et al. 1992). The frequency of the EGFRvIII expression in human gliomas is around 20 to 40% (Frederick et al. 2000).
  • Several strategies have already been tested as means for binding the EGFRvIII receptor using peptides and antibodies.
  • a peptide (PEPHC1) has been synthesized and tested for binding to EGFRvIII and EGFR (Campa et al., 2000, which is incorporated herein by reference in its entirety).
  • PEPHC1 bound the recombinant EGFRvIII extracellular domain or full-length EGFRvIII (solubilized from cell membranes) in preference to native EGFR.
  • Monoclonal antibodies have been developed with specific activity against this mutant receptor (Lorimer et al. 1996). These antibodies are internalized into the cell after receptor binding. Therefore, this receptor is a desirable target for adenoviral tropism since the receptor-binding molecules are efficiently internalized and the mutant form offers the opportunity to develop tumor-selective targeting strategies.
  • Rb is a tumor suppressor gene whose loss of function is associated with tumor formation.
  • Retinoblastoma protein or Rb refers to the polypeptide encoded by the retinoblastoma gene (Rb).
  • the retinoblastoma gene is located at 13q14 in humans and encodes a protein of approximately 110 kiloDaltons (kD).
  • Unphosphorylated Rb inhibits cell proliferation by sequestering transcription factors (e.g., E2F) and arresting cells in G 1 of the cell cycle. Transcription factors are released from Rb when Rb is phosphorylated.
  • E1A Several viral oncoproteins target Rb for inactivation in order to facilitate viral replication. These proteins include adenovirus E1A, SV40 large T antigen, and papillomavirus E7.
  • the E1A protein is one of the first virus-specific polypeptides synthesized after adenoviral infection and is required for viral replication to occur (Dyson and Harlow, 1992; Flint and Shenk, 1997). Interaction of the Rb protein and the E1A protein results in release of E2F from pre-existing cellular E2F-Rb complexes. E2F is then free to activate transcription from E2 promoters of adenovirus and E2F regulated genes of an infected cell. The transcriptional activation of these cellular genes in turn helps to create an environment suitable for viral DNA synthesis in otherwise quiescent cells (Nevins, 1992).
  • E1A Two segments of E1A are important for binding Rb; one includes amino acids 30-60 and the other amino acids 120-127 (Whyte et al., 1988; Whyte et al., 1989). Deletion of either region prevents the formation of detectable E1A/Rb complexes in vitro and in vivo (Whyte et al., 1989).
  • An adenovirus containing a Delta 24 mutation produces an E1A protein that cannot bind Rb, causing an infected cell to remain in G 0 .
  • a mutant Rb pathway and a mutant E1A, along with E2F activation are necessary for ⁇ 24 adenoviral transcription.
  • Retinoblastoma (Rb) pathway refers the interaction of a group of regulatory proteins that interact with Rb or other proteins that interact with Rb in regulating cell proliferation (for review see Kaelin, 1999). Proteins within the Rb pathway include, but are not limited to, Rb, the E2F family of transcription factors, DRTF, RIZ286, MyoD287, c-Ab1288, MDM2289, hBRG1/hBRM, p16, p107, p130, c-Abl tyrosine kinase and proteins with conserved LXCXE motifs, cyclin E-cdk 2, and cyclin D-cdk 4/6.
  • E2F Phosphorylation of Rb releases E2F, which is bound to unphosphorylated Rb.
  • E2F stimulates cyclin E transcription and activity, which results in more Rb phosphorylation.
  • Unphosphorylated Rb acts as a tumor suppressor by binding to regulatory proteins that increase DNA replicaiton, such as E2F (The Genetic Basis of Human Cancer, Vogelstein and Kinzler eds., 1998).
  • Defective retinoblastoma pathway refers to inactivation, mutation, or deletion of the Rb or the inability of the upstream or downstream regulatory proteins that interact with Rb to regulate cell proliferation due to a mutation or modification of one or more proteins, protein activities, or protein-protein interactions. Mutations causing a defective Rb pathway include, but are not limited to inactivating mutations in Rb, INK4 proteins, and CIP/KIP and activating mutations in the cyclin genes, such as cyclin D/cdk 4, 6 and cyclin E, cdk 2.
  • Rb associated tumors include gliomas, sarcomas, tumors of the lung, breast, ovary, cervix, pancreas, stomach, colon, skin, larynx, bladder and prostate.
  • the present invention involves the treatment of hyperproliferative cells, preferably a cell with a disrupted Rb pathway. It is contemplated that a wide variety of tumors 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 hyperplasia.
  • glioma refers to a tumor originating in the neuroglia of the brain or spinal cord.
  • Gliomas are derived form 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, ependymomas, 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 are deficient in the Rb and/or p53 pathway.
  • the present invention is directed at the treatment of diseases, including but not limited to retinoblastomas, gliomas, sarcomas, tumors of lung, ovary, cervix, pancreas, stomach, colon, skin, larynx, breast, prostate and metastases thereof.
  • Glioblastoma multiforme is the most common malignant primary brain tumor of adults. More than half of these tumors have abnormalities in genes involved in cell cycle control. Often there is a deletion in the CDKN2A or a loss of expression of the retinoblastoma gene.
  • Other types of brain tumors include astrocytomas, oligodendrogliomas, ependymomas, medulloblastomas, meningiomas and schwannomas.
  • 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 nonexhaustive 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 ⁇ 10 4 , 1 ⁇ 10 5 , 1 ⁇ 10 6 , 1 ⁇ 10 7 , 1 ⁇ 10 8 , 1 ⁇ 10 9 , 1 ⁇ 10 10 , 1 ⁇ 10 11 , or 1 ⁇ 10 12 infectious particles to the 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 know 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.
  • Preferred 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. Such 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.
  • in vivo administration includes all manipulations performed on cells within an animal.
  • 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 to selectively kill tumor cells.
  • 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 advantageously be contacted by administering multiple injections to the tumor, 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 also is 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.
  • limb perfusion may be used to administer therapeutic compositions of the present invention, particularly in the treatment of melanomas and sarcomas.
  • 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 also may be administered parenterally or intraperitoneally.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • compositions of the present invention are advantageously administered 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 also may 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. Examples of non-aqueous solvents are 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 neoplasic cell by attaching antibodies to the liposome that bind specifically to a cell surface marker on the neoplastic cell. 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.
  • lipid bilayers form spontaneously when phospholipids are suspended in an excess of aqueous solution.
  • the lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991).
  • a nucleic acid construct complexed with Lipofectamine (Gibco BRL).
  • 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 oncolytic 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 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 radiodiagnositc techniques. It also may prove effective to combine oncolytic virus therapy with immunotherapy.
  • a “target” cell contacting a mutant oncolytic virus 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 the expression construct and the 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 the oncolytic adenvirus and the other includes the second agent.
  • Oncolytic 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 oncolytic adenovirus treatment may precede or follow the second agent or treatment by intervals ranging from minutes to weeks.
  • the second agent and oncolytic adenovirus are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the second agent and oncolytic adenovirus would still be able to exert an advantageously combined effect on the cell.
  • oncolytic adenovirus is “A” and the other agent is “B”, as exemplified below: A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A B/B/A B/A/B B/B/B/A A/A/A/B B/A/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/B/A A/B/B B/A/B/B B/B/A/B/B B/B/A/B/B/A/B
  • both agents are delivered to a cell in a combined amount effective to kill 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, CPT11, temozolomide, platin compounds and pro-drugs such as 5-FC.
  • Examples of alkylating agents include, inter alia, chloroambucil, cis-platinum, cyclodisone, flurodopan, methyl CCNU, piperazinedione, teroxirone.
  • Topoisomerase I inhibitors encompass compounds such as camptothecin and camptothecin derivatives, as well as morpholinodoxorubicin. Doxorubicin, pyrazoloacridine, mitoxantrone, and rubidazone are illustrations of topoisomerase II inhibitors.
  • RNA/DNA antimetabolites include L-alanosine, 5-fluoraouracil, aminopterin derivatives, methotrexate, and pyrazoflirin; while the DNA antimetabolite group encompasses, for example, ara-C, guanozole, hydroxyurea, thiopurine.
  • Typical antimitotic agents are colchicine, rhizoxin, taxol, and vinblastine sulfate.
  • Other agents and factors include radiation and waves that induce DNA damage such as, ⁇ -irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, 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.
  • the cells of a precancerous lesion or tumor cells with an agent in addition to the oncolytic adenovirus. This may be achieved by irradiating the localized tumor site with radiation such as X-rays, Wv-light, ⁇ -rays or even microwaves.
  • the cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound such as, adriamycin, bleomycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, podophyllotoxin, verapamil, or more preferably, cisplatin.
  • the agent may be prepared and used as a combined therapeutic composition, or kit, by combining it with an oncolytic adenovirus.
  • Cisplatinum agents such as cisplatin, and other DNA alkylating agents may be used.
  • Cisplatin has been widely used to treat cancer, with efficacious doses used in clinical applications of 20 mg/m 2 for 5 days every three weeks for a total of three courses.
  • Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.
  • Bleomycin and mitomycin C are other anticancer agents that are administered by injection intravenously, subcutaneously, intratumorally or intraperitoneally.
  • a typical dose of bleomycin is 10 mg/m 2 , while such a dose for mitomycin C is 20 mg/m 2 .
  • Agents that damage DNA also include compounds that interfere with DNA replication, mitosis and chromosomal segregation.
  • chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m 2 at 21 day intervals for adriamycin, to 35-50 mg/m 2 for etoposide intravenously or double the intravenous dose orally.
  • nucleic acid precursors and subunits also lead to DNA damage.
  • nucleic acid precursors have been developed.
  • agents that have undergone extensive testing and are readily available are agents that have undergone extensive testing and are readily available.
  • agents such as 5-fluorouracil (5-FU) are preferentially used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic cells.
  • 5-FU is applicable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg/kg/day being commonly used or as alternative 5-FC may be administered and converted in a target tissue or target cell.
  • ⁇ -rays X-rays
  • X-rays X-rays
  • UV-irradiation UV-irradiation
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • Immunotherapy may be used as part of a combined therapy, in conjunction with mutant oncolytic virus-mediated therapy.
  • the general approach for combined therapy is discussed below.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155.
  • Antibodies specific for CAR, integrin or other cell surface molecules may be used to identify cells that the adenovirus could infect well.
  • CAR is an adenovirus receptor protein. The penton base of adenovirus mediates viral attachment to integrin receptors and particle internalization.
  • the inventors propose that local, regional delivery of oncolytic adenovirus to patients with retinoblastoma-linked cancers, pre-cancers, or hyperproliferative conditions will be a very efficient method for delivering a therapeutically effective gene.
  • the chemo- or radiotherapy may be directed to a particular, affected region of the subjects body.
  • systemic delivery of expression construct and/or the agent may be appropriate in certain circumstances, for example, where extensive metastasis has occurred.
  • oncolytic adenovirus therapies with chemo- and radiotherapies
  • combination with other gene therapies will be advantageous.
  • targeting of a oncolytic adenovirus in combination with the targeting of p53 at the same time may produce an improved anti-cancer treatment.
  • Any tumor-related gene or nucleic acid encoding a polypeptide conceivably can be targeted in this manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, p16, FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf, erb, src, fins, jun, trk, ret, gsp, hst, bcl and abl.
  • 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 oncolytic 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 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.
  • Animal models may be used as a screen for tumor suppressive effects of oncolytic adenoviruses.
  • orthotopic animal models will be used so as to closely mimic the particular disease type being studied 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.
  • tissue flap means any incision in the flesh of the animal that exposes the target tissue. It is generally preferred that an incision be made in the dorsal flank of an animal, as this represents a readily accessible site. However, it will be understood that an incision could well be made at other points on the animal, and the choice of tissue sites may be dependent upon various factors such as the particular type of therapeutics that are being investigated.
  • cancer cells either individually or in tumors, are contacted with the tissue site. Cancer cell application may be achieved simply using any convenient applicator. Naturally, this procedure will be conducted under sterile conditions.
  • 1 ⁇ 10 7 cells are inoculated into the exposed tissue flap of a nude mouse.
  • the number of cells will be dependent upon various factors, such as the size of the animal, the site of incision, 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. Although 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.
  • 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 following resealing of the tissue flap. Naturally, administering larger numbers of cells will result in a larger population of residual tumor cells. Those skilled in the area of animal testing will appreciate that such optimization is required.
  • Rb pathway is required to be defective in the sense that it is not able to repress the transcription-activating activity of E2F.
  • E2F activates the transcription of cellular genes and adenoviral DNA if its activity is not repressed. Examples of ways in which E2F could escape repression include, but are not limited to, Rb not being able to bind E2F (i.e., E1A binding to Rb), overexpression of E2F, less Rb than E2F and situations in which Rb remains phosphorylated.
  • Antibodies can be used to detect adenoviral proteins (e.g., E1A), Rb, and other proteins of the Rb pathway.
  • adenoviral proteins e.g., E1A
  • Rb e.g., Rb
  • antibodies may be produced that are immunoreactive with multiple antigens. These antibodies may be used in various diagnostic or therapeutic applications, described herein below.
  • antibody is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE.
  • 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. Pat. No. 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.
  • Certain embodiments of the invention provide antibodies to antigens and translated proteins, polypeptides and peptides that are linked to at least one agent to form an antibody conjugate.
  • a reporter molecule is defined as any moiety which may be detected using an assay.
  • Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.
  • antibody conjugates are those conjugates in which the antibody is linked to a detectable label.
  • Detectable labels are compounds and/or elements that can be detected due to their specific functional properties, and/or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and/or further quantified if desired.
  • Another such example is the formation of a conjugate comprising an antibody linked to a cytotoxic or anti-cellular agent, and may be termed “immunotoxins”.
  • Antibody conjugates are generally preferred for use as diagnostic agents.
  • Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and/or those for use in vivo diagnostic protocols, generally known as “antibody-directed imaging.”
  • Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated herein by reference).
  • the imaging moieties used can be paramagnetic ions; radioactive isotopes; fluorochromes; NMR-detectable substances; X-ray imaging.
  • radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 cobalt, copper 67 , 152 Eu, gallium 67 , 3 hydrogen, iodine 123 , iodine 125 , iodine 131 , indium 111 , 59 iron, 32 phosphorus, rhenium 186 , rhenium 188 , 75 selenium, 35 sulphur, technicium 99m and/or yttrium 90 .
  • Radioactively labeled monoclonal antibodies of the present invention may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
  • Monoclonal antibodies according to the invention may be labeled with technetium 99m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column.
  • direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNCl 2 , a buffer solution such as sodium-potassium phthalate solution, and the antibody.
  • Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylene diaminetetracetic acid
  • fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.
  • antibody conjugates contemplated in the present invention are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate.
  • suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase.
  • Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter and Haley, 1983).
  • 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens and Haley, 1987; Atherton et al., 1985).
  • the 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; and Dholakia et al., 1989) and may be used as antibody binding agents.
  • Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3 ⁇ -6 ⁇ -diphenylglycouril-3 attached to the antibody (U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein by reference).
  • DTPA diethylenetriaminepentaacetic acid anhydride
  • ethylenetriaminetetraacetic acid ethylenetriaminetetraacetic acid
  • N-chloro-p-toluenesulfonamide N-chloro-p-toluenesulfonamide
  • tetrachloro-3 ⁇ -6 ⁇ -diphenylglycouril-3 attached to the antibody
  • Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.
  • Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
  • imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.
  • derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated.
  • Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).
  • Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation.
  • Adenoviral gene expression in a population of cells will be determined by western blot analysis using antibodies as probes to adenoviral proteins. The level of viral proteins detected would indicate whether viral protein expression is occurring in the cell. Immunodetection using monoclonal antibodies that recognize various epitopes within the Rb protein or another protein of the Rb pathway can be used to see if Rb or a protein in the Rb pathway has been mutated.
  • 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 or the cellular Rb or p53 pathways.
  • 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 an antigen, 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 antigen-containing 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.
  • an antigen 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 antigen-containing 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.
  • Immunoassays in their most simple and/or direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and/or radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and/or western blotting, dot blotting, FACS analyses, and/or the like may also be used.
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • an antibody that recognizes an antigen is immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the antigen, such as a clinical sample, is added to the wells. After binding and/or washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection is generally achieved by the addition of another antibody that recognizes the antigen and is linked to a detectable label. This type of ELISA is a simple “sandwich ELISA”. Detection may also be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes.
  • Antibodies may also be used in conjunction with both fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • the method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and/or is well known to those of skill in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990, all of which are incorporated herein by reference).
  • frozen-sections may be prepared by rehydrating 50 ng of frozen “pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in ⁇ 70° C. isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections.
  • PBS phosphate buffered saline
  • OCT viscous embedding medium
  • Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections.
  • the nucleic acid sequences disclosed herein have a variety of uses. For example, they have utility as probes or primers for embodiments involving nucleic acid hybridization. They can be used to determine whether viral genes are being transcribed. In certain embodiments of the invention adenoviral genes may be transcribed in cells with a mutant Rb or p53 pathways. Nucleic acid detection may be used to determine if there is a mutation within the Rb gene, p53 gene or other genes encoding proteins of the Rb pathway. The DNA sequences of genes of the present invention may be determined by methods known in the art to identify mutations within the sequence.
  • a probe or primer of between 13 and 100 nucleotides preferably between 17 and 100 nucleotides in length, or in some aspects of the invention up to 1-2 kilobases or more in length, allows the formation of a duplex molecule that is both stable and selective.
  • Molecules having complementary sequences over contiguous stretches greater than 20 bases in length are generally preferred, to increase stability and/or selectivity of the hybrid molecules obtained.
  • Such fragments may be readily prepared, for example, by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.
  • Hybridization conditions are preferred. Under these conditions, hybridization may occur even though the sequences of the hybridizing strands are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and/or decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. to about 55° C. Hybridization conditions can be readily manipulated depending on the desired results.
  • nucleic acids of defined sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization.
  • an appropriate means such as a label
  • appropriate indicator means include fluorescent, radioactive, enzymatic or other ligands, such as avidinibiotin, which are capable of being detected.
  • Nucleic acids used as a template for amplification may be isolated from cells, tissues or other samples according to standard methodologies (Sambrook et al., 2001). In certain embodiments, analysis is performed on whole cell or tissue homogenates or biological fluid samples without substantial purification of the template nucleic acid.
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to first convert the RNA to a complementary DNA.
  • primer is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded and/or single-stranded form, although the single-stranded form is preferred.
  • the amplification product may be detected or quantified.
  • the detection may be performed by visual means.
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals (Affymax technology; Bellus, 1994).
  • PCRTM polymerase chain reaction
  • a reverse transcriptase PCRTM amplification procedure may be performed to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al., 2001.
  • Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641.
  • Polymerase chain reaction methodologies are well known in the art. Representative methods of RT-PCR are described in U.S. Pat. No. 5,882,864.
  • LCR ligase chain reaction
  • European Application 320308 incorporated herein by reference in its entirety.
  • U.S. Pat. No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.
  • a method based on PCRT and oligonucleotide ligase assay (OLA), disclosed in U.S. Pat. No. 5,912,148, may also be used.
  • Qbeta Replicase described in PCT Application PCT/US87/00880, may also be used as an amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence which may then be detected.
  • nucleic acid amplification procedures include Strand Displacement Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779; transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al., PCT Application WO 88/10315, incorporated herein by reference in their entirety).
  • SDA Strand Displacement Amplification
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Zaoh et al., 1989; Gingeras et al., PCT Application WO 88/10315, incorporated herein by reference in their entirety.
  • Davey et al. European Application 329 822 disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which
  • Miller et al., PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter region/primer sequence to a target single-stranded DNA (“ssDNA”) followed by transcription of many RNA copies of the sequence.
  • Other amplification methods include “race” and “one-sided PCR” (Frohman, 1990; Ohara et al., 1989).
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al., 2001). Separated amplification products may be cut out and eluted from the gel for further manipulation. Using low melting point agarose gels, the separated band may be removed by heating the gel, followed by extraction of the nucleic acid.
  • Separation of nucleic acids may also be effected by chromatographic techniques known in art.
  • chromatographic techniques There are many kinds of chromatography which may be used in the practice of the present invention, including adsorption, partition, ion-exchange, hydroxylapatite, molecular sieve, reverse-phase, column, paper, thin-layer, and gas chromatography as well as HPLC.
  • the amplification products are visualized.
  • a typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light.
  • the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the separated amplification products can be exposed to x-ray film or visualized under the appropriate excitatory spectra.
  • detection is by Southern blotting and hybridization with a labeled probe.
  • the techniques involved in Southern blotting are well known to those of skill in the art. See Sambrook et al., 2001.
  • One example of the foregoing is described in U.S. Pat. No. 5,279,721, incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids.
  • the apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
  • DGGE denaturing gradient gel electrophoresis
  • RFLP restriction fragment length polymorphism analysis
  • SSCP single-strand conformation polymorphism analysis
  • U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavage assay that involves annealing single-stranded DNA or RNA test samples to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNase A. For the detection of mismatches, the single-stranded products of the RNase A treatment, electrophoretically separated according to size, are compared to similarly treated control duplexes. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as positive.
  • a tumor may be biopsied and the above tests performed upon it to determine whether the cells have a functional Rb pathway.
  • An example of a biopsy protocol is as follows. The stereotactic biopsy is the precise introduction of a metal probe into the brain tumor, cutting a small piece of the brain tumor, and removing it so that it can be examined under the microscope. The patient is transported to the HI or CAT scan suite, and the frame is attached to the scalp under local anesthesia. The “pins” of the frame attach to the outer table of the skull for rigid fixation (frame will not and can not move from that point forward until completion of the biopsy). The scan (MRI or CT) is obtained. The neurosurgeon examines the scan and determines the safest trajectory or path to the target. This means avoiding critical structures.
  • the spatial co-ordinates of the target are determined, and the optimal path is elected.
  • the biopsy is carried out under general anesthesia. A small incision is created over the entry point, and a small hole is drilled through the skull. The “dura” is perforated, and the biopsy probe is introduced slowly to the target. The biopsy specimen is withdrawn and placed in preservative fluid for examination under the microscope. Often the pathologist is present in the biopsy suite so that a rapid determination of the success of the biopsy can be made.
  • the oncolytic adenoviruses of the present invention may be used in diagnostic assays to detect the presence of cells with a defective Rb and/or p53 pathway.
  • a sample of cells could be infected with the oncolytic adenovirus of the present invention and after an incubation period, the number of cells exhibiting adenovirus replication can be quantified to determine the number of neoplastic cells in the sample. This may be useful to determine if the adenovirus would be effective in treating the tumor from a patient from which a cell sample was taken.
  • Other uses are to diagnose a neoplasm as having a defective Rb and/or p53 pathway and to evaluate tumor cell load following treatment.
  • Alternate diagnostic uses and variations include an adenovirus with a Rb binding mutation in the E1A or an E1B55 kD—mutation and a reporter gene to score whether cells have been transformed by detecting reporter gene expression.
  • Expression of the reporter gene can be correlated with a phenotype of adenoviral replication indicating a lack of a functional Rb and/or p53 pathway.
  • Amino acid sequence variants of the polypeptides discussed above and throughout this application 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 non-terminal point in the polypeptide. This may include the insertion of 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, 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 is well understood in the art and is further defined in detail herein. Accordingly, sequences that have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of a particular polypeptide, such as E1A, provided the biological activity of the protein is maintained.
  • flanking codon refers to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids.
  • 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, i.e., introns, which are known to occur within genes.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic finction 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, DNA, 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 one derived from the Ang1 gene, Ang-2 gene, NIS gene or a yCD gene.
  • a nucleic acid segment or polynucleotide encoding a Ang1 gene, Ang-2 gene, NIS or yCD polypeptide refers to a nucleic acid segment or polynucleotide comprising a polynucleotide encoding wild-type, polymorphic, or mutant Ang1 gene, Ang-2 gene, NIS or yCD polypeptide.
  • nucleic acid a polynucleotide or polynucleotides, DNA segments, and recombinant vectors.
  • Recombinant vectors may include plasmids, cosmids, phage, viruses, and the like.
  • recombinant adenoviruses are contemplated.
  • an adenovirus comprising an expression cassette encoding a Ang1 gene, Ang-2 gene, NIS or a yCD.
  • Mutation may be a substitution, insertion, or deletion.
  • a mutation introduces a stop codon or introduces a frame shift that results in a premature stop codon.
  • nonfunctional polypeptides may be encoded by polynucleotides, such as truncated polypeptides.
  • polynucleotides of the invention may be mutated to produce a polypeptide that lacks 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, 460, 470, 480, 490,
  • polynucleotide refers to a nucleic acid molecule of greater than 3 nucleotides. Therefore, a “polynucleotide encoding a Ang1 gene, Ang-2 gene, NIS or yCD” refers to a DNA segment that contains a wild-type, a polymorphic, or a mutant Ang1 gene, Ang-2 gene, NIS or yCD polypeptide; similarly, a “polynucleotide encoding wild-type Ang1 gene, Ang-2 gene, NIS or yCD” refers to a DNA segment that contains wild-type Ang1 gene, Ang-2 gene, NIS or yCD polypeptide coding nucleic acid or DNA sequences.
  • a polynucleotide comprising an isolated or purified wild-type, polymorphic, or mutant nucleic acid encoding a Ang1 gene, Ang-2 gene, NIS or a yCD polypeptide refers to a nucleic acid segment comprising wild-type, polymorphic, or mutant Ang1 gene, Ang-2 gene, NIS or yCD polypeptide coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences.
  • the term “gene” is used for simplicity to refer to a functional 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.
  • the nucleic acid encoding Ang1 gene, Ang-2 gene, NIS or yCD may contain a contiguous nucleic acid Ang1 gene, Ang-2 gene, NIS or yCD sequence encoding all or a portion of Ang1 gene, Ang-2 gene, NIS or yCD of the following lengths: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 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, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660
  • isolated substantially away from other coding sequences means that the nculeic acid of interest, for example the polynucleotide encoding a wild-type, a polymorphic, or a mutant Ang1 gene, Ang-2 gene, NIS or yCD 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 coding 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.
  • the DNA segments used in the present invention may encompass biologically functional equivalent Ang1 gene, Ang-2 gene, NIS or hyCD and derivative peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
  • functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by human may be introduced through the application of site-directed mutagenesis techniques, e.g., to decrease the antigenicity of the protein or to inhibit binding to a given protein.
  • 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 also are defined.
  • the nucleic acid encoding a transgene product 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, 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, that is the promoter is associated with a nucleic acid that is not associated with its natural location.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
  • Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • At least one module in each promoter functions to position the start site for RNA synthesis.
  • the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.
  • 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 promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of a polynucleotide of interest, which may or may not encode a polypeptide of interest. Promoters that permit expression of a protein of interest generally under most conditions and in most cell types is termed constitutive, and an example of this is the CMV promoter.
  • a tissue-specific promoter is a regulatable promoter that is allows expression only in particular tissues or cells. Tables 2 list several elements/promoters that may be employed, in the context of the present invention, to regulate the expression of the nucleic acid of interest. This list is not intended to be exhaustive of all the possible elements involved in the promotion of polynucleotide expression but, merely, to be exemplary thereof.
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. Enhancer/Promoters inlcude but are not limited to enhancers/promoters of Immunoglobulin Heavy Chain, Immunoglobulin Light Chain, T-Cell Receptor, HLA DQ ⁇ and DQ ⁇ , ⁇ -Interferon, Interleukin-2, Interleukin-2 Receptor, MHC Class II 5, MHC Class II HLA-DR ⁇ , ⁇ -Actin, Muscle Creatine Kinase, Prealbumin (Transthyretin), Elastase I, Metallothionein, Collagenase, Albumin Gene, ⁇ -Fetoprotein, ⁇ -Globin, ⁇ -Globin, c-fos, c-HA-ras, Insulin, Neural Cell Adhesion
  • enhancers The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • a cDNA insert where 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 Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • the cells contain nucleic acid construct of the present invention, a cell may be identified in vitro or in vivo by including a marker in the expression vector. 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 internal ribosome binding sites
  • WES elements are used to create multigene, or polycistronic, messages.
  • WES 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. Pat. No. 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.
  • U87MG and U251MG glioma cell human xenograft models utilize human tumor cell lines obtained from patients, which are placed into short or long-term tissue culture.
  • the advantages of this system are that the tumor cells are of human origin and have readily identifiable characteristics such as expression levels of various growth factors and receptors.
  • These studies require immunocompromised animals to prevent tumor rejection, eliminating analysis of the role of the immune system in tumor biology and response to treatments.
  • Most xenograft models are well established and form predictable and reliable intracranial tumors with fairly uniform animal deaths occurring at 20-22 days. However, these tumors lack the heterogeneity seen in the clinical setting.
  • most xenograft models do not demonstrate extensive invasion of the surrounding brain parenchyma.
  • the implanted tumors tend to grow spherically and animal death is related to intracranial pressure associated with ventricular system compression and herniation, as seen after necropsy.
  • the localized and spherical growth of the xenograft implant may be an advantage and has been used extensively for this purpose.
  • the defined localization of the tumor simplifies the correlation of histopathologic findings with imaging results ( FIG. 4 ).
  • U87MG human xenograft model implanted intracranially into the mouse putamen has a demonstrated reliability (nearly 100% tumor production) and highly predictable growth and survival parameters (median survival 21 days rt 1 day).
  • use of the intracranial screw technique ensures reproducible placement of tumor and the ability to access tumor either for sampling or for injecting treatments (local delivery of viruses) or markers into the tumor mass.
  • An additional feature that improves the reproducibility of this technique is the use of a pump that simultaneously injects prepared tumor cells into 10 animals. This technique produces uniform injection times and consistent quality of the cell preparations.
  • Lung cell lines H1299 and ⁇ 549 when injected through tail veins, develop very reproducible systemic animal tumor burdens. In the case of H1299, strictly bilateral lung tumors are produced. In contrast, when an A549 cell line is used, all systemic organs (except the CNS) are at risk for tumor development.
  • the Rb pathway status has been evaluated in various cell lines, including the cell lines described herein: (U87MG, U251MG, H1299, and A549).
  • the Saos-2 cell line is used for the Rb -null and Rb restoration studies.
  • CAR Coxsackie Adenoviral Receptor
  • Nuclear imaging may detect radiation signals from radiopharmaceuticals that have been systemically administered to experimental animals.
  • Conventional nuclear imaging is performed using a gamma camera that can detect photons with energies between 60 and 600 eV (usually between 80 and 400 keV).
  • One of the advantages to whole body scanning is rapid sequential imaging (typically 1 image per 30-60 seconds) and the ease by which animals can be anesthetized and placed in a lane-prone position.
  • the utility of this system is also evident by its widespread use in clinical medicine, thus creating a convenient bridge between animal and human studies.
  • the conditionally replication selective adenovirus ⁇ 24 has been shown to selectively replicate in tumor cells that have a functional defect in the p16/Rb/E2F pathway, a defect that occurs in more than 90% of malignant glioma tumors.
  • ⁇ 24 replicates in vivo studies were conducted in which human U87MG glioma cells are injected (5 ⁇ 10 5 ) intracranially in nude mice. Three days later, 1 ⁇ 10 8 plaque forming units of ⁇ 24 are injected into the tumor using a screw-guided system. The animals are sacrificed arbitrarily on day 25 post-treatment to examine the extent of ⁇ 24 adenoviral dissemination within the tumor.
  • the infected cells are easily identifiable because they display viral nuclear inclusions, surround the area of necrosis, and are separated from normal brain tissue by a zone of minimally infected versus uninfected cells ( FIG. 5 ). This pattern of clearly different areas suggests that the virus spreads from the center of the tumor to its periphery.
  • the tumors are examined after immunostaining with an antibody specific for the adenovirus hexon protein (an encoded adenoviral late gene).
  • adenovirus hexon protein an encoded adenoviral late gene.
  • the presence of hexon protein indicates and correlates well with viral replication.
  • the characteristics of the staining in both nuclear and cytoplasmic regions, are due to the presence of capsid proteins produced in the cytoplasm and transported to the nucleus for viral assembly.
  • Transgene Expression by ⁇ 24 Virus The expression of an exogenous transgene mediated via ⁇ 24 transport (as an effective delivery vector), is described herein. Others have shown exogenous transgene expression with the use of other oncolytic viral vectors. Based on western blot analysis and quantitative RT-PCR analyses ( FIG. 6 ), which demonstrated that a tumor-selective oncolytic adenovirus ( ⁇ 24-hyCD) can be used to efficiently transduce high levels of an exogenous hyCD gene, resulting in a potent anti-glioma effect in vitro and in vivo. This finding is important because gene therapy has been hampered by various obstacles (Roth and Cristiano, 1997).
  • gene therapy is hampered by inadequate nucleic acid delivery to a large number of tumor cells.
  • Targeting of tumor cells and extension of lethality to tumor cells that are a modest distance away from the major tumor mass by a “bystander effect” is one aspect of this invention.
  • Viral constructs were designed to gain tumor selectivity by targeting the defective p I6/Rb/E2F pathway in malignant gliomas by using ⁇ 24 adenovirus that contains a deletion in the E1A gene.
  • This technique is designed to improve exogenous nucleic acid delivery to tumor cells by using a replication-competent adenovirus and to obtain a bystander effect via transgene expression.
  • FIG. 6 shows that an exogenous nucleic acid product (hyCD encoding polynucleotide) can be effectively expressed in target tumor cells by using the ⁇ 24-hyCD oncolytic virus.
  • the hyCD encoding polynucleotide is expressed at very high levels and is able to actively convert 5-FC into 5-FU, which exhibits superior activity against glioma cell lines.
  • En-Bloc Tumor resection Adenoviral gene therapy trials have been recently completed, adenoviral P53 (Ad-P53) phase I clinical trial. Patients with recurrent malignant glioma were enrolled after meeting entry criteria and underwent stereotactic biopsy with subsequent injection of Ad-p53. The injection catheter was cut at the level of the dura and ligated to maintain a geographic marker of the actual injection site. Two weeks after injection the patient returned for en bloc tumor resection under the direction of Dr. Frederick Lang, M.D. Anderson Cancer Center. The tumor mass was carefully dissected circumferentially, preserving the architecture along with the injection catheter and internal injection site.
  • the en bloc tumor specimen was sectioned perpendicular to the catheter placement to allow inspection and measurement of the distance of viral spread. As demonstrated in FIG. 12 , this technique allows one to correlate the distance of oncolytic viral spread with data obtained from the Ad-P53 clinical trial, as well as correlating the results with imaging findings obtained prior to tumor resection.
  • FIG. 12 shows a specimen from a patient treated with a single injection of Ad-P53 at a dose of 3 ⁇ 10 10 vp in 1 ml.
  • FIG. 12A is a photograph of a surgical specimen that was removed en bloc. The injection catheter is protruding from the tumor.
  • FIG. 12B are formalin-fixed tumor blocks. Specimen shown in panel a has been cut to the catheter. The hole created by the catherter is evident.
  • FIG. 12C is a low-power view (300 ⁇ ) of the same immnunostained with antibody to p53 protein. The hole from the catheter is at the top of the photograph. Transfected tumor cells stain darkly and are distributed within 5 mm of the injection site.
  • FIG. 12A is a photograph of a surgical specimen that was removed en bloc. The injection catheter is protruding from the tumor.
  • FIG. 12B are formalin-fixed tumor blocks. Specimen shown in panel a has been cut to the catheter. The hole created by the cat
  • FIG. 12D is a view (500 ⁇ ) of the same section as FIG. 12C demonstrating positive immunostaining for P53 within the transfected cells.
  • FIG. 12E is a view of adjacent section of that shown in FIG. 12 C demonstrating staining for p21/waf in same distribution as P53 staining.
  • FIG. 12F shows a low power (10 ⁇ ) view of cross-section. The catheter was within the central hole. Blue staining around hole shows distribution of Ad-P53.
  • Materials and Methods used in these studies include the construction and characterization of a replication-competent ⁇ 24 adenovirus encoding a therapeutic or diagnostic transgene, e.g., ⁇ 24-hyCD or ⁇ 24-NIS, see schematic representation shown in FIG. 13 . Also described are the material and methods used for assessing transgene expression in various tumor types and under the controls of diverse promoters to demonstrate its applicability to multiple, varied tumors. Exemplary materials and methods include”
  • NIS in vitro activity Cloning of the human NIS into pcDNA3.1-Zeo (invitrogen) will be preformed by RT-PCR of human thyroid mRNA. U251 MG and U87 MG glioma cell lines will be transiently transfected, then incubated with 99m TcO 4 . These in vitro techniques have already been carried out in our laboratory and modified based on an assay developed by Petrich et al. Briefly, cells will be plated into 24 well plates and allowed to achieve 90% confluence. Various titers of ⁇ 24-NIS will be added, incubating and infecting the cells for 2 hrs. The cells will then be washed, and the assay performed at 24, 48, and 72 hrs.
  • the cells will be pre-incubated at 37° C. for 30 min in 1 ml of HEPES-buffered Hanks balanced salt solution. 18.5 to 37 kBq per ml (0.5-mCi per ml) of 99m TcO 4 will be added after a 1 hr incubation time. The medium will be removed and the cells washed twice with ice-cold bHSS. Cellular radioactivity will be released with 1 ml ice-cold 100% ethanol for 20 min, and counted in a cross-calibrated ⁇ counter. Protein content of the 6 wells per cell line will be determined using a BCA assay kit.
  • E2F or hTERT Driven Transgene Exemplary E2F-driven NIS expression vectors are constructed in a fashion similar to the construction of cytomegalovirus (CMV) driven NIS. Specifically, the CMV promoter on pcDNA3.1-Zeo (Invitrogen) is excised using restriction enzymes Apa I and Nhe I, and the promoters for E2F and hTERT are amplified by PCR with primers that contain these restriction sites at the 5′-ends of their sequence. These promoters are cloned into the immediate proximal region of the NIS gene. Replication competent viruses are produced by cloning into the appropriate shuttle vectors. These shuttle vectors are then co-transfected into 293 cells for recombination to active virus. These viral constructs are used in subsequent studies to determine their efficiency of expression against various cellular backgrounds.
  • CMV cytomegalovirus
  • hTERT-NIS Plasmid p-hTERT was kindly provided by Dr. Fang, M.D. Anderson Cancer Center
  • NIS encoding polynucleotide inserted between the Xhol and HindIII sites just proximal to the NIS encoding polynucleotide.
  • This cassette is amplified by PCR and cloned into a shuttle vector. The methods culminating with co-transfections into 293 cells.
  • E2F promoter will be obtained from plasmid pE2F1-neo (kindly provided by Dr. Ta Jen Liu, M.D. Anderson Cancer Center): however, both the promoter and final cassettes will typically be cloned by PCR amplification. The steps described above are repeated to obtain ⁇ 24 containing NIS or hyCD under the control of E2F or hTERT.
  • U87MG cells, A549 cells, and H1299 cells obtained from the American Type Culture Collection, Manassas, Va.
  • U251 MG human glioma cell lines obtained from the American Type Culture Collection, Manassas, Va.
  • U251 MG human glioma cell lines obtained from the American Type Culture Collection, Manassas, Va.
  • Saos-2 cell line obtained by Dr. Fueyo's laboratory
  • Dulbecco's modified Eagle/F12 medium (1:1, vol:vol) (Media Tech, Hermdon, Va.) containing 5% fetal bovine serum (DIFCO) and 2 nM glutamine.
  • DIFCO fetal bovine serum
  • Adenoviruses Construction of the ⁇ 24 adenovirus has been described elsewhere (Fueyo et al., 2000, incorporated herein by reference). This construct has a 24 bp deletion in the CR2-region of the E1A gene (nucleotides 923 to 946, both included) corresponding to the amino acids L 122 TCHEAGF 129 .
  • Oligonucleotide primers (Midland Certified Reagent Co., Midland, Tex.) with the following sequences are used: Forward primer: 5′-AGCCTGTGCAATCAGGGTC-3′ (SEQ ID NO:7) and Reverse primer: 5′GGGTACCATATGCGCT-3′ (SEQ ID NO:8).
  • viruses are propagated in 293 cells and purified by ultracentrifuigation in a cesium chloride gradient. All viruses are titered using a plaque method as well as optical density measurements. Viruses are maintained at ⁇ 80° C. until used. Single lots of adenovirus ⁇ 24, adenovirus ⁇ 24-hyCD and adenovirus ⁇ 24-NIS are used in the following studies. As controls, non-replication-competent Ad-5 is used as a control (E1A-deleted with the NIS encoding polynucleotide cloned into the E1A region). Additional controls of wild-type adenovirus as well as ⁇ 24-NIS that had been inactivated by UV light and cells that had been mock-infected with culture medium are also used.
  • Cells are prepared and assayed at 24, 48, and 72 h.
  • the cells are pre-incubated at 37° C. for 30 min in 1 ml of HEPES-buffered Hanks-balanced salt solution (bHBSS).
  • bHBSS Hanks-balanced salt solution
  • 18.5 to 37.0 kBq per ml (0.5 to 1 mCi per ml) of 99m TcO 4 is added after 1 h of incubation time.
  • the medium is removed and the cells are washed twice with ice-cold bHBSS.
  • Cellular radioactivity is released with 1 ml ice-cold 100% ethanol for 20 min, and counted in a cross-calibrated gamma counter. Protein content of the 6 wells per cell line will be determined using a BCA assay kit.
  • Sodium perchlorate (NaClO 4 ) is used to inhibit the NIS pump and as a negative control.
  • PCR Real-time Quantitative Polymerase Chemical Reaction
  • the virus is aspirated and cells are washed twice twice with phosphate-buffered saline (PBS). Fresh complete medium containing 10% fetal bovine serum (FBS) is replaced and the cells incubated at 37° C. for 24, 48, 72, or 96 h. The cells are washed twice with PBS. Floating cells are centrifuged, immediately frozen, and stored at ⁇ 80° C. before harvesting mRNA. The cell pellets are lysed with Trizol reagent (Life Technologies) and the RNA is purified according to the manufacturer's recommendations for subsequent amplification by TAQ-Man.
  • PBS phosphate-buffered saline
  • FBS fetal bovine serum
  • a forward primier sequence of 5′-CAACATGAGGTTCCAGAAGGG-3′ (SEQ ID NO:9), a reverse primier sequence of 5′-CAGTTCTCCAGGGTGGAGATCT-3′ (SEQ ID NO:10) and a TaqMan probe with a seqeunce of 5′-TCCGCCACCCTG CACGGC-3′ (SEQ ID NO:11) are used for the amplification of NIS.
  • the primers or probe are typically labeled with a FAM label at the 5′ end and TAMRA label at the 3′ end for human NIS. Control primers and probes are used for the S9 housekeeping gene.
  • the expression of mRNA for NIS is quantified and reported relative to a stable NIS expression clone of the U251 MG glioma cell line. Expression levels are determined by using the ABI 7000 sequence detection system (Applied Biosystems, Foster City, Calif.).
  • U251MG and U87MG cell lines are prepared in 6-well plates and treated with ⁇ 24, ⁇ 24-NIS, nonreplication competent Ad-NIS, or PBS (mock-treatment), as described above.
  • the cells are harvested at 24, 48, 72, or 96 h after treatment.
  • Total cell lysates are prepared by incubating cells with 1 ⁇ sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris-HCl pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM dithiolthreitol). Protein concentration are typically quantified using a bicinchoninic acid (BCA) method (Pierce, Rockford, Ill.).
  • BCA bicinchoninic acid
  • Protein samples (20 ⁇ g) are boiled at 98° C. for 5 min, and lysates separated on a 15% SDS-Tris glycine polyacrylamide gel, while being subjected to electrophoresis at 95 V for 2 h.
  • the seperated proteins are then transferred to a nitrocellulose membrane.
  • the membrane is blocked with 3% nonfat milk, 0.05% Tween 20, 150 mM NaCl, and 50 mM Tris (pH 7.5) and incubated with primary antibody for hNlS (provided by Brahms Institute, Germany).
  • primary antibody for hNlS provided by Brahms Institute, Germany
  • the secondary antibody is horseradish peroxidase-conjugated goat antimouse IgG (Pierce, Rockford, Ill.).
  • the membranes are developed according to Amersham's enhanced chemiluminescence protocol (Amersham Corp., Arlington Heights, Ill.).
  • U251MG and U87MG cell lines are grown to 95% monolayer confluence, trypsinized and harvested with 0.25% trypsin/EDTA, plated in 6-well tissue culture plates, and allowed to adhere overnight at 37° C. in 5% CO 2 in humidified incubators.
  • the cell lines are then treated with various concentrations of virus, either with ⁇ 24, ⁇ 24-NIS, nonreplication competent Ad-NIS, UV-inactivated ⁇ 24-NIS or PBS (MOI ranging from 0.1 to 100).
  • MOI ranging from 0.1 to 100.
  • the addition of radionuclides at various concentrations will not exceed a 2 mCi total dose.
  • the cells will be incubated at 37° C. for 5 days.
  • Cell viability is determined by cellular respiration using 3-(4, 5-methylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium) (Promega, Madison, Wis.) as suggested by manufacturer.
  • the cell survival fraction is measured at each drug concentration as the ratio of absorbance at 490 nm. This calculation is normalized for background absorbance of the culture medium alone.
  • the cell survival fraction is plotted against the logarithm of the drug concentration, and ICW values extrapolated via linear regression into the drug concentrations producing a 50% reduction of normalized absorbance. Crystal violet assays are performed, as described Fueyo et al. (2000), to determine the oncolytic potential of the various constructs with virus and isotopes as described for MTT assays.
  • Tumor kill derived from oncolytic virus versus radiotherapy is disteinguished by FACS analysis, as well as detection of apoptotic markers, since cell death from adenovirus is mediated through cell lysis rather than apoptosis.
  • RB-null and RB-restored Cell Lines To confirm that ⁇ 24-NIS adenovirus replicates in a cell-cycle restricted manner when the Rb pathway is functionally normal, the ability of the mutant adenovirus to replicate in arrested cells expressing wild-type Rb is evaluated.
  • Saos-2 osteosarcoma cell line is used. Saos-2 cells have a well-characterized disruption of the Rb pathway, have a well-characterized response to the transfer of exogenous Rb, and can be easily infected with adenovirus. Saos-2 cells are infected with 100 MOI of an adenoviral vector carrying the exogenous wild-type Rb cDNA or the Ad5CMV-pA adenovirus.
  • the cells are infected with 10 MOI of either the E1A-mutant adenovirus or the UV- ⁇ 24-NIS adenovirus. It is contemplated that cells pretreated with a vector control, Ad5CMV-pA will be permissive for adenoviral replication and expression of the reporter gene. By contrast, cells infected with the Ad5CMV-Rb vector should acquire an oncolytic-resistant phenotype with an inability to display disseminated NIS expression. Because the effect of the E1A-mutant adenovirus is theoretically restricted by cell-cycle factors, flow cytometric analyses of DNA content is used to monitor the changes in the cell-cycle profile of Saos-2 cells in parallel to the studies described herein.
  • D54MG cells are infected with 100 MOI of an adenoviral vector carrying the exogenous wild-type p21 cDNA or Ad5CMV-PA, and 3 days later is infected with the ⁇ 24-NIS virus at 10 MOI.
  • ⁇ 24-NIS The human form of NIS (HNIS) has been cloned and its activity demonstrated by transient transfection in glioma cell lines.
  • HNIS human form of NIS
  • the ⁇ 24-NIS adenoviral construct has recently been obtained and superior radionuclide accumulation with this virus has been seen as compared to stably transfected cell lines.
  • a transgene may be put under the control of an inducible promoter system, such as the Tet/on or Teffoff systems.
  • the possibility of tracking the oncolytic virus ⁇ 24-NIS both after direct intratumoral injection (the intracranial glioma model) and following a method of systemic delivery using DOTAP: cholesterol encapsulated in ⁇ 24-NIS (in the systemic lung cancer models) is studied. These studies will establish a basis for clinical trials for effectively imaging the progression of oncolytic therapies.
  • the objective of this study is to determine the level of ⁇ 24-NIS propagation in intracranial glioma tumor-bearing animals and systemic lung cancer models through imaging radionuclide expression using different delivery methods.
  • U251MG and U87MG glioma cell lines will be injected intracranially into 4 to 6 week old nude mice. After 5 days of tumor growth ⁇ 24-NIS virus will be injected intracranially. After 5 days, the animals will be anesthetized and injected through the tail vein with a radioisotope (e.g., 99m TcO 4 or 131 I). The mice will then be positioned on a gamma camera with a 140 kev-high resolution colimator. The images will be obtained at 5, 15, 30, 60, 90 and 120 min after injection of the radioisotope. These time points have been selected because the optimal signal to noise ratios (SNR) will not initially be known. Because the clearance of isotope and uptake by the NIS pump may vary in different animals, the maximum SNR observed for each animal (M obs SNR) for calculations at any given time point is used.
  • SNR signal to noise ratios
  • U87MG and U251MG glioma cells are seeded in 6-well mictrotiter plates (5 ⁇ 10 5 cells per well) and incubated for 24 h at 37° C. with 5% CO2 and 10% FBS. On the following day, cells are washed twice with PBS and replaced with fresh media without FBS. The replacement media will contain either 2.0 ⁇ Ci of Na 123 I along with 5 ⁇ M of NaI as a carrier. The cells will then incubate for 1 h and then washed with ice-cold Hanks-balanced salt solution (HBSS). The cells will be lysed with 95% ethanol, counted, and measured using a gamma counter (Packard Instruments, Ill.). All studies will be performed in triplicate (Haberkorn, 2001; Cho et al., 2002).
  • mice Five days post-viral injection, the mice will have radioisotopes (e.g., I 123 or I 131 ) injected through a tail vein at a dose of about 0.5 ⁇ Ci per gram of animal body weight.
  • radioisotopes e.g., I 123 or I 131
  • the animals Two hours after tail-vein injection the animals are euthanized and tissues perfused with formalin/saline for autoradiography.
  • the brain and other tissues are harvested and sectioned for gamma-autoradiography using a counter Cobra E5003 device (Packard instruments).
  • the radioactivity is expressed in (counts of tissue of interest per milligcam of tissue of interest)/(the counts obtained in the liver per milligram of liver tissue).
  • Whole body gamma camera imaging will be performed in the small animal imaging facility at M.D. Anderson, Houston, Tex., which includes a dedicated 4.7 Tesla animal MRI, animal CT, animal Single Photon Emitted Computed Tomography (SPECT), as well as a purchased animal Positron Emission Tomography (PET) scanner.
  • PET Positron Emission Tomography
  • multiple imaging techniques will be used both to gain information regarding the spread of ⁇ 24-NIS and to correlate and validate the findings from the various imaging techniques used.
  • small animal MR imaging with a 4.7 Tesla Brucker magnet will be performed in the small animal facility to ensure uniform tumor take.
  • whole animal gamma camera imaging will be performed with a dedicated mouse gamma camera unit, also in the small animal imaging facility.
  • the Emission Tomography nuclear imaging technique measures emitted radiation signals from different locations and reconstructs the images based on the geometry of detection to provide the exact locations of the initial source's signals.
  • this method takes advantage of compounds that emit positrons that undergo very rapid annihilation with neighboring electrons and yields 2 gamma photons, which exit at 180° from each other. More complete geometric information is obtained by the simultaneous capture of gamma photons emitted in opposite directions.
  • 124 I which has a half-life of approximately 4 days, is ideally suited for the evaluation of the ⁇ 24-NIS construct.
  • the measured radiation is detected from different projections, using a conventional gamma camera.
  • This approach typically involves a dual-head gamma camera system, which allows for the detection and geometrical calculation of its emission source.
  • SPECT imaging is the reduced cost of using more readily available gamma-emitting radioisotopes, such as 99m TcO 4 , 123 I, or 131 I.
  • Another advantage of using nuclear imaging to detect oncolytic viral spread is that such small quantities of radioisotope are required for adequate imaging (typically much less than microgram quantities) and only minimal pharmacodynamic perturbation or toxic effects occur in the organ systems of the animals or the patients. When the level of chemical toxicity is minimal, it allows a large variety of useful radiopharmaceuticals to be produced for assessing physiologic and pathophysiologic processes in tumor biology.
  • a reproducible malignant glioma animal model is a component of the studies described herein.
  • the U87MG model was selected for comparing tissues and evaluating the presence of adenoviral replication, as well as for correlating these changes with alterations observed using radioisotope imaging techniques.
  • This cell line has been well characterized. It emulates many of the characteristics of de novo human gliomas including response to growth factors (Pollack et al., 1990), and is readily and reproducibly used to create an intracranial model of human glioblastoma.
  • the studies described herein also utilize the U87MG model to assess if there is an improved therapeutic potential when therapeutic doses of radionuclides are used in conjunction with ⁇ 24-NIS administration.
  • the U87MG cell line is a stable, immortalized human glioblastoma cell line.
  • U87G tumor cells readily grow in the brain of nude mice. For example, in initial studies there was a 100% tumor take in mice, which without treatment results in the need for sacrifice at 21 days ( ⁇ 2 days).
  • Molecular characteristics of the U87MG model include marked expression of coxsackie and adenoviral receptor (CAR), as well as Rb, p16, and p53.
  • CAR coxsackie and adenoviral receptor
  • the reproducibility of the intracranial animal model is based on the implantable screw-guide system developed by Frederick Lang, M.D., M.D. Anderson Cancer Center.
  • a guide screw is implanted into a small drill hole in the skull 2.5 mm laterally and 1.0 mm anteriorly to the bregma.
  • Tumor cells (5 ⁇ 10 5 ) are then slowly injected using an automated injection system, the depth of injection controlled by a collar on the syringe.
  • Early studies showed a 97% success rate with successful delivery of agents into an established tumor.
  • Each animal will be sacrificed by CO 2 inhalation after adequate sedation is confirmed by the toe pinch technique.
  • the tumor will be resected en bloc while maintaining careful attention to preserving the orientation of the tumor with respect to the imaging scan plane and range.
  • the MRI scan plane that traverses the maximum diameter of the tumor will be marked, and the tumor will be sectioned into two pieces along this plane.
  • One section of the tumor will be embedded in OCT (Miles Inc., Elkart, Ind.), frozen in liquid nitrogen, and stored at ⁇ 70° C.
  • the other sections will be fixed in formalin and embedded in paraffin.
  • the tumor specimens resected from the animals injected with Evans Blue will be similarly embedded in paraffin.
  • Cell implantation and adenoviral treatment are performed as described previously. Animal studies are conducted in the veterinary facilities of M.D. Anderson Cancer Center in accordance with institutional guidelines.
  • Non-viral Delivery of NIS is accomplished by liposomal delivery by synthesis of liposome:adenovirus complexes (LAdC).
  • LAdC liposome:adenovirus complexes
  • Liposome (20 mM DOTAP:Chol) are synthesized and extruded through Whatman filters (Kent, UK) of decreasing sizes (1.0, 0.45, 0.2, and 0.1 nm).
  • Whatman filters Karl, UK
  • For preparation of LAdC liposomes will be mixed with varying concentrations of adenoviurs particles (10 2 , 10 3 , and 10 4 ) in 5% dextrose to yield a final concentration of 4 mM liposome containing the appropriate viral particles.
  • Freshly prepared LAdC will be analyzed for mean particle size using a N4 particle size analyzer (Coulter, Miami, Fla.).
  • In vivo studies involves the systemic delivery of LAdC in an experimental lung metastasis model.
  • Human lung cancer cell lines H1299m and A549 are used to establish an experimental lung metastasis model in nude mice or in SCID/Beige mice. These tumor models are well established in the inventor's laboratory and routinely used.
  • A549 and H1299m tumor cells (1 ⁇ 10 6 ) are inoculated intravenously via the tail vein of nude mice and SCID/Beige mice, respectively, to establish experimental lung metastasis. Treatment will be initiated at 6 to 10 days after tumor cell injection at which time microscopic tumors in the lungs are established (data not shown).
  • Treatment for macroscopic tumors will be initiated on day 21 to 25, at which time large macroscopic tumors in the lungs (A549) and other organs (H1299m) are established. Animals are divided into groups and injected intravenously with the virus alone, liposome alone, and LAdC via tail vein (100 ⁇ l/animal). The amount of virus to be complexed with the liposome and injected is determined as above. Animals are subsequently monitored for transgene expression in the lung tumors by imaging. This study will allow the assessment of (a) the possible use of ⁇ -NIS complexed to liposomes for systemic delivery to treat disseminated metastases and (b) possible treatment-related toxicity.
  • the main analytical goal of these studies is to determine at which timepoint the signal-to-noise ratio (M obs SNR) is maximized for the two radioisotopes being examined.
  • the signal is defined as the number of gamma counts in the tumor/labeled tumor cells and the noise is defined as a ratio between the number of gamma counts in the liver/the number of liver cells.
  • Graphic displays of the mean SNR by time are to be provided for each isotope. Futhermore, likelihood-based methods are used to model the relationship between the SNR and time, and to determine the timepoint at which the SNR is the greatest.
  • Radioisotope uptake in tumors infected with ⁇ 24 versus ⁇ 24-NIS are quantified through gamma camera imaging and autoradiography.
  • the infected tumors are harvested at multiple time points and the amount of radiation is normalized for the amount of radiation expressed as counts per minute (CPM) against CPM of spleen.
  • CPM counts per minute
  • the inventors propose to use 30 mice per group and provided power estimates for various effect sizes so that i is mean maximum SNR for iodine-133, ⁇ 2 is the mean maximum SNR for 99m TcO 4 and s is the common standard deviation for both groups.
  • the imaging component of these studies relies on the ability of NIS to concentrate radioisotopes at a level sufficient for them to be visualized above background.
  • Current hNlS clones are able to concentrate 99m TcO 4 pertechnetate approximately 7-fold with only transient transfection experiments in U251 MG cells. This cell line usually does not support very high levels of transfection (somewhere in the range of 15% to 20%), and it is anticipated that the increased transfection efficiency that is afforded by adenoviral constructs will significantly improve this SNR.
  • Cell-mixing experiments with ⁇ 24-NIS demonstrate a much higher level of radionuclide accumulation at 1000% (10-fold) over control cells at only a 10% infection rate.
  • organification of the anion is required. Such organification improves the accumulation of these isotopes.
  • Cho et al., (2000) have shown that the U1240 glioma cell line was able to organify iodide to approximately 5%-6% within the cells compared with approximately 10%-12% radioiodide organification in cultured thyroid cells (a 50% retention would still allow 500% accumulation at a 10% rate of infection). If signal is not adequate in the cell lines, similar washout experiments will be performed to gauge the retention characteristics of U87MG and U251 MG cells. Retention/efflux of isotopes may also be addressed by treating animals with sodium perchlorate 15-30 min after the isotope is given to the animals.
  • Characterization of ⁇ 24-NIS as a therapeutic will include assessing the effectiveness of ⁇ 24-NIS plus therapeutic radionuclide delivery in improving animal survival, and assessing the dosimetry models of radionuclide therapy of a P-emitter through imaging using NIS-accumulated gamma emitter radionuclides.
  • ⁇ 24-NIS may be used for concentrating useful isotopes such as 131 I as well as 188 ReO 4 .
  • Use of the ⁇ 24 system will be studied in an animal model system. Specifically, improvement in animal survival will be assessed after infection with either ⁇ 24 or ⁇ 24-NIS with subsequent administration of therapeutic concentrations of these radioisotopes. Any potential improvement in the “bystander effect” needs to be assessed in animal models prior to future contemplation of using these strategies in human clinical trials. Additionally, these studies will also help to apply and modify current dosimetry models by using gamma emitter isotopes for predicting accumulated dosing within tumors by NIS for eventual use of beta-emitter radioisotope therapy.
  • any additive effects of oncolysis and radionuclide accumulation via concentration by the NIS transgene translates into improved therapy will be assessed, particularly for the intracranial glioma mouse xenograft model.
  • the endpoint will be survival and since the glioma model used has such a short and steep death curve, assessing modest increments in animal survival is feasible.
  • an adoptive randomization design will be employed. This method will limit the number of animals needed and increase the chances of successfully identifying therapeutically active combinations.
  • matched sets of animals will receive pure gamma-emitters at doses appropriate for imaging but not therapy. These will simultaneously act as controls for tumor kill associated with radioactivity and also allow for dosimetry model confirmation.
  • 99m TcO 4 is a routinely used radiopharmaceutical. Its low radiotoxicity makes it suitable for imaging and pharmacokinetic measurements.
  • the radionuclide Na 131 I is used routinely by the inventors and is preferred for many of the studies because of its short half-life and ease of use.
  • the radionuclide 188 ReO 4 has about 4 times the radiotoxicity of 131 I and is also suitable for external imaging. After initial experiments to establish the efficiency of the transfected tumor cells in animal models, treatment with 99m TcO 4 and Na 123 I will be performed.
  • the current tumor model of oncolytic virus transgene expression of NIS in tumor cells does not provide a mechanism for organification and the producer cells are lysed by the virus after a few days post-infection. Thus the retention time of exposure to radionuclides is transient. Because of the relatively short exposure times (about 0.5 h) compared with the long physical half-lives (6 to 1440 h), the relative potency of radionuclides for therapy depends predominantly on the absorbed dose rates of radiation.
  • the dose rates, or S-values for a 1- or 2-gram tumor are provided by MIRDose in Table 1.
  • radionuclides with long half-lives are not as convenient for this system compared to radionuclides such as 131 I.
  • 131 I is a convenient agent of choice for treatment because of its ready availability whereas 188 Re-perrhenate is the radionuclide with the highest dose rate for treatment.
  • 99m TcO 4 pertechnetate is the radionuclide of choice for detection purpose because of its low dose rate.
  • the thyroid is the critical organ for assessing the effect of radio-iodide usage.
  • the GI tract especially the stomach will be the critical organ (0.2 cGy/mCi for 99m TcO 4 or 6 cGy/mCi for 188 ReO 4 ).
  • the safety of thyroid ablation with radio-iodide treatment is well established for patients with Grave's disease as well as for patients with thyroid carcinoma. Doses between 100-200 mCi are routinely used for this purpose with limited to no systemic toxicity. In fact doses as high as 400 mCi have been administired to patients with only minimal grade I-II hematologic toxicity being seen as the major problem.
  • mice will receive 1 of 5 doses of virus (3 ⁇ 10 8 , 10 9 , 10 10 , 3 ⁇ 10 10 , 10 11 ) combined with 1 of 2 therapeutic radionuclides.
  • Three dose levels of the radionuclides will be used, Na 131 I (1.0 mCi, 1.5 mCi and 2.0 mCi) and 188 ReO 4 (1.0 mCi, 2.0 mCi and 3.0 mCi).
  • Na 131 I 1.0 mCi, 1.5 mCi and 2.0 mCi
  • 188 ReO 4 1.0 mCi, 2.0 mCi and 3.0 mCi.
  • Based on this design 30 different dose/virus/radionuclide combinations and a control arm will be used (a total of 31 groups). This high number of combinations will require between 240 and 300 mice to be sacrificed (assuming there are 8 to 10 mice per treatment).
  • An alternative strategy is to use Bayesian outcome-adaptive randomization.
  • the 9 treatment combinations with the greatest probability of being better than the control will be used. This will be followed by randomizing another 8 mice to the same treatment combinations while randomizing 8 other mice to the control arm. After the second stage the posterior probability for the data accumulated thus far in the experiment will be calculated. Once data from the second stage are accumulated the best two treatment/combinations will be selected based on the value of the calculated posterior probability. A confirmatory experiment comparing the best two combinations with the control will be conducted.
  • the first group is the best treatment and has only one arm.
  • the second group is moderately effective treatments after 0, 3, or 6 treatments, and the last and largest group use placebo like substances with 23, 27, or 30 treatments for the group.
  • the best treatment follows a shifted exponential with distribution with expected survival time of 60 days.
  • the moderately effective treaments follow a shifted exponential distribution with an expected survival of 45 days; and with control like treatments follow an exponential distribution with an expected survival of 30 days.
  • the inventors will validate the favored combination with an additional test sets of animals. The best combination will be compared with the “next best” and “least best” conditions. Expectation based on projected models is that the inventors would have selected the best combination with a probability of 94%.
  • a third cohort of patients to receive ⁇ 24-NIS will be added to those patients in an already approved phase I clinical trial using ⁇ 24-RGD. This will be coupled with administration of 99m TcO 4 pertechnetate for gamma camera assessment, a necessary step in the implementation of a phase I toxicity trial to assess the propagation potential of oncolytic viral therapy.
  • a phase I clinical trial is designed that is intended (1) to provide for evaluation of the safety, tolerability, and feasibility of administering ⁇ 24-RGD to patients with malignant glioma, and a wide basic information about the biologic effect of injecting ⁇ 24-RGD into human brain tumors in situ.
  • This study has been approved by M. D. Anderson's IRB. To achieve these goals, the study willinvolve two groups of patients. A first group (Group A) will undergo a standard dose escalation study in which ⁇ 24-RGD is administered by direct intratumoral injection and patients are followed for clinical and radiographic toxicity.
  • the second group includes only patients with resectable tumors. These patients will first undergo stereotactic injection of ⁇ 24-RGD via a catheter that has been permanently implanted into the center of the tumor. After 14 days, the tumor will be resected with the catheter in place, providing a biologic specimen for pathologic and molecular analysis. After tumor removal, ⁇ 24-RGD will be injected into the microscopic residual tumor surrounding the resection cavity. This will allow patients to be followed for toxic effects of injection into a brain infiltrated with microscopic tumor cells.
  • Group C an additional cohort (Group C) will be included to evaluate the suitability of ⁇ 24-NIS for imaging. Although the small numbers of patients will preclude statistical analysis, results from both Group A and B will provide valuable information about response and efficacy.
  • a stereotactic headframe (such as the CRW or Leksell systems) will be attached to the patient.
  • a stereotactic MRI will be performed to localize the tumor mass and a stereotactic biopsy will be carried out.
  • An initial specimen will be sent for frozen section (OCT block) to be analyzed by a certified neuropathologist to provide histologic confirmation of the presence of tumor.
  • a second specimen will be sent for routine fixing and H&E staining (paraffin-embedded fixed block).
  • the third specimen will be snap frozen in liquid nitrogen.
  • biopsy confirms the presence of recurrent glioma
  • the patient will then undergo stereotactic-guided placement of an injection needle.
  • Investigators will be supplied with a vial containing ⁇ 24-RGD or ⁇ 24-NIS.
  • One ml containing the appropriate dose of ⁇ 24-RGD or ⁇ 24-NIS will be injected over 10 min at 1 to 4 sites at the surgeon's discretion.
  • the needle will be flushed with normal saline to assure delivery of the virus (prior to needle placement the volume of the needle must be determined so that only the 1 ml of virus and not the flushing solution is delivered). For this injection the volume will be fixed regardless of tumor size.
  • stereotactic injection patients will be observed in the hospital for a period of observation determined by the treating investigator.
  • a non-contrast CT will be performed to verify the injection site and to identify any acute asymptomatic hematomas. Patients will be evaluated daily for adverse signs and symptoms while in the hospital. Patients will be discharged at the physician's discretion and in accordance with biosafety standards.
  • Group A Intratumoral Maximum Tolerant Dose (MTD). Cohorts of 3 patients will receive an intratumoral injection of ⁇ 24-RGD to determine the MTD. Cohorts of patients will be entered at each dose level as follows: Dose Level ( ⁇ 24-RGD in pfu) 3 ⁇ 10 8 , 1 ⁇ 10 9 , 1 ⁇ 1 10 , 3 ⁇ 1 10 , or 1 ⁇ 10 11 . The total volume of injection will depend upon the size of the tumor, but the dose within the volume will be fixed. The maximal injection volume for each injection site will be 200 ⁇ l per site, evenly distributed. If the calculated volume is greater than 200 ⁇ l per injection, additional injection sites should be established.
  • a stereotactic biopsy will be carried out, and an initial specimen will be sent for frozen section to be analyzed by a neuropathologist to provide histologic confirmation of the presence of tumor.
  • a second specimen will be sent for a routine paraffin-embedded fixed block.
  • the third specimen will be snap frozen in liquid nitrogen.
  • Group B Biologic Effects of ⁇ 24-RGD and Maximum Tolerant Dose (MTD) After Intramural Injection.
  • MTD Maximum Tolerant Dose
  • the goal of this second group is to assess the biologic effect of ⁇ 24-RGD within a tumor and to determine the toxicity resulting from administering ⁇ 24-RGD into the post-resection cavity containing infiltrating tumor cells.
  • a two-stage approach will be undertaken. The first stage will be a stereotactic injection of ⁇ 24-RGD into the tumor via an implantable permanent catheter.
  • the second stage will be an open craniotomy 2 weeks later with en bloc resection of the previously injected tumor mass (to provide a specimen that can be evaluated for ⁇ 24-RGD effects) followed by injection of ⁇ 24-RGD into the walls of the resection cavity (for assessment of toxicity).
  • Stage 1— ⁇ 24-RGD will be stereotactically injected using a silastic catheter. After injection, the catheter will be cut at the level of the skull, closed with a hemoclip and be left in place until the craniotomy is performed.
  • Stage 2 Fourteen days after the initial stereotactic injection, a long enough time to for adequate uptake and replication of ⁇ -24-RGD, patients will undergo open craniotomy (Day 15).
  • the tumor will be removed as a single mass with particular effort to avoid internal debulking and suctioning of the site of prior ⁇ 24-RGD injection.
  • the previously placed catheter will be used for localization of the injection sites.
  • ⁇ 24-RGD will be administered by injections into the wall of the resected tumor cavity.
  • the goal of injection is to distribute ⁇ 24-RGD throughout the tumor wall.
  • a grid of approximately 1 cm 2 squares will be established. Each 1 cm 2 square will be injected with ⁇ 24-RGD.
  • Injection will be performed using a 20 g blunt tip Dandy needle attached to a 1 cc syringe. The needle will be inserted 1-2 cm within the parenchyma and ⁇ 24-RGD infused for 1 min per injection. Minimal irrigation will be used after injection.
  • the total volume of injection will depend upon the size of the tumor, but the dose within the volume will be fixed.
  • the maximal injection volume for each injection site will be 200 ⁇ l per site evenly distributed. If the calculated volume is greater than 200 ⁇ l per injection, then additional injection sites should be established by adjusting the grid.
  • the biological data obtained from stage 2 will be reviewed, and the protocol may be modified, including but not limited to the interval between stereotactic injection and open craniotomy, as well as the number, volume, and depth of stereotactic and open craniotomy injections.
  • patients After the craniotomy and ⁇ 24-RGD injection, patients will be monitored according to customary standards with additional biosafety level precautions. Patients will be evaluated daily for adverse events while in the hospital and will be discharged at the discretion of the treating physician. Patients in Group 2 will not be entered into the study until the toxicity profile from intratumoral injection in Group A patients is established. To further ensure safety, Group 2 patients will not be evaluated until the cohort of patients from Group A has been analyzed. Thus, patients in Group 2 will lag behind Group 1 by at least one level. The entry criteria will be similar to a recently completed NABTC phase I clinical trial of adenovirus p53
  • Group C Intratumoral MTD with Intratumoral Imaging.
  • the third group includes only patients with resectable tumor as in the Group B patients. These patients will first undergo a stereotactic biopsy followed by stereotactic injection of ⁇ 24-NIS through a catheter that has been permanently implanted in the center of the tumor. After 14 days the tumor will be resected in an en-bloc with the catheter left in place. This will provide pathologic specimens for molecular analysis and immunohistochemical staining for assessing viral spread. After the tumor is removed, ⁇ 24-NIS will be injected again into the microscopic residual tumors surrounding the resection cavity. The patients will then be followed for toxic effects from the injection site intruding into the infiltrated surrounding brain.
  • the invenors will be able to accurately determine the extent and geometry of active viral spread throughout the tumor bed. These measurements will be compared to nuclear imaging of patients with isotopes injected at days 3, 7, 10 and 14 prior to the 14-day surgical resection interval. Again, analysis of tumor volume measurements at this dose level will be compared to the imaging results from day 14 and characterization of this material will be related to those patients having M obs SNR at that time point.
  • the primary statistical analysis for this cohort of patients is to determine at which time point (day) the signal-to-noise (STN) ratio is maximized (M obs STN).
  • M obs STN signal-to-noise ratio
  • a M obs SNR will be obtained for each patient on each day of imaging, obtained by chosing the time point which yields the best SNR (in minutes after giving the radionuclide tracer). This measurement will be used to calculate the relative functional uptake of the radionuclide over the observed time points (in intervals of days post viral infection).
  • the variable of interest is M obs STN.
  • the inventors plan to report the mean and standard deviation of maximum STN by day and dose level.
  • the general linear model for modeling M obs STN as a quadratic function of day will be used.
  • the inventors will generate a large number of bootstrap samples from patients' vectors of M obs STNs.
  • the M obs STN will be calculated for each day.
  • the proportion of times a given day is chosen for having the maximum STN ratio will be calculated. This proportion approximates the probability that a given day has the highest signal-to-noise ratio.
  • the M obs STN ratios of 10 or greater are expected on the optimum day after inoculation (which presumptively will be on day 14 for each dose level assuming 10% of the tumor cells are infected and the potential susceptible tumor cells ate not limiting). Concomitantly a minimal STN ratio of 1 is expected for days 1 through 3 (first-pass viral incubation time).
  • the mean M obs STN ratios for days 3, 7, 10, and 14 were 1, 3, 8, and 10 respectively.
  • the mean M obs STN ratios were 1, 3, 10, and 8
  • the mean M obs STN ratios were 1, 10, 8, and 3.
  • M.D. Anderson Cancer Center has experience harvesting en bloc resected tumors specimens with anatomical markers maintained along with an intact injection catheter in place.
  • Patients who are selected for this study with recurrent GBM that requires surgical debulking have tumors that are sizable enough to be adequate for imaging, but it is recognize that this group of patients may not be representative of all patients with tumor recurrence who are not surgical candidates.
  • ⁇ 24-NIS the objectives of trying to initially visualize the tumor through the use of ⁇ 24-NIS, it is believe that the hypothesis can be adequately tested.
  • U87MG cells obtained from the American Type Culture Collection, Manassas, Va., cat. # HTB-14
  • U251MG human glioma cell lines were cultured in Dulbecco's modified Eagle/F12 medium (1:1, vol:vol) (Media Tech, Herndon, Va.) containing 5% fetal bovine serum (DIFCO) and 2 nM glutamine.
  • DIFCO fetal bovine serum
  • Adenoviruses Construction of ⁇ 24 has been described elsewhere (Fueyo, 2000). This construct has a 24-bp deletion in the E1A gene (nt 923 to 946, both included), a region known to be necessary for Rb protein binding (Whyte, 1989), corresponding to the amino acids L 122 TCHEAGF 129 .
  • yeast CD yeast cytosine deaminase
  • oligonucleotide primers (Midland Certified Reagent Co., Midland, Tex.) with pairs that were sequentially elongated by PCR was used to construct ⁇ 24-hyCD. The process was repeated with progressively longer pieces until the full-length gene was obtained.
  • the 5′ most distal oligonucleotide contained an idealized Kozac consensus sequence, a proximal HindIII, and a distal XbaI restriction site for cloning.
  • the nucleotide sequence of the synthesized polynucleotide was also changed significantly (102 of 460 coding base pairs) to optimize a human codon rather than a yeast codon preference.
  • the full-length synthesized polynucleotide was cloned into pcDNA3.1 (Invitrogen) and clones were isolated and subsequently sequenced. Several clones with the DNA sequence of interest were then transiently and stably transfected into U87MG and U251 MG glioma cell lines and assayed for enzyme activity, as described below. Suitable clones expressing enzyme activity were then cloned into the E3 region of pBHG10 (Microbix). pBHG10-hyCD and pXC1- ⁇ 24 were cotransfected into 293 cells to allow homologous recombination, as previously described (Fueyo, 2000).
  • viruses were propagated in 293 cells and purified by ultracentrifugation in a cesium chloride gradient. All viruses were titered using a plaque method as well as optical density measurements and were maintained at ⁇ 80° C. until use. Single lots of adenovirus ⁇ 24 and adenovirus ⁇ 24-hyCD were used in the experiments. As controls, ⁇ 24-hyCD that had been inactivated by UV light and cells that had been mock-infected with culture medium were used.
  • 5-FU was purchased from Sigma Chemical Company (St. Louis, Mo.) and 5-FC from SP Pharmaceuticals (Albuquerque, N. Mex.).
  • PCR U251MG and U87MG cell lines were grown to 95% confluence, harvested with 0.25% trypsin/EDTA, replanted into T25 flasks to a total of 2 ⁇ 10 6 cells, and then incubated overnight.
  • Media were aspirated and 2 ml of adenovirus ⁇ 24-hyCD was added at 0.1, 1, 5, 10, or 100 pfu/cell to duplicate samples from a viral stock of 1 ⁇ 10 11 pfu/ml, and the flasks were incubated for 1 h with continuous shaking.
  • the virus was aspirated and cells were washed twice with PBS. Fresh complete medium containing 10% FBS was replaced and the cells were incubated at 37° C.
  • the following primers and probe were used for the amplification and detection of the hyCD transgene: Forward Sequence: 5′-CAACATGAGGTTCCAGAAGGG-3′ (SEQ ID NO:12); Reverse Sequence: 5′-CAGTTCTCCAGGGTGGAGATCT-3′ (SEQ ID NO:13); TaqMan probe: 5′-TCCGCCACCCTG CACGGC-3′ (SEQ ID NO:14).
  • the primers were labeled with FAM label at the 5′ end and TAMRA label at the 3′ end for yeast CD mRNA.
  • Control primers and probes were used for the ribosomal RNA housekeeping gene S9, a gene with little expression variability in human gliomas (Blanquicett, 2002).
  • the expression of mRNA for hyCD was quantified and reported relative to a stably expressing hyCD clone of the glioma cell line U251MG. Expression levels were determined with the ABI 7000 sequence detection system (Applied Biosystems, Foster City, Calif.).
  • Cytosine deaminase enzymatic assays Separation of uracil from cytosine or 5-FU from 5-FC was achieved by thin layer chromatography as modified from Rubery and Newton (1971). Briefly, aluminum-backed silica gel sheets were used (silica gel 60-F-254, EM Science, Germany). Each sheet was spotted with a total of 5 ⁇ l of a reaction mix or standards at 1 ⁇ l successive spots, with drying before additional spotting. The gel sheets were then resolved in a chromatography tank containing a mixture of 80% chloroform and 20% methanol. The solvent front was quite rapid, with the sheets being resolved within 2-3 min. The separated cytosine and uracil or 5-FC and 5-FU were then visualized with UV excitation at 254 nm. For quantitative enzyme assays, these resolved spots were cut out, placed in scintillation vials, and counted.
  • reaction mixtures were allowed to incubate for 1, 5, 10, or 15 min at 37° C., after which they were quenched by the addition of 1 M acetic acid and placed on ice.
  • the extent of reaction conversion was based on the fraction of produced 5-FU divided by the total counts of both the 5-FC and 5-FU bands. The percentage converted was used to calculate the production of 5-FU per ⁇ g of protein extract per min of reaction time.
  • the apparent K m and apparent V max values were based on nonlinear regression analysis using Graph Pad's Prism program (Graph Pad Software, San Jose, Calif.). All assays were done in triplicate.
  • U251MG and U87MG cell lines were prepared in 6-well plates and treated with ⁇ 24, ⁇ 24-hyCD, or PBS (mock-treatment condition) as described above. The cells were harvested at 24, 48, 72, or 96 h after treatment. Total cell lysates were prepared by incubating cells with 1 ⁇ SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM dithiothreitol), and protein concentration was quantified by using the bicinchoninic acid (BCA) method (Pierce, Rockford, Ill.) and read on a Beckman spectrophotometer.
  • BCA bicinchoninic acid
  • Protein samples (20 ⁇ g) were boiled at 98° C. for 5 min, and lysates were separated on a 15% SDS-Tris glycine polyacrylamide gel, subjected to electrophoresis at 95 V for 2 h, and transferred to a nitrocellulose membrane.
  • the membrane was blocked with 3% nonfat milk, 0.05% Tween 20, 150 mM NaCl, and 50 mM Tris (pH 7.5) and incubated with primary antibody for yeast CD (1:500; Biogenesis Inc., Singer, N.H.).
  • the secondary antibody was horseradish peroxidase-conjugated anti-sheep IgG (Pierce, Rockford, Ill.).
  • the membranes were developed according to Amersham's enhanced chemiluminescence protocol (Amersham Corp., Arlington Heights, Ill.).
  • cell viability by cellular respiration was determined using 3-(4,5-methylthiazole-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium) (Promega, Madison, Wis.) according to the manufacturer's protocol.
  • the cell survival fraction was measured at each drug concentration as the ratio of absorbance at 490 nm relative to untreated cells. This calculation was normalized for background absorbance of the culture medium alone.
  • the cell survival fraction was plotted against the logarithm of the drug concentration, and IC 50 values were calculated using a sigmoidal dose-response curve with variable slope in GraphPad Prism 3.01.
  • the crystal violet assay was performed as described previously (Fueyo, 2000). Briefly, cells were seeded at 10 5 cells per well in 6-well plates, allowed to grow for 20 h, and then infected with ⁇ 24-hyCD, ⁇ 24, or UV-inactivated ⁇ 24-hyCD at 10 MOI. Either 5-FU (at 0.25 mM) or 5-FC (at 0.5 mM) was added to the cultures at different times after infection (0 to 6 days). Cell monolayers were washed with PBS and fixed and stained with 0.1% crystal violet in 20% ethanol. Excess dye was removed with several water rinses.
  • In vitro cytotoxicity was quantified by using the tetrazolium salt 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to measure cell viability.
  • MTT tetrazolium salt 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • 10 4 cells were seeded in 96-well microtiter plates and infected 24 h later with 0, 2, or 5 pfu/cell of ⁇ 24-hyCD or ⁇ 24.
  • Sixteen wells were seeded with untreated glioma cells as a viability control, and 16 wells containing only complete medium were used as a control for nonspecific dye reduction.
  • 5-FC at 0.5 mM
  • 5-FU at 0.25 mM
  • 100 ⁇ l/well (1 mg/ml) of MTT was added to each well.
  • the plates were incubated for an additional 4 h and then read on a microplate reader at a test wavelength of 570 nm. Quadruplicate wells were used for each condition.
  • mice were given i.p. PBS or 5-FC at 500 mg/kg once daily, Monday thru Friday, until the mice displayed signs of neurologic dysfunction (primarily a lack of avoidance behavior or being hunched over in a posterior position) or until they were killed. Mice were killed by CO 2 inhalation and the brains were collected for histopathologic examination and immunohistochemical staining. Animal studies were conducted in the veterinary facilities of the M. D. Anderson Cancer Center in accordance with institutional guidelines.
  • Sections were then incubated with secondary antibodies at a 1:50 dilution at room temperature for 1 h. Staining was performed with acid-fast 3, 3′ amino diaminobenzidine tablets (Sigma). The sections were counterstained with 0.01% methanol green.
  • the anticancer effect in vivo was assessed by plotting survival curves according to the Kaplan-Meier method, and survivals among treatment groups were compared by using the logrank test in GraphPad Prism.
  • the ⁇ 24-hyCD adenovirus To generate ⁇ 24-hyCD the ⁇ 24 adenovirus genome, which includes a 24-base-pair deletion in the Rb-binding region of the E1A gene (Fueyo, 2000), was modified and inserted an expression mini-cassette in lieu of the deleted E3 region.
  • the expression cassette is driven by the human cytomegalovirus promoter placed immediately proximal to the hyCD sequence.
  • a bovine growth hormone polyadenylation region is immediately distal to the stop codon of the synthesized CD gene. This construct was confirmed by sequencing the mini-cassette using a series of primers that covered the entire sequence with some overlap (data not shown). The altered nucleotide sequence that confers human codon preference was confirmed by sequence analysis.
  • ⁇ 24-hyCD expressed a functional exogenous gene
  • the inventors initially assessed yeast CD messenger (mRNA) expression by using real-time quantitative PCR.
  • mRNA yeast CD messenger
  • U251MG and U87MG glioma cell lines were exposed to various concentrations of ⁇ 24-hyCD and allowed to incubate for various periods of time Table 4.
  • the production of mRNA was compared to the amounts of mRNA produced by a U251MG stable clone expressing hyCD (the table displays some negative values which reflects less mRNA production than the stable clone at the early time points and at initial low titer experiments).
  • hyCD enzyme activity was qualitatively assessed by thin layer chromatography. No identifiable conversion of cytosine to uracil was evident in uninfected cells, a finding consistent with the lack of this pyrimidine salvage pathway in human cells. However, enzymatic activity in the ⁇ 24-hyCD-treated U251MG cells rapidly converted cytosine into uracil. Enzymatic activity was verified by using a tritiated cytosine radioisotope.
  • ⁇ 24-hyCD is an efficient vector that can be used to deliver an enzymatically active form of hyCD to glioma cells in vitro.
  • TABLE 3 Percentage conversion of 5-FC to 5-FU by 0.75 mg cell extract previously incubated for 24 h with 10 pfu of ⁇ 24-hyCD per cell Cell Conversion of 5-FC to 5-FU, % Type at 0 min at 5 min at 10 min K m V max U251MG 1.4 ⁇ 0.9 48.6 ⁇ 4.2 82.7 ⁇ 5.6 0.63 ⁇ 0.04 8.4 ⁇ 1.0 U87MG 1.3 ⁇ 0.7 49.7 ⁇ 4.8 90.3 ⁇ 6.1 0.67 ⁇ 0.05 8.3 ⁇ 1.2 V max is expressed as ⁇ mol/min/mg lysate; K m is expressed as the reciprocal of mM.
  • the inventors infected human glioma cells with ⁇ 24-hyCD or ⁇ 24, with or without 5-FC or 5-FU, to analyze adenovirus- and/or drug-induced cell death. Cell death was determined by crystal violet staining of viable cells. The effect of 5-FU on cell killing was no different for ⁇ 24 versus ⁇ 24-hyCD in U251MG ( FIG. 16A ) or U87MG glioma cell lines (not shown). However, the addition of 5-FC to ⁇ 24-hyCD-transduced cells improved cell killing, presumably because of the hyCD enzymatic activity provided by ⁇ 24-hyCD. The addition of 5-FC to ⁇ 24-transduced cells did not affect cell killing at these incubation times and viral titers.
  • U87MG xenografts were implanted intracranially in athymic nude mice.
  • the U87MG cell line was selected because it produces glioma tumors in nude mice with highly predictable pathologic features and growth dynamics.
  • the inventors have previously shown (Miller, 2002) that among three intracranial xenograft models of human glioma, including D54MG, U251MG, and U87MG, U87MG is the most refractory to CD/5-FC GDEPT.
  • An implantable guide-screw system developed in the M. D.
  • the anticancer effect of a single injection of 1 ⁇ 10 8 pfu of ⁇ 24 was compared with a single injection of 1 ⁇ 10 8 pfu ⁇ 24-hyCD, which is designed to minimize the oncolytic effect on survival by this rapidly expanding tumor system.
  • Control animals were concomitantly treated with phosphate-buffered saline (PBS). Some of the treated animals were given 5-FC to determine if an added benefit would result.
  • the median survival time for control groups (PBS or PBS+5-FC) was consistently between 30 and 36 days. For animals treated with ⁇ 24 or with ⁇ 24+5-FC, the median survival times were 35 and 32 days, respectively.
  • the experiment was arbitrarily terminated at day 98 by killing the long-term survivors: 4 animals (40%) in the early-5-FC treatment group and 1 animal (10%) from the late-5-FC treatment group. Median survival time was no different in the early-versus late-treated groups.
  • mice Histopathologic examination of the tumors and brains. After the mice were killed, the brains were removed, fixed in formalin, embedded in paraffin, and sectioned. Microscopic examination of control animal brains revealed non-infiltrative tumors growing in a sphere-like pattern, with a high level of cell proliferation, hypervascularity, and absence of necrotic areas. Lateral displacement of hemispheric structures and collapse of the ipsilateral ventricle indicated that a mass effect was responsible for the animals' deaths. The brains of animals that had survived for long periods, in contrast, showed complete tumor regression. Tumor sequelae, including dystrophic calcification and microcyst formation, were identified at the tumor implantation site in the right caudate nucleus (data not shown).
  • immunohistochemical staining for hyCD showed that ⁇ 24-hyCD efficiently transduced exogenous hyCD protein into human glioma cells in vivo.
  • Expression of the enzyme was detected in the cytoplasm of the glioma cells and correlated with cells infected with the adenoviral vector, as demonstrated by anti-yeast-CD antibody staining of glioma cells within the field of cells characterized by the presence of viral inclusion bodies.
  • two ⁇ 24-hyCD-treated animals were killed at 7 and 14 days after tumor implantation.
  • Ang-2 cDNA (1,504-bp; Gene Bank, accession # AF 004327) which are incorporated herein by reference was amplified by RT-PCR using the following primers: 5′-TACTGAAGAAAGAATGTGG-3′ (forward) (SEQ ID NO:15) and 5′-TTAGAAATCTGCTGGTCGG-3′ (backward) (SEQ ID NO:16) from HUVEC cells. Subsequently, Ang-2 cDNA was cloned into an expression cassette (CMV promoter, SV40pA) in the shuttle adenoviral vector p ⁇ E1sp1A (Microbix Biosystems).
  • CMV promoter SV40pA
  • p ⁇ E1sp1A-Ang-2 was cotransfected with the plasmid pJM17 (Microbix Biosystems) into human embryonic kidney cell line 293 (ATCC, Rockville, Md.). After cotransfection, individual viral plaques were isolated, and AdAng-2 identified by PCR and restriction enzyme digestion, and propagated in 293 cells.
  • adenoviral plasmid which has an expression cassette consisting of the CMV promoter, Ang-2 cDNA, and the SV40-pA in the deleted-E3 region of the adenovirus, was cotransfected with pXC1-Delta-24 encompassing the 24-bp deletion of the E1A region (923-946), corresponding to the region required for Rb binding- in 293 cells (Fueyo et al., 2000). PCR and restriction analyses confirmed the E1A deletion and the insertion of the Ang-2 cDNA.
  • Exogenous Ang-2 expression Western blot analysis was used to confirm the expression of the Ang-2 protein in U-87 MG, D54 MG and U-251 MG (data not shown) glioma cell lines. Secreted Ang-2 was detected by immunoblot analyses of conditioned media (CM) from AdAng-2 or AdCMV-treated U-87 MG cells. Exogenous Ang-2 was secreted in the media, mimicking the dynamics of the endogenous Ang-2 protein.
  • CM conditioned media
  • RNA and protein expression were analyzed using RT-PCR analysis. RT-PCR analysis was performed on mRNA extracted from human glioma cell lines and cultures. Primers and conditions for the PCR reaction were published previously (Poncet et al., 2003).
  • VEGF-A U-87 MG, U-251 MG, LN229, SNB19, and D54 MG
  • D54 MG D54 MG cells
  • Infection of the cell lines will be carried out by dilution of viral stock to particular concentrations, addition of viral solutions to cell monolayers (0.5 ml per 60 mm dish), and incubation at 37° C. for 30 min with brief agitation every 5 min. This procedure will be followed with the addition of culture medium and return the infected cells to the 37° C. incubator.
  • Enzyme-linked Immunosorbent Assay Human VEGF ELISA analysis will be performed to quantify secretory VEGF165 in the conditioned media according to the manufacturer's instructions (R & D Systems, Minneapolis, Minn.).
  • VEGF-A, PCNA, ⁇ SMA will be detected by immunostaining. Similar procedures will be used for the detection of CD34 (Novocastra, Newcastle, UK) and Ang-2 (Santa Cruz, Calif.). Alternative in situ hybrization methods are described in Brown et al., 2000. The presence of E1A and hexon adenoviral proteins in the treated xenografts will be assessed through immunohistochemistry. Paraffin-embedded sections from the mice tumors will be de-paraffinized and rehydrated through xylene and ethanol into PBS. Endogenous peroxidase activity will be quenched by incubation for 30 min in 0.3% H 2 O 2 in methanol.
  • Sections will be treated with goat anti-hexon (Chemicon Inc., Temecula, Calif.) or goat anti-E1A (Santa Cruz Inc., Santa Cruz, Calif.). Immunohistochemical staining will be performed using diaminobenzidine according to the manufacturer's instructions with the Vector laboratories ABC kits (Amersham).
  • HIF-1 DNA-binding activity was determined using an ELISA-solid phase system. Briefly, the procedure was performed as following: U-87 MG cultures were plated at a density of 10 6 cell/100-mm dish; 20 h later cultures were treated with AdAng-2 or the adenovirus control AdCMV-pA at a dose of 80 MOIs, or were mock-infected. Two days after treatment parallel cultures were placed in a sealed modular incubator under hypoxic conditions (0.5% O 2 ) for 6 h. Nuclear extract was prepared as described in Gomez-Manzano et al. (2001).
  • TransAM HIF-1 transcription factor assay kit from Active Motif (Carlsbard, Calif.) was used. This consists of an ELISA-format assay where oligonucleotides containing a HRE motif are immobilized in a 96-well plate. The addition of anti-HIF-1 ⁇ antibody, followed by a secondary HRP-conjugated antibody was assessed by spectrophotometry.
  • HIF-1 protein translation assay Cells will be plated in 6-well plates and pretreated overnight with 25 ⁇ M 2ME2 or DMSO (0.025% vol/vol). Then, medium will be changed to methionine-or cysteine-free as well as serum free medium for 2 hrs. After this time, cells will be labeled by incubation with methionine-or cysteine-free medium containing 35 S-methionine at a final concentration of 100 ⁇ Ci/well at 37° C. for the proposed times. Subsequently, cells will be washed twice with ice-cold PBS, lysed, and subjected for immunoprecipitation using anti-HIF antibody and protein G-agarose beads.
  • Endothelial cell growth assay and endothelial cell migration assay These studies will be performed as described in Gomez-Manzano et al., 2003.
  • Tube Formation Assay This assay will be performed using an in vitro angiogenesis kit (Chemicon, Temecula, Calif.) according to the manufacturer's instructions. Wells in a 96-well plate will be coated with ECMatrix solution, and 5 ⁇ 10 3 cells will be plated in triplicate wells in a volume of 50 ⁇ l of EGM (Clonetics Corp.) containing 2% FBS. The cells will be incubated for 18 h at 37° C., and tube formation will be evaluated by phase-contract microscopy.
  • cells will be stained with Hoechst 33342 (5 ⁇ g/ml, Sigma) and propidium iodide (2.5 ⁇ g/ml, Sigma) for 5 min at 37° C. and analyzed by fluorescence microscopy.
  • Angiogenesis assay in chicken embryos Fertilized chicken eggs (SPAFAS; Charles River Lab., Wilmington, Mass.) will be incubated at 37° C. at 55% humidity for 9 days. An artificial air sac will be created over a region containing small blood vessels in the CAM as described (Brooks et al., 1999). A small window will be cut in the shell after removing 3 ml of albumen. Filter disks (6 mm in diameter) will be coated with cortisone acetate in absolute ethanol (3 mg/mL).
  • the CAM will be locally treated with filter disks saturated with a solution containing bFGF (50 ng/disk; R&D Systems, Minneapolis, Minn.) and VEGF121/rGel (at 1 or 10 nM), rGel (at 1 or 10 nM), or buffer (PBS).
  • the filter will be placed on the CAM in a region with the lowest density of blood vessels, and in the vicinity, as reference, of a large vessel.
  • Angiogenesis will be monitored by photography 3 days after treatment. Images will be captured using an Olympus stereomicroscope (SZ ⁇ 12) and SpotTM Basic software (Diagnostic Instruments, Inc.). The relative vascular area will be determined by measuring the area taken up by blood vessels.
  • Ang-2 is responsible for disrupting the angiogenic process, primarily reducing branching.
  • the inventors will study this effect further by stimulating angiogenesis using rhVEGfl65 and bFGF, prior to Ang-2 treatment. It will be determined if Ang-2-mediated modulation of the angiogenic process in the CAM model overrides the VEGF or bFGF stimulus.
  • CAMs will be pictured using a stereomicroscope and a Sony Digital camera. Images will be analyzed using Adobe Photoshop 6.0. and number of branching points will be quantified. Vessel density will be quantified by using Scion Image 1.63.
  • Regulatable vector constructs are based on the BD Tet-On gene expression system (Clontech).
  • a switchable Ang-2 expression system will be developed, based on the BD Tet-On Expression System from Clontech (Palo Alto, Calif.).
  • a Tet-responsive Ang-2-expression cassette will be made by cloning Ang-2 cDNA into pTRE2 between BamHI and EcoRV (Clontech). Cell lines will be first transfected with the pTet-On vector and selected clones will be then transfected with pTRE2-Ang-2. Clones will be selected by determining Ang-2 expression under doxycycline (Dox) control, selecting optimal induction and low background.
  • Dox doxycycline
  • Doxycycline administration determination of the effective concentration of Dox (Clontech) in isogenic U-87 MG cells will be performed in a titration experiment using different dilutions (e.g., 1, 0.1, 0.001, 0.0001, and 0 ⁇ g/ml).
  • PCI Quantification of PCI and MPI. At least 5 independent microscopic fields per tissue section will be analyzed to count PCNA-positive tumor cells and endothelial cells. Tumor cell proliferation and endothelial cell proliferation will be quantified in vascular hot spots that are identified by screening for areas with highest vessel density at low magnification. PCI will be determined by calculating the ratio between the number of microvessels that colocalized endothelial cell staining (CD34) and pericyte staining ( ⁇ -SMA).
  • TUNEL histochemistry will be performed using an In Situ Cell Detection Kit, POD (Roche Diagnostics Co.).
  • LSC Laser Scanning Cytometry
  • CPE assays The MOI resulting in the destruction of 50% of the cell monolayer (MOI50) will be determined for each virus (wild-type Ad300, UV-inactive-Ad, Delta-24, Delta-24-Ang-2, AdAng-2) in vitro. At indicated time points post-infection, the cells are either stained with crystal violet or photomicrographed. For staining with crystal violet, the medium is removed and cells are fixed for 3 min in 3.7% formaldehyde at room temperature. The formaldehyde is discarded, and the cells are incubated for 3 min in 1% crystal violet. After staining, the crystal violet solution is removed, and the cells are rinsed twice in 3 ml of water and then air-dried. A Trypan blue exclusion test will be performed as described in Fueyo et al., 2000.
  • Viral production will be quantified by TCID50.
  • the cells will be scraped into culture medium and lysed with 3 cycles of freezing and thawing.
  • the Tissue Culture Infection Dose50 method to determine the final viral titration. Briefly, the cell lysates are clarified by centriftigation and the supernatants are serially diluted in medium for the infection of 293 cells in 96-well plates. The cells are analyzed for CPE 10 days after infection. Final titers are determined as plaque-forming units, according the validation method developed by Quantum Biotechnology (Carlsbad, Calif.). Exemplary methods are described in Fueyo et al. (2003).
  • viral titers will be calculated at 48 and 96 h after infection in a diverse panel of proliferating normal cells and tumor cells (Fueyo et al., 2000). These time points should test the consistency of the ability or inability of the adenoviruses to replicate. Viral titers will be compared between Delta-24-Ang-2, wild-type adenovirus Ad300, and Delta-24 in each cell line.
  • Therapeutic index of Delta-24-Ang-2 adenovirus The inventors will assess the effect of adding Ang-2 on the replication profile of Delta-24 in gliomas in vitro. Studies will be performed in panel of glioma cells (Tie2(+): U-87 MG, D54 MG, LN229 and SNB19; Tie2( ⁇ ): U251MG) and in quiescent and proliferating normal human astrocytes (NHA).
  • the inventors have constructed a replication-deficient adenoviral vector, AdAng-2 that efficiently transduces Ang-2 into human glioma cells, and produced and secreted Ang-2.
  • AdAng-2 a replication-deficient adenoviral vector
  • Expression of ectopic Ang-2 results in a significant downregulation of VEGF protein and RNA levels in U-87 MG cell line.
  • the data derived under normoxic and hypoxic conditions indicate that this modulation occurs on the transcriptional level, probably by regulating HIF-Ic protein levels, and subsequently the DNA-binding activity of this transcription factor.
  • Tie2 mRNA and protein are expressed in glioma cultures FIG. 18 .
  • a 503-bp fragment amplification was obtained from RNA extracted from HU-VEC-C, U-87 MG and D-54 MG cells; however transcript amplification was not detected in U-251 MG cells or NIH3T3 cells (negative control).
  • Anti-human Tie2 antibody recognized a 140-kDa band in the membrane proteins subfraction of HU-VEC-C, U-87 MG and D-54 MG, but not in U-251 MG. Cytosol fraction proteins were negative for Tie2 expression. Immunoprecipitation analysis confirmed these results (data not shown).
  • Ang-2-mediated modulation of VEGF-A ELISA was performed to measure secreted VEGF protein levels in the media from Ang-2-treated U-87 MG, D54 MG and U-251 MG human glioma cell cultures.
  • VEGF levels in Ang-2-treated cells were reduced by 40% in U-87 MG and D54 MG cell lines, compared with mock- and AdCMV-treated cells (P ⁇ 0.001, t-test, double sided). However, no significant decrease compared to control-treated cells (P>0.5, t-test, double sided) was detected in U-251 MG cells treated with Ang-2, which is correlated with the pattern of Tie2 expression.
  • Ang-2 decreases the level of secreted VEGF protein at the transcriptional level and points to the existence of a regulatory loop between Ang-2 and VEGF.
  • HIF-1 ⁇ is one of the most important transcriptional regulators of VEGF expression
  • the inventors examined its expression after transfer of Ang-2. Results showed that the exogenous expression of Ang-2 in U-87 MG or D54 MG decreased HIF-1 ⁇ at the protein level. However, and consistent with null Tie2 expression, the expression of HIF-la was not significantly modified when U-251 MG were treated with AdAng-2.
  • transcription experiments using the luciferase reporter gene driven by the VEGF promoter showed that ectopic Ang-2 decreased promoter activity when the fragment containing the HIF-1 ⁇ binding site was tested. These data suggest that Ang-2 decreases HIF-1 ⁇ stimulation of VEGF expression.
  • Northern Blot analyses showed that HIF-1 ⁇ RNA levels were not significantly changed in AdAng-2-treated U-87 MG cells compared with mock- or AdCMV-treated cells.
  • Ang-2 regulation of p53 expression Because p53 is a regulator of VEGF, the inventors determined if the transfer of Ang-2 regulates p53 expression.
  • Western blot analyses of U-87 MG human glioma cell lysates (wild-type p53) three days after treatment with AdAng-2 did not affect the basal levels of p53 (data not shown).
  • a second line of evidence was provided from Luciferase experiments, which showed that the VEGF promoter construct containing an Sp1/p53-binding site is not sensitive to Ang-2.
  • FIG. 21 shows Ang-2 inhibits Ang-1-mediated MEK/ERK phosphorylation in glioma cultures.
  • the Ang-2/Ang-1/Tie2 system has been studied in endothelial cells, and different pathways have been reported as involved in the cascade signaling; these include phosphotidylinositol 2-kinase (PI3-K), focal adhesion kinase, Raf/Ras/mitogen-activated protein kinase (MAPK), and Dok-R/Dok-2/Nck/Pak (Yoon et al., 2003).
  • PI3-K phosphotidylinositol 2-kinase
  • MAPK Raf/Ras/mitogen-activated protein kinase
  • Dok-R/Dok-2/Nck/Pak Dok-R/Dok-2/Nck/Pak
  • tube formation was also compromised when endothelial cells were incubated with conditioned media from AdAng-2-treated U-87 MG cells, (compared to HU-VEC-C cells incubated with conditioned media from AdCMV-treated U-87 MG cells).
  • VEGF-A Modulation of VEGF expression by Ang-2 and antisense VEGF-A. It has been shown that after U-87 MG malignant glioma cells were infected with Ad ⁇ VEGF incorporating the cDNA of VEGF in an antisense orientation the level of the endogenous VEGF mRNA was reduced and the production of the targeted secretory form of the VEGF protein was drastically decreased (Im et al., 1999). ELISA experiments were performed to analyze the effect on secreted levels of VEGF-A from a combined treatment combining Ang-2 transfer with antisense VEGF.
  • U-87 MG cell lines were co-infected with AdAng-2 (50 MOIs) and AdxVEGF (50 MOIs), and levels of secreted VEGF-A were quantified in the conditioned media.
  • AdAng-2 50 MOIs
  • AdxVEGF 50 MOIs
  • the combined treatment of Ang-2 and ⁇ VEGF resulted in a greater decrease of secreted VEGF (33.1%), compared with the results from a single treatment (Ang-2 or aVEGF alone, 71.4% or 79%, respectively), or control (AdCMV;100 MOIs, equal to 100%).
  • VEGFs and the angiopoietins seem to play complementary and coordinated roles in vascular development (Yancopoulos, 2000). Tumor cells can initially home in and grow by co-opting host vessels. However, this diversion of the host vessels is sensed as inappropriated and the vessels regress (Holash et al., 1999a and 1999b). As vessels die, the tumor becomes secondarily avascular and hypoxic, resulting in marked induction of tumor-derived VEGF and robust new angiogenesis (Holash et al., 1999a and 1999b).
  • Ang-2 expression seems to correlate with vessel destabilization, apparently leading to vessel regression in the absence of VEGF, or robust new angiogenesis following induction of VEGF (Yancopoulos, 2000).
  • VEGF and Ang-2 in angiogenesis implies certain mechanisms regulating temporally and spatially the expression of both molecules, affording their concurring or subsequential effect on tumorigenesis.
  • Ang-2 regulates the expression of VEGF through transcriptional mechanisms and data support the fact that downregulation of HIF-1 ⁇ by Ang-2 is the main factor in the regulation of VEGF (Forsythe et al., 1996, Carmeliet and Jain, 2000).
  • the inventors have demonstrated the existence of the Tie2 receptor in glioma cells, and have collected data suggesting the involvement of transduction signaling in the Ang-2 effect.
  • Tie2 in glioma cell lines offers the possibility of 1) testing the effect of Ang-2 in two different scenarios, cells expressing Tie2 (Tie2+) and cells not expressing Tie2 (Tie2 ⁇ ), and 2) the possibility of confirming Ang-2-mediated effects in glioma cell lines not only by adenoviral-transduced Ang-2, but also by treatments using CM from AdAng-2-treated cells and/or direct treatment with rhAng-2.
  • HIF-1 ⁇ protein levels in glioma cells Previous results show that Ang-2 downregulates the protein levels of HIF-1 ⁇ but not the RNA levels in U-87 MG cells. This is consistent with the inventors current understanding that the primary regulation of HIF-1 ⁇ is posttranscriptional (Semenza, 2002). Preliminary data show that treatment of Tie2 positive cells (U-87 MG and D54 MG) with AdAng-2 decreased HIF-1 ⁇ nuclear protein levels, but no significant decrease in HIF-1 levels was seen when U-251 MG (undetectable Tie2) was treated.
  • AdCMV vector control
  • mock vehicle control
  • Adp53 positive control
  • AdGFP specificity control
  • HIF-1 ⁇ inhibition of Ang-2 the effect of Ang-2 on HIF-1 ⁇ postranscritional regulation will be studied.
  • CHX protein translation inhibitor cycloheximide
  • new protein synthesis is inhibited, so that HIF-1 ⁇ protein levels predominantly reflect the degradation process of HIF-1 ⁇ .
  • AdAng-2-treated glioma cells or treated with control adenoviruses, as above will be exposed to CHX for 0 to 40 min and HIF-1 ⁇ protein levels will be analyzed by Western blotting. Densitometry studies of HIF-1 ⁇ protein levels normalized with actin protein levels will allow us to obtain results on the role of Ang-2 in the stability of HIF-1 ⁇ protein.
  • AdAng-2 acceleration of HIF-1 ⁇ ubiquitination and degradation AdAng-2-treated U-87 MG cells will be studied in the presecence or absence of the proteosome inhibitor (10 ⁇ M for 4 hours). It is expected that in AdCMV-treated cells, MG132 will result in enhanced HIF-1 ⁇ protein levels and multiple higher molecular weight species (poly-ubiquinated HIF-1 ⁇ protein conjugates). If Ang-2 is involved in HIF-1 ⁇ protein degradation, MG132 will restore the inhibitory effect of Ang-2 on HIF-1 ⁇ protein levels.
  • Ang-2 effects on HIF-1 ⁇ protein translation.
  • U-87 MG cells will be labeled with 35 S methionine when treated with AdAng-2 (or control adenoviruses) for 0 to 120 min. After 15 min of labeling, HIF-1 ⁇ protein levels will be compared by immunoprecipitation (HIF-1 ⁇ antibody) and autoradiography. If Ang-2 decreases the synthesis of HIF-1 ⁇ , the signal will be higher in Ang-2-treated than in the adenovirus control-treated cells.
  • Ang-2-mediated regulation of HIF-1 ⁇ The Ang-2/Ang-1/Tie2 system has been studied in endothelial cells, and different pathways have been reported as being involved in the cascade signaling; these include P13-K/Akt/mTor, FAK, Raf/Ras/MAPK, and Dok-R/Dok-2/Nck/Pak (Yoon et al., 2003).
  • receptor-mediated HIF-1 regulation has been shown to occur via Ras/MEK/MAPK and PI3K/Akt/FRAP kinase cascades (Bilton and Booker, 2003). These cascades modulate protein synthesis and/or transcriptional activation.
  • the phosphorylation status of MEK/ERK will be examined (as well as the phosphorylation of Akt, it plays a role in the glioma system). It is comtemplated that blocking the effect of Tie2 will have a similar result as the effect of Ang-2 on inhibiting the phosphorylation status of MEK/ERK. These data directly connect Ang-1 to MEK/ERK in human glioma cells through Tie2, and indirectly will connect Ang-2 in this system.
  • Ang-2 Effect of the overexpression of Ang-2 on angiogenesis in vitro. Studies will be performed on the biologic significance of Ang-2 overexpression in human glioma cells to elucidate the role of Ang-2 in the glioma-mediated process of angiogenesis. Both human glioma and human endothelial cells will be used. Firstly, Ang-2 will be transferred, using AdAng-2, to human glioma cells and collect the conditioned media. Then, endothelial cells will be incubated with this conditioned media, and different assays will be used to analyze their phenotypes. Conditioned media from glioma cells treated with AdCMV, AdGFP, or mock treated, will be used as controls.
  • rhAng-2 will be used to examine the effect of Ang-2 on the dynamic phenotype of endotheial cells (growth, migration, formation of channels) independently from other players in angiogenesis, such as VEGF.
  • vehicle BSA
  • rhVEGF positive control
  • rhAng-1 control for specificity
  • Endothelial growth, endothelial migration, and channel formation Endothelial cells will be incubated with conditioned media from Ang-2-treated glioma cells to analyze endothelial growth and tube formation assays, their migration towards conditioned media from Ang-2-treated cells will be quantified (migration assay), and the modulation of the effect of Ang-2 will be also assessed by using stimulators of angiogenesis, such as VEGF, or by inhibiting VEGF.
  • VEGF protein and anti-human VEGF monoclonal antibody (0 to 1 mg/ml, mouse IgG2B isotype, R&D Systems) will be used as controls.
  • studies will be performed by pretreating endothelial cells with rhAng-1, prior to the incubation of the cells with conditioned media from glioma-treated with Ang-2. This procedure will allow one to ascertain if Ang-1 rescues the Ang-2-mediated regulation of the endothelial cell phenotype.
  • Ang-2-mediated effect on endothelial growth, migration, and channel formation Because Ang-2 binds the Tie2 receptor and inhibits pro-angiogenic signals, strategies blocking Tie2 may result in an effect similar to that caused by the overexpression of Ang-2.
  • the inventors will analyze the phosphorylation status of the Tie2 receptor (compared with basal levels) in endothelial cells after incubating them with rhAng-2 or with conditioned media from glioma cells treated with AdAng-2.
  • conditioned media from AdAng-2-treated glioma cells should contain low levels of VEGF protein
  • the phosphorylation status of the VEGFR-2 receptor (compared with basal levels of the protein) on endothelial cells will be assessed after being incubated with conditioned media.
  • rhAng-1 protein will be used as positive control for Tie2, and rhVEGF165 for VEGFR-2 phosphorylation analyses.
  • a dose-dependence study will be conducted using Tie2 blocking antibody (R&D Systems) and examine Tie2's effect on the growth and migration of endothelial cells, and on the ability of endothelial cells to form channels in vitro. Studies will also be performed with Ang-2 and Ang-1 treatment to determine if Ang-1 rescues the Ang-2-mediated regulation of the endothelial cell phenotype.
  • a switchable Ang-2 expression system was developed, based on the Adeno-X Expression System from Clontech (Palo Alto, Calif.).
  • a Tet-responsive Ang-2-expression cassette was made by cloning Ang-2 cDNA into pTRE-Shuttle2 (Clontech). Recombinants were identified using restriction analyses and PCR.
  • the Ang-2 expression cassette was excised from pTRE-Shuttle2 and ligated to Adeno-X Viral DNA (the adenoviral genome).
  • the “Tet-On” expression system was employed, the expression of Ang-2 by tumor cells was induced when doxycycline is added to the culture.
  • Ang-2 inhibits angiogenesis in vivo (CAM model).
  • the inventors also wanted to know if Ang-2 overexpression interferes with the process of angiogenesis in vivo.
  • CAMs were treated either with rhVEGF (200 ng/ml per egg) or rhAng-2 (200-800 ng/ml per egg).
  • Control CAMs were treated with a 0.5% BSA solution (vehicle for Ang-2).
  • Vascular responses were assessed 72 h later.
  • CAMs treated with BSA displayed the typical vascular pattern of a 12-day-old normal CAM, with thin vessels running parallel to each other in a leaf-like pattern.
  • Ang-2 treatment dramatically decreased newly sprouting angiogenic vessels, with no hemorrhagic areas.
  • VEGF stimulated a clearly visible angiogenic response in CAMs (data not shown). This study suggests that the overexpression of Ang-2 disrupts the formation of new vessels.
  • vascular development and growth kinetics in a human glioma intracranial animal model The inventors have performed a time point analysis of the evolution of angiogenesis in a U-87 MG animal model and established correlations between histology and growth patterns in the xenografts. The inventors have studied the Ang-2 expression pattern in this intracranial glioma model. After intracranial injection of 5 ⁇ 10 5 U-87 MG cells into the right basal ganglia of nude mice, the tumors grew from 0.02 mm 3 on day 4 to 100 mm 3 by day 20. All animals died by day 30 post-implantation.
  • the tumors were ellipsoid masses that compressed anatomical structures in the ipsilateral and contralateral hemispheres of the brain and were similar to those formed in other experiments using U-87 MG cells (Fueyo et al., 2003).
  • Ang-2 Effects of Ang-2 on the growth kinetics and angiogenesis in glioma.
  • the inventors contemplate that the VEGF blockade stage of tumor development is, at least in part, due to overexpression of Ang-2.
  • the inventors will use the U-87 MG and D54 MG models growing under different conditions of high VEGF expression and low or high Ang-2, and low VEGF expression and low or high Ang-2. Modulation of Ang-2 will be effected by use of isogenic cell lines expressing Ang-2 under a tetracycline regulatable system.
  • U-87 MG cell lines will be used: parental (high VEGF) and stably transfected with antisense VEGF (Cheng et al., 1998), as well as isogenic D-54 MG cell lines: parental (low VEGF) and stably transfected with VEGF165 cDNA.
  • parental (high VEGF) and stably transfected with antisense VEGF Choeng et al., 1998)
  • isogenic D-54 MG cell lines parental (low VEGF) and stably transfected with VEGF165 cDNA.
  • a tetracycline-regulatable system has been utilized in mammalian cells and in transgenic mice. Benjamin and Keshet (1997), among others, have tested a tetracycline-inducible system in gliomas in vivo.
  • Conditional switching on of Ang-2 correlates with survival.
  • a time point analysis of the evolution of angiogenesis in the U-87 MG animal model has been performed and the findings correlated with the histology and growth patterns of the xenografts.
  • the data showed that it is possible to delineate a well-characterized in vivo tumor model that is designed specifically for the study of antiglioma therapy.
  • specific expression of different angiogenesis-related markers at different stages of tumor growth can be used as a guide for assessing the modulation of angiogenesis by Ang-2.
  • Regulatable-Ang-2 expressing U-87 MG cells will be injected intracranially in nude mice. Three groups of animals will be established for every cell line, in which Ang-2 will be turned on by administration of Dox (antibiotic dissolved in the drinking water) through different periods. The particular time points selected for examining the timing of Ang-2 expression were derived from results obtained in the study of the intracranial U-87 MG xenograft.
  • Dox antibiotic dissolved in the drinking water
  • Ang-2 the expression of Ang-2 will be turn on at different times: (A) the same day of cell implantation (tumorigenicity study); (B) 4 days after cell implantation (angiogenesis switching; early vasculature); or (C) 10 days after cell implantation (establishment of intratumoral vasculature). Animals will be sacrificed if and when they demonstrate signs of general toxicity, or neurological signs or, in the case of long survivors, animals will be sacrified 60 days post-implantation, and brains will be extracted. Similar experiments will be performed in the D54 MG cell line.
  • Statistical analyses will analyze if changes in VEGF expression modified the effect of Ang-2 on the survival of U-87- or D54 MG tumor-bearing animals. Tumors will be examined using H/E staining and immunohistochemistry, as described herein.
  • tumor cells will be analyzed for PCNA (proliferation marker), apoptosis (TUNEL), VEGF, and Ang-2.
  • PCNA proliferation marker
  • TUNEL apoptosis
  • VEGF vascular endothelial growth factor
  • Ang-2 apoptosis
  • CD34,% TUNEL ratio CD34/TUNEL-positive cells
  • SMA pericytes
  • microvascular density (MVD) (measured by staining with antibodies against CD34) will be inversely proportional to the expression of Ang-2 and directly proportional to the expression of VEGF.
  • the microvessel pericyte coverage index (vessels positive for both endothelial and pericyte markers) will be inversely related to Ang-2 expression, indicating that overexpression of Ang-2 destabilized vessel formation.
  • the endpoints are to correlate overexpression of Ang-2 with (a) decreased production of tumor angiogenesis; (b) decreased tumor proliferation; (c) increased tumor and endothelial cell apoptosis; (d) decreased formation of mature vessels; and (e) reduced tumor production of VEGF.
  • Quantification of the data obtained will be by three-color immunofluorescence analysis of CD31 and TUNEL quantified by LSC (CompuCyte Corporation, Cambridge, Mass.) (Davis et al., 2004).
  • the LSC is an instrument designed to enable fluorescence-based quantitative measurements on cellular preparations at the single cell level.
  • the objectives include the collection of data on the timing of Ang-2 overexpression and the response to that (survival) in each cell line.
  • the studies will be performed to analyze survival in glioma-bearing nude mice with and without Dox treatment.
  • Two isogenic U-87 MG cell lines will be used for these studies that have been stable transfected with the regulatable Ang-2 system.
  • Survival curves will be estimated using the Kaplan-Meier method.
  • Cox proportional hazards regression analysis will be used to estimate the hazard ratio between groups along with a 95% confidence interval for this ratio and a likelihood ratio p-value for testing if the ratio is different from 1 (the value of the ratio if the groups have the same survival distributions).
  • the hazard ratio quantifies the relative rates of death between the groups.
  • Ang-2 and/or VEGF-based antiangiogenesis therapy In addition to mechanistic obstacles, the efficiency of gene therapy in general, has been halted by the inability of the replication-deficient adenovirus to transduce a sufficient number of cells to induce a significant anticancer effect in vivo.
  • Adenovirus typically delivers a functional p53 protein in the vicinity of the injection site, but that the majority of the tumor remains uninfected (Lang et al., 2003). For that reason, replication-competent adenoviral system will be used as a delivery tool, preferably Delta-24 adenovirus, which replicates efficiently in human glioma cells but does not replicate in normal cells (Fueyo et al., 2000).
  • VEGF transfer of antisense-VEGF inhibits tumor growth in vivo.
  • VEGF is potentially an optimal target for therapeutic strategies because it is essential for tumor growth and progression.
  • the recombinant adenoviral vector Ad5CMV- ⁇ VEGF incorporates the coding sequence of wild-type VEGF165 cDNA in an antisense orientation.
  • Infection of U-87 MG malignant glioma cells with Ad ⁇ VEGF reduced the amount of endogenous VEGF mRNA and drastically decreased the production of the targeted secretory form of the VEGF protein.
  • Intratumoral treatment with Ang-2 was assessed for modification of the survival of mice bearing U-87 MG intracranial tumors.
  • the inventors implanted 5 ⁇ 10 5 U-87 MG human glioma cells intracranially into nude mice. Three days later, treatment with 1.5 ⁇ 10 8 p.f.u. of AdAng-2 or AdCMV was injected into the tumor using a guide-screw system. Treatment was repeated twice weekly until mice, whose tumors were treated with AdCMV, died (day 32). In contrast, 50 days after cell implantation, 70% of the animals whose tumors were treated with AdAng-2 were still alive, without showing any signs of general or local toxicity.
  • Delta-24 expresses Ang-2 and replicates in vivo. 5 ⁇ 10 5 U-87 MG human glioma cells were injected intracranially into nude mice. Three days later 1.5 ⁇ 10 8 pfu of Delta-24-Ang-2 (2 mice) or UV-inactivated -Delta-24-Ang-2 (2 mice) were injected into the tumor using a guide-screw system. Animals were sacrified 11 days after implantation and brains were collected. Fresh sections were stained with H&E, and permanent paraffin sections were deparaffinized and stained to detect the expression of Ang-2.
  • Optical microscopy of the UV-inactivated Delta-24-Ang-2 treated tumors showed a tumoral mass (>20 mm3) that was highly vascularized and that had no areas of necrosis. In contrast, Delta-24-Ang-2 tumors were significantly smaller ( ⁇ 0.5 mm 3 ), and showed necrotic areas.
  • Immunohistochemical staining of Ang-2 in U-87 MG human tumor xenografts treated with Delta-24-Ang-2 demonstrated high levels of transduced protein compared with the control-treated xenograft. Of importance, staining with anti-hexon protein (structural viral protein) was positive in xenograft treated with Delta-24-Ang-2, suggesting adenoviral replication.
  • U-87 MG cell line was infected with Delta-24 (10 MOIs) or UV-inactivated Delta-24 (10 MOIs), and the replication-deficient AdGFP (25 MOIs) or the control AdCMV (no exogenous cDNA). Three days later cultures were imaged under a fluorescence microscope.
  • LN229 and SNB19 human cell lines exhibit invasive phenotype in vivo.
  • several human glioma cell lines have been tested that will have more clinical significance.
  • LN229 and SNB19 human glioma cell lines are tumorogenic when injected intracranially and exhibit an invasive phenotype in vivo.
  • both cell lines expressed Tie2, which makes them likely candidates for an Ang-2-based strategy.
  • PTK787 or Imatinib was administered at a dose of 25 mg/kg/ip/day or 20 mg/kg/ip/twice/day, respectively, until the animals showed signs of general or local tocixity. Treatment with these drugs did not significantly improve the survival of these animals (P>0.5, logrank test) compared with control-treated animals (vehicle). Neither group treatment had long-term survivors.
  • the inventors have designed an Ang-2/VEGF-based antiangiogenesis therapy with tumor selectivity and efficient delivery.
  • the efficiency of gene therapy is abrogated by the inability of replication-deficient adenoviruses to transduce a number of cells in vivo sufficient to induce a significant anticancer effect. This limitation is underscored by results obtained in a clinical trial using Ad-p53 in gliomas.
  • This cellular context will allow the dissection of the effect of E1A on HIF-10 activity, independently from other adenoviral proteins and from adenovirus replication.
  • the second approach will involve the use of glioma cultures (U-87 MG), which have constitutively high levels of HIF-1. After infecting both cultures with Ang-2 under hypoxic conditions (Zoltan et al., 1996), HIF activity will be assessed by detection of luciferase to quantify the transcriptional activity of HIF-1 and its DNA-binding capacity. If Delta-24 downregulates HIF-1 activity, additional oncolytic adenovirus will be studied.
  • One of these oncolytic adenovirus incorporates a deletion that encompasses the p300-binding region of E1A (Delta-39; aa. D 48 LDVTAPEDPNEE 60 ) (SEQ ID NO:18).
  • the other adenoviral construct has a combination of the E1A deletions present in both Delta-24 and Delta-39 (Delta-24/39) constructs. If the mutant Delta-39 does not have any effect on HIF-1 ⁇ activity, an expression cassette of Ang-2 will be inserted in the E3 region of the Delta-24/39. Thus, the tumor selectivity produced by the 24-bp deletion will be preserved and will have impaired the ability of E1A to interact with p300.
  • Delta-24-Ang-2 replication in vivo and modification of the angiogenic process 5 ⁇ 10 5 human glioma cells (U-87 MG and LN229) will be injected intracranially into 5 nude mice. Three days later, 1.5 ⁇ 10 8 plaque-forming units of Delta-24-Ang-2 or UV-inactivated-Delta-24-Ang-2 will be injected into the tumor using a guide-screw system. Animals will be sacrificed 20 days after treatment to examine the spread of the Delta-24-Ang-2 adenovirus within the tumor. Fresh sections will be stained with H&E. Permanent paraffin sections from the brain will be deparaffinized and stained for the detection of viral proteins such as hexon and E1A. Angiogenesis analyses will be performed. Tumor size, necrosis areas, MVD, PCI, endothelial or tumor apoptosis, and expression and localization of Ang-2 and VEGF will be determined.
  • Ad ⁇ VEGF replicative properties when it co-exists with Delta-24 Infection of Delta-24 with AdGFP (has a similar structure as Ad ⁇ VEGF, but with GFP cDNA) (Fueyo et al., 2000) will allow us to indirectly analyze the replication of the adenoviral vector.
  • U-87 MG, D54 MG, and LN229 cells will be infected with Delta-24 at an MOI of 10 (UV-inactivated Delta-24 as control) and AdGFP (AdCMV as control) at an MOI of 25 or 50. Two and four days later GFP-positive cells will be scored in a total of 500 cells.
  • Infection of Delta-24 with Ad ⁇ VEGF will allow one to directly analyze the replication of the adenoviral vector.
  • U-87 MG, D54 MG, and LN229 cells will be infected with Delta-24 at an MOI of 10 (UV-inactivated Delta-24 as control) and Ad ⁇ VEGF (AdCMV as control) at an MOI of 25 or 50. Two, four, and six days later, quantitative PCR amplification of a fragment of the Ad ⁇ VEGF containing the insert will be performed.
  • Delta-24-Ang-2 may be used in combination with other antiangiogenic agents to abolished mature, preexisting vasculature in established tumors.
  • pXC1-D24 containing the D-24 deletion (Fueyo et al., 2000) was used as the template DNA.
  • Two 5′ phosphorylated primers consisting of sequences of 20 nucleotides on either side of the deletion (sequence DLDVTAPEDPNEE, 48-60 aa of E1A protein (SEQ ID NO:18)) were used for linear mutagenesis primer-incorporating PCR.
  • the template was linearized and the PCR product was recircularized by ligation and transformed into E. coli to produce the vector pXC1-D24/300 encompassing deletions disabling binding of E1A protein with both Rb and p300.
  • pXC1-D24/300 and pBHG10 were co-transfected by liposome-mediated method into 293 cells for homologous recombination and individual plaques were isolated and amplified.
  • Delta-24-300 U-87 MG, U-251 MG and D54 MG human glioma cells were infected with Delta-24-300, Ad300 (wild-type adenovirus), Delta-300, Delta-24, or UV-inactivated Ad300 and assessed cell viability by crystal violet assay and then quantified with MTT tests. Both analyses showed a consistent dose-response effect of Delta-24-300 on the three human glioma cell lines. MTT analyses further showed that the decreased viability observed in the crystal violet assays was highly reproducible and dose dependent in the three cell lines tested. Delta-24-300 is able to induce cell death in glioma cells in vitro at a dose of less than 10 MOI and causes a 50% decrease is cell viability.
  • E1A and fiber protein were significantly down-regulated in the Delta-24-300-treated NHA as compared to the Delta-24-300-treated U-251 MG glioma cells.
  • Delta-24 has been shown to replicate inefficiently and wild-type adenovirus replicates efficiently in quiescent normal cultures (Fueyo et al., 2000, 2003).
  • An E2F-1-promoter construct driving the reported luciferase gene (Johnson et al., 1994) is used to determine if adenoviral-mediated S-phase induction would be impaired following infection of proliferating NHA with Delta-24-300 while wild-type adenovirus would efficiently induce S phase.
  • E2F-1-activity in NHA cultures infected with Delta-24-300 was similar to the mock-treated cultures and lower than that induced by Delta-24.
  • Delta-24-300 the replication phenotypes in glioma cultures and actively-dividing NHA infected with Delta-24-300, Delta-24, Delta-300, wild-type or UV-inactivated wild-type adenovirus were compared. Analysis of viral titers 3 days after infection revealed that the replicative ability of Delta-24-300 is greatly attenuated in the dividing normal cell population and that the ability of Delta-24-300 to acquire a replication phenotype in normal dividing cells is impaired compared to Delta-24 or Delta-300.
  • Delta 24-vIII contains Delta-24 deletion and the chimeric fiber targeting EGFR vIII.
  • the chimeric fiber was designed having the N-terminal of AdS fiber protein (1-83aa), T4 febritin (bacteriophage T4 fibritin halican domain and fold (233-487 aa), linker (e.g., G4SG4SG4S linker), and PEPHC1 ligand (HFLIIGFMRRALCGA (SEQ ID NO.:19)) (Krasnykh et al. 2001; Campa et al. 2000).
  • the tail and T4 fibritin moieties ensure the formation of the trimeric structure of the fiber as well as the correct insertion of the fiber into the virion particle through the tail.
  • the linker joins the fiber and the anti-EGFR vIII ligand.
  • oligonucleotides of cDNAs for the linker and ligand were synthesized according to their amino acid sequences. Complementary oligonucleotides were annealed and inserted into plasmids.
  • the pQE-trisystem (Qiagen, Valencia, Calif.) was used to construct the chimeric fiber so that the protein can ultimately be expressed in E. coli or mammalian cells to verify fiber trimerization. Trimerization was assessed by expression in E. coli M15 (pREP4) (Qiagen) and human cancer cells. The proteins collected from the E.
  • coli or human cancer cells were denatured or not denatured, and then separated by SDS-PAGE and immunoblotted with anti-Ad fiber tail MAb 4D2 (NeoMarkers, Fremont, Calif.).
  • MAb 4D2 anti-Ad fiber tail MAb 4D2 (NeoMarkers, Fremont, Calif.). The protein was successfully able to trimerize, showing a shift in the non-denatured sample to approximately threefold the size of the denatured sample (data not shown).
  • D24-30bvIII The whole fiber cDNA (1745 bp) has been deleted from pAB26 (Microbix Biosytem, Ontario, Canada) and created a Pac I site at the position where the chimeric fiber will be inserted. Three deletions (fiber, Delta-24 and p300) will be made within the Bst B1/Xba I fragment of the pFG173 plasmid (Microbix Biosytem, Ontario, Canada). In particular, these deletions will be made in a pBluescript KS+ backbone and the Bst BI/Xba I fragment containing the deletions will be ligated back to the remaining of pFG173 to obtain pFG ⁇ f ⁇ 24-300.
  • an expression minicassette for Enhanced Green Fluorescence Protein will be inserted into the deleted E3 region of the adenovirus and will be used as a reporter of adenoviral infection ability.
  • the EGFP expression cassette will also be inserted into multiple cloning sites of the plasmids pAB26 and pAB ⁇ f to yield pAB-EGFP and pAB ⁇ f-EGFP.
  • the chimeric fiber FFL which contains the peptide targeting EGFRvIII, will be inserted into pAB ⁇ f or pAB ⁇ f-EGFP in the same direction as the original fiber to obtain pAB-FFL or pAB-FFL-EGFP.
  • FF6H a chimeric fiber containing a peptide control
  • FF6H will also be cloned into pAB ⁇ f or pAB ⁇ f-EGFP to yield pAB-FF6H or pAB-FF6H-EGFP.
  • the shuttle plasmids and pFG ⁇ f ⁇ 24 will be co-transfected into 211B cells which constitutively express fiber protein to allow homologous recombination.
  • the media from the co-transfected 211B cells suspected of demonstrating cytopathic effect will be collected and tested via a PCR assay to verify the recombinant viral genomic region for the specific virus.
  • the confirmed recombinant virus from the cell lysates will be plaqued and each individual plaque will be amplified and verified in 211B cells.
  • the virions will have both the Ad5 fiber and chimeric fiber.
  • the modified adenoviruses will be propagated in A549 cells (ATCC) where the virions should have only chimeric fibers.
  • A549 cells do not express adenoviral genes, the production of wild-type adenovirus is highly unlikely.
  • PCR amplification of E1A followed by enzyme digestion will be performed to detect the E1A mutation for each batch of the virus.
  • U87MG.wtEGFR and U87MG. ⁇ EGFR cell lines were obatined (Nishikawa et al. 1994; Mishima et al. 2001). These cell lines stably over-express either wild-type EGFR (U87MG.wtEGFR) or EGFR vIII (U87MG. ⁇ EGFR). EGFRs were detected by immunoblotting and immunohistochemistry.
  • the anti-EGFR antibody (Cell Signaling, Beverly, Mass.) recognizes both EGFR and EGFRvIII and the anti-EGFRvIII antibody (Zymed Laboratories Inc., San Francisco, Calif.) specifically recognizes EGFRvIII. These cell lines represent a bona fide system to test selectivity of the targeted adenovirus infectivity and will be used for in vitro and in vivo studies.
  • EGFR and EGFRvIII in Heterotransplants from tumors.
  • Heterotransplants of human gliomas expressing EGFR or EGFRvIII will be used to study the targeting of the adenoviral constructs.
  • irrunohistochemical analyses detected expression of EGFR and EGFRvIII.
  • the heterotransplants exhibited an invasive pattern when injected intracranially in nude mice.
  • Infectivity of Mesenchymal Stem Cells The ability of adenovirus to infect human stem cells, will be assessed using human mesenchymal stem cells and two GFP-adenoviral vectors. One of the vectors was redirected to infect through a CAR-independent pathway. Studies showed that adenovirus poorly infects mesenchymal stem cells.
  • Flow cytometry was used to quantify infected cells (GFP-positive cells). Cells were plated to a density of 10 4 and 24 hours later were infected with AdGFP or AdGFP-RGD at an MOI of 10. 72 hours after infection, cells were examined for green fluorescence by flow cytometry.
  • Delta-24-300 Replication in vivo In order to determine if Delta-24-300 replicates in vivo, a study was performed in two animals bearing intracranial U-87 MG xenografts (5 ⁇ 10 5 cells inoculum) infected with Delta-24-300 or UV-inactivated Delta-24-300 (control) as a single dose of 10 8 pfiu/tumor. Brain MRl was performed on Days 7 and 14 after cell implantation and then sacrificed. The MRI scans, shown in FIG. 30 , showed growth progression in the control tumors, but no growth progression in the tumors infected with Delta-24-300.
  • glioma cell lines To test the effect of D24-300 and D24-300vIII in vitro, glioma cell lines have been selected that have 80-100% transduction efficiency with replication-competent adenoviral vectors (U-87 MG, U-251 MG, D-54 MG) (Fueyo et al., 1996a), and where Delta-24-mediated anti-cancer effect has been already tested.
  • U-87 MG cell line ATCC was developed by Nikshikawa et al. (1994).
  • U-87 MG cells have been stably transfected with wt-EGFR (U-87 MGwtEGFR) or mutant EGFR (U-87 MG ⁇ EGFR), expressing wt EGFR or EGFRvIII, respectively.
  • Normal cell cultures from ATCC that will be tested for infectivity and for the effect of the adenoviral construct include Normal Astrocytes (Clonetics), CDD-29Lu and CDD-33Lu (human lung fibroblasts); CDD-33Co, CDD-18Co, and CDD-1 12Co N (human colon fibroblasts), BUD-8 (human skin fibroblasts); HU-VEC-C (human endothelial cells).
  • Human stem cultures will be represented in the in vitro experiments by mesenchymal stem cells and neural progenitors cultures (Cambrex, Walkersville, Md.). Culture conditions will be performed as recommended by manufacturers.
  • Cell Synchronization Cells will be serum-starved for 3 days by culturing in MCDB-105 serum-free medium (Sigma St. Louis, Mo.). To stimulate synchronous cell cycling progression, medium will be replaced with DMEM containing 10% fetal calf serum.
  • Infection of cell lines will be carried out by dilution of viral stock to particular concentrations, addition of viral solutions to cell monolayers (0.5 mL per 60 mm dish) and incubation at 37° C. for 30 min with brief agitation every 5 min. The infected cells will be returned to the 37° C. incubator.
  • PCR Analysis This test will be used to detect the presence of wild-type adenoviruses, adenoviruses carrying wild-type E1A (Fueyo et al., 2000).
  • BrdU Analysis Cells will be pulse-labeled with BrdU for 2 h before collection. After BrdU labeling, adherent and nonadherent cells will be combined, resuspended in 0.5 mL PBS, and fixed in 5 mL 70% ethanol, 50 mM glycine (pH 2.0). Samples will be subjected to denaturation in 0.5 mL 4M HCl for 20 min and then incubated at 37° C. for 1 h in 0.1 mL PBS containing 0.5% BSA, 0.1% Tween and 20 ⁇ L FITC-conjugated anti-BrdU antibody (clone BMG 6H8, Roche Molecular Biochemicals, Indianapolis, Ind.).
  • samples After incubation, samples will be centrifuged and resuspended in a 0.5 mL solution containing 500 ⁇ g/mL RNase A and 10 ⁇ g/mL propidium iodide in PBS. Cells will be analyzed by two-color flow cytometry for BrdU incorporation and DNA content.
  • Cells will be seeded at 10 5 cells per well in two-well chamber slides, allowed to grow for 20 hours and then infected with Delta-24-300. 24 hours later, cells will be fixed with methanol for 4 minutes at ⁇ 20° C. The slides will be incubated for 10 minutes with DAKO protein block serum-free and then incubated with primary antibodies (anti-E1A, Santa Cruz Inc., Santa Cruz, Calif.; anti-BrdU (Biogenics, San Ramon, Calif.). Cells will be pulse-labeled with BrdU for 2 hours before collection. The slides will be washed twice for 5 min with PBS and fluorescent antibody (FICT and Rho) will be added. Slides will be covered with mounted media and fluorescence detected under a fluorescent microscope.
  • Viral Replication Viral production will be quantified by plaque-forming assay. 36 hours after infection, cells will be scraped into culture medium and lysed by three cycles of freezing and thawing. Cell lysates will be clarified by centrifugation and the supernatant will be serially diluted in medium for infection of 293 cells in 6-well dishes. After 1 hour of incubation at 37° C., the infected cells will be overlaid with 3 mL 1.25% SeaPlaque agarose (FMC, Rockland, Me.) in DMEM/F12 with 10% fetal bovine serum. Additional agarose will be added to each dish 4 days later. Plaques will be visualized 7 days after infection.
  • SeaPlaque agarose FMC, Rockland, Me.
  • Immunohistochemistry The presence of adenoviral E1A and hexon proteins in the treated xenografts will be assessed by immunohistochemistry. Paraffin-embedded sections from tumors will be de-paraffinized and rehydrated through xylene and ethanol into PBS. Endogenous peroxidase activity will be quenched by incubation for 30 minutes in 0.3% H 2 O 2 in methanol. Sections will be treated with goat anti-hexon (Chemicon Inc., Temecula, Calif.) or goat anti-E1A (Santa Cruz Inc., Santa Cruz, Calif.). Immunohistochemical staining will be performed according to the manufacturer's instructions with diaminobenzidine by using Vector Laboratories ABC kits (Amersham).
  • Infectivity analyses (GFP detection): Briefly, 48 hrs after infection, cells will be collected by trypsinization, washed with PBS, and stained with 50 mg/mL propidium iodide and 20 mg/mL RNAse for 15 min at room temperature. Samples will be analyzed with an EPICS XL-MCL flow cytometer (Beckman-Coulter, Inc., Miami, Fla.) by using a 488-nm argon laser for excitation. Fluorescence will be detected through 520 band pass filter. All cytometric data will be analyzed with the System II software (Beckman-Coulter, Inc.).
  • PEPHC1 peptide and 6H (control) peptide will be produced synthetically. Cells will be cultured in 96-well plates. 20-24 hours later, PEPHC1 peptide (1 mg/ml) will be added to the culture. Ten minutes later, cultures will be treated with D24-300vIII-EGFP, D24-300-FF6H-EGFP, or D24-300-EGFP. 48 hours later, a direct examination of the cells with fluorescence microscopy will take place, followed by determination of the percentage of green cells (fluorescence) after scoring 500 cells using phase contrast.
  • Detection of EGFR or EGFR vIII (Immunohistochemistry): An anti-EGFR antibody (Cell Signaling, Beverly, Mass.) will be used that recognizes both EGFR and EGFRvIII and an anti-EGFRvIII antibody (Zymed Laboratories Inc., San Francisco, Calif.) that specifically recognizes EGFRvIII.
  • Intracranial implantation and treatment of the tumors The implantable guide-screw method described above (Lal et al, 2000) will be used. PET scan will be used to determine the antiglioma efficiency of the oncolytic system targeting EGFRvIII.
  • Viral particles will be detected by immunostaining for viral proteins as hexon and E1A, as well, as detection of viral inclusions. Because the proliferative potential of precursors cells, and thus, the possibility of supporting viral replication, the subventricular zone, the subgranular zone, the hippocampus and cortex (Gage, 2000) will be examined systematically for the expression of adenoviral proteins. Double immunoflourescence for E1A/hexon and nestin will be performed to elucidate the existence of viral particles in precursor cells.
  • volumes of 50-200 ⁇ l of this processed tissue will then be implanted into the right flank of the recipient animals. Serially passed tumors will be followed until a volume of at least 1000 mm 3 is achieved. Volume doubling times will be calculated from sequential measurements once exponential growth begins. A volume of 500 mm 3 will be tacked as a measure of successful growth as volumes will probably fluctuate somewhat below this level, and progressive growth is expected to occur 500 mm 3 will be on the linear portion of the growth curve in all instances.
  • To asses tumor morphology portions of subcutaneous lesions from all mice that die spontaneously or will be killed will be fixed for at least 48 hours in 10% buffered formalin, embedded in paraffin, cut into 5/7 micron sections, and stained with H&E. These slides will be coded and evaluated for the presence of neoplastic cells.
  • the Delta-24 oncolytic system will be modified to improve the ability of the adenovirus to identify cancer cells.
  • the inventors will target the oncolytic adenoviruses to specific cell-surface receptors.
  • the proposed adenoviral constructs are designed to infect through a mutant form of EGFR, known as variant III or EGFRvIII (Kuan et al., 2001). This mutant receptor has been found expressed only in cancer cells, including approximately 30% of glioblastoma multiforme, which is the most frequent and most common form of gliomas.
  • EGFRvIII encompasses an in-frame deletion of exons 2 through 7 (amino acid residues 6-273) in the extracellular domain. Due to the presence of this deletion, peptides can be designed to bind specifically to EGFRvIII, but not to any of the wild type forms of EGFR. Since malignant gliomas express low levels of adenoviral receptors, redirection of adenoviruses to cancer-related receptors, such as EGFRvIII, should result in a high therapeutic index through the improvement of efficiency in the infection of cancer cells and inability of infection of normal cells.
  • the inventors will replace the wild-type fiber structure of the protein with a chimeric fiber that has been constructed using a commercially available T4 fibritin DNA (Krasnykh et al., 2001), a DNA linker, and finally, the binding peptide.
  • the peptide will be the “contact area” with the host cell.
  • An adenovirus which has been modified to express a chimeric fiber to bind the EGFRvIII receptor, will be able to bind and internalize exclusively into human glioma cells.
  • an adenovirus directed to bind EGFRvIII should be able to infect human glioma cells more efficiently than a wild-type adenovirus because glioma cells express low levels of the natural, main receptor for adenovirus (CAR).
  • CAR main receptor for adenovirus
  • EGFRvIII was selected because it is one of the best-examined systems in gliomas.
  • at least one peptide has already been described that is able to bind EGFRvIII.
  • a system will be used that limits the differences between cells in regards to EGFRvIII expression.
  • This system consists of a human glioma cell line, U-87 MG, which expresses low levels of CAR and EGFRvIII and has been genetically modified to constitutively express high levels of EGFRvIII (U-87 MG- ⁇ EGFR) or EGFR (U-87 MGwtEGFR) (Nikshikawa et al. 1994). Comparisons between the wild-type fiber and fiber-modified adenoviruses in this system should yield clear information about the infectivity capability of both constructs.
  • urokinase plasminogen activator receptor uPAR
  • uPAR urokinase plasminogen activator receptor
  • FGF fibroblast growth factor
  • Glioblastomas also express an alternatively spliced form of FGFR1 containing two immunoglobulin-like disulfide loops (FGFR1 beta), whereas normal human adult and fetal brain tissues express a form of the receptor containing three immunoglobulin-like disulfide loops (FGFR1 alpha) (Yamaguchi et al., 1994).
  • FGFR1 beta immunoglobulin-like disulfide loops
  • FGFR1 alpha immunoglobulin-like disulfide loops
  • the intracranial model of human glioma xenografts implanted in nude mice is one of the most representative procedures for the exploration of novel therapies for brain tumors.
  • the inventor will check the pathology of the tumors, the expression of viral proteins, and the spread of the virus throughout the tumor at several time points in every experiment. The study is designed to ascertain whether or not there is a correlation between the kinetics of the viral spread, the tumor suppression and the improvement of survival. Survival data and pathological observations support the hypothesis that one can efficiently examine the adenovirally-mediated anti-cancer effect in vivo. Furthermore, these data are consistent with the prediction that tropism-modified adenoviruses are more powerful than wild-type fiber adenoviruses, such as Delta 24, in a cell line expressing low levels of CAR.
  • the inventors will perform studies with two different animal models. First, the inventors shall use the U-87 MG system. Glioma cells will be injected intracranially into nude mice. In the second study, the inventors shall use heterotransplants of human gliomas expressing EGFR or EGFRvIII implanted subcutaneously in nude mice. This model is required because the expression of EGFRvIII is generally lost in human glioma cell lines (Bigner et al. 1990). In addition, data from this model will complement those obtained from the U-87 MG model which is both more accurate to analyze the dependence on the expression of EGFRvIII for adenoviral infection, but more artificial than the heterotransplant system.
  • the U-87 system and the heterotransplants are two complementary models in the way that in the U-87 MG system, all cells have a similar genetic makeup with the exception of the artificial expression of EGFR or EGFRvIII.
  • the common characteristic between all the tumors should be the abnormal expression of EGFR or EGFRvIII with most probably different genetic abnormalities.
  • the cell line model will be used to examine in detail adenoviral mediated anti-cancer effect.
  • the second model will be used to determine the specificity in a more realistic setting closer to the clinical scenario.
  • Heterotransplants are also useful to examine the degree of homogeneity in the ability of the targeted adenovirus to infect tumors with different degrees of EGFRvIII expression.
  • Tie2 expression in gliomas To examine the relationship of Tie2 expression in gliomas and the possibility of Tie2 of being involved in tumor formation, the inventors are applying two different and complementary approaches.
  • One approach consists of the isolation of Tie2+ cells from established human glioma cultures, and then characterize these populations for tumor stem-cell like and/or tumorigenic properties.
  • a second approach is based on the isolation of tumor spheroids (stem-like cultures) from human glioma specimens, and subsequent analysis of the expression of Tie2, and the tumorigenic properties of Tie2+ populations in these tumor neuro-spheroids.
  • the information can be obtained from the stable cell lines.
  • Tie2 expression was not detectable in human normal brain.
  • Tie2 transcript was present in tumor neurospheres derived from human glioma tumors in co-existence with CD133 (stem cell marker).
  • CD133 stem cell marker
  • a small population of partially differentiated neural precursors was positive for Tie2 expression.
  • A172 glioma cell line was divided into two populations: Tie2+ and Tie2 ⁇ .
  • FIG. 18 Expression of Tie2 in human malignant gliomas is shown in FIG. 18 .
  • RT-PCR analysis was performed on mRNA extracted from human glioma cell lines and cultures. Primers and conditions for the PCR reaction were published previously (Poncet et al., 2003). A 503-bp fragment amplification was obtained from RNA extracted from the majority of the cell lines and high Tie2 RNA levels were present in A172, U-87 MG, and D54 MG. Sequencing of the amplified product in these cell lines confirmed the presence of the Tie2 transcript. Futhermore, Tie2 expression was detected by western blotting using the membrane subfraction lysates from different cell lines. Anti-human Tie2 antibody recognized a 140-kDa band in the membrane proteins subfraction of HU-VEC-C, U-87 MG and D-54 MG, Al 72, but not in U-251 MG.
  • Cytosol fraction proteins were negative for Tie2 expression. Because expression of RTKs have been reported being different in vitro than in vivo, U-87 MG cells were implanted in the brain of nude mice and Tie2 expression was assessed in sections of those xenografts. Tie2 levels were analyzed using two different antibodies (Santa Cruz, R&D), and competitive inhibition of the epitope/antibody reaction, was obtained with Tie2 peptide. Tie2 expression was in the endothelial cells of some tumoral vessels and peritumoral vessels, as well as in the glioma compartment.
  • Tie2 is present in primary tumor neurosphere culture. Cultures were established from 3 GBMs tumors, which were acutely dissociated into individual cells. Culture conditions were used that favored stem cell growth, established previously for isolation of neural stem cells as neurospheres (Galli et al., 2004). SFM allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF induced the proliferation of multipotent, self-renewing, and expandable neural stem cells (Reynolds et al., 1996). Within 7-14 days of primary culture a subset of the GBMs tumors (3 out of 5) yielded a minority fraction of cells that demonstrated growth into neurosphere-like clusters, or tumor neurospheres.
  • nestin transcript an intermediate filament protein found in undifferentiated central nervous system cells and a characteristic neural stem cell marker
  • CD133 transcript a novel putative neural stem cell marker
  • Bmi1 a molecule necessary for neural stem cell renewal and early neural progenitors (Valk-Lingbeek et al., 2004).
  • the tumor spheres did express Tie2 (please note that tumor should contain glial and endothelial Tie2).
  • RT-PCR was used to detect the presence of CD133 and nestin transcript in A172 and U-87 MG glioma cells lines (Tie2+ cancer cells).
  • Tie2 + populations were isolated from A172 human glioma cell line. To better define the significance of Tie2 expression on glioma cells, the expression of Tie2 was analyzed using flow cytometry, and Tie2 positive and negative cell populations were sorted. Flow cytometric quantification of Tie2 expression in A172 glioma cultures was 11.2%. When tumor cell cultures were sorted for Tie2 expression, Tie2 positive (1.2% total population) and negative (16.5%) cell populations were collected and cultured separately in serum-free neural stem cell medium. Tie2 ⁇ and Tie2+ A172 cells were cultured in suspension in this media. Although some cells died in this culture condition, approximately 50% of the Tie2+ cells remained viable showing with morphological signs of active mitosis, at the moment of that submission.
  • the tumors were large (41.2 ⁇ 6.3mm 3 ) and hypervascularized with large vessels (36.7 ⁇ 3.4 and 53.1 ⁇ 12.9 vessels/0.5 mm 2 ). After day 20 and (104.66 ⁇ 7.1 mm 3 ) the vessels were numerous (53.1 ⁇ 12.9 vessels/0.5 mm 2 ). Staining for proliferating cell nuclear antigen revealed a high proliferative activity ranging from a few hours after implantation (>80% cells).
  • Tie2 was immunoprecipitated from membrane fractions of U-87 MG cells treated with rAng1 (500 ng/ml) for 10 min, and phosphorylation of Tie2 was addressed using anti-phosphotyrosine antibody.
  • treatment of Ang1 induced phosphorylation of Tie2 in U-87 MG cells, suggesting that Tie2 receptor is functional in U-87 MG cells. Therefore, the antagonizing effects of rAng-2 on Ang1-induced signaling pathway in U-87 MG cells was investigated. Inhibition of Tie2 phosphorylation by Ang1 treatment in rAng-2 treated U-87 MG cells was observed.
  • Ang-2/Ang1/Tie2 The Ang-2/Ang1/Tie2 system has been studied in endothelial cells, and different pathways have been reported as involved in the cascade signaling.
  • the inventors have obtained results that suggest that Ang1 induces MEK/ERK stimulation in glioma cells.
  • MAPK activity was measured by the degree of phosphorylation of two MAPKs, ERK1 (p44 MAPK ) and ERK2 (p42 MAPK ). Consistant with previous reports (Kim et al., 2002; Toumaire et al., 2004), rhAng1 (100 ng/ml) increased ERK1/2 phosphorylation of HU-VEC-C endothelial cells.
  • Ras activity which detects active GTP-bound Ras, was performed by immunoprecipitation of Ras-Raf complexes (contains only active Ras), and consequent western blotting detection of Ras-GTP molecule.
  • the inventors results show that Ras activity increases after Ang1-stimulation, suggesting the idea of Ang1/Tie2 signalling through Ras/MAPK in gliomas.
  • Ang-1/Tie signaling in endothelial cells in vitro involves PI3K/Akt activation (DeBusk et al., 2004; Papapetropoulos et al., 2000). Similar experiments were performed by the invnetors confirming the Ang1 effect on Akt phosphorylation in HUVEC (endothelial cells); however, treatment of U-87 MG glioma cells with rhAng1 (100-500 ng/ml) did not modify the pattern of phosphorylation of the Akt molecule.
  • Tie2 can also recruit additional signaling molecules that participate in cellular pathways that affect the shape and migratory properties of cells.
  • the Tie2-associated docking protein Dok-R can potentiate NCK-dependent endothelial cell migration in response to Ang1 (Master et al., 2001; Jones et al., 2003).
  • Our preliminary results performed in stable transfected Tie2-U-251 MG showed that stimulation of Tie2 with Ang1 results in Tie2 association with Dok-R protein.
  • the inventors In order to generate a cell system suitable for the analyses of the Tie2's effect in cell growth, cell invasion, and tumorigenicity, the inventors have constructed an isogenic U-251 MG that constitutively expresses Tie-2.
  • the generation of the cell line involved the stable transfection of Tie-2 cDNA (Audero et al., 2004).
  • Transfected cells have been characterized by immunoblotting for basal and p-Tie2 expression, as well as flow cytometry studies.
  • the invnetors planned to modify the Tie2-positive U-87 MG (ATCC).
  • Tie2 siRNA has been used (Santa Cruz Biotechnology) (Niu et al., 2004). Since both U-251 MG and U-87 MG express a mutant PTEN and therefore are characterized by high levels of p-AKT, a LNN29 isogenic cell line expressing Tie2 will be generated. This cell line will be used to examine the effect of the Tie-2 pathway in a glioma cell line expressing a wild type PTEN protein.
  • Ang1 promotes adhesion of Tie2+ hematopoietic stem cells to fibronectin and collagen and also promotes the interaction of endothelial cells with surrounding mesenchymal cells and the extracellular matrix (Davis et al., 1996; Suri et al., 1996; Arai et al., 2004). Therefore, the inventors analyzed the effects of Ang1/Tie2 signalling on glioma adhesion. Briefly, 96-well dishes were coated for 2 hr at room temperature (RT) with rAng1 diluted in PBS. Wells were then blocked for 30 min at RT with 0.5% heat-inactivated BSA in PBS (80° C.
  • Chemotactic Migration Witzenbichler et al. (1998) have reported the chemotactic properties of Ang1 for Tie2-endothelial cells.
  • a chemotaxis assay was performed in the presence or absence of Ang1. Incubation with various concentrations of Ang1 enhanced chemotactic migration of U-87 MG cells. Migration assay was performed as described previously (Gomez-Manzano, 2003). Briefly, the lower surface of the filter was coated with gelatin. 600 ⁇ l of DMEM/F-12 (1:1) containing rAng1 and 0.1% BSA was added into lower compartment of the Transwell chamber, and 100 ⁇ l of cell suspension was added in the upper chamber.
  • the tumors were ellipsoid masses that compressed anatomical structures in the ipsilateral and contralateral hemispheres of the brain and were similar to those formed in other experiments using U-87 MG cells (Fueyo et al., 2003). Conversely, animals bearing intracranial Ang-2-treated U-87 MG cells had a longer survival (P ⁇ 0.0001, log-rank test); and importantly, 3 of them (3/17; in controls: 0/17) longer than 100 days without signs of neurological disease. These studies showed sustained expression of Ang-2 lead to significant increased on survival. Examination of the brains of long-term survivor animals did not revealed residual tumor. Time point analyses were performed were animals were sacrified 36 h and 10 days after cell implantation.
  • NLLMAAS SEQ ID NO:20 peptide specifically interacts with Tie2.
  • One peptide, NLLMAAS (T4) completely abolished by binding to the Tie2 receptor, the binding of both Ang1 and Ang-2.
  • T4 One peptide, NLLMAAS (T4), completely abolished by binding to the Tie2 receptor, the binding of both Ang1 and Ang-2.
  • T4 the inventors synthesized the peptide linked to FAM signal that was suitable for flow cytometric detection.
  • Peptide was synthesized by automatic solid phase chemistry using Fmoc strategy.
  • N-terminal aminohexanoic acid was labeled by coupling 5(6)-carboxyfluorescein.
  • Peptides were purified to >98% purity by reverse phase HPLC.
  • the inventors have obtained data confirming by flow cytometric methods, that the peptide T4 binds preferentially to the membrane of cells overexpressing Tie2, using U-251 MG isogenic system, and A172.
  • Tie2+ populations derived from cell lines The inventors have isolated Tie2+ and Tie2 ⁇ populations from A172 human glioma cell line by flow cytometric sortening. Similarly, Tie2+ and Tie2 ⁇ populations will be isolated from U-87 MG cell line. Sorted Tie2+ and Tie2 ⁇ aliquots from each cell line will be examined by flow cytometry to evaluate the efficiency of sorting and purity of both populations will be analyzed. Parallel studies will be performed by immunohistochemistry analysis of Tie2 expression.
  • Tue2 expression in tumor spheres derived from human glioma tumors Cultures have been established from 3 glioblastoma multiforme tumors, which were acutely dissociated into individual cells, and cultured. Each brain tumor yielded a minority fraction of cells that demonstrated growth into neurosphere-like clusters, termed tumor spheres. These tumor neurospheres showed some properties related with stem cell populations, as self-renewal (formation of secondary and tertiary neurospheres) and were positive for stem-cell markers. Of interest, Tie2 expression was positive in these cultures.
  • glioma tumor stem cells Purity of brain tumor stem cell population. Because normal stem cells can migrate to sites of injury, and brain tumor cultures may potentially be contaminated with some normal neural stem cell, a conventional cytogenetic analysis will be conducted to demonstrate that the glioma tumor stem cells being isolated are indeed transformed and are not normal brain cells.
  • Tie2/CD133 population The co-existance of Tie2+ and CD133+ cells will be analyzed by using double immunostaining and flow cytometric analysis to detect these markers in the membrane of the isolated population from both, Tie2+ cells from glioma cell cultures, and tumor neurospheres from GBMs specimens.
  • Neurosphere-initiating cells will be assessed for self-renewal activity by examining the replating activity of single viable cells from the Tie2+-sorted/expanded neurosphere cells.
  • Cells derived from neurophere cultures (single neurospheres in 96-well dishes) initiated from Tie2+-sorted cells should consistently reinitiate the formation of secondary neurospheres.
  • the morphology of secondary tumor spheres and the maintenance of expression of the neural stem cell markers nestin, CD133, Brml, will be assessed, as well as maintenance of Tie2 expression.
  • Proliferation ability of Tie2+cells will be assessed by plating 1000 cells/well, and quanifying the number of viable cells on days 0, 3, 5, and 7 after plating using a colometric assay.
  • MIB-1 index will be analyzed for the tumor neuropheres.
  • Tumorigenicity in the initial tumor Tie2 expression in human glioma cell lines: correlation with tumor formation.
  • the main question for this experiment is the importance of Tie2+ populations in tumorigenesis or glioma initiation.
  • a time point analysis of the evolution of the histology and growth patterns of intracranial U-87 MG-derived xenografts has been performed.
  • Tie2+ and Tie2 ⁇ U-87 MG cells will be injected intracranially in nude mice. Two weeks after cell implantation animals will be sacrified and brains collected and analyzed for incorporation of transplanted cells into the brain. Analysis will be based on the formation of tumor. In the case of tumor formation the inventors will examine if tumors maintain similar characteristics of U-87 MG parental-derived tumors, such as vascular proliferation (factor VIII, CD31), MIB index, and/or Tie2 expression.
  • mice will be randomized to a combination of one of three cell concentrations and also to Tie2 ⁇ or Tie2+ cell lines.
  • the response variable for this model is the presence or absence of tumor within a given mouse. This methodology models association between pairs of responses for a given patient with log odds ratios.
  • the Fisher's Exact test will have 97% power to detect a difference of 70% tumorigenesis rate in the 10 5 Tie2 ⁇ cells group vs. a 10% tumorigenesis rate in the 10 3 Tie2 ⁇ cells group.
  • survival curves will be estimated using the Kaplan-Meier method.
  • the inventors will use Cox proportional hazards regression analysis to estimate the hazard ratio between groups along with a 95% confidence interval for this ratio and a likelihood ratio p-value for testing if the ratio is different from 1 (the value of the ratio if the groups have the same survival distributions).
  • the hazard ratio quantifies the relative rates of death between the groups. Based on historic data the inventors expect the control animals to have a median survival of 20 days.
  • a second group of experiments will be focused in analyze the anticancer effect with viral spread (Fueyo et al., 2003).
  • the inventors will perform similar studies as above, however animals will be sacrified at different time points after cell implantation: 10 and 20 days, and brains will be extracted and analyzed for: (a) tumor size, (b) areas of necrosis, (c) viral spread, and (d) localization of the virus around necrosis/microcystic areas, and compare with U-87 parental-derived tumors.
  • the study will reflect adenoviral infection and replication within tumor cells.
  • Spearman rank correlation analysis which is sensitive to general monotonic relationships and is robust to outliers in the data, will be performed.
  • Tie2+ population in tumor-stem cell like populations will transplant Tie2+ sorted/expanded neurosphere cells into nude mice. Briefly, expanded Tie2+ and Tie2 ⁇ sorted neurosphere cells at passage 6-10 will be harvested and gently dissociated with collagenase. 10 3 , 10 4 , 10 5 cells will be transplanted from every group (Tie2+ and Tie2 ⁇ ) into the brain of nude mice. Three weeks after cell implantation animals will be sacrified and brains collected and analyzed for incorporation of transplanted cells into the brain. Analysis will be based on the formation of tumor.
  • Modulation of Tie2 pathway will be studied using two different approaches. First, acute stimulation using recombinant protein Ang1 (rAng1) will be studied using time-dependent and dose-dependent studies. Counterpart experiments will consist on competitive inhibition by treatment with rAng-2, soluble Tie2 receptor (Tie2-Fc), or T4 peptide (as Toumaire et al., 2004).
  • rAng1 recombinant protein Ang1
  • Tie2-Fc soluble Tie2 receptor
  • T4 peptide as Toumaire et al., 2004.
  • the inventors have tested the expression of endogenous Tie2 in a panel of glioma cell lines cell lines. Two Tie2-positive glioma cell lines, U-87 MG, D54 MG, and two Tie2-negative glioma cell lines, U-251 MG and LN229 have been choosen for further study. The inventors have generated an isogenic system consisting of parental U-251 MG and Tie2-expressing U251. Similarly, U-87 MG cell line has been cloned and several clones have been isolated that differ in the levels of Tie2 transcript expression. The inventors have demonstrated that siRNA-Tie2 can inhibited more than 80% of the Tie2 transcript. This strategy will be extended to the Tie2-positive cell line D54 MG and the Tie2-negative cell line LN229.
  • Ang1/Tie-2 signaling pathway in glioma cells Ang1/Tie2 system in endothelial cells has been related with migration, sprouting, and survival. In these cell lines, several groups have demonstrated that these processes are mediated through pathways that include PI3K/Akt, FAK, Raf/Ras/MAPK, and Dok-R/Dok-2/Nck/Pak. Although the significance of Ang1 and Tie2 in vasculogenesis is well established, the signal transduction cascades initiated by the binding of Ang1 to the Tie2 receptor have not been completely characterized. In addition, the transduction signal trigger by Ang1-mediated activation of Tie2 is unknown in glioma cells.
  • Ang1/Tie2 is involved in MAPK activation, and no Akt activation, in U-87 MG cell line.
  • active Tie2 recruits Dok-R in glioma cells, leading to the hypothesis of Tie2 regulating glioma migration.
  • Ang1/Tie2 The role of Ang1/Tie2 in the activation of the Ras/MAPK pathway. Ang1 had been previously shown to activate the MAPK signaling cascade in HUVECs. The inventors have data suggesting the presence of an active Tie2/Ras/MAPK pathway in U-87 MG glioma cells. Studies will be performed using a panel of glioma cells, including Tie2 ⁇ isogenic system. For these studies the inventors will determine whether Ang1-trigerred p42/p44 phosphorylation can be completely abolished by recombinant soluble Tie2 receptor (sTie2), by the presence or absence of a series of concentrations of the Tie2-binding peptide T4, and by Ang-2.
  • sTie2 recombinant soluble Tie2 receptor
  • Ras an intermediary signaling mediator between receptors and ERK1/2.
  • Ras-Raf complexes (containining only active Ras) will be immunoprecipitated, and consequent western blotting detection of Ras-GTP molecule.
  • the inventors have shown that Ras activity increases after Ang1-stimulation, suggesting that Ang1/Tie2 signaling through Ras/MAPK in gliomas.
  • Studies will involve pre-treatments with Tie2 blockers, as well as inhibitors of Ras (FTIs) and MAPK (PD98059) to ascertain whether there is a concatenation of signaling from Ang1/Tie2 to Ras/MAPK.
  • Studies will be directed to ascertain whether Ang1 induced phosphorylation of the Tie2 receptor results in the activation of the p85 subunit of PI 3′-kinase and increased the activity of PI 3′-kinase in the above isogenic cell lines.
  • Cells will be seeded and grown for 24 hours. Then, the medium will be change to medium containing wortmannin. Two hours later, rAng1 will be added to the cells at the indicated amounts, and the cells will be incubated for the indicated times.
  • a phosphorylation assay of the Tie2 or p85 subunit of PI 3′-kinase will be performed with anti-Tie2 antibody (Santa Cruz Biotechnology) or anti-p85 subunit of PI 3′-kinase (Upstate Biotechnology, Inc) according to the method described by Maisonpierre and collaborators (1997) and Hu and coworkers (1996).
  • the phosphorylation of AKT will be assessed comparing basal and phosphorylated levels.
  • PI3K/Akt pathway is activated in glioma cell lines upon Tie2 activation
  • complementary studies to confirm the cellular responses to that pathway will involve blockade of that signaling by LY294002, and PTEN expression (Gomez-Manzano et al., 2003).
  • Dok-R will be immunoprecipitated after Ang1 stimulation followed by immunoblotting the precipitates for phosphotyrosine to establish whether Ang1-mediated activation of Tie2 results in the phosphorylation of Dok-R.
  • Phosphorylation of DokR establishes binding sites for the Ras GTPase-activating protein and the adaptor protein Nck, both of which have been implicated in cell motility and actin rearrangement (Jones et al., 2003). Coimmunoprecipitation studies will then be performed to ascertain whether Ang1 triggering induces significant association of Dok-1 with PAK and Nck.
  • Ang1/Tie2 stimulates the activation of PAK kinase in glioma cells
  • the inventors will stimulate Tie2 positive glioma cells with mock, Ang1 or EGF, and PAK immunoprecipitates will be subjected to an in vitro kinase assay to measure the ability of PAK to phosphorylate MBP (myelin basic protein). If the experiments show that Ang1 stimulates PAK activity, relationship between PAK and migration can easily tested by overexpressing PAK and then, analyzing migration abilities of glioma cells.
  • MBP myelin basic protein
  • Ang1/Tie2 Cellular responses elicited by activation of the Ang1/Tie2 pathway. Studies will be performed to analyze the impact of Ang1/Tie12 signaling in the glioma phenotype. For specificity purpose sTie2, T4 peptide, and Ang-2, will be used, as well as integrin-blocking agents (RGD; see Fueyo et al., 2003). Signaling pathways will be modulated by specific inhibitors (wortmannin or LY294002 for PI3K pathway; PD98059 for MAPK activity) to correlate signal pathways with cellular responses.
  • wortmannin or LY294002 for PI3K pathway
  • PD98059 MAPK activity
  • Ang1 does not stimulate the proliferation of endothelial cells, it promotes survival of those cells (Koblizek et al., 1998; Kim et al., 2000).
  • the inventors have shown that blockade of Tie2 by sustained expression of Ang-2, leads to a decrease of viability in U-87 MG cell line.
  • Studies will be performed using isogenic cell lines exposed to Ang-2, sTie2 and T4, with or without Ang1 treatment. Population doubling time, cell cycle analysis, and BrdU incorporation will be assessed for the Ang1-treated isogenic systems and then compared. Involvement of signaling pathways will be studied by analysis of Tie2 phosphorylation, activation of MEK or Akt (western blotting), and the use of specific inhibitors.
  • Colony forming assay The effect of Ang-1 on colony formation ability under anchorage independent conditions in the above glioma isogenic cell lines will be assessed.
  • the inventors contemplate an enhanced ability to form colonies in the isogenic cell lines expressing Tie2 and a reduction in the number and the size of the colonies in cells treated with soluble Tie2-Fc, Ang-2. It is also contemplated that exogenous Ang-I will support colony formation in a dose-dependent manner.
  • Ang1 has little effect on proliferation, but that it potently stimulates endothelial cell adhesion (Carlson et al., 2001; Xu et al., 2001). It was reported that Ang1 promotes adhesion of Tie2+ hematopoeitic stem cells to fibronectin and collagen (Sato et al., 1998; Takakura et al., 1998), and also promotes the interaction of endothelial cells with surrounding mesenchymal cells and the extracellular matrix (Davis et al., 1996; Suri et al., 1996).
  • Tie2 is expressed in human glioma cells, the inventors contemplate that Tie2 is critical for maintenance of the neoplastic phenotype via cell adhesion to the ECM of the tumor. Therefore, the effects of Tie2/Ang-1 signaling on cell adhesion will analyzed.
  • First Tie2+ cell lines (U-87 MG and A172) will be plated onto wells of a tissue culture dish that had been precoated with either Ang-1 or Ang-2 or various other known ECM proteins. These experiments will be performed also with isogenic cell lines that differ only in their Tie2 status (U-251 MG). The requirement of Tie2 for adhesion will be assessed by blocking the positive reactions with sTie2 and or T4 peptide.
  • ECM-associated Ang1 will be studied in glioma adhesion.
  • Glioma cells will be seeded onto plastic dishes with or without Ang1 or the culture dish containing the ECM components deposited by confluent U-87MG expressing Ang1 (infected with AdAng1).
  • Adherence of glioma cells to ECM-associated Ang1, as well as the activation of Tie2 receptor in their surfaces will be assessed by western blot.
  • Competitive experiments will include the blockade of the Tie2 receptor binding (sTie2, T4), as well as integrin binding (RGD).
  • Ang1 will be applied at different doses with and without a 10-fold excess to reach maximal saturated concentration of either sTie2 or T4 to the lower chambers.
  • a modified checkerboard analysis will be performed in which the concentration of the chemoattractant above and below the filter will vary. If Ang1 is a chemoattractant factor, glioma cells only will migrate when a concentration gradient is present.
  • Ad-E1A-COX-Tie2 Generation of Ad-E1A-COX-Tie2.
  • the inventors will construct an adenovirus carrying the GFP cDNA in the E3 deleted region, driven by the CMV promoter.
  • T4 (Tie2 ligand) coding sequence will be inserted in the HI loop of the fiber protein.
  • the modified adenoviruses will be propagated in A549 cells (ATCC) where the virions should have only chimeric fibers. Since A549 cells do not express adenoviral genes, the production of wild-type adenovirus is highly unlikely.
  • PCR amplification of E1A, and knob fiber-encoding sequence, followed by enzyme digestion will be performed for each batch of the virus (See Fueyo et al., 2003).
  • An Ad-E1A-cTie2 will be generated as a control, having a similar backbone structure than Ad-E1A-Tie2 but having a random peptide in the HI loop.
  • Both adenoviruses will carry a minicassette of expression for the GFP gene (CMV-GFP-SV40 polyadenylation signal) that will be cloned in the deleted E3 region.
  • the strategy will consist of introducing the Tie2 ligand into the HI loop of the fiber protein.
  • pXKdeltaHI is a fiber shuttle vector designed to introduce the ligands into the HI loop of the fiber protein and will be used in the construction of retargeted adenoviruses of the invention.
  • the T4 peptide encoding sequence will be cloned into the EcoRV site of the vector.
  • DNA fragment to be cloned should contain two Thr codons upstream from the ligand and a CC sequence downstream of it (Thr-Thr-ligand-CC). This way, the ligand will be positioned between the Thr-546 and Pro-547 residues of the fiber protein, that is—within the HI loop sequence.
  • Thr-Thr-ligand-CC CC sequence downstream of it
  • Ad-E1A-COX-Tie2 Replication-selective Tie2 targeted adenovirus.
  • Ad-E1A-COX-Tie2 will contain the adenovirus E1A gene fused with the PTGS2 3′ UTR, as Ahmed et al., 2003.
  • the inventors will repeat several of the in vitro experiments performed with Ad-E1A-Tie2, and then it will be tested for anti-cancer effect in vivo.
  • Adenoviral constructs used as controls will include adenovirus with a mutant peptide (cTie2) in the HI loop (Ad-E1A-Cox-cTie2), Ad-E1A-Cox (non-retargeted virus), Adwt, Adwt-Tie2, and PBS.
  • cTie2 adenovirus with a mutant peptide
  • Ad-E1A-Cox-cTie2 Ad-E1A-Cox (non-retargeted virus)
  • Adwt Adwt-Tie2
  • PBS PBS.
  • isogenic systems from high-MAPK activity cell lines U-87 MG and U-251 MG
  • U-118 MG low-MAPK activity cell line
  • the inventors will use GFP (cell quantification) and E1A expression (cell quantification by immunohistochemistry) to quantified the infectivity of the different constructs.
  • GFP cell quantification
  • E1A expression cell quantification by immunohistochemistry
  • the Ad-cTie2 construct, which is expressing in the HI loop of the fiber the control peptide, should infect all these cultures poorly.
  • Tie2 is used only by the Ad-Tie2 construct for internalization, the inventors contemplate a decrease in the infectivity of this vector in the U-251-Tie2 cells pretreated with the T4 peptide. On the contrary, pretreatment with this peptide should not result in a modification of the infectivity of the control adenoviruses.
  • Ad-GFP-Tie2 and Ad-GFP-cTie2 possess similar replication systems but the insertion of the Tie2-targeted peptide and ablation of the CAR binding enhances the selectivity of the former to infect Tie2 expressing glioma cells. Since the ability to replicate is inherently related to the ability of the virus to infect, the inventors are interested in determining whether Ad-GFP-Tie2 infection leads to an increased viral production in Tie2-expressing cancer cells. For viral production analysis the inventors will use TCID50 methodology and an hexon-expressing quantitative assay (BD Bioscience).
  • Oncolytic power of Ad-E1A-Cox-Tie2 Studies will be performed to compare the oncolytic effects of Ad-E1A-Cox-Tie2, Ad-E1A-Cox-cTie2, Ad-E1A-Cox (non-retargeted virus), Adwt, Adwt-Tie2 in normal (toxicity) and cancer cell cultures. These studies will be design to demonstrate that the new construct enhances oncolytic effect in the cultures with Tie2 and low CAR expression (U-87 MG and HUVEC).
  • Crystal Violet and Trypan Blue Exclusion Test will be performed in the panel of glioma cells described above after infection with Ad-E1A-Cox-Tie2, and the explained controls. Dose dependence experiments will be performed (Gomez-Manzano et al., 2004).
  • Toxicity in normal cultures The expression of Tie2 and CAR in Normal Human astrocytes and Neuronal Precursors will be assessed by RT-PCR, Western blot and Immunohistochemical analyses. Transduction will be assessed by GFP detection analyzed after infection with the GFP-constructs (Ad-GFP-Tie2, Ad-GFP-cTie2, and Ad-GFP). Cell viability will be assessed by trypan blue exclusion test after viral infection. The inventors contemplate that although neuronal precursors express Tie2, these cells would not support a efficient viral replication due to low levels of MAPK activity.
  • MAPK-dependent replication Although the correlation between MAPK activity and Ad-E1A-COX ability to replicate has been shown (Ahmed et al., 2003), the inventors have designed studies to assess the ability of retargeted constructs to replicate in glioma cells with high or low MAPK activity. To characterize the effects of the insertion of the 3′ UTR on E1A expression, inhibitors of kinases signaling, PD98059 (p42/p44 MAPK inhibitor), and for specificity testing: SB203580 (p38 MAPK inhibitor) and LY294002 (PI3K inhibitor) will be used.
  • PD98059 p42/p44 MAPK inhibitor
  • SB203580 p38 MAPK inhibitor
  • LY294002 PI3K inhibitor
  • the inventors will use an isogenic U-87 MG system, containing a stably integrated, IPTG-inducible activated Ha-rasVal12 cDNA (Sheng et al., 2000).
  • This model system is based in cells that only differ in the expression of an activated variant of the Hras oncogene, Ha-rasVal12 (Sheng et al., 2000).
  • The will assess whether PTGS2 3′ UTR-mediated Ad-E1A-COX-Tie2 depends on the P-MAPK pathway.
  • the inventors have shown that PD98059 effectively inhibited P-MAPK expression in U-87 MG cells.
  • Ad-E1A-COX-Tie2 The replication capability of Ad-E1A-COX-Tie2 in U87-MG cells untreated and treated with PD98059 (and the control inhibitors) will be assessed.
  • the inventors will use histologically closely matched glioma tumor lines that differ in P-MAPK activity (Ahmed et al., 2003).
  • U-87 MG parental cells will be implanted into the brain of nude mice animals (Lal et al., 2000), and then treated with Ad-E1A-Cox-Tie2 (T4 peptide), Ad-E1A-Cox-cTie2 (control peptide), Ad-E1A-Cox (non-retargeted virus), Adwt, Adwt-Tie2, and PBS (to examine the effect of the technique in survival).
  • Ad-E1A-Cox-Tie2 T4 peptide
  • Ad-E1A-Cox-cTie2 control peptide
  • Ad-E1A-Cox non-retargeted virus
  • Adwt Adwt-Tie2
  • PBS to examine the effect of the technique in survival

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