WO1999031118A1

WO1999031118A1 - Inhibition of tumor cells proliferation using ribozymes

Info

Publication number: WO1999031118A1
Application number: PCT/US1998/017292
Authority: WO
Inventors: Frank Czubayko; Anton Wellstein
Original assignee: Georgetown University
Priority date: 1997-08-22
Filing date: 1998-08-21
Publication date: 1999-06-24

Abstract

Transient adenovirus-mediated transduction of HER-2/neu ribozymes inhibit HER-2/neu expression in cancer cells. Adenovirus constructs containing the appropriate vectors can be administered parenterally for systemic effect or be administered directly into the tumor and/or into the specific arteries which supply the tumor for purposes of inhibiting tumor growth. Hence, the invention provides a method for inhibiting growth of a malignant tumor by administration of recombinant adenovirus which expresses ribozymes that target PTN mRNA or Her-2/neu in pharmaceutically acceptable carriers.

Description

Title: INHIBITION OP TUMOR CELLS PROLIFERATION USING RIBOZYMES

5 Inventors: Anton Wellstein and Frank Czubayko

Field of the Invention;

This invention relates to use of ribozymes to inhibit growth and metastatic spread of cancer. Background of the Invention:

Tumor growth and, ultimately, metastasis is a complex 5_. precess regulated in part by factors controlling cellular proliferation and death as well as tumor angiogenesis.

One of the factors related to oncogenic transformation, HER-2/neu, a member of the epithelial growth factor (EGF) tyrosine kinase receptor family has been implicated in 0 mediating growth factor signals as well as in modulating hormone responsiveness of breast cancer cells. HER-2/neu is frequently over-expressed in a variety of adenocarcinomas, including those of lung, breast and ovarian cancer. In these tumors, HER-2/neu overexpression serves as a marker of faster 5 tumor progression and poor prognosis. Currently targeting of HER-2/neu expression with anti-HER-2/neu antibodies is being evaluated as a potential therapeutic approach in breast cancer patients. Despite its useful role as a diagnostic marker for disease progression, it remains unclear if HER-2/neu expression 0 is mostly a marker or a rate-limiting factor in cancer cell growth.

Tumor-angiogenesis, a process whereby factors stimulating the ingrowth of blood vessels into the tumor are secreted into the local tumor milieu by cancer and stroma cells, also plays 5 a critical role by regulating the balance between cell proliferation and cell death and by providing a route for distant spread.³ Both clinical and laboratory evidence suggests that spread of malignant cells from a localized tumor is directly related to the number of microvessels in the primary 0 tumor. Of the multitude of factors secreted by tumor and stroma cells which are potentially angiogenic, two have been confirmed as angiogenic factors which are rate-limiting in in vivo tumor models. The importance of one of these, vascular endothelial growth factor / vascular permeability factor 5_. (VEGF/VPF) , was demonstrated through functional knockout through use of blocking antibodies. A critical role for the other factor, pleiotrophin (PTN) , was shown in angiogenesis and metastasis associated with melanoma using a hammerhead-ribozyme PTN mRNA depletion strategy. 0 Summary of the Invention:

It has now been discovered that transient adenovirus- mediated transduction of HER-2/neu ribozymes inhibit HER-2/neu expression in cancer cells. Adenovirus constructs containing the appropriate vectors can be administered parenterally for 5 systemic effect or be administered directly into the tumor and/or into the specific arteries which supply the tumor for purposes of inhibiting tumor growth. Hence, the invention provides a method for inhibiting growth of a malignant tumor by administration of recombinant adenovirus which expresses 0 ribozymes that target PTN mRNA or Her-2/neu in pharmaceutically acceptable carriers. Brief Description of the Figures:

Figure 1 shows the overall genomic organization of the recombinant adenovirus ribozyme expression vectors. 5 Description of the Invention:

This invention involves constructed adenoviruses expressing hammerhead-ribozymes targeted to two factors: (1) the tyrosine kinase receptor HER-2/neu or (2) the growth factor pleiotrophin (PTN) . Adenovirus- ediated transduction of either 0 HER-2/neu or PTN - targeted ribozymes depleted the respective RNAs and inhibited protein expression significantly in three different human cancer cell lines. This resulted in almost complete abrogation of HER-2/neu or PTN dependent cancer-cell proliferation. 5 The availability of ribozyme gene transfer technologies to biologically down-regulate expression of specific genes coupled with efficient gene delivery vehicles provides anti- cancer strategies to block expression of potentially rate- limiting tumor promoting factors such as HER-2/neu or PTN. Hammerhead-ribozymes can be targeted to destroy specific mRNA transcripts by binding to and cleaving specific ribonucleotide sequences. Such ribozymes targeted to specific oncogenes have been used successfully to down-regulate oncogene expression in both in vitro and in vivo models. To efficiently deliver ribozyme expression constructs into tumor cells in vivo, adenovirus vectors are well suited as an alternative to plasmid-based gene transfer technologies. Adenovirus vectors provide highly efficient means for gene transfer and expression in vitro and in vivo, can be easily produced in high titers and have a broad host range. Further, adenovirus vectors have been used for the efficient delivery and expression of ribozymes into tumor cells in culture and mouse liver cells in vivo.

Using the methods described here, adenoviruses expressing hammerhead ribozymes for either HER-2/neu or PTN were constructed and evaluated in four separate human cancer cell lines. Each vector led to high level gene transfer and hammerhead ribozyme expression and to significant depletion of the respective gene products. Importantly, inhibition of either HER-2/neu or PTN gene expression in tumor cells abrogated the proliferative effects of these proteins, thus demonstrating the feasibility of these therapeutic approaches. Ribozyme expressing vectors were derived from Ad-dl327 by homologous recombination as described in the Materials and methods section. The deletion within the El-region resulted in a replication-defective virus and further deletion of the E3 gene region provided space for the incorporation of transgenes. Ribozymes are expressed under the control of a viral CMV promoter and contain a SV40 polyadenylation signal to enable processing and increase transcript stability in eukaryotic cells. The HER-2/neu ribozyme (RzHER) targets the HER-2/neu mRNA 1991 nucleotides (nt) downstream of the translation initiation site. HER-2/neu specific antisense flanking regions of 9 nt and 8 nt on the 5' and 3' ends of the ribozyme were positioned around a minimized catalytic hammerhead ribozyme core of 22 nt, a sequence which has been used successfully in previous studies. The specificity of this HER-2/neu ribozyme construct was demonstrated in in vitro cleavage assays as well as in transient co-transfection studies in NIH/3T3 cells using 5_. the pRc/CMV plasmid as a ribozyme expression vector. A catalytically inactive mutant ribozyme and an unspecific ribozyme were used as controls in those experiments and had no effects. The PTN ribozyme (RzPTN) cleaves the PTN mRNA 66 nt downstream of the translation initiation site and contains PTN 0 specific antisense flanking regions of 12 nt and 11 nt at the 5' and 3' ends, respectively. The specificity and efficacy of the RzPTN construct was previously demonstrated in in vitro cleavage assays, in transient co-transfection studies in SW-13 cells, in two human melanoma cell lines stably expressing the 5 ribozyme in cell culture, and finally, in in vivo experiments in nude mice. In addition, two recombinant adenoviruses expressing either β-Galactosidase (AvlLacZ4) or Luciferase (AvlLuc) were used as controls in some of the experiments.

Recombinant adenoviruses expressing ribozymes were 0 generated by a homologous recombination method. (Berkner, K.L. , BioTechnique 1988; 6: 616-624.) They were plaque- purified, amplified and titrated in 293 cells as described under Materials and Methods section of this specification. Correct transgene integration and genomic organization of the 5 recombinant adenoviruses was confirmed by Southern analysis. A PCR assay for Ela with a sensitivity to detect contaminations of one copy of wild-type adenovirus in 10⁷ copies of recombinant adenovirus was used to assure the absence of replication competent wild-type adenovirus. 0 In a first set of experiments the levels of ribozyme expression in human cancer cells after adenoviral infection were compared to expression levels from plasmid-expression vectors stably transfected into the same cell lines. Human SW- 13 adrenal carcinoma cells, human 1205 melanoma and human U87 5 glioblastoma cells were infected with either Av-RzPTN or Av- RzHER at multiplicity of infection (MOI's) of 100 and total RNA was harvested one and three days after the infection. Northern analysis revealed very high expression levels of both ribozymes in SW-13 and U87 cells. Ribozyme expression was easily detectable after one day and increased significantly after three days. (A 50 nt difference in transcript size between the PTN and HER-ribozymes was due to a slightly modified cloning procedure.) Both ribozymes were also expressed in 1205 melanoma cells although at somewhat lower levels. This was in stark contrast to the expression levels in SW-13 and 1205 cells stably transfected with RzPTN expression plasmids. Ribozyme transcripts could not be detected in 1205 cells and only at very low levels in SW-13 cells in a Northern blot that was exposed to film for three days.

In a more detailed time course experiment in human MDA-MB- 361 breast cancer cells, ribozyme expression peaked 3 days after adenoviral infection, decreased rapidly after day 5 and was not detectable by Northern analysis after day 10.

In order to evaluate the influence of different MOI' s on ribozyme expression, RzHER levels in human SK-OV-3 ovarian cancer cells were examined three days after the infection with Av-RzHER at MOI' s from 100 to 500. An increase in the MOI from 100 to 500 was followed by a linear, approximately 5-fold increase in RzHER expression. To determine the optimum non- toxic MOI for the subsequent experiments the cell lines were infected with the β-Galactosidase expressing AvlLacZ4 at different MOI' s and stained for β-Galactosidase expression 48 hours after the infection. Since in all cell lines 100 % of the cells stained positive at a non-toxic MOI of 100, this virus titer was used in the following studies unless indicated otherwise. MATERIALS AND METHODS

Cell culture, viruses and plasmids

Human adrenal carcinoma (SW-13) , glioblastoma (U87) , ovarian cancer (SK-OV-3) and embryonic kidney epithelial (293) cells were obtained from American Type Culture Collection (ATCC) and were grown as adherent cells in IMEM (Life Technologies, Gaithersburg, MD) with 10 % fetal bovine serum (FBS; Life Technologies) ; human melanoma cells (1205LU; gift from M. Herlyn, Wistar Institute, Philadelphia, PA) were maintained in KSFM/L15 media (Life Technologies) mixed at a ratio of 3:1 and supplemented with 5% FBS. 32D mouse hematopoietic stem cells which were stably co-transfected with HER-2/neu and HER-3 5_. expression plasmids (32D/H2+3; gift from J. Pierce, NCI, NIH) were grown in IMEM with 10% FBS. Recombinant adenoviruses expressing either β-Galactosidase (AvlLacZ4) or Luciferase (AvlLuc) as well as Ad-dl327 virus DNA and PAVS6A plasmid DNA which were used for the subsequent adenovirus (Av) -vector 0 constructions were described earlier. (Trapnell, B.C., "Adenoviral vectors for gene transfer", Advanced Drug Delivery Reviews 1993; 12: 185-199; and Mittereder, N. , et al . , "Evaluation of the Efficacy and Safety of In Vitro, Adenovirus- Mediated Transfer of the Human Cystic Fibrosis Transmembrane 5 Conductance Regulator cDNA" , Hum Gene Ther 1994; 5: 717-729.)

Construction of recombinant adenoviral vectors

In order to generate a recombinant adenovirus that expressed hammerhead-ribozymes (Rz) targeted to Pleiotrophin 0 (PTN) , the Rz minigene expression cassette from the pRc/CMV- Rz66 expression plasmid was used. (Czubayko F., Riegel A.T., and Wellstein A. , "Ribozyme-targeting Elucidates a Direct Role of Pleiotrophin in Tumor Growth", J Biol Chem 1994; 269: 21358-21363.) The expression cassette was cut out as a Nrul- 5 Xbal fragment (826 nucleotides (nt) ) and blunt-end ligated into the EcoRV side of the PAVS6A Av-shuttle plasmid. The generation of the eukaryotic expression plasmid that contains the Ribozyme targeted to HER-2/neu will be described in detail elsewhere. In brief, the following Rz-coding sense and 0 antisense oligonucleotides: sense: 5 ' -agcttcaagaccacctgatgagtccgttaggacgaaaccagcaga-3 ' (Seq. #1) ; and antisense: 5 ' -agcttctgctggtttcgtcctaacggactcatcaggtggtcttga-3 ' (Seq. #2) 5 were annealed together and ligated into the Hindlll site of the pRc/CMV plasmid (Invitrogen, San Diego, CA) . This ribozyme contains HER-2/neu specific antisense flanking regions of 9 nt and 8 nt on the 5' and 3' ends of the 22nt catalytic Rz-core, that target the Rz to its cleavage site 1991 nt downstream of the translation initiation site in the HER-2/neu mRNA (GenBank accession no. M11730) . The RzHER minigene expression cassette was excised as a NruI-EcoRV fragment (778 nt) and ligated into 5_. the EcoRV site of PAVS6A. The correct insertion of the PTN and HER-2/neu expression cassettes into PAVS6A was verified by DNA sequencing.

Recombinant replication-deficient adenoviral-vectors were constructed by a homologous recombination method (Berkner, 0 K.L., Development of Adenovirus Vectors for the Expression of Heterologous genes", BioTechnigue 1988; 6: 616-624.) using PAVS6A-RZ shuttle plasmids and the 35 Kb Clal fragment of Ad- d!327. The 293 cells were co-transfected with the respective PAVS6A-plasmid and virus DNA and individual plaques containing 5 recombinant virus were purified and further amplified in 293 cells as described (Metereder, supra) . Recombinant adenoviral- DNA was analyzed by restriction enzyme digestions with EcoRI, Ndel and Xbal followed by Southern analysis using radio-labeled oligonucleotides specific for RzPTN or RzHER as probes. 0 Adenoviruses with the correct restriction pattern were produced in large amounts and purified in a two-step CsCl ultracentrifu- gation procedure. Viral titers were obtained by plaque forming assays in 293 cells and high titer virus stocks were aliquoted and stored at -80 °C. To rule out minor contaminations with 5 replication competent wild-type-adenovirus, PCR reactions with El specific primer pairs were performed using lμg of purified recombinant AvRz-HER-2/neu or AvRz-PTN DNA as templates and 0.1 pg of purified Ad-dl327 DNA as positive control (sensitivity 1 in 10⁷) . 0 Adenoviral-infections of cell lines

If not indicated otherwise, cells were grown until they reached a confluency of 50 to 70%. They were then infected with the different recombinant adenoviruses at various MOI' s in infection medium (IMEM plus 2% FBS) for 2 hours with 5 rocking. After that, infected medium was replaced with normal growth medium and cells were used in the experiments as indicated. Northern analysis

Total cellular RNA was isolated with the RNA STAT-60 method (Tel-Test, Friedenswood, Tx) , separated and blotted as described y Fang, W.J., et al., (J Biol Chem 1992; 267: 25889- 25897.) A HER-2/neu cDNA probe (1.5 Kb EcoRI fragment) was hybridized, washed and exposed to film for 16 hours. To correct for variability in loading, blots were stripped and reprobed with a Glyceraldehyde-3 phosphate dehydrogenase (G3PDH, Clontech, Palo Alto, CA) cDNA probe. Relative band intensities were measured by densitometry. PTN or HER-2/neu ribozyme transcripts were detected with [γ-³²P]-T4-Kinase labeled oligonucleotides (45 nt long) as probes. Hybridization and washing conditions were modified as follows: the hybridization buffer contained only 25% formamide and the final washing step was done at 50 °C, 0.5 x SSC/0.1% SDS for 30 minutes. Fluorescence activated cell sorting (FACS)

To quantitate HER-2/neu protein levels by FACS-analysis, cells were trypsinized, washed once with serum containing growth medium, twice with PBS (SIGMA) and resuspended in PBS at 5xl0⁵ cells/100 μl . The cells were incubated for 30 minutes at 4°C with 1: 100 dilution of a primary anti-human HER-2/neu mouse monoclonal antibody (Clone 9G6.10; Neomarkers, Fremont, CA) . Cells were washed twice with PBS and incubated for 30 minutes at 4°C in the dark with a 1:200 diluted FITC-labeled goat anti-mouse secondary antibody (Boehringer, Mannheim) . After two final washes with PBS the mean value of fluorescence intensity of 10,000 cells was determined by FACS (FACStar plus; Becton and Dickinson) . Unlabeled cells and cells labeled with secondary antibody alone served as negative controls. Growth assays

SW-13/PTN cells (Fang, supra) were infected with Av-RzPTN or Av-RzHER at MOI' s of 100 for two hours. Infected cells were then trypsinized and 2xl0⁴ cells were plated in 35 mm soft agar dishes in triplicates. Soft agar colony formation was evaluated after a 10 day incubation period as described previously. The 32D cell proliferation/survival assay was performed. (Goldstein, D.J. et al., "The Bovine Papillomavirus Type 1 E5 Transforming Protein Specifically Binds and Activates the β- Type Receptor for the Platelet-Derived Growth Factor but Not Other Related Tyrosine Kinase-Containing Receptors to Induce Cellular Transformation", J Virol 1994; 68: 4432-4441.) In brief, 32-D/H2+3 cells were plated overnight at 50 - 70 % confluence in twelve-well plates and then infected with Av- RzHER or AvlLacZ4 at MOI' s of 100 for two hours. Infected or non-infected cells were then grown for 48 hours in the absence or presence of Interleukin-3 , or in the presence of recombinant heregulin (Neomarkers) at 10^"9 M without addition of IL3. Proliferation was then assessed by counting surviving cells manually using a hemocytometer.

Adenovirus-mediated transduction of HER-2/neu ribozymes down- regulates HER-2/neu expression and inhibits HER-2/neu-mediated proliferation

To examine whether a transient adenovirus-mediated transduction of HER-2/neu ribozymes was sufficient to inhibit HER-2/neu expression of cancer cells, SL-OV-3 cells were infected with Av-RzHER or Av-RzPTN as control virus at MOI ' s of 100. total RNA was harvested one and three days after the infection and HER-2/neu mRNA levels were quantified by Northern analysis. The Av-RzHER-specific ribozyme infection led to a specific and significant depletion of HER-2/neu mRNA by approximately 75% one day after the infection when compared to the Av-RzPTN control infection.

In order to further evaluate the specificity and efficacy of the Av-RzHER vector, human MDA-MD-361 breast cancer cells were infected with Av-RzHER and Av-lLacZ4 as control at MOI ' s of 100. HER-2/neu protein levels were determine by FACS analysis over a period of 7 days after adenoviral infection and the mean fluorescence intensities were noted. Infection with Av-RzHER led to a significant reduction of HER-2/neu protein expression compared to the control virus that had no effect on HER-2/neu levels throughout the entire study. HER-2/neu levels were reduced three days after infection and remained low until day five, at which time the levels started to increase again. This time course correlated with the ribozyme expression levels described above.

In the next study, the biological significance of a Av- RzHER-mediated HER-2/neu depletion was addressed. To that end, 32D mouse hematopoietic progenitor cells which do not endoge- 5_. nously express any member of the EGF receptor family and require exogenously added Interleukin-3 (IL3) for continuous proliferation in tissue culture were used. The 32D cells were stably transfected with HER-2/neu and HER-3 expression plasmids (32D/H2+3) were also used. In contrast to the parental cell 0 line, these cells survive in the absence of IL3 when recombinant heregulin, the ligand for HER-3 , is added to the media. Since only the HER-2/neu/HER-3 heterodimer becomes an active signal transducting receptor upon ligand binding, it was believed that down-regulation of HER-2/neu would abrogate the 5 heregulin-mediated cell survival in these cells. To test this hypothesis, 32D/H2+3 cells were infected with either Av-RzHER or AvlLacZ4 and cell survival was compared to uninfected control cells. As expected, withdrawal of IL3 led to almost complete cell death in control and infected cells, whereas 0 cells proliferated at comparable levels in the presence of IL3. Heregulin promoted cell survival in the absence of IL3 in approximately 40% of the non-infected and AvlLacZ4 control cells. In contrast, heregulin was not sufficient to prevent cell death significantly in the Av-RzHER infected cells. These 5 results clearly indicated that the HER-2/neu ribozymes were effective in abating the heregulin signal transduction, and that HER-2/neu is the limiting factor for the mitogenic response in these cells. Efficacy of adenovirus-mediated PTN ribozyme expression: 0 The specificity and efficacy of this particular PTN ribozyme was characterized earlier at the mRNA and protein level in various cell lines in vitro and in vivo. It was possible to directly assess the efficacy of adenovirus-mediated transduction of PTN ribozymes in a bioassay. As a model, SW- 5 13/PTN human adrenal carcinoma cells were used. These were stably transfected with a PTN expression plasmid. In contrast to parental SW-13 cells, which do not express PTN endogenously and are not colonogenic in soft agar, SW-13/PTN cells form colonies spontaneously at a high rate. Consequently, depletion of PTN expression by adenovirus-mediated ribozymes targeting should reverse colony formation in these cells. The SW-13/PTN 5_. cells were infected with Av-RzPTN or AvlLacZ4 at MOI's from 1 to 100 plated in soft agar. Colony formation was measured after a 10 day incubation period. Av-RzPTN infection at an MOI of 25 led to a dramatic reduction in colony formation by >85% compared to the control infection. 0 The recombinant adenovirus containing the ribozymes which target PTN mRNA or HER-2/neu may be administered by means which will result in contact with the target tumor. If administered systemically , doses of about 10³ to about 10¹⁰ plaque-forming units (pfu) may be administered. When injected locally into 5 an artery that carries blood to the tumor, slightly higher doses on the order of up to about 10¹¹ pfu may be appropriate. When injected into a tumor, one would try to estimate the number of tumor cells (1 gram of tumor tissue contains approximately 10° cells) and would adjust the viral dose in 0 order to achieve at least a multiplicity of infection of 1 pfu/tumor cell into the tumor.

Appropriate carrier include the usual carriers used for administration such as saline, buffered saline, glucose in saline, Ringer's lactate, etc. 5 The constructs of the invention have been deposited in the

American Type Culture Collection located in Manassa, Virginia, United States of America.

Claims

What we claim is:

1. In a method of manufacture of a composition for the inhibition of tumor cells growth, said method comprising

5_. preparing ribozymes which target PTN mRNA or HER-2/neu in a pharmaceutically acceptable carrier.

2. A method of claim 1 wherein the ribozymes target HER- 2/neu. 0

3. A method of claim 1 wherein the expressed ribozymes target PTN MRNA.

4. A method of claim 1 wherein the recombinant adenovirus is 5 formulated for intravenous administration.