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WO1996025507A9 - Procedes de preparation et d'utilisation de vecteurs adenoviraux recombines - Google Patents

Procedes de preparation et d'utilisation de vecteurs adenoviraux recombines

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Publication number
WO1996025507A9
WO1996025507A9 PCT/US1996/002336 US9602336W WO9625507A9 WO 1996025507 A9 WO1996025507 A9 WO 1996025507A9 US 9602336 W US9602336 W US 9602336W WO 9625507 A9 WO9625507 A9 WO 9625507A9
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WIPO (PCT)
Prior art keywords
cells
cell
vector
adwtp53
human
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PCT/US1996/002336
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English (en)
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WO1996025507A3 (fr
WO1996025507A2 (fr
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Priority to AU52974/96A priority Critical patent/AU5297496A/en
Publication of WO1996025507A2 publication Critical patent/WO1996025507A2/fr
Publication of WO1996025507A3 publication Critical patent/WO1996025507A3/fr
Publication of WO1996025507A9 publication Critical patent/WO1996025507A9/fr

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  • compositions and methods of the invention are suitable for treatment of a subject afflicted with a tumor and are also useful in cancer gene therapy as a mechanism for purging bone marrow cells of contaminating tumor cells, and preventing the development of cancer cells and tumors.
  • adenoviral vectors have been rendered replication- deficient by replacing the replication regulating Ela nucleotide sequence located at the 5' end of the adenovirus genome with foreign gene expression cassettes.
  • adenoviral genome To replace the portion of the Ela nucleotide sequence in the adenoviral genome with the foreign gene expression cassette, one must modify the adenoviral genome so that adenovirus virions are not produced. These modifications are known in the art, and have been achieved through the application of two methods. One method utilizes the presence of a unique Clal restriction site to excise 900 base pairs of the 5' end of the adenovirus genome (2.6 map units of the adenovirus genome) . The remaining nucleotide sequence of the adenovirus genome, devoid of its' 5' end, does not produce virions as it has lost two essential elements critical for the replication of adenovirus genome:
  • Another approach for modification of the adenovirus genome is by the replacement of DNA sequences in the adenoviral genome by another DNA fragment, resulting in a adenoviral genomic sequence large enough to exceed the packaging limit of the adenovirus virions.
  • This problem has been addressed through a method which replaces the 2.2 kb fragment of pFG140 (a circular DNA derived from dl309 genome with 4.4-kb DNA fragment) containing an ampicillin resistance gene and a bacterial origin of replication (See Graham, F. L. and Prevec, L. (1991) Manipulation of adenovirus vectors, p. 109-128 In Murray, E. J.
  • pJMl7 The resulting plasmid, designated pJMl7, may then be propagated as a plasmid.
  • pJMl7 can be rescued as infectious virions when the foreign 4.4 kb fragment of pJM17 is replaced by homologous recombination with another DNA fragment small enough for the resulting genome to package the adenovirus virion.
  • the pJM17 system solves the background problem present during the screening of recombinant adenoviruses, the difficulty in the quality control of the plasmids and the recombinant vectors still remains. This may be due to the large size and structure of pJM17.
  • pJMl7 is a very large circular DNA molecule (about 40 kb) and has a tendency to undergo undefined rearrangements. After such homologous recombination, specific DNA sequences are occasionally kicked out from the recombinant constructs in vivo, thus rendering the expression of the DNA inserts impossible.
  • the present invention provides a novel method for the construction of adenoviral vectors.
  • the invention provides a technique of constructing an adenoviral vector whereby an additional Clal restriction site is introduced upstream of the original Clal site.
  • the introduction of the second Clal restriction site greatly reduces the chances of obtaining undigested DNA genome. Because only one of the two Clal sites must be cut to prevent the production of the non- recombinant background infectious virions, the addition of a second Clal site greatly increases the chances of generating fully cut DNA and thus reducing the parental genome background.
  • the adenoviral vectors are made using viral genomic DNA as the starting point, there is no need to utilize a plasmid based vector.
  • Adenoviral vectors are generally the preferred vector for the expression of DNA fragments. Although plasmids and retroviruses have been used to express DNA fragments, the efficiency of transfection is generally low. (See Chen, et al., Science. 250:1576-1579, (1990): Shaw, et al. , Proc. Natl. Acad. Sci. USA, 89:4495-4499, (1992); and Casey, et al., Onco ⁇ ene, 6:1807-1811, (1991)). Adenoviral vectors are the preferred vector because they possess certain characteristics which allow for a high efficiency of transfection.
  • Adenovirus based vectors are capable of a high efficiency of transfections because 1) they can grow to high titers; 2) they are internalized into cells with an efficient receptor-mediated endocytosis; 3) are replication incompetent; and 4) they express a transgene to high levels in epithelial cells.
  • Ginsberg, H. S. Virology, eds. Dulbecco, R. and Ginsberg, H. S., (Lippincott, Co., Philadelphia) pp 147-160 (1988); Graham, et al., In Gene transfer and expression protocols, (Murray ,E. J. ed.) pp.
  • adenoviral vectors capable of expressing of cDNA fragments such as wild- ype p53, WAFl/Cipl/p21, wild-type pl6, p27/kipl, and E. coli cytosine deaminase are provided by the present invention.
  • the utilization of adenoviral expression vectors capable of producing high levels of proteins in cells allows for the study of the roles of these proteins in the control and regulation of cell growth in both normal and malignant cells. Furthermore, this strategy has implications in gene therapy for cancers.
  • the protein encoded by the wild-type p53 gene affects cell proliferation by recognizing DNA damage to a cell, resulting in either a delay in progress through the cell cycle to allow for the repair processes of the cell to proceed, or by the initiation of programmed cell death, and/or the induction of apoptosis.
  • WAFl/Cipl/p21 which was also independently isolated as a negative growth regulatory gene.
  • WAFl/Cipl/p21 the protein encoded by WAFl/Cipl, then binds to cyclin-dependent kinases and inhibits their activity. This event enables the cell cycle to be arrested before DNA synthesis, giving the cell the opportunity to repair the damaged DNA.
  • an expression vector capable of producing high levels of WAFl/Cipl/p2l protein in cells would be useful in the determination of the exact relationship between p53 and WAF1-mediated cell growth regulation causing cell cycle arrest and/or apopt ' osis. Further, utilization of this vector would be useful in the determination of the effects of WAFl/Cipl/p21 gene expression in the absence of other p53-mediated signal transducing agents.
  • vascular smooth muscle cells constitute the medial layer of arterial walls that maintains the normal tonus and resistance of vessels.
  • the abnormality of vascular smooth muscle cells induces both functional and anatomical changes of the vessels (Ross, R. (1986) New Engl. J. Med. 314:488-500; Isoyama, S., et al. , (1989) J. Clin. Invest. 84:288-294; and Ogata, M. , et al. (1992) Am. J. Physiol.
  • Adenoviral vector constructs that would be useful in the understanding of the biochemical mechanisms underlying the cell cycle progression through various stages are those that express cyclin kinase inhibitors.
  • a key contribution has been the cloning of several cyclin kinase inhibitors, such as p21/WAFl/Cipl, p27/kipl, and pl6/INK4.
  • cyclin kinase inhibitors such as p21/WAFl/Cipl, p27/kipl, and pl6/INK4.
  • Recent wor has indicated that each one of these kinase inhibitors can potentially regulate one or more of the cyclin kinases leading to the dephosphorylation of Rb protein, which in turn can control the progression of the cell cycle into S phase.
  • RB-like proteins such as pl07 and pl30 can also potentially control this signal transduction pathway leading to the cell cycle arrest
  • pl6/INK4 mediated growth arrest is tightly associated with the status of Rb protein phosphorylation. Therefore, it would be extremely useful to have adenoviral vectors that express pl6/INK4 in order to investigate the association between Rb and pl6-mediated cell cycle arrest.
  • An additional adenoviral vector construct that would be useful in the understanding of the biochemical mechanisms that control growth regulation is an adenoviral vector that expresses the cyclin kinase p27/kipl.
  • Adenoviral vector constructs that would be useful in exploring the clinical utility of suicidal enzymes for the gene therapy of breast cancer is one which expresses E. coli cytosine deaminase. While adenoviral vectors have many attractive features, a key problem with adenoviral vectors is that they can only infect a small population of cancer cells within a tumor mass, leaving many of the cells uninfected. Thus, there is a need to develop adenoviral vectors which should induce cytotoxicity across the whole tumor should adenovirus infect only a small number of tumor cells. One approach would be to use adenoviral vectors which under certain circumstances can be made to produce cytotoxic products which are smaller in size and hence will have opportunities to escape the cells and kill the uninfected cells.
  • adenoviral vectors would prove to be extremely useful. For example, during acute chemotherapy, many breast cancer patients often acquire resistance to various drugs. Although a great deal is known about the molecular mechanisms by which tumor cells acquire drug resistance, there are relatively limited approaches to treat cancers once the drug resistance has been acquired. Thus, there is a need for approaches for treating drug resistant cancers.
  • the invention provides for methods of constructing recombinant adenoviral vectors capable of expressing human cDNAs, such as wild-type p53 cDNA, called herein AdWTp53; WAFl/Cipl cDNA, called herein AdWAFl; p27/kipl, called herein Adp27; E. coli cytosine deaminase, called herein AdCD; wild-type pl6, called herein Adpl6; TAM 67 (a jun/fos dominant negative mutant) , called herein AdTAM67; and B7-1 and B7-2, called herein AdB7-l and AdB7-2, respectively.
  • AdWTp53 wild-type p53 cDNA
  • WAFl/Cipl cDNA called herein AdWAFl
  • Adp27/kipl called herein Adp27
  • AdCD E. coli cytosine deaminase
  • Adpl6 E. coli cytosine
  • the invention further provides a method of inhibiting the growth and/or the cell cycle of proliferating cells.
  • This method comprises contacting the cells with a recombinant adenovirus vector capable of expressing human cDNAs in an amount effective to inhibit cell proliferation.
  • the invention also provides a composition for contacting cells with an amount of a recombinant adenovirus vector capable of expressing human cDNAs in an amount effective to inhibit cell proliferation.
  • the invention additionally provides a method of treating a subject afflicted with a tumor which comprises contacting the tumor with an effective amount of a recombinant adenovirus vector capable of expressing human cDNAs so as to inhibit proliferation of the tumor cells.
  • the invention provides a method of treating a subject afflicted with a tumor which comprises contacting the tumor with an effective amount of a recombinant adenovirus capable of expressing human cDNAs in the presence of a chemotherapeutic agent so as to inhibit proliferation of the tumor cells.
  • the invention provides a method of treating a subject afflicted with a tumor which comprises contacting the tumor with an effective amount of a recombinant adenovirus capable of expressing human cDNAs in the presence of an amount of irradiation so as to inhibit proliferation of the tumor cells.
  • the invention also provides for the use of adenoviral vectors in cancer gene therapy as a mechanism for purging bone marrow cells of possible breast cancer contaminants.
  • the invention also provides for the use of adenoviral vectors in combination with toxins and cytotoxic drugs as a mechanism for purging bone marrow cells of possible breast cancer contaminants.
  • the invention further provides for the use of adenoviral vectors to eradicate cancer cells and tumors by contacting the cancer cells with an amount of adenoviral vector sufficient to eradicate the cancer cells.
  • the invention provides for the use of adenoviral vectors as a preventative mechanism for the development of cancer in subjects who are at risk of developing cancer.
  • the invention also provides for a method of treating a subject afflicted with a tumor that has shown resistance to drugs which comprises contacting the tumor with an effective amount of a recombinant adenovirus capable of expressing human cDNAs so as to inhibit proliferation of the tumor cells.
  • FIG. 1 Structure of the Recombinant Adenoviral Vector AdWTp53.
  • a diagrammatical representation of the structure of the recombinant adenoviral vector AdWTp53 is shown.
  • the top, hatched segment of the diagram represents the adenovirus type 5 genome, consisting approximately of 9.24- 100 map units (mu) .
  • the bottom portion of the diagram represents an enlargement of the human-wild type- p53 expression cassette.
  • the human-wild type p53 expression cassette contains a left inverted terminal repeat (ITR) , an origin of replication, encapsidation signals, and an Ela enhancer derived from adenovirus type 5 (stippled segments) .
  • the expression cassette also contains human wild-type p53 cDNA (solid segments) and an SV40 maturation signal (right blank segment) .
  • Figure 2A I ⁇ nunoprecipitation of 3S S-labeled human wild- type p53 protein from H-358 cells exposed to varying doses of AdWTp53 or AdControl.
  • This figure shows the immunoprecipitation of 35 S-labeled human wild-type p53 protein from H-358 cells exposed to increasing doses of AdWTp53 or AdControl.
  • cell lysates were immunoprecipitated using anti-p53 antibody, solubilized protein samples loaded on 8% SDS-polyacrylamide gel electrophoresis, gels dried and exposed to X-ray film.
  • the left panel shows radioactive signals of p53 precipitates from H-358 cells exposed to AdControl at 0.1, 1, 10 and 50 pfu/cell.
  • the numbers 0.1 through 50 denoted above the lanes represent the pfu/cell.
  • the arrow indicates the position of migration of p53 protein.
  • Figure 2B Immunoprecipitation of 3S S-labeled p53 protein from various cell lines. Cell lines, MCF-10, MCF-7, MDA-MB- 231 and Adr R MCF-7 were exposed to AdWTp53 (50 pfu/cell) or AdControl (adenovirus alone; 50 pfu/cell) , and p53 protein was immunoprecipitated as set forth in Example 8.
  • the left panel shows the results of p53 immunoprecipitation of uninfected cells
  • the middle panel shows the immunoprecipitation of cell exposed to AdControl
  • the right panel indicates the results of immunoprecipitation of cells exposed to AdWTp53.
  • the arrow indicates the position of migration of p53 protein.
  • Figure 3A Effect of AdWTp53 and AdControl on H-358 cell growth. 5xl0 4 cells were plated in triplicate on 6 well tissue culture plates, exposed to AdWTp53 (10 pfu/cells) or AdControl (10 pfu/cell) , and the cell number counted on each day. Shown are cell number of H-358 cells: uninfected (9 ) , exposed to AdWTp53 (B) , and exposed to AdControl ( ⁇ ) . Values shown are mean+SE.
  • FIG. 3B Effect of AdWTp53 and AdControl on MDA-MB-231 cell growth. 5xl0 4 cells were plated in triplicate on 6 well ⁇ tissue culture plates, exposed to AdWTp53 (10 pfu/cells) or AdControl (10 pfu/cell) , and the cell number counted on each day. Shown are cell number of MDA-MB-231 cells: uninfected
  • Figure 3C Effect of AdWTp53 and AdControl on MCF-7 cell growth. 5xl0 4 cells were plated in triplicate on 6 well tissue culture plates, exposed to AdWTp53 (10 pfu/cells) or
  • AdControl (10 pfu/cell) , and the cell number counted on each day. Shown are cell number of MCF-7 cells: uninfected (•) , exposed to AdWTp53 ( ⁇ ) exposed to AdControl( ⁇ ) . Values shown are mean+SE.
  • FIG. 4 Effect of AdWTp53 on the survival rate of different cells.
  • Cells were exposed to different concentrations of AdWTp53 for 7 days and the survival rates calculated by the colorimetric method described in Example 3. Shown are the percent survival values for each cell line using different pfu/cell, as shown in Figure 3. The results for each cell line are represented by the following symbols:
  • MCF-7 (•), MCF-10 ( ⁇ ), 184B5 (*) , NMECs ( ⁇ ) , and represent the mean of triplicate determinations.
  • FIG. 5 Western blot analysis of p53, WAFl/Cipl, mdm2 and actin proteins in breast cancer cell lines (MDA-MB-157, MDA- MB-231, MDA-MB-453, MCF-7) , a lung cancer cell line (H-358) , immortalized mammary cells (MCF10, 184B5) and NMECs.
  • MDA-MB-157, MDA- MB-231, MDA-MB-453, MCF-7 a lung cancer cell line
  • H-358 immortalized mammary cells
  • NMECs NMECs.
  • Cells at a concentration of 0.5xl0 6 were plated in 6 cm tissue culture dishes and infected with either 10 or 50 pfu/cell of AdWTp53 or 50 pfu/cell of AdControl for 24 hours.
  • the cells were harvested and resuspended in 1 ml of IxSDS-poly acrylamide gel electrophoresis buffer and 15 ⁇ g of protein were separated in a 8% SDS polyacrylamide gel, electroblotted onto nitrocellulose, and the membranes reacted with antibodies corresponding to p53, WAFl/Cipl, mdm2 and actin. Protein bands were detected by autoradiography of X-ray film. The type of each cell line used is shown on the top of the panel. Numbers 10 or 50 on top of the lanes represent the amount of AdControl or AdWTp53 (pfu/cell) .
  • the antibodies used for detecting proteins are indicated on the left side of the panel. Protein molecular weight markers are indicated on the right side of the panel.
  • FIG. 6 Northern blot analysis of p53 mRNA in cells exposed to AdWTp53.
  • Cells (2xl0 6 ) were plated and 24 hours later infected with AdWTp53 or AdControl (10 pfu/cell) .
  • 24 hours after infection RNA was prepared and subjected to Northern blot analysis as described in Example 6. After transferring RNA to Magna NT membranes, blots were either probed with a p53 or a 36B4 cDNA probe. The results of autoradiograms obtained from different cells are shown on top of the lane, exposed to either AdControl or AdWTp53 as shown.
  • FIG. 7 Nucleosomal DNA fragmentation in AdWTp53-infected MDA-MB-231, MCF-7 and NMECs.
  • 2xl0 6 cells were plated in 10 cm dishes and exposed to either AdControl or AdWTp53. . 1 day after infection, the cells were collected, incubated with a lysis buffer. Low molecular weight DNA was then prepared and subjected to an agarose gel electrophoresis. The results shown are the DNA pattern observed in various cell lines (shown on top of the lane) infected with 50 pfu/cell of either AdControl or AdWTp53. The numbers on the left side of the panel indicate the position of molecular weight markers (bp) .
  • FIG. 8 Construction of the recombinant adenoviral vector AdWAFl.
  • a diagrammatical representation of the method of construction of the adenoviral vectors AdWTp53 and AdWAFl is shown.
  • the Clal restriction site which was added by the invention is the upstream site, located within the first 900 nucleotides from the 5'end of the adenovirus genome.
  • Figure 9 Structure of the recombinant adenoviral vectors AdWTp53 and AdWAFl.
  • This Figure represents the structure of the recombinant adenoviral vectors AdWTp53 and AdWAFl.
  • the hatched segment represents adenovirus type 5 genome of 9.24 mu-100 mu.
  • the bottom is shown the enlargement of human wild type p53 and WAFl/Cipl expression cassette.
  • the expression cassettes contains left inverted terminal repeat (ITR) , origin of replication, encapsidation signals and Ela enhancer derived from adenovirus type 5 (stippled segments) ; human cytomegalovirus immediate early promotor (left blank segment) ; human wild type p53 or WAFl/Cipl cDNA (solid segment) and SV40 RNA maturation signal (right blank segment) .
  • AdWAFl has the identical genomic structure as AdWTp53 except p53 cDNA is replaced by WAFl/Cipl cDNA.
  • FIG. 10 Western blot analysis of p53, WAFl/Cipl and actin proteins in various cell lines following AdWTp53 and AdWAFl infection.
  • This Figure sets forth a Western blot analysis of p53, WAFl/Cipl and actin proteins in various cell lines following AdWTp53 and AdWAFl infection.
  • the cells were infected with 50 pfu/cell of AdControl, AdWTp53 or AdWAFl for 48 hours and subjected to Western blot analysis.
  • the cell lines used are indicated on the top of the panel and the antibodies on the left side of the panel.
  • the protein molecular weight markers are indicated on the right side of the panel.
  • Figures llA-C Effect of AdWTp53 and AdControl on cell growth. These Figures set forth the effect of AdWTp53 and AdControl on cell growth. The cells were exposed to AdWTp53 (10 pfu/ cell) , AdWAFl (10 pfu/cell), and AdControl (10 pfu/cell) , and the cell number was counted on each day. Shown are the cell number of H-358 cells ( Figure 11A) , MDA- MB-231 cells ( Figure 11B) and MCF-7 cells ( Figure 11C) . The cell number in AdWTp53 infected cells are shown by (B) ,
  • AdWAFl infected cells by ( ⁇ ) AdControl infected cells by
  • Figure 12A-D Cell cycle analysis of MDA-MB-231 cells infected with AdWTp53 and AdWAFl. These Figures set forth a cell cycle analysis of MDA-MB-231 cells infected with AdWTp53 and AdWAFl. MDA-MB-231 cells were infected with AdControl, AdWTp53 or AdWAFl (50 pfu/cell) and 24 hours later were subjected to cell cycle analysis as described in Example 13. The results shown are the cell cycle analysis of uninfected cells ( Figure 12A) ; AdControl infected cells (Figure 12B) ; AdWTp53 infected cells ( Figure 12C) ; and AdWAFl infected cells ( Figure 12D) .
  • Figures 13A-D Percentage distribution of cells in different cell cycle stages following AdWTp53 and AdWAFl infection. These Figures set forth the percentage distribution of cells in different cell cycle stages following AdWTp53 and AdWAFl infection. The cells were exposed to AdWTp53, AdWAFl or AdControl and subjected to cell cycle analysis as described in Example 16. Shown are the percentage of cells in Gl (solid bars) , S (hatched bars) and G2 + M (dashed bars) in MDA-MB-231 cells ( Figure 13A) ; H-358 cells ( Figure 13B) ; MCF-7 cells ( Figure 13C) ; and NMECs ( Figure 13D) .
  • Figures 14A-E Detection of Apoptosis in AdWTp53 and AdWAFl- infected Cells. These Figures set forth the detection of apoptosis in AdWTp53 and AdWAFl-infected cells. Two days after infection, adherent and floating cells were collected and incubated with a lysis buffer. Low molecular weight DNA was then prepared and analyzed by agarose gel electrophoresis. The results shown ( Figures 14A-C) are the DNA pattern observed in various cell lines (as indicated on top of the lane) infected with 50 pfu/cell of either AdControl, AdWTp53 or AdWAFl.
  • FIG. 15 Ad.RSV ⁇ gal expression in MDA-MB-231 and human bone marrow cells.
  • This Figure sets forth Ad.RSV ⁇ gal expression in MDA-MB-231 and human bone marrow cells.
  • the cells (2xl0 4 ) were infected with different moi of Ad.RSV ⁇ gal (0-10,000 pfu/cell) and ⁇ -gal activity determined as described in Example 7.
  • the results show the ⁇ -gal activity obtained in MDA-MB-231 (•) and human bone marrow cells (o) .
  • FIG. 16 Effect of AdWTp53 on the MDA-MB-231 and human bone marrow cell's viability.
  • This Figure indicates the effect of AdWTp53 on the MDA-MB-231 and human bone marrow cell's viability.
  • Freshly trypsinized MDA-MB-231 cells were mixed with CD34 + bone marrow cells and infected with different pfu/cell of AdWTp53. The cells were then assayed for colonogenicity as described in Example 19. The results shown are the percentage number of colonies formed after each treatment, assuming uninfected cells to be 100 %.
  • the results of MDA-MB-231 cells are shown by (•) and of CD34+ cells by (o) and are the average of the triplicate determinations.
  • FIG 17 Ad-mediated transfection of CMV ⁇ -gal plasmid in MDA-MB-231 and human bone marrow cells.
  • Cells (2 xlO 5 ) were transfected using CMV ⁇ -gal plasmid and different moi of dl312. After a 24 hour incubation at 37° C, the cells were lysed and ⁇ -gal activity measured, ⁇ -gal activity obtained in MDA-MB-231 cells is shown by (•) and in bone marrow cells by (o) . The results are of the mean of the triplicate determinations.
  • Figure 18 Ad-mediated enhancement in the cytotoxicity of a plasmid pULIlOO in breast tumor and bone marrow cells.
  • MDA-MB-231 500
  • CD34+ bone marrow cells 1000
  • pULIlOO plasmid 10 pfu/cell
  • dl312 10 pfu/cell
  • lipofectamine lipofectamine
  • Cell survival was estimated for MDA-MB-231 by a calorimetric assay and for CD34+ bone marrow cells by the colony forming assay described in Example 15. Uninfected cells were treated as 100% survival.
  • Results of MDA-MB-231 cells are shown by solid bars, and bone marrow cells by the hatched bars. Results shown are the average of the triplicate determinations.
  • Figures 19A and 19B AdWTp53-induced apoptosis in human breast cancer cells. Shown are cells infected with AdControl ( Figure 19A) and with AdWTp53 ( Figure 19B) .
  • MDA-MB-231 xenografts in nude mice MDA-MB-231 xenografts in nude mice.
  • MDA-MB-231 cells were injected subcutaneously in nude mice. 2 weeks after injection (day 0) tumors were given weekly injections of either AdControl (10 9 pfus) or AdWTp53 (10 9 pfus) . Tumor sizes were measured on the days shown in Figure 20 and are represented by a solid bar for AdControl and a hatched bar for AdWTp53 infected tumors.
  • Figures 21A and 21B Nude Mice Photos.
  • Figure 19A shows a photograph of the animal that received an injection of AdWTp53, indicating that the tumor size disappeared completely.
  • Figure 19B shows a photograph of the animal that received an injection of AdControl, indicating that the tumor size increased further.
  • FIG 23 Cell Cycle Analysis of Human Aortic Vascular Smooth Muscle Cells Infected with AdWTp53 and AdWAFl.
  • 2xl0 5 of human aorta vascular smooth muscle cells were infected with AdWTp53, AdWAFl and AdControl (50 pfu/cell) for 48 hours and subjected to cell cycle analysis. Results shown are the cell cycle analysis of uninfected cells (Panel A) , AdControl-infected cells (Panel B) , AdWAFl-infected cells- (Panel C) and AdWTp53-infected cells (Panel D) .
  • the arrow indicates the population of cells in Gl subgroup.
  • Figure 24 Cytotoxicity of AdWTp53, AdWAFl and AdControl to human aortic vascular smooth muscle cells.
  • the cytotoxicity of each adenovirus on human aortic vascular smooth muscle cells (250/well) was determined in triplicate on each wells of 96 well plates, exposed to AdWTp53 ( ⁇ ) , AdWAFl (•) and AdControl (D) of up to 40000 pfu/cell and after 7 days the number of cells was analyzed by colorimetric assay. Values shown are mean ⁇ S.E.
  • Figure 25 sets forth a schematic diagram of Adp27.
  • Figure 26 shows Adp27-mediated p27 Expression in Human Breast Cancer Cells.
  • Figures 27A and 27B Effect of Adp27-mediated expression of 27 on DNA Cell Cycle Histograms.
  • Figure 28 Effect of Adp27 Infection on Apoptosis.
  • Figure 29 sets forth a schematic diagram of AdCD.
  • Figures 30A and 30B Figure 30A sets forth AdCD-mediated cytotoxicity of MCF-7 cells in the presence of 5-FC.
  • Figure 30B sets forth AdCD-mediated cytotoxicity of MDA-MD-231 in the presence of 5-FC.
  • Figure 31 sets forth a schematic diagram of Adpl6.
  • Figure 32 shows Adpl6-mediated pl6 expression in various cell cancer lines.
  • Figure 33 sets forth a schematic diagram of AdTAM67.
  • Figure 34 sets forth a schematic diagram of AdB7- 1.
  • Figure 35 sets forth a schematic diagram of AdB7- 2.
  • Figure 36 Figure 36 depicts AdWTp53-mediated Expression.
  • Figure 37 Figure 37 illustrates the effect of AdWTp53 Infection on Cell Cycle.
  • Figure 38 sets forth the Effects of AdWTp53 on Apoptosis. DETAILED DESCRIPTION OF THE INVENTION:
  • the present invention provides novel methods for the construction of recombinant adenoviral vectors capable of expressing human cDNAs.
  • human cDNAs are wild-type p53, WAFl/Cipl/p21, p27/kipl, E. coli cytosine deaminase, wild-type pl6, TAM 67 (a jun/fos dominant negative mutant) and B7-1 and B7-2.
  • the present invention affords a novel method for the construction of the adenoviral vectors by the addition of a second Clal site for utilization in excising the 5' end of the adenoviral genome.
  • the present invention additionally provides a method of inhibiting the proliferation of cells.
  • This method comprises contacting the cells with an adenoviral vector, capable of expressing human cDNAs, in an amount effective to inhibit cell proliferation.
  • adenoviral vector capable of expressing human cDNAs
  • Particular non-limiting examples of such vectors are AdWTp53, AdWAFl, Adp27, AdCD, Adpl6, AdTAM67, AdB7-l and AdB7-2.
  • the invention provides a method of inhibiting the cell cycle of proliferating cells.
  • This method comprises contacting the cells with an adenoviral vector, capable of expressing human cDNAs, in an amount effective to inhibit DNA synthesis.
  • the present invention also provides a method of eradicating cancer cells by contacting the cells with an adenoviral vector, capable of expressing human CDNAs, in an amount effective to eradicate the cancer cells.
  • adenoviral vector as described herein can be used and engineered to contain and express other genes (i.e. cDNAs) that may be useful for eradicating tumor cells in which the vector is expressed via the toxic effects of the expressed genes.
  • cDNAs genes that can be used in the adenovirus vectors of the invention are HSVTK, No-synthetase, GADD 45, pl5, mdm2, Rb, BAX, IL2, GMCF, p53-antisense, Her/Neu2 antisense, and Erb4 antisense.
  • the present invention also provides for a method of treating a subject afflicted with a tumor that has shown resistance to drugs which comprises contacting the tumor with an effective amount of a recombinant adenovirus capable of expressing human cDNAs so as to inhibit proliferation of the tumor cells.
  • AdWTp53 A preferred recombinant adenovirus vector expressing human wild-type p53 cDNA is AdWTp53.
  • AdWTp53 possesses a human-wild type p53 expression cassette consisting of a left inverted terminal repeat, an origin of replication, encapsidation signals, an Ela enhancer derived from adenovirus type 5, a 1.7 kb human wild-type p53 cDNA, and an SV40 maturation signal.
  • the amount of the recombinant adenovirus vector expressing human wild-type cDNA effective to inhibit cell proliferation of actively proliferating cells will vary according to the cell type. Maximal inhibition of cell proliferation by the recombinant adenovirus vector is achieved on cancer cells that are either deficient in the p53 protein or those that express a mutant p53 protein.
  • the present invention further provides a method of treating a subject afflicted with a tumor which comprises contacting the tumor with an amount of a recombinant adenovirus vector capable of expressing human cDNAs which is administered to the subject previous to, simultaneously, or subsequent to, administration of a chemotherapeutic agent or to an amount of irradiation effective to treat the tumor.
  • chemotherapeutic agents are known to those skilled in the art and include, but are not limited to, bleomycin, mitomycin, cyclophosphamide, doxorubicin, paclitaxel, and cisplatin (See El-Deiry, et al., Cell. 75:817-825 (1993) ) .
  • the recombinant adenovirus vector containing a gene or cDNA of interest is administered in a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers such as sterile solution, tablets, coated tablets and capsules. Such carriers may typically contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stensic acid, talc, vegetable fats or olis, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives and other ingredients.
  • the administration of the composition may be effected by any of the well known methods, including but not limited to, oral, intravenous, intramuscular, and subcutaneous administration.
  • the preferred method of administration of the composition is local, i.e. at the site of the tumor.
  • the amount of a recombinant adenovirus vector capable of expressing human cDNAs incorporated in the composition may vary widely. Methods for determining the precise amount depend upon the subject being treated, the specific pharmaceutical carrier, the route of administration being employed, the frequency with which the compound is to be administered, and whether the composition is administered in conjunction with a chemotherapeutic agent and/or irradiation treatment.
  • the preferred amount of the vectors which may be administered for effective inhibition of proliferation of cells ranges from 10 8 to 10 10 pfu/tumor.
  • the invention provides a novel method for the construction of adenoviral vectors. This method is not hampered by the problems which presently exist in the construction of adenoviral vectors.
  • the invention provides a construction technique whereby an additional Clal site is introduced at the 5' end of the adenovirus genome.
  • the introduction of the second Clal site at the 5' end of the adenovirus genome greatly reduces the chances of obtaining undigested genome as only one of the two Clal sites will have to be cut to prevent the production of the non- recombinant background infectious virions.
  • the novel adenovirus vectors will be made using viral genomic DNA as the starting point, there is no need to utilize a plasmid based vector.
  • an adenovirus vector expressing human wild-type p53 cDNA (AdWTp53) was constructed.
  • AdWTp53 human wild-type p53 cDNA
  • AdWTp53 was used as a parental adenovirus genome to generate future recombinant adenoviruses.
  • AdWAFl a recombinant adenovirus which contains WAFl/Cipl cDNA
  • an adenovirus vector expressing human WAFl/Cipl/p21 cDNA (AdWAFl) was constructed. Recently, radiation-inducedp53 effects were shown to be associated with WAFl/Cipl induction (El-Deiry, ' et al., Cancer Res.. 54:1169-1174 (1994)). However, whether WAFl/Cipl overexpression in the absence of other p53- mediated signals induces apoptosis was not clear.
  • AdWTp52 and AdWAFl of the present invention which can both induce WAFl/Cipl gene overexpression, allowed the direct investigation of the role of WAFl/Cipl induction independent of p53 overexpression. It appears that AdWTp53-mediated p53 overexpression induces expression of WAFl/Cipl in cells resulting in cell cycle arrest in all cells studied and apoptosis in those cells lacking expression of endogenous p53 or cells expressing mutant p53. In contrast, AdWAFl- mediated WAFl/Cipl expression in infected cells resulted in cell cycle arrest without inducing apoptosis.
  • p53 overexpression can transcriptionally activate several genes, including MDM2 (Katayose, et al. , Clin. Cancer Res. (Submitted 1995); Momand, et al. Cell 69:1237-1245 (1992)) GADD45 (Smith, et al. Science 266, 1376-1379 (1994)).
  • MDM2 Keratayose, et al. , Clin. Cancer Res. (Submitted 1995); Momand, et al. Cell 69:1237-1245 (1992)
  • GADD45 Smith, et al. Science 266, 1376-1379 (1994)
  • the p53-mediated apoptotic pathway could involve induction of these or previously unidentified genes.
  • AdWTp53 and AdWAFl infection produces marked differences in cell cycle arrest. Infection with AdWTp53 resulted in a decline in the S phase and an increase in the proportion of G2+M. In contrast, AdWAFl infection caused a marked increase in the proportion of cells in the Gl phase and a decline in S phase cells.
  • H-358 lung cancer cell line
  • MDA- MB-231 breast cancer cell line
  • apoptosis in response to p53 overexpression
  • MCF-7 NMECs
  • AdWTp53-mediated effects are not necessarily at the transcriptional level but perhaps involve other post transcriptional regulation as previously suggested (Caelles, et al., Nature 370:220-223 (1994)) and could also depend upon the cell's DNA repair ability (Modrich, P., Science 266:1959-1960 (1994) ) .
  • the AdWTp53 vector may be clinically useful in tumors expressing mutant p53, which includes many human cancers (Nigro, et al., Nature 342:705-708 (1989); Takahashi, et al., Science 246:491-494 (1989); Srivastava, et al., Nature 348:747-749 (1990); Katayose, et al. , Clin. Cancer Res. (Submitted 1995); Liu, et al. , Cancer Res. 54: 3662-3667 (1994); Fujiwara, et al. , Cancer Res. 54:2287-2291 (1994) ) .
  • the AdWAFl vector may be useful in clinical settings wherein growth arrest of cells is an effective treatment.
  • the present invention also provides for the use of the novel AdWTp53 vector in order to elucidate the role of p53 in inducing growth inhibition and apoptosis in vascular smooth muscle cells.
  • the role of p53 was analyzed by observing the effects of AdWTp53 on the proliferation and apoptosis of cultured human aortic vascular smooth muscle cells (AoVSMC) (See Example 25) .
  • AdWAFl vector of the present invention expressing p53-inducible p21 cyclin-dependent kinase inhibitor, was also used to further elucidate the role of p53 in cancer.
  • adenovirus vector expressing p53 induced marked cytotoxicity in primary cultured AoVSMC. This cytotoxicity was associated with cell cycle arrest in Gl and G2/M boundary, accumulation of cells in Gl subgroup and perhaps apoptosis. Examining the mechanisms of p53-mediated cytotoxicity to AoVSCM is an important issue because as to date, there is no report to indicate that p53 induces apoptosis in normal cells, including vascular smooth muscle cells. This examination is made possible by the use of the vectors of the present invention. In this context, the involvement of p21 was studied by examining the overexpression of p21 in AoVSMC infected with either AdWTp53 or AdWAFl.
  • AdWaFl was about 200 times less toxic than AdWTp53, it is therefore unlikely that induction of p21 due to p53 overexpression mediated the cytotoxicity of AdWTp53 to AoVSMC. Further, the data herein shows that AdWTp53 induced not only Gl arrest but also G2/M arrest in AoVSMC, whereas AdWAFl only induced Gl arrest.
  • the present invention further provides for the use of adenoviral vectors in cancer gene therapy.
  • adenoviral vectors in cancer gene therapy.
  • the presence of mutated p53 is widespread in different human cancers.
  • reconstituting tumor suppressor p53 gene expression by adenoviral vectors is an attractive strategy for gene therapy. Since the adenovirus enters human epithelial cells with an efficient low-pH endosomes mediated endocytosis (Seth, et al. , (1986) Pathway of adenovirus entry into /25507 PC 7 S96/0 3
  • tumors of mammary epithelial origin will be especially amenable to treatment by AdWTp53.
  • the present invention demonstrates that normal mammary epithelial cells are resistant to apoptosis by AdWTp53, while tumor cells null for p53, or expressing mutant p53, readily undergo apoptosis.
  • Human adenoviruses have been used previously in gene transfer techniques in eucaryotic cells either by infecting the cells with a recombinant adenovirus expressing the transgene, or by adenovirus-mediated transfection of the plasmid DNAs.
  • the present invention shows that breast tumor cells are a much better target for Ad-mediated gene transfer as compared to bone marrow cells.
  • the present invention also indicates that adenoviruses, in combination with appropriate toxic genes, can kill breast tumor cells while sparing bone marrow cells.
  • the present invention allows a comparison of the infectability using a replication-deficient adenovirus expressing ⁇ -galactosidase ( ⁇ -gal gene) .
  • the invention can be used to determine or measure the relative cytotoxicity of the adenoviral vector encoding the catalytic domain of the Pseudomonas exotoxin gene.
  • the vectors of the invention cause an increase in the infection and transfection efficiencies of plasmid DNA in the presence of adenovirus in human breast tumor and bone marrow cells. Following infection of breast tumor cells with an adenovirus expressing ⁇ -galactosidase gene, high levels of ⁇ -galactosidase activity were observed, while normal bone marrow cells expressed about 500-fold less ⁇ - galactosidase activity.
  • AdWTp53 a recombinant adenovirus expressing human wild-type p53 protein
  • adenoviruses are effective for delivering genes to breast tumor cells as compared to bone marrow cells may relate to the high number of adenovirus receptors present on the surface of the cell. Therefore, other tumors found to have high levels of adenoviral receptors could be used in accordance with the invention.
  • Human breast tumor cells tested were found to have fairly high number of adenovirus receptors (in the range of 5-10 x 10 3 ) while bone marrow cells are relatively deficient in cell surface adenovirus receptors (less than 5 x 10 Vcell) .
  • breast cancer cells expressing adenoviral receptors would be a better target for adenoviruses than the adenovirus receptor deficient bone marrow cells.
  • adenoviral-mediated transfections may be enhanced by using a replication- deficient adenovirus mutant dl312, and even further enhanced by the addition of a liposome, such as lipofectamine and other mono and polycationic lipids.
  • a liposome such as lipofectamine and other mono and polycationic lipids.
  • the present disclosure indicates that human bone marrow cells treated under the same conditions expressed very low levels of the transfected ⁇ -galactosidase DNA. It was also determined that transfection of cells with plasmid DNA expressing Pseudomonas exotoxin gene in the presence of dl312 and lipofectamine resulted in greater than 90% breast tumor cell killing, while human bone marrow CD34+ cells were at least 500-times more resistant to this treatment.
  • human adenoviruses are useful for cancer gene therapy and for removing or "purging" cancer cells from bone marrow.
  • Human adenoviral vectors are also useful in cancer gene therapy for the treatment of lung, prostrate, and liver cancer cells, and may be useful in the treatment of leukemia.
  • Adenovirus vectors which express a toxic gene may be used in gene therapy, or, alternatively, a replication- deficient adenovirus in combination with plasmid DNAs coding for a toxin gene may be used.
  • the present disclosure indicates that since the use of both results in at least 100-fold increase in cytotoxicity to breast cancer cells compared to bone marrow cells, this approach offers a fairly wide efficacy window for purging.
  • the protocols for human bone marrow purging using recombinant adenovirus vectors appear to be simple and effective. Recombinant adenoviruses for use in the protocols are replication-deficient, which pose few problems to the bone marrow cells.
  • adenoviruses are used in combination with toxin genes, much lower dosage of adenoviruses will be required, thus making the bone marrow purging protocol even more safe. Because breast cancer cell contamination in human bone marrow which is to be used for marrow transplantation is potentially a serious problem, the adenoviral vectors of the present invention are necessary and useful tools for purging bone marrow cells of such contaminating tumor cells.
  • Adenoviral vectors may also be employed in the eradication of cancer cells.
  • MDA-MB-231 cells were injected subcutaneously in nude mice and two weeks later when the tumors were palpable, they were injected with AdWTp53 (10 9 pfus) or a control adenovirus (AdControl) (10 9 pfus) . Tumors which received a control virus continued to grow over the next 14-21 days, while tumors which received AdWTp53 exhibited inhibition of subsequent tumor growth.
  • adenoviral vectors of the present invention would be extremely useful in cancer gene therapy.
  • a preferred mode of administration is by direct injection. Therefore, the adenoviral vectors of the invention will be useful for the eradication of cancer cells by contacting the cancer cells of the tumor with an amount of the adenoviral vector sufficient for the eradication of the cancer cells.
  • a particular example of such a vector for use in this treatment is AdWTp53.
  • the adenoviral vector may also be administered to a subject previous to, simultaneously, or subsequent to, the administration of a chemotherapeutic agent or an amount of irradiation effective to eradicate the cancer cells.
  • the adenoviral vectors are also useful in the prevention of the development of cancer cells in those subjects who are at risk of developing cancer.
  • the preventative treatment involves the administration of an adenoviral vector expressing the desired DNA which eradicates the cancer, via the toxic effects of the expressed genes, in an amount effective to prevent or inhibit the development of cancerous cells.
  • the present invention also provides for the construction and use of an adenoviral vector capable of expressing pl6/INK4 kinase inhibitor.
  • an adenoviral vector capable of expressing pl6/INK4 kinase inhibitor.
  • pl6 and Rb expression were investigated in cells with different endogenous pl6 and Rb status. Different cells were infected with different doses (1-200 pfu/cell) of either Adpl6, AdRb or AdControl, and 24 hours later analyzed for cell cycle distribution. Following Adpl6 infection, in each cell line expressing endogenous wild type Rb protein (MCF-7, MDA-MB- 231) , an increase in cells in Gl and a decrease in cell number in S phase was observed, indicating that the cells were arrested at Gl/S. The accumulation of cells in Gl phase was dependent upon the concentration of Adpl6 used. However, in cells expressing non-functional form of Rb protein, Adpl6 failed to induce cell cycle arrest.
  • Adpl6 The effect of Adpl6 on cell cycle arrest was not dependent on the endogenous status of pl6. In each cell line used in this study (except those defective for Rb) , Adpl6 induced a strong cell cycle arrest, which was dependent on the dose of Adpl6 used.
  • the present invention also provides for the construction and use of an adenoviral vector capable of expressing p27/kipl, a cell cycle inhibitor that is involved in the signal transducing effects of TGF ⁇ .
  • an adenoviral vector capable of expressing p27/kipl a cell cycle inhibitor that is involved in the signal transducing effects of TGF ⁇ .
  • Adp27 of the present invention was used to study the relationship between p27/kipl effects on cell growth, cell cycle, cyclin kinases and apoptosis. It is herein shown that p27/kipl expression can regulate the cell cycle at both Gl/S and G2/M check points, and that these effects are associated with the inhibition of cdk2 kinase and cdc2 cyclin Bl-associated kinases.
  • the present invention further provides for adenoviral vectors for use in treating drug resistant cancers.
  • AdWTp53 cytotoxic effects of AdWTp53 in two drug resistant breast cancer cells; adriamycin resistant human breast cancer MCF-7 cells (MCVF-Adr) and mitoxantrone resistant MCF-7 cells, were herein investigated. (See Example 25) Following infection by AdWTp53, all the cell lines expressed high levels of p53 protein. However, MCF-Adr and MCF-Mito cells were much more sensitive to killing by AdWTp53 as compared to the parental MCF-7 cells.
  • the present invention further provides for adenoviral vector constructs that would be useful in exploring the clinical utility of suicidal enzymes for the gene therapy of breast cancer. While adenoviral vectors have many attractive features, a key problem with adenoviral vectors is that they can only infect a small population of cancer cells within a tumor mass, leaving many of the cells uninfected. Thus, there is a need to develop adenoviral vectors which should induce cytotoxicity across the whole tumor should adenovirus infect only a small number of tumor cells.
  • adenoviral vectors which under certain circumstances can be made to produce cytotoxic products which are smaller in size and hence will have opportunities to escape the cells and kill the uninfected cells; or diffuse across cell boundaries (e.g. through gap junctions) providing bystandard effect.
  • One such gene is E. coli deaminase which can convert a pro-drug 5-fluoro cytosine into a toxic species 5-fluoro uracil.
  • the present invention provides for the construction and use of an adenoviral vector capable of expressing E. coli cytosine deaminase.
  • AdCD is extremely cytotoxic to MDA-MB-231 and MCF-7 breast cancer cells, and show that bystander effects play a role in overall cytotoxicity of AdCD. Further, investigation of the cytotoxic effects of AdCD in vivo in the presence of 5-FC in human breast tumors grown as xenografts in nude mice. Thus, AdCD has potential clinical applications for the treatment of breast cancer. Because adenoviruses can express the transgenes to very high levels, coupled with the bystander effects of the suicidal genes, AdCD has advantages over retroviruses expressing E. coli CD. EXAMPLES
  • the Examples herein are meant to exemplify the various aspects of carrying out the invention and are not intended to limit the scope of the invention in any way.
  • the Examples do not include detailed descriptions for conventional methods employed in the construction of vectors, the insertion of cDNA into such vectors, or the introduction of the resulting vectors into the appropriate host. Such methods are well known to those skilled in the art and are described in numerous publications, including Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual. 2nd Edition, Cold Spring Harbor Laboratory Press, USA, (1989) .
  • EXAMPLE 1 Construction of an Adenovirus Vector Coding For the Expression of Human Wild-Type p53 Protein (AdWTp53) .
  • Plasmid AdWTp53 was constructed using co-transfection of shuttle vector pDKlO containing the wild type p53 expression cassette and a plasmid pJM17 containing the adenovirus type 5 genome.
  • pJM17 may be obtained from Microbix Biosystems, Inc., Toronto, Ontario Canada.
  • pDKlO was deposited with the American Type Culture Collection (ATCC) in Rockville, Maryland on February 17, 1995 under ATCC Accession No. 97064.
  • the shuttle vector pDKlO was constructed by inserting the cytomegalovirus (CMV) immediate early promotor and enhancer, a 1.7-kilobase Xbal fragment of human p53 cDNA
  • Figure l shows the schematic diagram of AdWTp53.
  • the 5' end of the genome contains the AdWTp53 expression cassette (10.3 mu) followed by the rest of the adenovirus genome.
  • the key elements of the expression cassette of AdWTp53 include the left inverted terminal repeat (ITR) , the adenoviral origin of replication, encapsidation signal, the Ela enhancer, the CMV immediate early promotor, the human wild type p53 cDNA and SV40 polyadenylation signal.
  • ITR left inverted terminal repeat
  • Plasmid pDKlO was co-transfected with pJM17- (See McGrory, et al., Virology. 163:614-617 (1988)) (kindly provided by F. Graham, McMaster Univ., Hamilton, Ontario) into the transformed human embryonic kidney cell line 293 (ATCC Accession No. CRL1573) by calcium phosphate mediated gene transfer technique (See Graham, et al. (1991) Manipulation of adenovirus vectors, p. 109-128 In Murray, E. J. (ed.), Gene transfer and expression protocols, Humana Press, Clifton, New Jersey; and Gilardi, et al., FEBS Lett..
  • the following primer was selected from exon seven of the p53 nucleotide sequence:
  • AdWTp53 was propagated in 293 cells grown in monolayers, purified by two cesium chloride density gradients, dialyzed against a buffer containing 10% glycerol, 1 mM MgCl 2 pH 7.5, and stored at - 70°C as described by Seth, et al . , J. Virol. 68:933-940 (1994) . PCR analysis of the purified AdWTp53 indicated that it contained p53 cDNA but was devoid of Ela sequences.
  • Control adenovirus vectors used in this study were: Ad.RSV ⁇ gal, an adenovirus vector containing ⁇ -galactosidase gene (Stratford-Perricaudet, et al . , J. Clin. Invest.. 90: 626-630 (1992)), and AdControl, a null adenovirus vector dl312 (Jones, et al., Cell. 17:683-689 (1979) (kindly provided by R. Crystal, NIH, Bethesda)
  • AdWAFl Human WAFl/Cipl Protein
  • AdWAFl was constructed by homologous recombination between a shuttle vector containing WAFl (p21 cyclin-dependent kinase inhibitor) cDNA expression cassette (pDK13, see Figure 8) and Clal cut genomic DNA derived from AdWTp53 using a previously described procedure (See Graham, F. L. and Prevec, L. (1991) In Gene transfer and expression protocols, (Murray ,E. J. ed.) pp. 109-128, Humana Press,
  • pDK13 was deposited with the American Type Culture
  • WAFl cDNA in AdWAFl was confirmed by polymerase chain analysis using the following primers:
  • AdControl used in this study was a replication-deficient adenovirus dl312 (Jones, N. and Shenk, T. , Cell. 17:683-689
  • Adenoviruses were propagated in 293 cells, purified by two cesium chloride density centrifugation, tittered and stored at -70°C as described previously (Katayose, et al. , Clin. Cancer Res. (Submitted 1995); Seth, et al., J. Virol. 68:933-940 (1994)).
  • Adp27 Human p27 Protein
  • Adp27 was constructed by co-transfection of a shuttle vector pCGl containing the p27 expression cassette and 35 kb fragment derived from an adenovirus expressing p53 (AdWTp53) .
  • the shuttle vector pCGl was constructed by inserting the human cytomegalovirus (CMV) immediate promoter and enhancer, a 1.7 kilobase Xbl fragment of p27 cDNA into a null shuttle vector described previously.
  • CMV human cytomegalovirus
  • the two DNAs were cotransfected into the transformed human embryonic kidney cell line 293 (ATCC CRL1573) by calcium phosphate mediated gene transfer technique (Gibco BRL, Gaithersberg, MD) .
  • Figure 25 shows the schematic diagram of Adp27.
  • the 5' end of the genome contains the Adp27 expression cassette (10.3 mu) followed by the rest of the adenoviral genome.
  • Adp27 The key elements of the expression cassette of Adp27 include the left inverted terminal repeat (ITR) , the adenoviral origin of replication, encapsidation signal, the Ela enhancer, the CMV immediate early promoter, the human wild-type p53 cDNA and SV40 polyadenylation signal.
  • ITR left inverted terminal repeat
  • Adp27 has been deposited with the American Type Culture Collection (ATCC) in Rockville, Maryland.
  • AdCD Addenoviral Vector Coding for the Expression of E. Coli Cytosine Deaminase
  • AdCD was constructed by co-transfection of a shuttle vector pPSl containing the E. coli cytosine deaminase expression cassette and 35 kb fragment derived from Clal cut adenoviral genomic DNA as previously described. Plasmid pPSl was constructed by inserting the human cytomegalovirus
  • CMV CMV early promoter and enhancer
  • a l.l kilobase Xbal fragment of CD cDNA into a null shuttle vector.
  • Co- transfection and isolation of viral plaques were performed by published methods.
  • Several plaques were screened for the presence of CD sequences by polymerase chain reactions (PCR) according to published methods (Katayose, et al., Am. J.
  • FIG. 29 shows the schematic diagram of AdCD.
  • the 5' end of the genome contains the AdCD expression cassette (10.3 mu) followed by the rest of the adenoviral genome.
  • the key elements of the expression cassette of AdCD include the left inverted terminal repeat (ITR) , the adenoviral origin of replication, encapsidation signal, the Ela enhancer, the CMV immediate early promotor, E. coli CD gene and SV40 polyadenylation signal.
  • AdCD has been deposited with the
  • a recombinant adenovirus expressing human pl6 was constructed by using homologous recombination methods described previously to construct other adenoviral vectors.
  • a shuttle vector pCG2 containing the pl6 expression cassette was co-transfected with a 35 kb fragment derived from Clal cut adenoviral genomic DNA in transformed human kidney 293 cells.
  • Adenoviral plaques were picked and subjected to another cycle of infection in 293 cells as described previously.
  • Several plaques were screened for the presence of pl6 sequences by polymerase chain reaction (PCR) according to published methods (Katayose, et al., Am. J. Physiol ..
  • Adpl6 has been deposited with the American Type Culture Collection (ATCC) in Rockville, Maryland.
  • adenoviral vectors were prepared as set forth above:
  • AdTAM67 See Figure 33 for the schematic diagram.
  • AdTAM67, AdB7-l and Adb7-2 have been deposited with the American Type Culture Collection (ATCC) in Rockville, Maryland.
  • MDA-MB-231 ATCC Accession No. HTB26
  • MCF-7 kindly provided by R. Buick, Univ. of Toronto
  • Adr R MCF-7 adriamycin resistant MCF-7 cells
  • MCF-Mito mitoxantrone resistant MCF-7 cells
  • H-358 a lung cancer cell line (kindly provided by J. Minna, NCI-Navy Medical Oncology Branch, Bethesda, MD) , and MDA-MB-453 cells, a breast cancer cell line (ATCC Accession No. HTB131) , were grown in RPMI containing 10% FBS.
  • MDA-MB- 157 ATCC Accession No. HTB 24
  • IMEM Gibco BRL
  • NMECs Normal Mammary Epithelial Cells
  • CC-201 6, Clonetic Corp., San Diego, CA reduction mammoplasties
  • 184B5 cells immortalized mammary epithelial cells (ATCC CRL10317) were cultured in Mammary Epithelial Basal Medium (MEBM, Clonetics, Corp.) supplemented with lx vitamins, 0.5% FBS, 20 ng/ml EGF, 5 ⁇ g/ml hydrocortisone and 52 ⁇ g/ml bovine pituitary extract (Gudas, et al., Cell Growth Differ.. 5:295-304 (1994)).
  • MEBM Mammary Epithelial Basal Medium
  • Immortalized MCF10 cells were cultured in DMEM/F12 (Gibco BRL) supplemented with 2.5% horse serum (Gibco BRL) 10 mM Hepes (Calbiochem, La Jolla, CA) , 2 mM glutamine (Biofluids, Rockville, MD) , 0.ImM nonessential amino acids, (Gibco BRL) 20 ng/ml EGF (Upstate Biotechnology) , 10 ⁇ g/ml insulin (Boehringer Mannheim, Indianapolis, IN), 0.5 ⁇ g/ml hydrocortisone.
  • 293 cells (ATCC Accession No. CRL 1573) , an adenovirus transformed human embryonic kidney cell line was cultured in improved minimal essential medium (Biofluids) supplemented with 2 mM glutamine (Biofluids), 2.5 mcg/ml fungizone
  • AoVSMC (CC-2023, MyoPack-AOSMC, Clonetics Corporation., San Francisco
  • H-358 lung cancer cells which are devoid of p53 protein (p53 null) (See Takahashi, et al . , Science. 246:491- 494 (1989));
  • MDA-MB-231 human breast cancer cells which express mutant p53 protein (Zakut-Houri, et al . , EMBO J.. 4:1251- 1255 (1985) ) ;
  • MCF-7 human breast cancer cells which express wild- type p53 protein (Casey, et al . , Oncogene. 6:1807-1811 (1991) ; Zakut-Houri, et al . , EMBO J.. 4:1251-1255 (1985)) .
  • 5xl0 cells of each respective cell line were plated in each well of 6 well tissue culture dishes. After 24 hours, the cells were exposed to 10 pfu/cell of AdWTp53 or AdControl in medium containing 2% fetal bovine serum. After an incubation of 2 hours at 37°C, the serum concentration in the medium was raised to 10% and the incubations continued at 37°C. The cells were trypsinized on each day and counted using a hematocytometer. The cytotoxicity of the adenovirus vectors was assessed using a colorimetric assay as described previously (Skehan, P., J. Natl . Cancer Inst.. 82:1107-1112 (1990)) .
  • the cells were fixed by the addition of ice-cold 50% trichloroacetic acid (TCA) , which was added onto the top of the medium in each well to a final concentration of 10%, the cells were incubated at 4°C for 1 hour, washed five times with water, and allowed to air dry. TCA-fixed cells were stained for 20 minutes with 0.4% (wt/vol) sulforhodamine B (Sigma, St. Louis, MO) dissolved in 1% acetic acid, followed by rinsing four times with 1% acetic acid. An O.D. 564 was obtained using a Bio Kinetics Reader EL340 (Bio-Tek Instruments) and was used as a measure of cell number. The percent survival rates of cells exposed to adenovirus vectors were calculated by assuming the survival rate of uninfected cells to be 100%.
  • TCA trichloroacetic acid
  • AdWTp53 Because differences in the sensitivity of various cell lines to AdWTp53 could result from either reduced uptake and/or decreased transgene expression, the expression of an adenoviral vector containing the marker gene, ⁇ - galactosidase was examined in the following cell lines: NMEC, MCF-7, MCF-10, MDA-MB-453, MDA-MB-231, MDA-MB-157, MDA-MB-453, and H-358.
  • the cells were plated at a concentration of 2xl0 4 cells in each well of a 96 well tissue culture plate. After 24 hours, the cells were exposed to various concentrations of Ad.RSV ⁇ gal (0.1-500 pfu/cell) in medium used by each respective cell line, however, the serum concentration (if required) was reduced to 2%. After a 2 hour incubation at 37°C, the serum concentration (if required) was raised to 10%, and the cells were then incubated at 37°C for an additional 24 hours. The cells were washed three times with phosphate buffered saline pH 7.5 (PBS) and lysed in 50 ⁇ l of 0.1M Tris pH 7.5 containing 0.1% Triton X-100.
  • PBS phosphate buffered saline pH 7.5
  • each of the cell lines expressed high amounts of p53 when they were exposed to AdWTp53. Therefore, the differences in the sensitivity of killing effects of AdWTp53 cannot be explained by alteration in viral uptake and/or differential expression of the transgene.
  • RNA was prepared from the following cell lines and used in the Western blot analysis:
  • Each respective cell line was plated in 6 cm tissue culture dishes at a concentration of 0.5xl0 6 and incubated with AdWTp53 or AdControl for 24 hours as described in Example 2. The cells were then washed three times with ice-cold PBS, scraped and resuspended in 1 ml of lx SDS-poly acrylamide gel electrophoresis buffer (62 mM Tris pH 6.8, 2 mM ethylenediaminetetraacetate (EDTA), 15% sucrose, 10% glycerol, 3% SDS, 0.7 M 2-mercaptoethanol) and boiled for 10 min.
  • lx SDS-poly acrylamide gel electrophoresis buffer 62 mM Tris pH 6.8, 2 mM ethylenediaminetetraacetate (EDTA), 15% sucrose, 10% glycerol, 3% SDS, 0.7 M 2-mercaptoethanol
  • Equal amounts (15 or 50 ⁇ g) of denatured protein were electrophoresed on SDS-polyacrylamide gels and transferred to nitrocellulose filters (Gudas, et al., Cell Growth Differ.. 5:295-304 (1994)). Filters were blocked with Tris-buffered saline containing 5% dried milk and 0.1% Tween 20 (Sigma). Blots were probed with 4 ⁇ g/ml of Ab-2; and Ab-6 for p53, 4 ⁇ g/ml of EA 10 for WAFl/Cipl, 3 ⁇ g/ml of IF2 for mdm2, with 3 ⁇ g/ml of Actin (Ab-1) antibody. All antibodies were obtained from Oncogene Science.
  • the blots were washed with Tris-buffered saline containing 0.1% Tween 20, incubated with horseradish peroxidase, conjugated to secondary antibody, and the specific complex was detected by the enhanced chemiluminescence technique according to the manufacturer's directions (NEN) .
  • the Western blot analysis demonstrates that low levels of endogenous p53 were detecte in all cell lines examined except MDA-MB-157 and H-358.
  • the level of p53 increased substantially (at leas 10-fold) in each cell line following AdWTp53 infection (1 or 50 pfu/cell) .
  • the amount of p53 increase little, if at all, above the endogenous p53 protein level i cells exposed to 50 pfu/cell of AdControl. Because cell exposed to either AdControl or AdWTp53 expressed simila levels of actin protein, (See Figure 5) , increased levels o p53 following AdWTp53 infection can not be due to loading o different amounts of proteins or other non-specifi mechanisms.
  • AdWTp53 infection resulted in greater than 70 fold increase in WAFl/Cipl protein in cells that lacked endogenous p53 gene expression (MDA-MB-157, H-358) or expressed mutant p53 (MDA-MB-231, MDA-MB-453) (See Table 1) .
  • RNA was prepared from the following cell lines and used in the Northern blot analysis: (l) NMEC (normal mammary epithelial cells; (2) MCF-10 (breast cancer cells) ;
  • MCF-7 breast cancer cells
  • MDA-MB-453 breast cancer cells
  • MDA-MB-231 breast cancer cells
  • MDA-MB-157 (breast cancer cells) .
  • Each respective cell line was plated in 15 c tissue culture dishes at a concentration of 2xl0 6 , an incubated with 10 pfu/cell of adenoviral vectors. Afte incubation for 24 hours at 37 ° C, RNA was extracted b rinsing the cells three times with cold PBS and dissolvin them in a 2 ml solution of guanidine isothiocyanate. RN was purified by centrifugation over a 5.7 M cesium chlorid cushion (See Gudas, et al. , Cell Growth Differ..
  • NMECs, MCF-10 and MC 7 cells all contained endogenous wild-type p53 and express varying levels of WAFl/Cipl mRNA expression followi infection with AdControl.
  • AdWTp5 Following infection of AdWTp5 the WAFl/Cipl mRNA levels in cell lines null for p53 expressing mutant p53 were induced significantly; MDA-MB-453 showed a 7.4 fold increase, MDA-MB-231 showed a 21 fold increase, and MDA-MB-157 showed a 8.2 fold increase.
  • MDA-MB-453 showed a 7.4 fold increase
  • MDA-MB-231 showed a 21 fold increase
  • MDA-MB-157 showed a 8.2 fold increase.
  • MCF-7 cells showed a marked 6 fold increase in WAFl/Cipl mRNA levels.
  • MCF-10 showed only a 2 fold increase, and NMECs only a 1.2 fold increase in WAFl/Cipl mRNA following infection with AdWTp53.
  • the level of a control mRNA (36B4) was similar in cells infected with either AdControl or AdWTp53.
  • AdControl AdControl
  • AdWTp53 AdWTp53
  • NMECs which are the most resistant to killing by AdWTp53, despite the expression of high levels of p53 following AdWTp53 infection, did not undergo apoptosis and showed the smallest increase (1.2 fold) in WAFl/Cipl induction.
  • tumor cells deficient in wild-type p53 or expressing mutant p53 were quite sensitive to the cytotoxic effects of AdWTp53 and showed marked induction of WAFl/Cipl RNA and protein. Therefore, AdWTp53-mediated cytotoxic effects appeared to be associated with the high expression of WAFl/Cipl.
  • the WAFl/Cipl gene has been shown to bind to cellular cyclin CDK kinase and thereby inhibit their function (Katayose, et al., Am. J. Phvsiol.. 264: L100-L106 (1993); Stratford- Perricaudet, et al., J. Clin. Invest. 90:626-630 (1992); and Fairchild, et al., Cancer Res.. 47:5141-5148 (1987)). This inhibition is manifested in turn by a decrease in the level of phosphorylation of the Rb protein (Seth, et al. , Moi. Cell. Biol.. 4:1528-1533 (1984)).
  • AdWTp53-mediated inhibition of cell growth involved programmed cell -54- death (apoptosis)
  • the effect of AdWTp53 on nucleosomal DNA fragmentation was determined after infection of MDA-MB-231 cells (which express endogenous mutant p53) , H-358 cells (which are null for p53) , and in MCF-7 and NMECs (both of which express endogenous wild type p53) .
  • Each respective cell line was plated at a concentration of 2xl0 6 cells in 10 cm tissue culture dishes and incubated with adenoviral vectors (50 pfu/cell) for 24 hours. Both adherent and floating cells were collected together and pelleted by centrifugation at 1800 x g for 5 min (RT-6000B, Du Pont, Boston) . Cell pellets were rinsed with cold PBS and low molecular weight DNA was prepared by a modified Hirt extraction method as described in Rosenfeld, et al., Hum. Gene Ther.. 5:331-342 (1994).
  • pellets were lysed in 1 ml of 10 mM Tris, 10 mM EDTA disodium pH 7.4 (Research Genetics), 0.6% SDS (Research Genetics) and 0.2 mg/ml proteinase K (Boehringer Mannheim) . Samples were incubated at 55°C for 5 hours, low molecular weight DNA was prepared by the Hirt extraction method as described i Rosenfeld, et al., Hum. Gene Ther.. 5:331-342 (1994) and th evaluated by electrophoresis on a 2% agarose gel.
  • AdWTp53 expresses p53 protein in tumor cells
  • the lung tumor cell line H-358 which lacks endogenous p53 (Takahashi, et al., Science. 246, 491-494 (1989)) was exposed to various concentrations of either AdControl or AdWTp53 for 24 hours.
  • immunoprecipitation of p53 was performed as described in Example 4.
  • Figure 2A there was no detectable p53 in H358 cells infected with AdControl.
  • p53 protein was easily detected by immunoprecipitation in cells infected with 1 pfu/cell of AdWTp53.
  • the amount of immunoprecipitable p53 protein increased with increasing concentrations of AdWTp53 vector.
  • MCF-7, MCF-10, Adr R MCF-7, MDA-MB-231 were exposed to AdWTp53, and the synthesis of p53 protein assessed by immunoprecipitation.
  • MCF-10, MCF-7, Adr R MCF-7 and MDA-MB-231 cells expressed low levels of endogenous p53.
  • AdWTp53 following exposure of the cells to 10 pfu/cell of AdWTp53, a marked increase in the rate of p53 protein synthesis was observed in the infected cells.
  • AdControl did not result in any increase in p53 expression above that present in uninfected cells.
  • AdWTp53-mediated p53 expression and its consequence on WAFl/Cipl induction were examined in cells infected with AdWTp53.
  • AdWAFl-mediated WAFl/Cipl expression was also examined in cells infected with AdWAFl.
  • p53 null (p53 null) (Takahashi, et al., Science 246:491-494 (1989)); MDA-MB-231 human breast cancer cells which express mutant p53 (Casey, et al., Oncogene 6:1807-1811 (1991)); MCF-7 human breast cancer cells which express wild type p53
  • the blot were washed with Tris-buffered saline containing 0.1% Twee 20, incubated with horse radish peroxidase conjugated t secondary antibody and specific complex detected by th enhanced chemiluminescence technique according to th manufacturer's directions (Amersham, Arlington Heights, IL)
  • the Western blot analysis demonstrates that t level of p53 increased substantially (at least 10-fol following AdWTp53 (50 pfu/cell) infection in each cell li
  • adenoviral vectors 10 pfu/cell
  • the cells were trypsinized on each day and counted using a hematocytometer. Cytotoxicity of adenovirus vectors was also assessed after plating cells in 96 well plates (500 cells/well) .
  • the cells were incubated with various doses (0-10 4 pfu/cell) of adenovirus vectors for 7 days at 37°C.
  • the cells were fixed with trichloroacetic acid and stained with 0.4% (wt/vol) sulforhodamine B (Sigma, St.
  • AdWAFl The growth inhibitory effect of AdWAFl was much weaker than AdWTp53 in H-358 and MDA-MB-231 cells, but was comparable to that of AdWTp53 in MCF-7 cells ( Figures 10A-10C) .
  • AdWTp53 was about 30-100 times more toxic than AdWAFl in H-358 and MDA- MB-231 cells, and nearly equally toxic in MCF-7 and NMECs.
  • the AdWTp53 infection had a more profound growth inhibitory effect as compared to AdWAFl on cells that were either deficient in p53 or expressed a mutant endogenous p53.
  • the growth inhibitory effects of AdWTp53 and AdWAFl on cells expressing wild-type endogenous p53 were similar, though much weaker.
  • Cells were plated in 6 well tissue culture dishes (2xl0 5 cells/well) and infected with adenoviral vectors (50 pfu/cell) for 48 hours. The cells were harvested b trypsinization and resuspended at a concentration of 2 x 10 cells/ml in medium containing 10% FBS, and stored froze until analyzed.- Samples were stained for DNA cell cycl analysis using the rapid nuclear isolation procedur (Wersto, R. P., and Stetler-Stevenson, M. A., Cytometry (i press. 1995) ) . DNA content was measured using a FACSan flo cytometer (Becton-Dickinson, Mountain view, CA) .
  • FACSan flo cytometer Becton-Dickinson, Mountain view, CA
  • AdWTp53 and AdWAFl produced similar effects in H-358 cells.
  • AdWTp53 produced a reduction in S phase and increase in G2 + M phase; while AdWAFl produced a reduction in S phase and increase in Gl phase ( Figure 12B) .
  • AdWTp53 and AdWAFl infection resulted in declines in the percentage of cells in S phase in MCF-7 and NMECs.
  • no significant change in proportion of cells in G2 + M or Gl was apparent following AdWTp53 or AdWAFl infection of these cells ( Figures 12C and 12D) .
  • Cells were plated in 6-well dishes (2 x 10 5 cells/well) and infected with adenoviral vectors (50pfu/cell) for 48 hours. Cells were harvested by trypsinization and resuspended at a concentration of 2 x 105 cells/ ml in 100% FBS and stored frozen until analyzed. Samples were stained for DNA cell cycle analysis using the previously described procedure. DNA content was measured using a FACSan flow cytometer (Becton-Dickenson, Mountain View, CA. ) . Cell cycle analysis of the resulting DNA histograms of cell number versus integrated red fluorescence was performed with Multicycle (Phoenix Flow Systems, San Diego, CA.
  • cell lysates were incubated with 1 ug primary antibody for 1 hour at 4oc Immune complexes were collected on protein A-Sepharose beads.
  • the beads were washed three times with EBC buffer, three times with kinase reaction buffer (20 mM Tris-HCL pH 7.5, 4 mM MgCl 2 ) .
  • the beads were then resuspended in kinase assay mixture containing 80 uM ( 32 P-ATP) , histone HI (2 ug) (Gibco-BRL) .
  • Af er incubation at 37°C the reaction was stopped by the addition of 2X Laemli SDS sample buffer. Proteins were separated on 10% SDS-polyacrylamide gels, and the gels were then dried and autoradiographed.
  • Ad.RSV ⁇ gal vector a recombinant Ad vector expressing ⁇ -gal gene (Dulic", et al., Cell 76:1013-1023 (1994))
  • AdWTp53 a replication- deficient recombinant adenoviral vector expressing human wild-type p53 were propagated in 293 cells and purified by two rounds of CsCl 2 density centrifugation.
  • Adenovirus titers were determined from viral stocks and stored in Tris- Cl pH 7.5 buffer containing 20% glycerol.
  • a plasmid expressing the bacterial ⁇ -galactosidase gene driven by cytomegalovirus early gene promoter, (CMV ⁇ -gal) (Nelson, W. G. and Kastan, M. B., Moi. Cell. Biol. 14:1815- 1823 (1994)) was used to measure the transfection efficiency of cells.
  • Adenoviral-mediated enhancement of toxicity of plasmid DNA was determined using plasmid pULIlOO, containing the Pseudomonas exotoxin catalytic domain (minus secretory signals) driven by a CMV promoter (Michalovitz, D., Halevy, 0. and Oren, M. , Cell. 62:671-680 (1990)).
  • Human breast cancer cells were plated at the appropriate density. After 24 hours, the medium was changed to OPTI-MEM (Gibco-BRL, Gaithersberg, MD) and the cells were infected with various multiplicity of infection (moi) of adenovirus. After 2 hours, FBS was added to a final concentration of 10% and the incubation continued at 37°C. For human bone marrow cells and human CD34 + cells, freshly isolated cells were plated in OPTI-MEM and infected with increasing moi of adenoviral vectors. After 2 hours, FBS was added to the final concentration of 10% and the incubation continued at 37°C. Other experimental details such as the number of cells, moi of adenovirus, and the length of incubations are described below for each experiment.
  • Adenovirus Receptor Number for adenovirus were determined by scatchard analysis of 35 S-dI312 binding to cells as described previously. (See Seth, P., Rosenfeld, M. , Higginbotham, J. and Crystal, R. G., J. Virol. 68:933-940 (1994)). In.brief, 0.2 x 10 6 cells were used to bind 35 S-dI312 adenovirus (10 4 cpm, 0.1 ⁇ g adenovirus protein) at 4oC for 1 hour in the presence of unlabeled dI312 (0-100 ⁇ g) . Scatchard plots were drawn as described previously.
  • Binding assays were conducted i triplicate and the mean taken. Table 2 sets forth the receptor numbers for adenoviruses on breast cancer cells an human bone marrow cells.
  • Freshly trypsinized breast cancer cells were mixed with CD34 + human bone marrow cells and exposed to different moi of AdWTp53 for 2 hours. Cell cultures were then split; half of the cells (500 cells) were plated in 60 mm dishes and grown in IMEM containing 10 % FBS to form colonies of breast cancer cells. After 14 days the colonies were stained with 5 mM methylene blue, and counted. The other half (500 cells) of the cell suspension was cultured in 0.8 % methyl cellulose medium containing 5 % PHA-LCM (Stem Cell Technologies, Vancouver, BC) to determine colony forming units of granulocytes/macrophages.
  • methyl cellulose medium containing cells was placed in a 35 mm gridded tissue culture dish (Nunc, Napervile, IL) , incubated at 37°C, and the CFU-GM colonies counted after 14 days.
  • AdWTp53 an Ad vector expressing human wild type p53
  • AdWTp53 was shown to be cytotoxic to breast cancer cells.
  • the cytotoxicity of AdWTp53 was measured by colony forming assays. As shown in Figure 16, following infection of MDA- MB-231 cells with AdWTp53, significant cytotoxicity (about 55% decline in colony numbers) was observed at an moi of 8 pfu/cell. At moi 100 pfu/cell or higher, essentially no colony formation was observed.
  • Bone marrow cells were much more resistant to killing by AdWTp53. At an moi of up to 1000 pfu/cell, there was essentially no decline in CFU-GM. However, at a very high moi (>10,000 pfu/cell) of AdWTp53 there was about 50% reduction in colony numbers ( Figure 16) . These results indicate that MDA-MB-231 breast cancer cells are at least 3 orders of magnitude more sensitive to the cytotoxic effects of AdWTp53 compared to human CD34 + human bone marrow cells.
  • CMV ⁇ -gal plasmid DNA (1 ⁇ g) was pre-incubate with different concentrations of lipofectamine (1 ⁇ g) a room temperature for 20 min and used for transfectio assays. Following exposure of cells to these reagents fo 2 hours, FBS was added for a final concentration of 10% and the cells incubated at 37°C for 24 hours.
  • ⁇ -galactosidase activity was undetectable in bone marrow cells in the presence of CMV ⁇ - gal plasmid alone or in the presence of CMV ⁇ -gal plasmid and low concentrations of dl312. Only at a moi of 100 pfu/cell of dl312 was there any detectable ⁇ -gal activity (0.007 unit) observed in bone marrow cells ( Figure 17) . Thus, at an moi of 100 pfu/cell, dl312 infection enhanced the expression of ⁇ -gal plasmid DNA in MDA-MB-231 cells 100-fold more than the expression in bone marrow cells.
  • bone marrow cells showed very little ⁇ - galactosidase activity (0.007 and 0.023 units respectively) in the presence of either dl312 (100 pfu/cell) or lipofectamine alone. Even in the presence of both dl312 and lipofectamine, only 0.04 unit of ⁇ -gal activity was obtained (Table 3) . This level of activity is approximately 100-fold less than that obtained in MDA-MB-231 cells.
  • dl312 could enhance the delivery of a plasmid DNA coding for a toxin gene as measured by the cytotoxicity.
  • MDA-MB-231 breast tumor cells were transfected with pULIlOO DNA, a plasmid containing the cDNA encoding for the catalytic component of Pseudomonas exotoxin which is cytotoxic to cells. Transfections were conducted in the absence and presence of dl312 and/or lipofectamine, and cell survival was examined.
  • EXAMPLE 22 AdCD-Mediated Cytosine Deaminase Assays and Cytotoxi Effects of AdCD in Breast Tumor Cells. A. Cytotoxicity Assays.
  • Cytotoxicity of adenovirus vectors was assayed afte plating cells in 96 well plates (500 cells/well) . Cell were incubated with various doses (0-10 4 pfu/cell) o adenovirus vectors in the absence and presence of differen concentrations of 5-Fluoro-cytosine (5-FC) for 7 days a 37°C. The cells were fixed with tricholoacetic acid an stained with 0.4% (wt/vol.) sulforhodamine B (Sigma, St Louis, Mo.) essentially as described. An O.D. 564 wa obtained using a Bio Kinetics Reader EL340 (Bio-Te Instruments) and used as a measure of cell number.
  • MDA-MB-231 cells were infected with AdCD (10 pfu/cell) and mixed in different ratios with mock infected cells. Briefly, cells were plated in 10 cm dishes (l x 10 6 cells/dish) . 24 hours later the cells were infected with either AdControl (10 pfu/cell) or AdCD (10 pfu/cell) . The next day, AdCD-infected cells were mixed with uninfected cells in varying ratios (0.0001 to 100%) and plated in 96-well dishes. A total 20,000 cells were plated in each well.
  • 5-FC was added (1-5 mM) and the cells were incubated at 37°C for 5 days.
  • the cell viability was estimated using a MTT assay as described previously. In the presence of 5 mM 5-FC alone, the uninfected cells were 100% viable. However, when the cells were incubated with 5-FC, and either 100, 50, 20 or 10% of the infected cells; the viability of the total cell population was reduced to 0%. However, when the proportion of infected cells was less then 10%, some viable cells were still present at the end of the 5-day period. From these results it appears that to kill 100% of the population, only about 10% of the cells need to be infected by AdCD, which is likely to be due to bystandard effects of the AdCD in the presence of 5-FC.
  • MDA-MB-231 human breast cancer cells were plated in 10 cm dishes (2 x 10 6 /dish) and 24 hour later, medium was changed to IMEM containing 2% FBS. Cells were infected with either AdControl or AdWTp53 (50 pfu/cell) . After 2 hours, the serum concentration was raised to 10% and incubation continued at 37°C for another 24 hours. Cells were harvested, fixed in 70% ethanol, and apoptotic cells detected via a terminal transferase reaction using Apotag kit (Oncor, Gaithersberg, Maryland) .
  • Apotag kit Oncor, Gaithersberg, Maryland
  • MDA-MB-231 grown as zenografts in nude mice.
  • 2 week old athymic mice (nu/nu) (Frederick Cancer Research Facility, Frederick, Maryland) were used in this study as an animal model for tumor growth.
  • MDA-MB-231 cells were grown in monlayers. Prior to injections, cells were trypsinized, washed, and suspended in phosphate buffered saline. The mice were injected with 10 7 MDA-MB-231 cells in 0.1 PBS subcutaneously (5 x 10 6 cell/site) using a 21 g needle. Tumors were allowed to develop for 14 days.
  • tumors were either injected with AdWTp53 or AdControl (10 9 pfu/tumor) in the middle of the tumor using a 25G needle.
  • An additional injection of either AdWTp53 or AdControl was administered on day 21 and the animals photographed on day 28. (See Figures 21A and 21B) .
  • the results of this study indicate that those tumors of nude mice that were injected with AdWTp53 disappeared completely over the course of the treatment (See Figure 21A) , while the tumors of the nude mice that were injected with AdControl (adenovirus only) increased in size to a ⁇ final volume of 913 mm 3 in 21 days (See Figure 21B) .
  • adenoviral vectors are useful for the eradication of cancer cells by contacting the cancer cells of the tumor with an amount of the adenoviral vector sufficient for the eradication of the cancer cells.
  • adenoviral vectors are useful in the prevention of the development of cancer cells in those subjects who are at risk of developin cancer.
  • the preventative treatment involves th administration of an adenoviral vector expressing th
  • AdWTp53 was studied in two human breast cancer MCF-7 cel lines which are resistant to adriamycin (MCF-Adr) an
  • Cytotoxicity of adenovirus vectors was assessed after plating cells in 96 well plates (500 cells/well) . The cells were incubated with various doses (0-10 4 pfu/cell) of adenovirus vectors for 7 days at 37°C. The cells were fixed with trichloroacetic acid and stained with 0.4% (wt/vol) sulforhodamine B (Sigma, St. Louis, MO) essentially as described previously (Katayose, et al., Clin. Cancer Res. (Submitted 1995)). An O.D. J64 was obtained using a Bio Kinetics Reader EL340 (Bio-Tek Instruments) and used as a measure of cell number.
  • the blots were washed with Tris-buffered saline containing 0.1% Tween 20, incubated with horse radish peroxidase conjugated to secondary antibody and specific complex detected by the enhanced chemiluminescence technique according to the manufacturer's directions (Amersham, Arlington Heights, IL) .
  • Cells were plated in 6-well dishes (2 x 10 5 cells/well) and infected with adenoviral vectors (50 pfu/cell) for 48 hours. Cells were harvested by trypsinization and resuspended at a concentration of 2 x 105 cells/ ml in 100% FBS and stored frozen until analyzed. Samples were stained for DNA cell cycle analysis using the previously described procedure. DNA content was measured using a FACSan flow cytometer (Becton-Dickenson, Mountain View, CA.) .
  • E. Cyclin Kinase Assays 4 x 10 6 cells were plated in 15 cm dishes. The next day the cells were infected with different doses (1-200 pfu/cell) of recombinant adenoviruses for 24 hours. The cells were then harvested and lysed in a buffer. For cdk2 kinase activity, lysates were immunoprecipitated by anti- cdc2. For cdc2 kinase, cell lysates were immunoprecipitated. For cdc2-cyclin B-l dependent kinase, lysates were precipitated with anti-cyclin Bl.
  • cell lysates were incubated with 1 ug primary antibody fo 1 hour at 4°C. Immune complexes were collected on protei A-Sepharose beads. The beads were washed three times wit EBC buffer and three times with kinase reaction buffer (2 mM Tris-HCL pH 7.5, 4 mM MgCl 2 ) . The beads were the resuspended in kinase assay mixture containing 80 uM ( 3 P ATP) , histone HI (2 ug) (Gibco-BRL) . After incubation a 37°C, the reaction was stopped by the addition of 2X Laeml SDS sample buffer. Proteins were separated on 10% SDS polyacrylamide gels, and the gels were then dried an autoradiographed.
  • each cell line has a certain percent distribution of cells in Gl, S and G2/M phase (Panel E,F,G).
  • a dose-dependent reduction in cell population in S phase was observed in parental MCF- 7 (Panel A-C) as well as the MCF-Adr (Panel D-F) and MCF- Mito (Panel G-I) cell lines.
  • MCF-7 MCF-7 and the drug resistant cells.
  • MCF-ADR and MCF-Mito cells following infection with AdWTp53, there appeared to be a population of cells in sub GO phase where apoptotic cells should accumulate. This accumulation of apoptotic cells in MCF-Adr and MCF-Mito increased with an increase in the dose of AdWTp53 used.
  • cdc2 kinase (cyclin Bl associated) was also expressed in all cell lines at a certain basal level. Following infection of these cells with a control adenovirus, the basal level of the enzyme activity was not reduced. I parental MCF-7 cells, cdc2 kinase was not much affecte following AdWTp53 infection. However, infection of MCF-Ad with AdWTp53 resulted in complete inhibition of cdc2 cycli Bl-associated activity. Similar results were obtained wit MCF-Mito.
  • AdWTp53-Mediated Apoptosis The effect of AdWtp53 infection on apoptosis was furthe examined in MCF-7 and the drug resistant cells by evaluatin the nucleosomal DNA degradation, as described in Example 17 While in MCF-7 cells, AdWTp53 infection (100 pfu/cell) failed to induce any nucleosomal DNA degradation, both the drug resistant cells showed DNA laddering following 24 hour infection with AdWTp53. Even increasing the concentration of AdWtp53 up to 500 pfu/cell or increasing the length of incubation with AdWTp53 up to 48 hours did not induce any specific DNA laddering in the parental MCF-7 cells. Control adenovirus (100 pfu/cell) did not induce apoptosis in any of these cells. These results indicate that induction of apoptotic pathway may play an important role in determining the overall cytotoxicity of AdWTp53.
  • AdWTp53 The amount of virus required to kill 50% cells (IC50) of AdWTp53 was 4 pfu/cell, whereas those of AdWAFl and AdControl were 800 pfu/cell and 1200 pfu/cell, respectively. These results indicate that AdWTp53 was 200 and 300 times more toxic to AoVSMC than AdWAFl and AdControl, respectively. This data is compatible with earlier results showing the existence of Gl subgroup in AoVSMC infected with AdWTp53. To determine whether AdWTp53-mediated cell death of AoVSCM occurred by apoptosis, DNA fragmentation analysis was performed. DNA fragmentation in AoVSMC infected with AdWTp53 was not detected.
  • AdWTp53 (50 pfu/cell) showed high levels of p53 expression, whereas AoVSMC infected with AdWAFl, AdControl (50 pfu/cell) or uninfected cells showed the low base level.
  • AdWTp53 induced p2l expression probably by direct transcriptional transactivation of the p21 gene.
  • AoVSMC infected with AdWAFl of 50 pfu/cell had much higher levels of p2l expressed compared to cells infected with AdControl or uninfected cells. This level of p21 expression is similar to p21 expressed by AdWTp53.
  • AdControl slightly increased p2l protein levels as compared to uninfected cells. The protein levels of whole actin were unchanged in AoVSMC infected by AdWTp53, AdWAFl, AdControl, or uninfected cells.
  • AdControl had no effect on the cell cycle distribution compared to uninfected cells.
  • infection with either AdWTp53 or AdWAFl 50 pfu/cell
  • AdWTp53 and AdWAFl infection decreased the number of S phase cells in dose dependent manner.
  • AdWTp53 and AdWAFl infection decreased S-phase cells
  • differential effects by AdWTp53 and AdWAFl were observed;
  • AdWTp53 induced the accumulation of G2/M phase cells ( Figure 37, D, G)
  • AdWAFl resulted in an increased number of Gl phase cells ( Figure 37, C, E) . From these results, it can be determined that p53 overexpression induced bot Gl and G2/M cell cycle arrest, whereas p21 overexpression arrested cells at Gl/S boundary.
  • AdWTp53- infected cells (50 pfu/cell) showed the population in Gl subgroup, indicating that some population of cells infected with AdWTp53 underwent apoptosis. No Gl subgroup was observed in cells infected with either AdWAFl or AdControl.
  • Adp27-mediated p27 expression was examined in MDA-MG-231 and MCF-7 human breast cancer cells following infection with Adp27. Western blot analyses demonstrated that the level of p27 increased substantially (at least 20-fold) following Adp27 (100 pfu/cell) infection in each cell line ( Figure 27, Top Panel) . In these experiments, AdControl vector did not increase p27 expression; and no change in actin protein level was detected in any of the cells lines following infection with either AdControl or Adp27.
  • Adp27 produced a similar dose-dependent effects on cell cycle in MCF-7 cells. These results indicate that lower amounts of p27 can induce a strong Gl/S arrest in breast cancer cells while higher amounts of p27 can either reverse this arrest or has additional check points at G2.M.
  • ADDRESSEE MORGAN & FINNEGAN, L.L.P.
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL No
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL No

Abstract

Cette invention se rapporte à de nouveaux procédés pour construire des vecteurs adénoviraux recombinés, capables d'exprimer des ADNc humains, tels que p53 de type sauvage, WAF1/Cip1/p21, p27/kip1, désaminase de cytosine de E. coli, p16 de type sauvage, TAM 67 (un mutant négatif dominant jun/fos) et B7-1 et B7-2. Cette invention se rapporte en outre à des procédés pour inhiber la prolifération de cellules, pour inhiber le cycle de cellules proliférantes, et à des procédés pour éradiquer des cellules, en particulier des cellules cancéreuses et malades, en infectant ces cellules avec un vecteur d'adénovirus recombiné capable d'exprimer des ADNc humains. Des compositions et des procédés objets de cette invention peuvent servir à traiter des sujets souffrant d'une tumeur, dont des cellules sont privées de l'allèle p53 de type sauvage et/ou possèdent un gène p53 ayant subi une mutation. Cette invention se rapporte en outre à un procédé d'utilisation de vecteurs adénoviraux dans le traitement de cellules cancéreuses, telles que des cellules du cancer des poumons et des cellules du cancer du sein. Cette invention se rapporte en outre à des procédés d'utilisation de vecteurs adénoviraux dans la thérapie génique du cancer, comme mécanisme pour purger les cellules de la moelle osseuse des cellules tumorales contaminantes, pour éradiquer les cellules cancéreuses et pour empêcher le développement de cellules cancéreuses et de tumeurs.
PCT/US1996/002336 1995-02-17 1996-02-16 Procedes de preparation et d'utilisation de vecteurs adenoviraux recombines WO1996025507A2 (fr)

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NZ313828A (en) * 1995-07-17 1999-02-25 Univ Texas Expression constructs containing the encoding region of p16 and its regulatory elements and their application in cancer therapy e.g. a replication deficient adenviral vector containing p16
US7087582B1 (en) 1995-09-26 2006-08-08 Regents Of The University Of Michigan Combination for site-specifically transforming cells in vivo comprising a double-balloon catheter and nucleic acid comprising a gene encoding P21
US5863904A (en) 1995-09-26 1999-01-26 The University Of Michigan Methods for treating cancers and restenosis with P21
FR2755148B1 (fr) * 1996-10-31 1999-01-29 Calvo Fabien Procede destine a mettre en evidence l'etat d'une cellule maligne et procede de traitement
US6177272B1 (en) * 1997-07-21 2001-01-23 The Regents Of The University Of Michigan Method for treating vascular proliferative diseases with p27 and fusions thereof
US6475481B2 (en) * 1997-11-18 2002-11-05 Canji Inc Purging of stem cell products
WO1999025195A1 (fr) * 1997-11-18 1999-05-27 Canji, Inc. Purge de produits de cellules souches
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