Poxviral oncolytic vectors

Abstract

The present invention relates to a poxvirus comprising a defective F4L and/or I4L gene, to composition comprising such poxvirus and to the methods and use of such compositions and poxviruses for therapeutic purposes, and more particularly for the treatment of cancer.

Claims

1. A method for treating cancer, tumors and diseases which result from unwanted cell proliferation comprising administering to a host organism or cell which is in need an oncolytic Orthopox poxvirus comprising a defective I4L and/or a defective F4L gene, and a defective J2R gene, or a composition comprising said poxvirus, with the proviso that said poxvirus is not NYVAC, and administering to said host organism or cell a topoisomerase I inhibitor which is irinotecan.

2. The method as defined by claim 1 wherein said poxvirus or composition is administered via the systemic route.

3. The method as defined by claim 1 wherein said poxvirus further comprises a suicide gene.

4. The method as defined by claim 3 wherein said suicide gene encodes a polypeptide comprising an amino acid sequence having at least 70% identity to that of SEQ ID NO: 1 or SEQ ID NO: 3.

5. The method as defined by claim 4 further comprising an additional step in which a pharmaceutically acceptable quantity of a prodrug is administered to said host organism or cell.

6. The method as defined by claim 5 wherein said prodrug is 5 fluorocytosine.

7. The method as defined by claim 5 wherein the administration of said prodrug takes place at least 3 days after the administration of said poxvirus or composition.

8. A method for treating cancer, tumors and diseases which result from unwanted cell proliferation comprising administering to a host organism or cell which is in need an oncolytic Orthopox poxvirus comprising a defective 14L and/or a defective F4L gene and a defective J2R gene or a composition comprising said poxvirus with the proviso that said poxvirus is not NYVAC and further comprising administering to said host organism or cell an antimitotic drug which is oxaliplatin.

9. The method as defined by claim 8 wherein said poxvirus or composition is administered via the systemic route.

10. The method as defined by claim 8 wherein said poxvirus further comprises a suicide gene.

11. The method as defined by claim 10 wherein said suicide gene encodes a polypeptide comprising an amino acid sequence having at least 70% identity to that of SEQ ID NO: 1 or SEQ ID NO: 3.

12. The method as defined by claim 11 further comprising an additional step in which a pharmaceutically acceptable quantity of a prodrug is administered to said host organism or cell.

13. The method as defined by claim 12 wherein said prodrug is 5 fluorocytosine.

14. The method as defined by claim 12 wherein the administration of said prodrug takes place at least 3 days after the administration of said poxvirus or composition.

Description

BRIEF DESCRIPTION OF FIGURES IN THE DRAWINGS

(1) FIG. 1. In vitro Sensitivities to 5-FC of vaccinia viruses infected human colorectal tumor cells (LoVo). LoVo cells, infected at a MOI of 0.0001 with the indicated viruses (mock (.circle-solid.) VVTK-/FCU1 (.square-solid.) or VVTK-I4L-/FCU1 ()) were exposed to various concentration of 5-FC. Cell survival was measured at 5 days post-infection. Results were expressed in percentage of cellular viability in the presence or not of drugs. Values are represented in meanSD of three individual determinations without the cell mortality due to the replication of the viruses.

(2) FIG. 2. In vitro Sensitivities to 5-FC of vaccinia viruses infected human colorectal tumor cells (LoVo). LoVo cells, infected at a MOI of 0.0001 with the indicated viruses (mock (.circle-solid.) VVTK-/FCU1 (.square-solid.) or VVTK-F4L-/FCU1 () were exposed to various concentration of 5-FC. Cell survival was measured at 5 days post-infection. Results were expressed in percentage of cellular viability in the presence or not of drugs. Values are represented in meanSD of three individual determinations without the cell mortality due to the replication of the viruses.

(3) FIG. 3. In vitro replication efficacy of VVTK-/FCU1 and VVTK-I4L-/FCU1 in LoVo infected at a MOI of 0.0001 with the indicated viruses at day 5 post infection. Values are represented in meanSD of three individual determinations.

(4) FIG. 4. In vitro replication efficacy of VVTK-/FCU1 and VVTK-F4L-/FCU1 in LoVo infected at a MOI of 0.0001 with the indicated viruses at day 5 post infection. Values are represented in meanSD of three individual determinations.

(5) FIG. 5. Mean tumor volumeSEM of s.c LoVo in Swiss nude mice after i.v injection of virus. 7 days after inoculation with tumor (palpable tumor), mice were treated by 10.sup.7 pfu of buffer+saline (), buffer+5-FC (.diamond-solid.), VVTK-I4L-/FCU1+saline () or VVTK-I4L-/FCU1+5-FC (.box-tangle-solidup.). The animals were treated by saline or 5-FC at 100 mg/kg/j twice a day by oral gavage, 7 days after virus injection during 3 weeks. Tumor volume was measured twice a week.

(6) FIG. 6. Mean tumor volumeSEM of s.c LoVo in Swiss nude mice after i.v injection of virus. 7 days after inoculation with tumor (palpable tumor), mice were treated by 10.sup.7 pfu of buffer+saline (), buffer+5-FC (.diamond-solid.), VVTK-F4L-/FCU1+saline () or VVTK-F4L-/FCU1+5-FC (a). The animals were treated by saline or 5-FC at 100 mg/kg/j twice a day by oral gavage, 7 days after virus injection during 3 weeks. Tumor volume was measured twice a week.

(7) FIG. 7. Mean tumor volumeSEM of s.c LoVo in Swiss nude mice after i.v injection of virus. 11 days after inoculation with tumor (palpable tumor), mice were treated by buffer+H.sub.2O(), or buffer+5-FC (.diamond-solid.), or one injection of 10.sup.7 pfu of VVTK-I4L-/FCU1+H.sub.2O(), or one injection of 10.sup.7 pfu of VVTK-I4L-/FCU1+5-FC (5-FC administrated 7 days after virus injection and during 3 weeks) (.circle-solid.), or two injections (day 11 and day 33) of 10.sup.7 pfu of VVTK-I4L-/FCU1+H.sub.2O (), or two injections (day 11 and day 33) of 10.sup.7 pfu of VVTK-I4L-/FCU1+5-FC (5-FC administrated from day 18 to day 32 and from day 40 to day 54) (.square-solid.). The animals were treated by 5-FC at 100 mg/kg twice a day by oral gavage. Tumor volume was measured twice a week.

(8) FIG. 8. Mean tumor volumeSEM of s.c U87-MG (glioblastoma tumor cells) in Swiss nude mice after i.v injection of virus. 11 days after inoculation with tumor (palpable tumor), mice were treated by buffer+H.sub.2O (), or buffer+5-FC (.diamond-solid.), or 10.sup.7 pfu of VVTK-I4L-/FCU1+H.sub.2O (), or 10.sup.7 pfu of VVTK-I4L-/FCU1+5-FC (.circle-solid.). The animals were treated by 5-FC at 100 mg/kg twice a day by oral gavage, 7 days after virus injection and during 3 weeks. Tumor volume was measured twice a week.

(9) FIG. 9. Ratio of virus yield in dividing cells versus in confluent cells. PANC1 (pancreatic human tumor), H1299 (Lungs human tumor) or U118MG (glioma human tumor) cells are infected with 100 pfu of () VVTK-/FCU1 or () VVTK-I4L-/FCU1. 48 h post-infection, viral titers were determined. Values are the ratio between yields of virus in dividing cells versus in confluent cells.

(10) FIG. 10. Ratio of virus yield in dividing cells versus in confluent cells. PANC1 (pancreatic human tumor), H1299 (Lungs human tumor) or U118MG (glioma human tumor) cells are infected with 100 pfu of () VVTK-/FCU1 or () VVTK-F4L-/FCU1. 48 h post-infection, viral titers were determined. Values are the ratio between yields of virus in dividing cells versus in confluent cells.

(11) FIG. 11. Viral titers (pfu/mg of tissue) in organs or tumors at day 6 and day 21 after i.v. infection into Swiss nude mice bearing subcutaneous human tumors with 110.sup.6 PFU of VVTK-/FCU1 () or VVTK-I4L-/FCU1 ().

(12) FIG. 12. Viral titers (pfu/mg of tissue) in organs or tumors at day 6 and day 21 after i.v. infection into Swiss nude mice bearing subcutaneous human tumors with 110.sup.6 PFU of VVTK-/FCU1 () or VVTK-F4L-/FCU1 ().

(13) FIG. 13. Survival of Swiss nude mice after treatment with 110$ pfu of VVTK-/FCU1 (.square-solid.) or VVTK-I4L-/FCU1 () by i.v injection.

(14) FIG. 14. Survival of immunocompetent B6D2 mice after treatment with 110.sup.7 pfu (A) or 110.sup.8 pfu (B) of VVTK-/FCU1 (.square-solid.) or VVTK-I4L-/FCU1 () by i.v injection.

(15) FIG. 15. Average quantity of pocks on tails after i.v injection of 110.sup.6 pfu VVTK-/FCU1 or VVTK-I4L-/FCU1 in Swiss nude mice at day 13 post-infection and at day 34 post-infection.

(16) FIG. 16. Average quantity of pocks on tails after i.v injection of 110.sup.6 pfu VVTK-/FCU1 or VVTK-F4L-/FCU1 in Swiss nude mice at day 13 post-infection and at day 34 post-infection.

(17) FIG. 17. Average quantity of pocks on tails after i.v injection of 110.sup.7 pfu VVTK-/FCU1 or VVTK-I4L-/FCU1 in Swiss nude mice at day 15 post-infection and at day 31 post-infection.

(18) FIG. 18. Average quantity of pocks on tails after i.v injection of 110.sup.7 pfu VVTK-/FCU1 or VVTK-F4L-/FCU1 in Swiss nude mice at day 15 post-infection and at day 31 post-infection.

MODE(S) FOR CARRYING OUT THE INVENTION

Examples

(19) Construction of Vector Plasmids

(20) A shuttle plasmid for deleting I4L was constructed using the DNA of vaccinia virus strain Copenhagen (accession number M35027) deleted on Thymidine Kinase gene and expressing FCU1 gene under the control of vaccinia synthetic promoter p11K7.5. The DNA flanking regions of I4L were amplified by PCR. Primers of the downstream flanking region of I4L were 5-TCC CCC GGG TTA ACC ACT GCA TGA TGT ACA-3 (SEQ ID NO:7; SmaI site underlined) and 5-GCC GAG CTC GAG GTA GCC GTT TGT AAT TCT-3 (SEQ ID NO:8; SacI site underlined). Primers for the upstream region were 5-GCC TGG CCA TAA CTC CAG GCC GTT3 (SEQ ID NO:9; MscI site underlined) and 5-GCC CAG CTG ATC GAG CCG TAA CGA TTT TCA-3 (SEQ ID NO:10; PvuII site underlined). The amplified DNA fragment were digested with restriction enzyme SmaI/SacI or MscI/PvuII and ligated into the corresponding sites in PpolyIII plasmid. A repeat region of the downstream flanking region of I4L was amplified by PCR using the primers 5-GCC GCA TGC ATC CTT GAA CAC CAA TAC CGA-3 (SEQ ID NO:11; SphI site underlined) and 5-GCT CTA GAG AGG TAG CCG TTT GTA ATC TG-3 (SEQ ID NO:12; XbaI site underlined) and inserted in PpolyIII plasmid. The repeat region is used to eliminate the selection cassette during the production of deleted viruses. The selection cassette, corresponding to the GFP/GPT fusion gene under the control of pH5R vaccinia promoter, was inserted into the SacI/SphI site in PpolyIII plasmid. The obtained plasmid is the recombinant shuttle plasmid named pI4L for deletion of I4L gene.

(21) A shuttle plasmid for deleting F4L was constructed using the DNA of vaccinia virus strain Copenhagen (accession number M35027). The DNA flanking regions of F4L were amplified by PCR. Primers of the downstream flanking region of F4L were 5-CGC GGA TCC TTT GGT ACA GTC TAG TAT CCA-3 (SEQ ID NO:13; BamHI site underlined) and 5-TCC CCC GGG TTA TAA CAG ATG CAG TAT CCA-3 (SEQ ID NO:14; SmaI site underlined). Primers for the upstream region were 5-GCC CAG CTG TTC AAT GGC CAT CTG AAA TCC-3 (SEQ ID NO:15; PvuII site underlined) and 5-GAA GAT CTA GTA TCG CAT CTA AAA GAT GG-3 (SEQ ID NO:16; BgIII site underlined). The amplified DNA fragment were digested with restriction enzyme BamHI/SmaI or BgIII/PvuII and ligated into the corresponding sites in PpolyIII plasmid. A repeat region of the downstream flanking region of I4L was amplified by PCR using the primers 5-GCC GAG CTC ACC CAC ACG TTT TTC GAA AAA-3 (SEQ ID NO:17; SacI site underlined) and 5-GCC GCA TGC TTA TAA CAG ATG CAG TAT CAA-3 (SEQ ID NO:18; SphI site underlined) and inserted in PpolyIII plasmid. The repeat region is used to eliminate the selection cassette during the production of deleted viruses. The selection cassette, corresponding to the GFP/GPT fusion gene under the control of pH5R vaccinia promoter, was inserted into the SacI/SmaI site in PpolyIII plasmid. The obtained plasmid is the recombinant shuttle plasmid named pF4L for deletion of F4L gene.

(22) The Generation of Recombinant Vaccinia Viruses.

(23) CEF cells were infected with VVTK-FCU1 (Vaccinia virus, defective for the J2R Kinase gene, expressing FCU1 gene under the control of synthetic promoter p11k7.5) strain Copenhagen at a MOI of 0.1 and incubated at 37 C. for 2 h, then transfected with a CaCl.sub.2 coprecipitate of the recombinant shuttle plasmid (0.2 g). The cells were incubated for 48 h at 37 C. Dilutions of virus emerging were then used to infect the CEF cells in selection medium containing Hypoxanthine at final concentration of 15 g/ml, xanthine at final concentration of 250 g/ml and mycophenolic acide at final concentration of 250 g/ml. Fluorescent (GFP) and positive (GPT selection) plaques were isolated and selected for a several round of selection in CEF cells in presence of GPT selection medium. The presence or not of VVTK-FCU1 was determined by 40 cycles of PCR with primers inside the deletion region. After the elimination of parental virus, the double deleted virus was used to infect CEF without GPT selection medium to eliminate the selection cassette. Non-fluorescent plaques were isolated and selected for 2 cycles in CEF. Final recombinant VV viruses were amplified in CEF, purified and virus stocks were titrated on CEF by plaque assay.

(24) In Vitro Cell Sensitivity to 5-FC.

(25) Human tumor cells were transduced by the respective recombinant VV at a MOI of 0.0001. A total of 310.sup.5 cells/well were plated in 6-well culture dishes in 2 ml of medium containing various concentrations of 5-FC. Cells were then cultured at 37 C. for 5 days, and the viable cells were counted by trypan blue exclusion. Results depicted in FIGS. 1, 2, 3 and 4 shows that the FCU1 activity is equivalent in viruses defective for the J2R gene than in viruses defective for the I4L and J2R gene or than in viruses defective for the F4L and J2R gene.

(26) In Vitro Replication in Cultured Cells.

(27) Dividing or confluent cells were infected, in 6-wells plaques, at 100 PFU of viruses (nearly MOI 0.0005). 2 mL of medium supplemented with 10% FCS for dividing cells and no supplemented for confluent cells were added. The cells were harvested at 48 hours post-infection. The cells were stored at 20 C. and sonicated to release the virus, virus was also quantified by plaque titration on CEF cells. The ratio between replication in dividing cells and confluent cells are similar in all cells. Both viruses VVTK-/FCU1, VVTK-I4L-/FCU1 and VVTK-F4L-/FCU1 replicate more in dividing cells than in confluent cells.

(28) As an indirect mean to assay for replication virus specificity, the yield of virus produced in dividing versus confluent tumor cells (pancreatic human tumor PANC1; lung human tumor H1299; glioma human tumor U118MG) was determined. Confluent cells were plated at 110.sup.6 cells/well and cultured in complete media for 7 days then 1 day before infection the cells were washed and cultured in media without serum. Dividing cells were plated at 310.sup.5 cells/well one day before infection. To evaluate the level of cell division, the amount of titrated thymidine incorporated into nucleic acid was measured 5 hours, 24 hours and 48 hours after plating cells. During this period thymidine incorporation was relatively constant in confluent cells whereas in dividing cells an increase in incorporation was seen over time. Then the cells were infected with 100 pfu of viruses, and 48 h post infection the ratio between the yield of virus produced in dividing tumor cells and in confluent tumor cells was determined by plaque titration on CEF. Results depicted in FIGS. 9 and 10 show that both viruses VVTK-/FCU1, VVTK-I4L-/FCU1 and VVTK-F4L-/FCU1 replicate more in dividing cells than in confluent cells. Results depicted in FIGS. 9 and 10 show moreover an increase of ratio in all the different types of cells for both viruses VVTK-I4L-/FCU1 and VVTK-F4L-/FCU1 by comparison with VVTK-/FCU1. This increase of ratio in all the different types of cells is due to a lower replication of both viruses VVTK-I4L-/FCU1 and VVTK-F4L-/FCU1 in confluent cells. These results demonstrate that both viruses VVTK-I4L-/FCU1 and VVTK-F4L-/FCU1 display an increased specificity toward dividing cells compared to VVTK-/FCU1.

(29) Subcutaneous Tumor Model.

(30) Female Swiss nude mice were obtained from Charles River Laboratories. Animals used in the studies were uniform in age (6 weeks) and body weights ranged from 23-26 g. Swiss nude mice were injected subcutaneously (s.c.) into the flank with 510.sup.6 LoVo cells. When tumors reached a diameter of 50-70 mm.sup.3, the mice were randomized in a blinded manner and treated with the indicated vectors for the in vivo experiments.

(31) Biodistribution of the Virus.

(32) The presence of the various viruses was evaluated by virus titration in tumors and organ samples. 110.sup.6 PFU of VV-FCU1 or VVTK-I4L-/FCU1 or VVTK-F4L-/FCU1 was injected intravenously (i.v.) by tail vein injection into nude mice bearing established s.c. LoVo tumors. Mice were sacrificed at indicated time points, and the tumors and other organs were collected and weighted. Tumors and organs were homogenized in PBS and titers were determined on CEF as described previously. Viral titers were standardized to milligram of tissue. Viral titers were standardized to milligram of tissue. Results depicted in Table 2, 3, 4 and 5 (the range of virus titers is presented in pfu/mg of tissue) show that after 14 days the virus according to the invention is mostly found in the tumor. Results depicted in FIGS. 11 and 12 show that both viruses VVTK-/FCU1, VVTK-I4L-/FCU1 and VVTK-F4L-/FCU1 target the tumor with about 1 000 to 10 000 fold more virus in the tumor than in the other organs analyzed except for tails in the case of VVTK-/FCU1. A small amount of VVTK-/FCU1 is detected in lungs, spleen, kidney and lymph nodes (less than 10 pfu/mg) and more in skin, tail and bone marrow at day 6, and skin and tail at day 21. In contrast, both VVTK-I4L-/FCU1 and VVTK-F4L-/FCU1 have higher tumor specificity with only a small amount in lymph nodes and tail at day 6, and only in tumor at day 21.

(33) TABLE-US-00002 TABLE 2 Tumor Lungs Spleen Kidney L.Nodes Heart VVTK- (0.2-3.3) 0.1-2 0-2.2 0-1.8 0-61 0-0.3 /FCU1 10.sup.5 VVTK- 25.6-2.2 0-0.1 n.d 0-1 n.d n.d I4L/FCU1 10.sup.5

(34) TABLE-US-00003 TABLE 3 Bone Ovaries Skin Tail Marrow Brain Muscles VVTK-/ 2.2-74 0.1-24 13.5-7 .Math. 10.sup.4 0-800 0-1.8 0-22 FCU1 VVTK- 0-102 n.d 26.3 n.d n.d n.d I4L/FCU1

(35) TABLE-US-00004 TABLE 4 Tumor Lungs Spleen Kidney L.Nodes Ovaries VVTK- (0.2-3.3) 0.1-2 0-2.2 0-1.8 0-61 2.2-74 /FCU1 10.sup.5 VVTK- 51.8-3.8 n.d n.d n.d 0-2.1 n.d F4L/FCU1 10.sup.4

(36) TABLE-US-00005 TABLE 5 Bone Tail Marrow Intestine Brain Muscles Heart VVTK- 13.5-7 .Math. 10.sup.4 0-800 n.d 0-1.8 0-22 0-0.3 /FCU1 VVTK- 0-7.9 n.d n.d n.d n.d n.d F4L/FCU1
Antitumor Activity of the Poxvirus of the Invention in s.c. Tumor Model.

(37) Nude mice bearing established s.c. LoVo tumors (50-70 mm.sup.3) were treated one time intravenously (by tail vein) with the indicated vectors at dose of 1.10.sup.7 PFU, respectively. Starting day 7 following viral injection, 5-FC was given by oral gavage at 100 mg/kg (0.5 ml 5-FC 0.5% in water) twice a day for 3 weeks. Tumor size was measured twice weekly using calipers. Tumor volume were calculated in mm.sup.3 using the formula (p/6) (lengthwidth.sup.2). The results depicted in FIGS. 5 and 6 show that the variouses viruses have a similar efficacy with an oncolytic activity (p<0.05) able to control the growth of tumor, and a combined activity (oncolytic of the virus and therapeutic of FCU1 gene) with administration of 5-FC which can further improve the control of the tumor growth (p<0.01).

(38) Nude mice bearing established s.c. LoVo tumors (50-70 mm.sup.3) were also treated intravenously (by tail vein) with the indicated vectors at dose of 1.10.sup.7 PFU according to the followings: 11 days after inoculation with tumor (palpable tumor), mice were treated by buffer+H.sub.2O, or buffer+5-FC, or one injection of 10.sup.7 pfu of VVTK-I4L-/FCU1+H.sub.2O, or one injection of 10.sup.7 pfu of VVTK-I4L-/FCU1+5-FC (5-FC administrated 7 days after virus injection and during 3 weeks), or two injections (day 11 and day 33) of 10.sup.7 pfu of VVTK-I4L-/FCU1+H.sub.2O, or two injections (day 11 and day 33) of 10.sup.7 pfu of VVTK-I4L-/FCU1+5-FC (5-FC administrated from day 18 to day 32 and from day 40 to day 54). The animals were treated by 5-FC at 100 mg/kg twice a day by oral gavage. Tumor size was measured twice weekly using calipers. Tumor volume were calculated in mm.sup.3 using the formula (pl6) (lengthwidth.sup.2). The results depicted in FIG. 7 show that no antitumoral activity of virus alone after one or two injections. The addition of 5-FC treatment shows statistically significant inhibition of tumor growth (p<0.05) when compared with vehicle groups and virus alone (without 5-FC) until day 50. As with one single injection, two i.v. injections of VVTK-I4L-/FCU1+5-FC demonstrates a significant antitumoral activity when compared with vehicule groups and two injections of virus alone (without 5-FC) (p<0.05). Moreover, a significant difference on tumor evolution is observed from day 56 between one and two injections of virus in combination of 5-FC treatment (p<0.05).

(39) Nude mice bearing established s.c. U87-MG (glioblastoma tumor cells) were treated intravenously (by tail vein) with the indicated vectors at dose of 1.10.sup.7 PFU according to the followings: 11 days after inoculation with tumor (palpable tumor), mice were treated by buffer+H.sub.2O, or buffer+5-FC, or 10.sup.7 pfu of VVTK-I4L-/FCU1+H.sub.2O, or 10.sup.7 pfu of VVTK-I4L-/FCU1+5-FC. The animals were treated by 5-FC at 100 mg/kg twice a day by oral gavage, 7 days after virus injection and during 3 weeks. Tumor size was measured twice weekly using calipers. Tumor volume were calculated in mm.sup.3 using the formula (/6) (lengthwidth.sup.2). The results depicted in FIG. 8 show a high oncolytic activity of the VVTK-I4L-/FCU1 on U87-MG cells which result in a strong antitumor activity (p<0.0001). The combined activity with addition of 5-FC, by oral gavage, results in similar activity (p<0.0001).

(40) Viral Pathogenicity.

(41) Viral pathogenicity was assessed with survival studies done on both Swiss nude mice (FIG. 13) and immunocompetents B6D2 mice (FIG. 14). Mice were injected I.V. with 1.10.sup.7 or 1.10.sup.8 PFU of all VVTK-/FCU1 and VVTK-I4L-/FCU1 in 100 L of Buffer per mouse. Mice were observed daily throughout the course of the experiment. In Swiss nude mice (FIG. 13), the injection of 110.sup.8 PFU of VVTK-/FCU1 results in the death of 40% of the animals 3 days after infection. The remaining mice died between day 50 and day 80 after infection. The administration of VVTK-I4L-/FCU1 was less pathogenic, the majority of the animals died between day 65 to 140 (p<0.01). No evidence of toxicity has been observed with both viruses at 10.sup.7 pfu (FIG. 14 (A)). All mice died after i.v injection of 10.sup.8 pfu of VVTK-/FCU1 (FIG. 14 (B)). The group with treatment of VVTK-I4L-/FCU1 had significantly prolonged survival to 70% compared with the VVTK-/FCU1 infected mice (FIG. 14 (B)). Therefore, this result demonstrates the decrease of toxicity with the double-deleted virus VVTK-I4L-/FCU1.

(42) Pocks Tail Lesion Model.

(43) Swiss nude mice were injected I.V. with 1.10.sup.6 (FIGS. 15 and 16) or 1.10.sup.7 (FIGS. 17 and 18) PFU of each virus. Tail lesions were enumerated once a week. Mice injected with 1.10.sup.6 PFU of VVTK-I4L-/FCU1 or VVTK-F4L-/FCU1 have less than 1 pock/mice compared with mice injected with VVTK-/FCU1 with a average of 8 pocks by mice in day 13 post-infection (p<0.001) as shown in FIG. 15 (A) and FIG. 16 (A). The results are similar at day 34 post-injection with an average of 4 pocks with VVTK-/FCU1 compared to nearly 1 for VVTK-I4L-/FCU1 or VVTK-F4L-/FCU1 (p<0.0001) as shown in FIG. 15 (B) and FIG. 16 (B). Mice injected with 1.10.sup.7 PFU of VVTK-I4L-/FCU1 or VVTK-F4L-/FCU1 have respectively an average of 3 pocks/mice and 2 pocks/mice compared to mice injected with 1.10.sup.7 PFU of VVTK-/FCU1 having an average of 10 pocks/mice at day 15 post-infection (FIG. 17 (A) and FIG. 18 (A)). At day 31 post-infection mice injected with VVTK-I4L-/FCU1 or VVTK-F4L-/FCU1 have respectively an average of 1.5 pock/mice and 2 pocks/mice compared to mice injected with VVTK-/FCU1 having an average of 7 pocks/mice (FIG. 17 (B) and FIG. 18 (B)). The difference in pock number between VVTK-/FCU1 and both VVTK-I4L-/FCU1 and VVTK-F4L-/FCU1 is statistically significant (p<0.01). The pocks formation is correlated with the replication of virus in the tail and so with virulence and toxicity. Injection in i.v of VVTK-I4L-/FCU1 or VVTK-F4L-/FCU1 is less toxic than with the single deleted TK virus.

(44) Combination VVTK-I4L-/FCU1 and Irinotecan

(45) The therapeutic effects of VVTK-I4L-/FCU1 treatment in combination with irinotecan were evaluated in vivo on human pancreas cancer MiaPaca2 cell line (ATCC number: CRL-1420) in the presence and absence of 5FC prodrug. More specifically, nude mice (n=12 mice/group) were injected subcutaneously with 5106 MiaPaca2 cells. When the tumors had 100-200 mm3, VVTK-I4L-/FCU1 was injected intravenously at 1106 PFU on days 24, 28 and 30 post tumor implantation. Irinotecan was administered intravenously at 33 mg/kg/day on days 28, 31, 35, 38, 42, 45. Starting on day 31 post tumor implantation, 5-FC was given by oral gavage at 100 mg/kg twice a day for 3 weeks. Tumor size was measured using calipers. Tumor volume was calculated in mm3 using the formula (/6) (lengthwidth2). Mice were killed when tumor size exceeded 4000 mm3 in volume. Mann-Whitney U-tests were used to determine tumor volume differences between the groups. A P-value <0.05 was considered significant.

(46) Tumor volume of human pancreatic MiaPaca2 implanted tumors were measured regularly (every 4-6 days) after systemic injection of VVTK-I4L-/FCU1+/5-FC and irinotecan over a period of 73 days post tumor implantation days. Table 6 provides the mean of tumor volumes in mm3 measured in the various mice groups at 24, 39, 50, 59, 63 and 73 days after implantation. iri represents irinotecan and VV VVTK-I4L-/FCU1.

(47) TABLE-US-00006 TABLE 6 Group D24 D39 D50 D59 D63 D73 Control 202 533 1395 1865 2876 Iri 195 245 122 118 157 719 5-FC 218 561 1165 2484 2832 Iri + 5FC 192 217 113 197 253 634 VV 205 438 572 1371 1639 2265 VV + iri 189 217 88 74 71 66 VV + 5FC 192 303 490 1211 2067 2451 VV + iri + 197 225 78 69 71 96 5FC : Mice euthanized when tumor volume is >4000 mm.sup.3.

(48) In MiaPaca2 model, in terms of tumor growth, there was no difference between the control (untreated) group and the group with 5-FC alone. Administration of VVTK-I4L-/FCU1 with or without 5-FC resulted in a slight antitumor effect. Administration of irinotecan with or without 5-FC displayed a significant tumor growth control compared with no treatment. The greatest benefit in terms of tumor growth control was seen with VVTK-I4L-/FCU1 in combination with irinotecan and this with or without 5-FC administration.

(49) In conclusion, treatment with the combination of VVTK-I4L-/FCU1 and irinotecan (with or without 5-FC) resulted in significant inhibition of tumor growth compared with the other groups.

(50) Combination VVTK-I4L-/FCU1 and Oxaliplatin

(51) The therapeutic effects of VVTK-I4L-/FCU1 treatment in combination with oxaliplatin were evaluated in vivo on colorectal cancer LoVo cell line in the presence and absence of 5FC prodrug. 5FU was also tested in these conditions.

(52) More specifically, nude mice (n=11 mice/group) were injected subcutaneously with 5106 LoVo cells. When the tumors had 100-200 mm3, VVTK-I4L-/FCU1 was injected intravenously at 1107 Pfu on day 21 post tumor implantation. Oxaliplatin was administered intraperitoneally at 2.5 mg/kg/day on days 24, 27, 31, 34, 38, 41. Starting on day 28 post tumor implantation, 5-FC was given by oral gavage at 100 mg/kg twice a day for 3 weeks. Starting on day 23 post tumor implantation, 5-FU was given intraperitoneally at 20 mg/kg/day for 3 weeks. Tumor size was measured using calipers. Tumor volume was calculated in mm3 using the formula (/6) (lengthwidth2). Mice were killed when tumor size exceeded 4000 mm3 in volume. Mann-Whitney U-tests were used to determine tumor volume differences between the groups. A P-value <0.05 was considered significant.

(53) Tumor volume of human colorectal LoVo implanted tumors were measured regularly (every 4-6 days) after systemic injection of VVTK-I4L-/FCU1+/5-FC and oxaliplatin over a period of 52 days post tumor implantation days. Table 7 provides the mean of tumor volumes in mm3 measured in the various mice groups at 19, 25, 32, 46, 49 and 52 days after implantation. oxa represents oxaliplatin and VV VVTK-I4L-/FCU1.

(54) TABLE-US-00007 TABLE 7 Group D19 D25 D32 D46 D49 D52 Control 167 379 746 1849 2077 2558 oxa 172 283 529 1313 1499 1768 5-FC 169 341 592 1511 1786 1987 oxa + 5FC 176 349 682 1476 1899 1828 5-FU 169 293 508 1362 1759 1558 Oxa + 179 309 517 1141 1323 1558 5FU VV 175 315 579 1554 1830 1724 VV + 5FC 172 316 573 790 836 974 VV + oxa 172 355 555 1024 1089 1215 VV + oxa + 165 328 559 657 623 760 5FC

(55) In LoVo model, in terms of tumor growth, treatment with oxaliplatin alone, 5-FC alone, oxaliplatin+5-FC, 5-FU alone, oxaliplatin+5-FU or VVTK-I4L-/FCU1 alone provided a slight control of tumor growth compared with control (untreated) mice. VVTK-I4L-/FCU1+ oxaliplatin or VVTK-I4L-/FCU1+5-FC resulted in significant tumor growth control compared with no treatment. The greatest benefit in terms of tumor growth control was seen with the combination VVTK-I4L-/FCU1+5-FC+oxaliplatin. Treatment with this combination resulted in significant inhibition of tumor growth compared with no treatment, oxaliplatin alone, 5-FC alone, oxaliplatin+5-FC, VVTK-I4L-/FCU1 alone, VVTK-I4L-/FCU1+oxaliplatin or VVTK-I4L-/FCU1+5-FC. Moreover the treatment with the combination VVTK-I4L-/FCU1+5-FC+oxaliplatin resulted in significantly antitumor effect compared with the chemotherapeutic drug 5-FU alone or in combination with oxaliplatin (5-FU was given intraperitoneally at the maximum tolerated concentrations).

(56) In conclusion, treatment with the combination of VVTK-I4L-/FCU1 and oxaliplatin notably in presence of 5-FC proved to provide an enhanced antitumor effect as compared to each of VVTK-I4L-/FCU1 and oxaliplatin as well as to the standard antitumor drug 5FU.

(57) Statistical Analysis.

(58) Statistical analyses were performed using the nonparametric Mann-Whitney Utest and STATISTICA 7.1 software (StatSoft, Inc.). A P<0.05 was considered to be statistically significant.

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