WO1999038992A1

WO1999038992A1 - Synergism between thymidine kinase gene therapy and topoisomerase i and topoisomerase ii inhibitors

Info

Publication number: WO1999038992A1
Application number: PCT/US1999/002302
Authority: WO
Inventors: Dirk G. Kieback; Xiao-Wen Tong
Original assignee: Baylor College Of Medicine
Priority date: 1998-02-03
Filing date: 1999-02-03
Publication date: 1999-08-05
Also published as: WO1999038992A9; AU2656299A

Abstract

The invention describes a method in which adenovirus mediated thymidine kinase gene therapy enhances the sensitivity of ovarian cancer cells to topoisomerase inhibitors chemotherapy. This combination demonstrates synergistic properties. The combination is most effective when the gene therapy treatment is administered first, followed by topoisomerase inhibitor chemotherapy.

Description

SYNERGISM BETWEEN THYMIDINE KINASE GENE THERAPY AND TOPOISOMERASE I AND TOPOISOMERASE II INHIBITORS

Field of the Invention

The present invention is in the field of cancer therapeutics. This invention is directed towards a cancer treatment combining gene therapy and chemotherapy after surgery. The invention is directed further to the combination use of thymidine kinase suicide gene therapy and topoisomerase I and/or II inhibitor chemotherapy after surgical

removal of cancerous tissue.

Background of the invention

Ovarian cancer is the most lethal female malignancy and has so far been treated with surgery followed by chemotherapy. The standard therapy for ovarian cancer often

includes radical debulking surgery and platinum-based combination chemotherapy.

McGuire, W.P. era/. , N. Engl. J. Med.334: 1 -6 (1996). Other chemotherapeutic agents that have been used to treat ovarian cancer include cyclophosphamide, taxol, cisplatin, carboplatin, and adriamycin. One distinct class of chemotherapeutic substances used is

based on the inhibition of the cellular enzyme topoisomerase involved in cellular damage

repair and proliferation control.

There are two forms of topoisomerase, topoisomerase I and topoisomerase II. Topoisomerase II is selectively inhibited by the compound Vepesid® (VP-16) which currently is supplied in an intravenous preparation and an oral dosage form. Topotecan

on the other hand is a selective topoisomerase I inhibitor. Both substances show pronounced anti-ovarian cancer effect and are substances of choice in second line

treatment at this point.

Topoisomerase I is an enzyme critical for cell growth and proliferation. It catalyzes the cutting and mending of a single DNA strand and is required for DNA replication, DNA repair, and gene expression. Boothman, D.A. etal., Cancer Research 49:605-612 (1989). Hycamtin is one example of a topoisomerase inhibitor. Hycamtin exerts its cytotoxic effect, not by inhibiting the enzyme, but by stabilizing the covalent DNA-enzyme complex thus blocking DNA repair. Potmesil, M. etal., Cancer Research 54: 1431 -1439 (1994); Hsiang, Y.H. et al., J. Biol. Chem. 260:14873-14878 (1985). When DNA replicates in the presence of the stabilized complex, double-strand DNA breaks occur, and the resulting fragmentation of DNA causes cell death. Id. Cells selected for resistance to hycamtin and

defective in topoisomerase I are hypersensitive to ionizing radiation. Mattern, M.R. etal., Cancer Research 51 :5813-5816 (1991). Increased expression of topoisomerase I has been found in cisplatin-resistant cell lines. Kotoh, S. et al., Cancer Research 54:3248-3252 (1994); Pratesi, G. et al., Br. J. Cancer 71:525-528 (1995). Radio-sensitizing properties of hycamtin with supra-additive cytotoxicity have been

observed in vitro and associated with a lower cellular expression of topoisomerase I. Marchesini, R. er a/., Int. J. Cancer 66:342-346 (1996).

Despite recent advances in the non-surgical part of the treatment, McGuire, W.P.

etal., N. Engl. J. Med. 334:1-6 (1996), a majority of patients eventually die of the disease. Adenovirus mediated thymidine kinase (ADV-TK) gene therapy of ovarian cancer has been

shown to improve long term survival of nude mice bearing xenotransplanted human ovarian cancer, Tong, X.W. era/., Anticancer Research 16:1611-1618 (1996); Tong, X.W. etal. , Gynecol. Oncol.61 : 175-179 (1996), with lasting remission after a single application. Tong, X.W. et al., Anticancer Research 17:811-813 (1997).

The transduction of ovarian cancer cells with Adenovirus (ADV) mediated Rous Sarcoma Virus (RSV) driven herpes simplex thymidine kinase (TK) gene in combination with ganciclovir (GCV) administration has been shown in vitro and in vivo to be of

therapeutic potential in this disease (Tong, X.W. etal. , Anticancer Research 16:1611-1618 (1996); Tong, X.W. et al., Gynecol. Oncol. 61:175-179 (1996). Treatment efficacy was shown to be dependent on the viral dose and on the concentration of GCV. The amount of GCV was especially important for the "bystander effect" that leads to cell death of non-infected cells by cell-to-cell transfer of toxic GCV-triphosphate (Tong, X.W. et al., Anticancer Research 16:1611-1618 (1996)). Encouraging results using this treatment concept have also been noted treating a variety of different tumors including brain tumors, colon cancer, liver cancer and head and neck cancer (Chen, S.H. et al., Proc. Natl. Acad. Sci. USA 91:3054-3057 (1994); Chen, S.H. et al., Proc. Natl. Acad. Sci. USA 92:2577- 2581 (1995); O'Malley, B.W.J. et al., Cancer Research 55:1080-1085 (1995)). This attractive treatment approach is currently under investigation in clinical trials in treating

brain tumors and prostate cancer. In adenovirus mediated thymidine kinase gene therapy of ovarian cancer, viral particles carrying the thymidine kinase gene are used to infect the tumor cells. Thereafter, a prodrug is administered to the patient which is metabolized to a highly toxic compound inside the infected tumor cells, which die as a result of this treatment. However, once again, this treatment is not one hundred percent effective and

an improved mode of treatment is highly desirable.

The antiviral agent GCV is the original part of this treatment concept. The drug is

approved for the treatment of CMV retinitis in the immunocompromised patient and for the

prevention of CMV disease in transplant patients at risk for CMV. Clinical studies of

intravenous GCV have been performed at the 10mg/kg/day dose level.

Acyclovir (ACV) is a synthetic purine nucleoside analog. It has in vitro inhibitory

activity against Herpes simplex virus (HSV) types 1 and 2 (HSV-1 and HSV-2), varicella

zoster, Epstein-Barr and cytomegalovirus. Clinical studies of intravenous ACV have been

performed at a concentration of 30mg/kg/day. This dose can be increased up to

45mg/kg/day.

The mechanism of the selective cell killing in ADV/TK positive cells with purine and

pyrimidine nucleosides can be considered to be a consequence of selective activation by

a viral specified TK, conversion of the mono- to the di- and tri-phosphates by the cellular

enzymes and termination of nascent DNA chains whenever an nucleosides-triphosphate

molecule is incorporated. ACV shares the same mechanism of selective cell killing in

ADV/TK positive cells as GCV (Elion, G.B. etal., Proc. Natl. acad. Sci. USA 74:5716-5720

(1977); Cheng, Y.C. etal., J. Virol. 31:172-177 (1979); Kit, S. etal., Int. J. Cancer 14: 598-

610 (1974); Kit, S. er a/., Prog. Med. Virol. 21:13-34 (1975). It is preferentially taken up

and selectively converted to the active triphosphate form by HSV-infected cells and cellular

enzymes (Miller, W.H. et al., J. Biol. Chem. 255:7204-7207 (1980); Miller, W.H. et al.,

Biochem. Pharmacol. 31 :3879-3884 (1982)). ACV-triphosphate can be incorporated into growing chains of DNA and terminate the DNA chain resulting in cell death (Cheng, Y.C.

etal., J. Biol. Chem.258:12460-12464 (1983); Culver, KW. etal., Science 256: 1550-1552

(1992); Ezzeddine, Z.T. et al., New Biol. 3:608-614 (1991 ); Moolten, F.L. et al., Human

Gene Therapy 1 :125-134 (1990)). The fact that administration of ACV in the clinic for

treating viral infections caused much less adverse reactions even used at a higher

concentration than GCV.

Summary of the Invention

The present invention combines surgical tumor reduction with gene therapy to

minimize the tumor burden before chemotherapy is to commence. A

3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide (MTT) based assay known in the

art is used to quantify the interaction between chemotherapeutic agents and adenovirus

mediated thymidine kinase gene therapy. (See e.g., Carmichael, J. et al., Cancer

Research 47:936-942 (1987); Denizot, F. etal., J. Immunol. Methods 89:271 -277(1986))

In contrast to p53 gene therapy, which also relies on cooperation between gene

therapy and chemotherapy in the treatment of tumors, the present invention includes the

component of the adenovirus mediated thymidine kinase gene therapy which in itself is a

very efficient antitumor treatment. The uniqueness of the present invention consists of the

combination of two components of treatment that each demonstrate a pronounced

antitumor effect and that add in a synergistic fashion. Furthermore, the mechanism of

action is not restricted to ovarian cancer cells, but is also present, for example, in bladder cancer cells. Additionally, the invention will function with any cell line that is capable of being targeted by ADV-TK. The synergistic effect is not a tumor specific effect, but rather,

is specific for the two components of the treatment combination. Topoisomerase inhibiting chemotherapeutic agents are enhanced in their treatment potential by combination with

gene therapy and visa versa.

One aspect of the present invention utilizes gene therapy as an integral part of

ovarian cancer treatment in combination with topoisomerase inhibitors. The specific synergistic effect between adenovirus mediated thymidine kinase gene therapy and topoisomerase inhibitors is novel, and improves treatment of ovarian and other cancers. There is never an antagonistic effect between chemotherapy and gene therapy when the gene therapy was followed by chemotherapy. A synergistic effect is observed with topoisomerase inhibitors such as Topotecan and Vepesid® (VP-16) are used follwoing gene therapy. However, there is a slight increase in resistance to gene therapy when the

chemotherapeutic agent is administered first. A complete recovery of susceptibility to gene therapy is observed when the chemotherapeutic agent is removed. The invention involves the specific interaction between thymidine kinase gene therapy and topoisomerase I and topoisomerase II inhibitors. Topotecan demonstrates this synergistic effect, even when the topotecan was added 72 hours after gene therapy was administered.

The ADV-TK therapy also enhances the effect of subsequent chemotherapy, even though up-front chemotherapy was disadvantageous.

An object of the invention is a method of arresting or slowing uncontrolled cellular division comprising the steps of: surgical reduction of the uncontrolled cells if possible; introducing an adenoviral vector into said cells wherein said vector is comprised of a DNA

sequence encoding ADV-TK operatively linked to a promoter and wherein said cells

express ADV-TK; administering a prodrug in amounts sufficient to cause regression of said

tumor when said prodrug is converted to a toxic compound by ADV-TK; and administering

a topoisomerase inhibitor.

A further object of the invention is a method which uses a retrovirus or a liposome

delivery system to deliver the viral thymidine kinase suicide gene to the target cells. The

skilled artisan will appreciate that any delivery system will suffice if it is capable of

delivering the TK gene to, and expressing the TK gene in, the target cells.

Another object of the invention is a method in which the promoter is a Rous

Sarcoma Virus - Long Terminal Repeat, cytomegalovirus promoter, murine leukemia virus

LTR, simian virus 40 early and late, or a herpes simplex virus.

A further object of the invention is a method of treating uncontrolled cell division

when that uncontrolled division is a cancer.

A further object of the invention is a method of treating ovarian cancer. Other

objects of the invention include the treatment of endometrial cancer, cervical caner,

pancreatic cancer, breast cancer, colon cancer, stomach cancer, liver cancer, bladder

cancer, prostate cancer, peritoneal cancer, lung cancer, kidney cancer, tube cancer, or

gallbladder cancer.

An additional object of the invention is a combination therapy in which there is

chemotherapeutic administration of a topoisomerase inhibitor such as Vepesid® (VP-16),

topotecan, irinotecan, or hycamtin. Another object of the invention is a combination therapy in which there is

administration of a prodrug such as ganciclovir, acyclovir, pamciclovir, valacyclovir,

famcirclovir, or FIAU where that prodrug is converted to a toxic compound by cells

expressing ADV-TK or other TK forms.

Another object of the invention utilizes any suitable enzyme-prodrug combination

as long as the combination produces a toxic substance capable of killing the targeted cells.

Still another object of the invention is a method wherein the ADV-TK, or other TK-

based suicide gene therapy is administered before the topoisomerase inhibitor

chemotherapy is administered.

Other and further objects, features, and advantages will be apparent from the

following description of the presently preferred embodiments of the invention which are

given for the purpose of disclosure when taken in conjunction with the accompanying

drawings.

Brief Description of the Drawings

Figure 1 shows a comparison of acyclovir (ACV) and ganciclovir (GCV) toxicity

assays in OV-CA-2774, OV-CA- 1225 and SKOV3 cells.

Figure 2 shows a comparison of cell killing efficiency of adenovirus mediated

thymidine kinase based gene therapy followed by exposure to acyclovir vs. conventional

ganciclovir in OV-CA-2774 cells. MOI represents multiplicities of infection. Figure 3 shows a comparison of bystander effects of adenovirus mediated

thymidine kinase gene therapy followed by exposure to acyclovir vs. conventional

ganciclovir in OV-CA-2774 cells (low percent ADV/RSV-TK positive cells).

Figure 4 shows a comparison of bystander effects of adenovirus mediated

ganciclovir in OV-CA-2774 cells (high percent ADV/RSV-TK positive cells).

Figure 5 shows the survival of treated mice as a percentage survival rate vs.

number of days survived.

Figure 6 shows the interaction between adenoviral vector alone and chemotherapy.

The percent of surviving cells is on the Y-axis, and multiplicity of infection (MOI) is plotted

on the X-axis.

Figure 7 shows the cell killing efficacy in ovarian cancer cells (OV-CA-2774) pre-

treated with chemotherapy (Cisplatin).

Figure 8 shows the interaction between adenovirus mediated thymidine kinase

gene therapy and chemotherapy (Cisplatin). C5 represents the serum concentration in

patients 2 hours after i.v. infusion of 100 mg/m² over two hours.

Figure 9 shows the interaction between adenovirus mediated thymidine kinase

gene therapy and chemotherapy (Taxol). C6 represents the serum concentration in

patients 2 hours after i.v. infusion of 135 mg/m² over 24 hours.

Figure 10 shows the interaction between adenovirus mediated thymidine kinase

gene therapy and chemotherapy (Hycamtin). C7 represents the serum concentration in

patients 2 hours after i.v. infusion of 1 mg/m² over 30 minutes. Figure 11 shows the time dependent synergistic effect between adenovirus

mediated thymidine kinase gene therapy and hycamtin. D₀ represents the concomitant

gene therapy and chemotherapy with hycamtin. D₁ represents chemotherapy with hycamtin

performed 24 hours after gene therapy. D₂ represents chemotherapy with hycamtin

performed 48 hours after gene therapy. D₃ represents chemotherapy with hycamtin

performed 72 hours after gene therapy.

Figure 12 shows the interaction between adenovirus mediated thymidine kinase

gene therapy and Chemotherapy with VP-16. C4 represents the serum concentration in

patients 2 hours after i.v. infusion of 100mg/m² over 3 hours.

Disclosure of the Invention

It will be readily apparent to one skilled in the art that various substitutions and

modifications may be made to the invention disclosed herein without departing from the

scope and spirit of the invention.

The compound "FIAU" is 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracii.

The term "prodrug" refers to any non-toxic chemical that can be converted to a toxic

compound, ie. toxic to the target ceils. The prodrug is converted to the toxic product by

the gene product of the therapeutic nucleic acid sequence in the vector of the present

invention.

The term "promoter" as used herein refers to a recognition site on a DNA strand to

which the RNA polymerase binds, The promoter is usually a DNA fragment of about 100 to 200 bp in the 5' flanking DNA upstream of the cap site or the transcriptional initiation

start site. The promoter forms an initiation complex with RNA polymerase to initiate and

drive transcriptional activity.

The term "vector" as used herein refers to some means by which DNA fragments

can be introduced into a host organism or host tissue. There are various types of vectors

such as adenoviruses, retroviruses, and liposomes.

Topotecan and other topoisomerase chemotherapeutic administration protocols are

well known in the art. It is expected that the skilled artisan may follow any suitable dosage

protocol, and the prcise dosage required will vary from patient to patient based on factors

known to the skilled artisan and on observation of the patient's response.

The present invention provides a method of arresting or slowing uncontrolled

cellular division comprising the steps of: surgical reduction of the uncontrolled cells if

possible; introducing an adenoviral vector into said cells wherein said vector is comprised

of a DNA sequence encoding ADV-TK operatively linked to a promoter and wherein said

cells express ADV-TK; administering a prodrug in amounts sufficient to cause regression

of said tumor when said prodrug is converted to a toxic compound by ADV-TK; and

administering a topoisomerase inhibitor.

The present invention may incorporate the use of retroviruses or liposomes as

alternative delivery systems to the adenoviral vector.

Various promoters may be used to drive the vector useful in the method of the

present invention. Representative examples of a useful promoter are selected from the

group consisting of Rous Sarcoma Virus - Long Terminal Repeat, cytomegalovirus promoter, murine leukemia virus LTR, simian virus 40 early and late, and herpes simplex

virus.

The present invention may be used to treat a variety of conditions in which it is

desirable to stop or inhibit cellular division, however, a preferred embodiment encompasses the treatment of cancer.

In another preferred embodiment the condition treated by the method is ovarian

cancer.

In other embodiments the invention is designed to treat endometriai cancer, cervical caner, pancreatic cancer, breast cancer, colon cancer, stomach cancer, liver cancer, bladder cancer, prostate cancer, peritoneal cancer, lung cancer, kidney cancer, tube cancer, or gallbladder cancer.

Any topoisomerase inhibitor is suitable for use in the invention. In preferred embodiments of the invention the topoisomerase inhibitor is one of VP-16 (Vepesid®),

topotecan, irinotecan, or hycamtin.

The present invention uses any prodrug that is converted to a toxic form by ADV- TK. In preferred embodiments of the invention the prodrug is ganciclovir, acyclovir, pamciclovir, valacyclovir, famcirclovir, or FIAU. The present invention can utilize any expressible enzyme and prodrug combination other that TK as long as that combination

that is capable of producing a toxic species.

In another preferred embodiment the ADV-TK suicide gene therapy is administered before the topoisomerase inhibitor is administered. The following examples are offered by way of illustration and are not intended to

limit the invention in any manner.

Example 1 ADV-TK Based Gene Therapy as a Chemotherapy Sensitizer

Three ovarian cancer cell lines with different growth patterns were used. Cell killing

efficacy of gene therapy, chemotherapy, and their combinations with different

concentrati on s and time i ntervals were measured using a

3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide (MTT) based assay. A slightly

increased resistance to gene therapy was observed in cells pre-treated with

chemotherapy. Removal of the drugs restored the previous susceptibility of the cells to

gene therapy. No antagonism was observed with gene therapy followed by chemotherapy.

The concomitant applications of gene therapy and chemotherapy resulted in a higher rate

of cell death than the interval therapy. A synergistic interaction was observed in the

combination of gene therapy and hycamtin. These results indicate that ADV-TK based

gene therapy can be used as a chemotherapy-sensitizer for treating ovarian cancer.

Example 2 Cell Culture

Three well-characterized human epithelial ovarian cancer cell lines; OV-CA-2774,

OV-CA-1225, and SKOV-3 (purchased from ATCC) were selected. Cells were maintained in HGDMEM (Dulbecco's Modified Eagle Medium with high glucose, Gibco#56-439-110)

supplemented with 1 % penicillin/streptomycin (100x, Gibco#600-5140AG), 1% glutamine

(1 OOx Gibco #320-5030AG), and 10% fetal bovine serum (Hyclon) at 37°C and 5% C0₂ in

a humidified incubator.

Example 3 Recombinant Adenovirus and Therapeutic Agents

Construction of recombinant adenovirus of both Rous Sarcoma Virus Promoter

driven versions of the thymidine kinase gene (ADV/RSV-TK) and cytomegalovirus driven

form (ADV/CMV-TK) are known in the art. (See e.g., Tong, X.W. et al., Anticancer

Research 16:1611-1618 (1996)). The viral titer is based on biological infection (plaque

forming units, PFU). ADV/RSV-TK and ADV/CMV-TK were compared in the gene therapy

component. The drugs chosen were cisplatin, carboplatin, doxorubicin, taxol,

mitoxantrone, VP-16, and hycamtin.

Example 4 Cell Killing Efficacy and Statistical Analysis

A MTT based assay was used to evaluate cytotoxicity as described in the art. (See

e.g., Carmichael, J. et al., Cancer Research 47:936-942 (1987) and Denizot, F. et al., J.

Immunol. Methods 89:271-277 (1986)). It was carried out in flat-bottomed 96-well

microtiter trays (Costar 3696, Cambridge, MA). In view of the rapid doubling time of

OV-CA-2774 cells, 500 cells were considered optimal to ensure logarithmic growth at the

end of the experiment while 2000 cells of SKOV3 and OV-CA-1225 were seeded. All results represent the median of 8 identically treated wells. The Wilcoxon Test for Paired

Samples was used for statistical analysis.

Example 5 Cell Killing Efficacy of Chemotherapeutic Agents

For cell killing efficiency assay, 500 cells (OV-CA-2774) or 2000 cells (OV-CA-1225

or SKOV3) in a volume of 10Oμl were plated in flat-bottomed 96-well microtiter trays for six

hours. After washing 1x with warm PBS, they were incubated with serum-free medium

containing ADV/RSV-TK at varying MOI (125, 61 , 30, 15, 7.5, 3.6, 1.8, 0.9 and 0 for

OV-CA-2774 and OV-CA-1225, 1000, 500, 250, 125, 61 , 30, 15, 7.5, 3.6, 1.8, 0.9 and 0

for SKOV3). Two hours later, the medium was replaced with complete medium containing

10% fetal calf serum (FCS) and the cells were incubated for another 12 hours. Cells were

then incubated with varying doses of each chemotherapeutic agent from 0 to a dose at

which 100% cell killing was reached (Table 1 ). For example, in one instance, the medium

was replaced with complete medium containing different doses of GCV or ACV (Oμg/ml,

10μg/ml, 25μg/ml, 50μg/ml, 100μg/ml) and the cells were incubated for three days. The

number of surviving cells was determined by MTT assay. The serum concentration of the

tested chemotherapeutic agents that is observed two hours after administration in patients

was covered by the concentration range tested for each substance (Table 1). The cells

were incubated for 3 days. The number of surviving cells was determined by MTT assay. Table 1 Concentration of therapeutic agents tested.

Agents Hycamtin Taxol Cisplatin Carbopiatin Doxorubicin Mitoxantrone

Dosage 1 mg/m² 135 mg/m² 100 mg/m² 240 mg/m² 70 mg/m² 12 mg/m²

Serum conc.(μg/ 46x10^'3 2x10^"1 25x10^"1 1 x10¹ 2.2 x10^"1 8x10^"2 ml)

C1 46x10^'6 1 x10^"3 25x10^"3 5x10^"2 2.2x10^"3 4x10^'3

C2 92x10^"6 2x10^"3 5x10^"3 1 x10^"1 44x10^"3 8x10^"3

C3 46x10^"5 1 x10^"2 5x10^"3 5x10^"1 2.2x10-² 1 x10-²

C4 9.2 x10^'5 2x10^"2 5x10^"2 1 44x10^"2 8x10^'2

C5 46X10^"4 1 x10^"1 2.5x10^"1 5 2.2x10^"1 1 x10^"1

C6 9.2x10^ 2x10^"1 5x10^"1 1 x10¹ 44x10^'1 8x10^"1

C7 46x10^"3 1 2.5 5x10¹ 2.2 4

C8 92x10^"3 2 5 1 x10² 44 8

Example 6 GCV and ACV Toxicity Assay

In order to determine the cytotoxicity caused by GCV and ACV alone, 500 cells (OV- CA-2774) or 2000 cells (OV-CA-1225 or SKOV 3) in a volume of 100μl were plated out in flat-bottomed 96-well microtiter trays for six hours at 37°C and 5% C0₂ in a humidified incubator Medium was replaced with serum-free medium for two hours Cells were then incubated with complete medium for 12 hours followed by incubation with GCV and ACV at varying concentrations (0, 10μg/ml 25μg/ml 50μg/ml 100μg/ml 200μg/ml, and 400μg/ml for 72 hours. The number of surviving cells was determined by MTT assay. Each data point represented the median value of eight samples.

GCV or ACV in doses of up to 200μg/ml did not inhibit cell growth in either cell line compared to untreated cells (Fig. 1 ). No obvious toxicity and morphological changes were observed under 200μg/ml of GCV concentration. A slight inhibitory effect of GCV and ACV on OV-CA-2774 and OV-CA-1225 cell growth was observed at a concentration of 400μg/ml without significant morphological changes. The difference of inhibitory effect caused by ACV and GCV was not significant (P>0.05). No obvious toxicity and morphological changes were observed up to 400μg/ml of either GCV or ACV concentration in SKOV3.

Example 7 Comparison of Cell Killing Efficacy

Cell killing efficacy was found to be dependent on multiplicities of infection (MOI) and GCV or ACV dose (Fig. 2). At fixed MOI cells exposed to ACV showed either higher or the same cell killing efficacy compared with the cells exposed to GCV at the same concentration. However, at certain fixed MOI range where 100% cell killing was not reached, cells treated with 2.5 or 5 times (25μg/mi or 50μg/ml) higher concentrations of

ACV always resulted in more effective cell killing than cells treated with 10μg/ml GCV

(P<0.01 ). For example, in OV-CA-2774 cells transduced at a MOI of 0.9, 58% cell death was observed after administration of GCV at a concentration of 10μg/ml and 59% cell death using ACV at the same concentration (1 Oμg/ml) while 73% and 80% cell killing were achieved by using ACV at a concentration of 25μg/ml and 50μg/ml respectively (Fig 2). Cell killing efficacy in the cells transduced at the same MOI (0.9) followed by

administration of GCV or ACV at different doses was significantly different (P<0.01 ). This GCV or ACV dose-dependent cell killing efficacy was also seen in other groups of cells transduced at higher MOIs. 98% cell death was achieved by using GCV or ACV at a concentration of 10μg/ml in the cells transduced at an MOI of 15 while 100% cell death was observed when cells exposed to ACV at a concentration of 25μg/ml or 50μg/ml.

Almost identical results were observed in cell line OV-CA-1225 and SKOV3 with some variation of cell killing efficacy as a result of the same combinations of ADV/RSV-TK and GCV or ACV as previously used in cell line 2774. Decreased cell killing efficacy by using ACV at the same concentration as GCV was not observed in all three ovarian cancer cell lines. Replacing GCV with ACV in the gene therapy increases the treatment effect without

increasing toxicity.

Example 8 In-vitro Bystander Effect Assay

The phenomenon that ADV/RSV-TK positive cells are toxic to nearby ADV/RSV-TK negative cells by cell-cell transfer of GCV-triphosphate was named the "bystander effect".

To quantify the "bystander effect" and to determine the factors affecting this phenomenon, cells of cell line OV-CA-2774, OV-CA-1225 and SKOV3 with different ratios of

ADV/RSV-TK positive plus ADV/RSV-TK negative cells were exposed to different doses of GCV. Cells were transduced in serum-free medium with ADV/RSV-TK at an MOI by which only 50% of the cells could be transduced (MOI of 7.5 for OV-CA-2774, MOI of 3.6 for OV-CA-1225 and MOI of 150 for SKOV3) and incubated for 2 hours at 37°C and 5%

C0₂ in a humidified incubator. Medium was thereafter replaced with fresh complete

medium. Twelve hours later, 500 cells (OV-CA-2774) or 2000 cells (OV-CA-1225 or

SKOV3) in a volume of 10Oμl containing varying mixing ratios of transduced and untreated

cells were seeded in 96-well tissue culture plates and incubated for another 12 hours (0,

2.5%, 5%, 10%, 25%, 50%, 75%, 90% and 100%). This was followed by incubation with

GCV or ACV at varying concentrations (0, 10μg/ml, 25μg/ml, 50μg/ml, and 100μg/ml) for

72 hours. The number of surviving cells was calculated by MTT.

The bystander effect in terms of cell killing efficacy was known in the art to be

dependent on the percentage of ADV/RSV-TK transduced cells as well as on the GCV

dose (See e.g., Tong, X.W. etal., Anticancer Research 16:1611-1618 (1996)). As shown

in Figures 3 and 4, similar cell killing efficacy was achieved in the cells containing same

percentage of ADV/RSV-TK positive cells by using GCV or ACV at the same

concentration. The influence of ACV or GCV concentration on the" bystander effect" are

shown in Figures 3 and 4). Cells with the same ratio of infected cells were exposed to

varying doses of GCV or ACV (0-100μg/ml). In the cells containing small portions of

ADV/RSV-TK positive cells (0.5 to 2.5), no significant changes in cell viability were seen

in the cells whether exposed to low doses of GCV or ACV (1 Oμg/ml) or to high doses of

GCV or ACV (100μg/ml). 58% and 60% of the cells were killed in the cells containing

2.5% positive cell after exposure to 10μg/ml of GCV and ACV respectively.63% cell death

was seen after exposure to 10Oμg/ml GCV and 62% cell death when exposed to 10Oμg/ml

ACV(P>0.05) (Fig. 3). Significant changes were observed in cells containing higher infection ratios after treatment with different doses of GCV or ACV (Fig. 4). 69% or 68%

of cell death was observed in assays consisting of 25% ADV/RSV-TK positive cells treated

with 10μg/ml of GCV or ACV (P>0.05), 92% and nearly 100% cell death resulting from

10Oμg/ml of GCV and ACV respectively (P<0.05). These data also suggest that in the cells

containing high proportion of infected cells a stronger "bystander effect" can be achieved

by using ACV.

Example 9 Comparison of GCV vs. ACV in Nude Mice

The human epithelial ovarian cancer cell line OV-CA-2774 was selected because

OV-CA-2774 cells are especially suited for the testing of novel gene therapy approaches,

as they known in the art to be cisplatin resistant and contain p53 mutations (See e.g.,

Freeman, R. et al., Cancer 42:2352-2359 (1978); Santoso, J.T. et al., Gynecolog.

Oncology 59: 171 -178 (1995)).

The ovarian cancer animal model was established in 6-8 weeks old female athymic

CD-1 nu/nu mice after intraperitoneal injection of 1 x 10⁸ OV-CA-2774 cells. The tumor

growth pattern still reflected the histology of the original tumor and resulted in

disseminated nodules throughout the abdomen and copious bloody malignant ascites.

The construction and isolation of recombinant adenovirus is known in the art (See

e.g., Tong, X.W. etal., Anticancer Research 16:1611-1618 (1996); Chen, S.H. etal., Proc.

Natl. Acad. Sci. USA 91 :3054-3057 (1994)). One preparation of ADV/RSV-TK containing 5.75 x 10¹¹ normal adjusted standard infectious units (NAS IU)/ml and 4.2 x 10¹² particles/mi was used for the whole experiment.

Administration of GCV started 20 hours after virus injection. The median survival of mice treated with ADV/RSV-TK(2 x 10⁸ to 2 x 10⁹) alone at the different time points and of mice treated with GCV(1 Omg/kg) or ACV(1 OOmg/kg) alone varied from 14.5 to 20 days. No liver toxicity was found in the animal model of ovarian cancer even in the subset of animals treated with the high ACV dose (1 OOmg/kg).

A prospective randomized treatment plan was designed to evaluate the therapeutic efficiency and toxicity of ADV/RSV-TK mediated gene therapy followed by administration of ACV vs GCV. Randomization was chosen as a means to eliminate potential confounding influences on the data by animal age, cell passage number, viral storage time and investigator. Survival was chosen as the study endpoint. Each individual treatment and control group was to be comparable to each other with a statistical power of 80%. A 1.5 fold increase in survival time was considered the lower limit of statistical discrimination.

In this design 25 mice were needed per treatment group. 75 female CD-1 nu/nu mice were divided into 3 groups. Three days after tumor inoculation 500μl PBS containing 6.67x10⁸ IU were injected intraperitoneally followed by administration of GCV (1 Omg/kg) at a concentration of 1 mg/ml (i.p., bid , plus Hanks one time a day) or ACV (33.3mg/kg or 66.6mg/kg i.p., tid) for six consecutive days. 6.67x10° IU were chosen because at this

dose no inhibitory effect on tumor growth was observed by viral vector alone. Treatment was started three days after tumor inoculation.25 mice inoculated with tumor were treated per week. The log-rank test was used to compare survival among the groups. Survival was significantly prolonged in all treated mice with a median survival of 35

± 1 days (Fig. 5). However, 28% and 24% of mice treated with ACV, 100mg/kg/day and

200mg/kg/day, respectively, survived more than 70 days in contrast to only 4% of GCV

treated mice. Two mice treated with ACV were still alive without evidence of disease 150

days after tumor inoculation. No long-term survival was observed in mice treated with

GCV. No liver toxicity was observed in either of the treatment groups.

ADV/RSV-TK in combination with ACV administration can achieve at least the same

therapeutic effect in the treatment of disseminated ovarian cancer in vivo as GCV.

Increased long term survival was observed in ACV treated animals, and two mice treated

with ACV were still alive at the end of the data collection period. This data indicated that

ADV-mediated gene therapy followed by the administration of ACV is superior to GCV

treatment.

Liver toxicity is a major concern of intraperitoneal ADV-mediated gene therapy in

combination with high dose GCV. No liver toxicity was observed in the animal model of

ovarian cancer, even in the subset of animals treated with high dose ACV. Therefore, ACV

gives this treatment approach a wider therapeutic safety margin.

The most common adverse events with GCV are renal impairment and bone marrow

suppression. These adverse reactions are also a major concern when using

chemotherapy for the treatment of ovarian cancer. By contrast, less than 1 % of the

patients showed myelosuppression after ACV therapy. Thus, replacing GCV by ACV in

ADV/RSV-TK gene therapy concept may improve the margin of treatment safety in patients whose renal and/or bone marrow function may have been impaired by previous

chemotherapy.

Example 10 interaction of Gene Therapy and Chemotherapy

For determining the efficacy of gene therapy after chemotherapy, ceils were treated

with the chemotherapeutic agents at a concentration at which 40% to 65% of cells were

killed. Cells were then transduced with ADV/RSV-TK on day 0, day 1 , day 2, or day 3 with

different multiplicities of infection (MOIs) followed by administration of ganciclovir at a

concentration of 10μg/ml.

For determining the influence of chemotherapy concomitantly with or after gene

therapy, cells were incubated with ADV/RSV-TK or ADV/CMV-TK at varying MOIs at which

20% to 80% cells were killed when exposed to GCV at a concentration of 10μg/ml. At the

same time, chemotherapeutic agents of different concentrations were added into the

medium and the cells were incubated for three days. Also, cells were treated with gene

therapy as described above and incubated with complete medium alone for one day, two

days or three days. The medium was thereafter replaced by medium containing

therapeutic agents with different concentrations. Cells were then incubated for another

three days.

Cells transduced with either ADV/RSV-TK or ADV/CMV-Tk alone in a dose range

of virus (from 0 to the beginning of vector toxicity) did not change their sensitivity to the

chemotherapeutic agents tested. The sensitivity to chemotherapy was mildly enhanced in the cells transduced with higher doses of virus (up to 61 MOI for OV-CA-2774 and OV-

CA-1225, up to 1000 for SKOV3). This effect is probably explained by viral toxicity alone.

No additive or synergistic effects were observed (Fig. 6).

A significantly decreased susceptibility to gene therapy was observed in the cells

pre-treated with chemotherapy (p<0.01 ), so that at constant concentration of GCV a 2-4

fold MOI had to be applied to achieve the same cell killing efficacy. Removal of the drugs

restored the previous sensitivity of the cells to gene therapy (Fig. 7). No antagonism was

observed between gene therapy and application of chemotherapy either simultaneously

or after a time interval (Figs. 8-10, 12). Using this protocol, the obtained cytotoxicity was at least equal, if not higher compared with gene therapy or chemotherapy alone. The

concomitant application resulted in a significantly higher rate of cell death than the interval

therapy (Fig. 11 ). This result was independent of the promotor used (RSV or CMV). At

a fixed concentration of chemotherapy the efficacy of combination therapy correlated with

the viral dose used in gene therapy.

A significantly synergistic interaction (P<0.01) was observed in simultaneous

application of ADV-mediated TK gene therapy and hycamtin or VP-16. (Figs. 5-7). With

gene therapy alone, 66% of OV-CA-2774 cells were killed at a given MOI. 100%

cytotoxicity was obtained using concomitant therapy of the same viral MOI and hycamtin

at a dose which itself resulted in only 17% cell death. A low dose of hycamtin at which no

cytotoxicity had been observed was combined with gene therapy, and yielded an 83%

reduction of cell viability was observed instead of the expected 66% (Fig. 5). This

synergism was inversely related to the time interval between gene therapy and treatment with hycamtin. Similar results were obtained in all cell lines. This phenomenon was

observed with these two chemotherapeutic compounds only.

To avoid this potentially antagonistic phenomenon, ADV-mediated gene therapy

should not be performed directly after chemotherapy. On the other hand, none of the 3

ovarian cancer cell lines showed a decreased sensitivity towards chemotherapy when

ADV-mediated gene therapy had been applied prior to chemotherapy. In contrast,

ADV-TK-pretreatment or simultaneous application enhanced the chemotherapeutic

vulnerability of the investigated cell lines. The application of gene therapy prior to or

simultaneous with chemotherapy represents the optimal protocol for combination

treatment.

A synergistic effect between gene therapy and hycamtin or VP-16 was evident in

all 3 ovarian cancer cell lines. All cell lines with different in vitro and in vivo behavior

responded to hycamtin and ADV therapy in a similar fashion. The 3-day exposure to

hycamtin or VP-16 used is considered sufficient to effectively inhibit even initially elevated

levels of topoisomerase.

ADV-TK gene therapy combined with GCV administration causes an accumulation

of the phosphorylated product of GCV in tumor cells, which can be incorporated into

nascent DNA chains of proliferating cells and act as a chain terminator thereby resulting

in cell death. Combination of gene therapy and chemotherapy with topoisomerase

inhibitors interferes with DNA replication processes at two different steps with the shared

aspect of damage to nascent DNA chains which might result in the observed synergism.

The lack of a synergistic interaction between other agents and thymidine kinase gene therapy is probably due to their different pharmacological effect. Hycamtin is an

advantageous choice when selecting chemotherapeutic agents to be administered in

conjunction with gene therapy.

Example 11

Impact of Combined ADV-TK Topotecan Therapy on the Long-Term

Survival of Nude Mice Bearing Epithelial Ovarian Cancer

Randomization was chosen as a means to eliminate potential confounding

influences on the data by animal age, cell passage number, viral storage time and

investigator. Survival was chosen as the study endpoint. Each individual treatment and

control group was comparable to each other with a statistical power of 80%. A doubling

of survival time was considered the lower limit of statistical discrimination.

153 female CD-1 nu/nu mice (Charles River Laboratories, Wilmington, MA) age

6-10 weeks were divided into 9 groups: Group I: 6.67 x 10⁸ ADV/RSV-TK + ganciclovir

(1 Omg/kg, bid for six days); Group II: 6.67 x 10⁸ ADV/RSV-TK + ganciclovir (1 Omg/kg, bid

for six days) + topotecan (using the corresponding dose used in human); Group III: 6.67

x 10⁸ ADV/RSV-TK + ganciclovir ( 10mg/kg, bid for six days) + topotecan (using 1 /10 of the

corresponding dose used in human); Group IV: topotecan alone (using the corresponding

dose used in human); Group V: topotecan alone (using 1/10 of the corresponding dose

used in human); Group VI: 6.67 x 10⁸ ADV/RSV-TK + ganciclovir (1 Omg/kg, bid for six

days) + Taxol (using the corresponding dose used in human); Group VII: 6.67 x 10°

ADV/RSV-TK + ganciclovir (1 Omg/kg, bid for six days) + Taxol (using 1/10 of the

corresponding dose used in human); Group VIII: Taxol alone (using the corresponding dose used in human); Group IX: Taxol alone (using 1/10 of the corresponding dose used

in human); Each mouse is injected with 1 x 10⁸ tumor cells of OV-CA-2774 in a volume of

2 ml Hanks buffer interaperitoneally. At that dosage all mice develop ovarian cancer.

Three days after tumor inoculation 500μl PBS containing 6.67 x 10⁸ infectious viral

particles were injected intraperitoneally or PBS alone followed by administration of GCV

(1 Omg/kg, bid) at a concentration of 1 mg/ml for six consecutive days with or without

Topotecan or Taxol. Seventeen mice inoculated with tumor were treated each week.

Cytological examination of the ascites and histological examination of heart, lung,

diaphragm, liver, spleen, kidney, bowel, uterus, ovary and omentum were performed

immediately after the tumor-induced death of the mice. The log-rank test is used to

compare survival among the groups.

10 mice were treated each week for up to six months. Another five months were

required for observing the treatment effects and daily recordings of animal weight and

body temperature and tissue analysis of dead mice. Topotecan was found to demonstrate

a pronounced overadditive effect when combined with ADV-TK gene therapy.

Example 12

Impact of Combined ADV-TK VP 16 Therapy on the Long-Term

Survival of Nude Mice Bearing Epithelial Ovarian Cancer

Randomization was chosen as a means to eliminate potential confounding

investigator. Survival was chosen as the study endpoint. Each individual treatment and control group was to be comparable to each other with a statistical power of 80%. A doubling of survival time was considered the lower limit of statistical discrimination.

In this design 17 mice were needed per treatment group. 102 female CD-1 nu/nu mice (Charles River Laboratories, Wilmington, MA) age 6-10 weeks were divided into 9

groups: Group 1: 6.67 x 10⁸ ADV/RSV-TK + ganciclovir (1 Omg/kg, bid for six days); Group

II: 6.67 x 10⁸ ADV/RSV-TK + ganciclovir (1 Omg/kg, bid for six days) + VP-16 (using the

corresponding dose used in human); Group III: 6.67 x 10⁸ ADV/RSV-TK + ganciclovir

(1 Omg/kg, bid for six days) + topotecan (using 1/10 of the corresponding dose used in

human); Group IV: VP-16 alone (using the corresponding dose used in human); Group

V: VP-16 alone (using 1/10 of the corresponding dose used in human); Group VI:

untreated mice as control group. Each mouse is injected with 1 x 10⁸ tumor cells of OV-CA-2774 in a volume of 2ml Hanks buffer intraperitoneally. At that dosage all mice develop ovarian cancer. Three days after tumor inoculation 200μl PBS containing 6.67

x 10⁸ infectious viral particles were injected intraperitoneally or PBS alone followed by administration of GCV (1 Omg/kg, bid) at a concentration of 1 mg/ml for six consecutive days

with or without VP-16. Ten mice inoculated with tumor were treated each week. Cytological examination of the ascites and histological examination of heart, lung, diaphragm, liver, spleen, kidney, bowel, uterus, ovary and omentum were performed

immediately after the tumor induced death of the mice. The log-rank test is used to

compare survival among the groups. 10 mice were treated each week for up to six months. Another five months were required for observing the treatment effects. Daily recordings of animal weight and body temperature and tissue analysis of dead mice were made.

Claims

We Claim:

1. A method of arresting or slowing uncontrolled cellular division comprising the steps

of: first performing a surgical reduction of the uncontrolled cells if possible;

followed by introducing a vector into said cells wherein said vector is comprised of

a DNA sequence encoding an enzyme operatively linked to a promoter and wherein said

cells express said enzyme;

next administering a prodrug in amounts sufficient to cause regression of said tumor

when said prodrug is converted to a toxic compound by said enzyme; and

then administering a topoisomerase inhibitor.

2. The method of Claim 1 wherein said enzyme is thymidine kinase.

3. The method of Claim 1 , wherein said vector is an adenoviral vector comprised of

a DNA sequence encoding ADV-TK operatively linked to a promoter and wherein said cells

express ADV-TK.

4. The method of Claim 1 , wherein said uncontrolled cell division is a cancer.

5. The method of Claim 4, wherein said cancer is a cancer with intraperitoneal spread.

6. The method of Claim 4, wherein said cancer is ovarian cancer, endometrial cancer, cervical cancer, pancreatic cancer, breast cancer, colon cancer, stomach cancer, liver cancer, bladder cancer, prostate cancer, peritoneal cancer, lung cancer, kidney cancer, tube cancer, or gallbladder cancer.

7. The method of Claim 1 , wherein said topoisomerase inhibitor is VP 16, topotecan, irinotecan, or hycamtin.

8. The method of Claim 1 , wherein said prodrug is ganciclovir, acyclovir, pamciclovir,

valacyclovir or FIAU.

9. The method of Claim 1 , wherein said promoter is Rous Sarcoma Virus - Long Terminal Repeat, cytomegalovirus promoter, murine leukemia virus LTR, simian virus 40 early and late, or herpes simplex virus.

10. The method of Claim 1 , wherein said topoisomerase inhibitor is administered within 72 hours of said prodrug administration.

11. The method of Claim 1 , wherein said mammal is a human.