PSEUDORABIES VIRUS FOR TREATING TUMORS

Abstract

A pseudorabies virus (PRV) or a modified form thereof, or a genome sequence or a cDNA sequence containing the PRV or the modified form thereof, or nucleic acid molecules of a complementary sequence of the cDNA sequence, for treating tumors of subjects and/or reducing or inhibiting tumor recurrence, and for preparation of a pharmaceutical composition used for treating the tumors of the subjects and/or reducing or inhibiting the tumor recurrence. A method for treating tumors and/or reducing or inhibiting tumor recurrence, comprising a step of administering, on a subject having a need, the PRV or the modified form thereof, or nucleic acid molecules of the genome sequence containing the PRV or the modified form thereof.

Claims

1. A method for treating a tumor and/or reducing or inhibiting tumor recurrence in a subject, the method comprising: administering to a subject in need thereof, an effective amount of a wild-type pseudorabies virus (PRV), a modified form of the virus, or a nucleic acid molecule; wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (1) a genomic sequence or cDNA sequence of the wiled-type PRY or the modified PRV; and (2) a complementary sequence of the cDNA sequence.

2. The method of claim 1, wherein the genomic sequence of the wild-type PRV has a sequence identity of at least 70%, to the nucleotide sequence of SEQ ID NO: 1.

3. The method of claim 1, wherein, as compared with the wild-type PRV, the modified PRV has at last one modification selected from the groups consisting of the following (1)-(4): (1) a deletion or mutation of at least one endogenous gene; (2) a mutation, deletion or insertion of at least one nucleotide in an untranslated region; (3) an insertion of at least one exogenous nucleotide sequence; and (4) a combination thereof.

4. The method of claim 1, wherein the modified PRY does not express a functional EP0 protein.

5. The method of claim 1, wherein the genome of the modified PRV comprises the following modification: an original promoter of at least one PRV gene is replaced with a tumor-specific promoter.

6. The method of claim 1, wherein the modified PRV comprises an exogenous nucleotide sequence; and wherein the exogenous nucleotide sequence encodes an exogenous protein selected from the group consisting of a fluorescent protein, immunomodulatory polypeptide, cytokine, chemokine, and anti-tumor protein or polypeptide.

7. The method of claim 1, comprising administering the wild-type PRV, the modified PRY or a combination thereof, to the subject.

8. The method of claim 1, wherein the nucleic acid molecule has a genomic sequence of the wild-type PRV or the modified PRV;

9. The method of claim 1, wherein the nucleic acid molecule is a vector comprising a cDNA sequence of the wild-type PRV, the modified PRV, or a complementary sequence of the cDNA sequence.

10. (canceled)

11. The method of claim 1, further comprising administering at least one additional pharmaceutically active agent having an anti-tumor activity to the subject.

12. The method of claim 1, wherein the tumor is at least one selected from the group consisting of neuroglioma, neuroblastoma, gastric cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colon cancer, lymphoma, ovarian cancer, cervical cancer, endometrial cancer, melanoma, pancreatic cancer, osteosarcoma, prostate cancer, nasopharyngeal cancer, squamous cell carcinoma of nasal septum, laryngeal cancer, thyroid cancer, ductal carcinoma of thyroid and bladder cancer.

13. The method of claim 1, wherein the subject is a human.

14. (canceled)

15. The method of claim 4, wherein a genome of the modified PRV comprises the following modification: (i) the EP0 gene comprising a loss-of-function mutation, or (ii) the EP0 gene which is deleted or substituted with an exogenous nucleotide sequence encoding an exogenous protein.

16. The method of claim 15, wherein the loss-of-function mutation is at least one selected from the group consisting of a missense mutation, nonsense mutation, frameshift mutation, base deletion, base substitution, base addition, and a combination thereof.

17. The method of claim 1, wherein a genomic sequence of the modified PRV has a sequence identity of at least 70% to a nucleotide sequence shown in SEQ ID NO: 4.

18. The method of claim 11, wherein the additional pharmaceutically active agent is at least one selected from the group consisting of an oncolytic virus, chemotherapeutic agent and immunotherapeutic agent.

19. The method of claim 18, comprising at least one of the following (i), (ii), and (iii): (i) the additional oncolytic virus is selected from the group consisting of adenovirus, parvovirus, reovirus, Newcastle disease virus, vesicular stomatitis virus, measles virus, and a combination thereof; (ii) the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil, mitomycin, methotrexate, hydroxyurea, cyclophosphamide, dacarbazine, mitoxantrone, anthracyclines, etoposide, platinum compounds, taxanes, and a combination thereof; (iii) the immunotherapeutic agent is selected from the group consisting of an immune checkpoint inhibitor, a tumor-specific targeting antibody, and a combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0132] FIGS. 1A to 1C show the resultant micrographs of the in vitro killing experiments in Example 2 of wild-type PRV on human lung cancer cell line H1299, human liver cancer cell line BEL7402, human gastric cancer cell line BGC823, human colon cancer cell line HCT-116, mouse breast cancer cell line 4T1, human melanoma cell line MPWO, human cervical cancer cell line SIHA, mouse kidney cancer cell line Renca, human ovarian cancer cell line A2780, human nasopharyngeal cancer cell line CNE1, neuroglioma cell line GBM, human laryngeal cancer cell line Hep-2, human pancreatic cancer cell line Panc-1, human lymphoma cell line A20, mouse prostate cancer cell line Tramp C2, and human embryonic lung fibroblast MRCS, wherein MOCK indicates cells uninfected with the virus. The results show that the PRV showed a significant oncolytic effect on human and mouse tumor cell lines, but had less killing effect on MRCS of human non-tumor cell, after 72 hours of infection at a multiplicity of infection (MOI) of 1.

[0133] FIG. 2 shows the killing effect of the wild-type PRV virus on mouse kidney cancer cell line Renca in Example 2. The results show that the Renca cells infected with PRV exhibited very obvious CPE in 24 hours, and they were almost all lysed to death by 48 hours.

[0134] FIG. 3 shows the results of in vivo anti-tumor experiments in Example 3 of PRV-WT on human nasopharyngeal carcinoma model CNE1 (A), human Burkitt's lymphoma model Raji (B) and human neuroglioma model GBM (C). The results show that in the challenge groups, the growth of tumors formed by subcutaneous inoculation of CNE1, Raji or GBM cells in SCID mice significantly slowed down and arrested, and the tumors were even lysed and disappeared; in contrast, the tumors in the negative group (Mock) that were not treated with the oncolytic virus maintained normal growth, and their tumor volumes were significantly larger than those of the challenge groups.

[0135] FIG. 4 shows the in vivo anti-tumor experiment results in Example 3 of PRV-WT on mouse colon cancer model CT26 (FIG. 4A), mouse liver cancer model Hep1-6 (FIG. 4B), mouse kidney cancer model Renca (FIG. 4C), mouse breast cancer model 4T1 (FIG. 4D). The results show that the PRV-WT had significant therapeutic effects in the above mouse tumor models.

[0136] FIG. 5 shows the safety evaluation results of PRV-WT in the mouse intravenous injection model in Example 4. By intravenous injection of PBS (A) or 1*10.sup.7 PFU virus (B) into Bab/c mice, the body weight and survival rate of the mice were monitored. The results show that the mouse body weight of the PRV-WT group and the PBS group showed the same trend, and none of the mice died, confirming that the wild-type PRV-WT had very good safety in the mouse intravenous model.

[0137] FIG. 6 shows the safety evaluation results of wild-type PRV-WT in the mouse intracranial injection model in Example 4. By intracranial injection of 2*10.sup.6, 2*10.sup.5, and 2*10.sup.4 PFU of PRV-WT into Bab/c mice, the survival rate of mice was monitored. The results show that the mice injected with the virus all died one after another, and showed a certain dose dependence. Such results suggest that the PRV-WT may have certain neurotoxicity.

[0138] FIG. 7 shows the in vitro killing activity evaluation results of PRV-del-EP0 to tumor cell lines and diploid cell lines (similar to normal cell lines) in Example 5. FIG. 7A shows the killing results of PRV-del-EP0 (BK61-dEP0) to various tumor cell lines, and FIG. 7B shows the killing results of PRV-del-EP0 (BK61-dEP0) to various diploid cell lines (similar to normal cell lines). The results show that PRV-del-EP0 had a tumor killing activity comparable to that of PRV-WT, and its killing activity to normal cells was reduced.

[0139] FIG. 8 shows the safety evaluation results of PRV-del-EP0 in the mouse intracranial injection model in Example 5. By intracranial injection of 1*10.sup.6, 1*10.sup.5, 1*10.sup.4, 1*10.sup.3, 1*10.sup.2, 1*10.sup.1 PFU of PRV-WT (A) or PRV-del-EP0 (B) into mice, the survival rate of mouse was monitored. The results show that PRV-del-EP0 had significantly improved in vivo safety as compared to the wild-type PRV.

[0140] FIGS. 9 to 10 show the therapeutic effects of PRV-del-EP0 on mouse liver cancer model in Example 5. Among them, FIG. 9 shows the effects of PRV-del-EP0 on the tumor size of the mouse liver cancer model; FIG. 10 shows the effects of PRV-del-EP0 on the survival rate of the mouse liver cancer model. The results show that PRV-del-EP0 had a significant antitumor activity comparable to that of PRV-WT.

[0141] FIG. 11 shows the evaluation results of the tumor recurrence rate of the mice cured by PRV-WT and PRV-del-EP0 in Example 5. The results show that PRV-WT and PRV-del-EP0 could prevent tumor recurrence.

[0142] Sequence Information

[0143] The information of parts of sequences involved in the present invention is provided in Table 1 below.

TABLE-US-00001 TABLE 1 Description of sequences SEQ ID NO: Description 1 Genome sequence of wild type PRV (PRV-WT) 2 Nucleic acid sequence encoding the early protein EP0 in the PRV genome 3 GFP gene sequence 4 Genome sequence of PRV-del-EP0

EXAMPLES

[0144] The present invention will now be described with reference to the following examples intended to illustrate the invention (not to limit the invention).

[0145] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention basically referred to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F M Ausubel et al., Short Protocols in Molecular Biology, 3.sup.rd Edition, John Wiley & Sons, Inc., 1995. The use of restriction enzymes was in accordance with the conditions recommended by the product manufacturers. If no specific conditions were indicated in the examples, the conventional conditions or the conditions recommended by the manufacturers should be followed. The used reagents or instruments, of which manufacturers were not given, were all conventional products that were commercially available. Those skilled in the art know that the examples describe the present invention by way of example, and are not intended to limit the claimed scope of the invention. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1: Acquisition and Preparation of PRV and Modified Forms Thereof

[0146] 1.1 Isolation of Pseudorabies Virus (PRV) from Specimens

[0147] (1) Pharyngeal swabs and anal swabs of sick pigs were from Xiamen Center for Disease Control and Prevention, China, and pig embryonic kidney cells (PK-15; ATCC NumberCCL-33™) were preserved by the National Engineering Research Center for Diagnostic Reagents and Vaccines for Infectious Diseases, Xiamen University, China, which were cultured in DMEM medium supplemented with 10% fetal bovine serum, glutamine, penicillin and streptomycin.

[0148] (2) Treatment of specimens: the pharyngeal swabs and anal swabs were placed in a specimen preservation solution and fully stirred so as to wash off the virus and virus-containing cells attached to the swabs, and then the specimen preservation solution was subjected to high speed centrifugation under 4000 rpm and 4° C. for 30 min;

[0149] (3) Inoculation and observation:

[0150] A. PK-15 cells were plated on a 24-well plate with 1×10.sup.5 cells/well. The growth solution (DMEM medium, 10% fetal bovine serum, and glutamine, penicillin and streptomycin) was aspirated, and then each well was added with 1 mL of maintenance solution (DMEM medium, 2% fetal bovine serum, and glutamine, penicillin and streptomycin). Then except for the negative control wells, each well was inoculated with 50 μL of sample supernatant, and cultured in an incubator at 37° C., 5% CO.sub.2.

[0151] B. The cells were observed under microscope every day for one week and the occurrence of specific cytopathic effect (CPE) in the inoculated wells was recorded.

[0152] C. If the pseudorabies virus-specific CPE appeared in the cells of the inoculated wells within 7 days, the cells and supernatant were collected and cryopreserved at −80° C.; if no CPE appeared after 7 days, the cells were subjected to blind passage.

[0153] D. If CPE appeared in the cells within 6 blind passages, the cells and supernatant were collected and cryopreserved at −80° C.; if CPE did not appear after 6 blind passages, the cells were determined as negative.

[0154] (4) Virus isolation and cloning:

[0155] The viruses isolated from clinical specimens were identified by PCR, the pseudorabies virus-positive cultures were selected and subjected to at least 3 virus plaque purification experiments; the clonal strains obtained from virus plaques in each round were also identified by PCR, and pseudorabies virus-positive clonal strains were selected and subjected to the next round of cloning; and a single strain of pseudorabies virus with strong growth viability was selected as the candidate strain of oncolytic virus.

[0156] 1.2 Gene Editing of PRV Based on CRISPER/CAS9 Technology and Acquisition of the Modified Form Thereof

[0157] This example used wild-type PRV strain that was also referred to as BK61-WT (SEQ ID NO: 1) as an example to show how to obtain the PRV and modified form thereof for the present invention through gene editing. The specific method was as follows.

[0158] (1) sgRNA design based on CRISPER/CAS9 technology: the PAM site near the site where an exogenous gene was to be inserted was found by virus genome analysis, and the sgRNA was designed according to the sequence near the PAM, so that the CAS9 protein could cleave the virus genome, and then homologous recombination was performed to obtain a new modified virus.

[0159] (2) Construction of donor target fragments: the target fragments were sent to Shanghai Shengong for synthesis. The acquisition of target gene fragments were also the key to successful modification, when homologous recombination was performed.

[0160] Modified form: The gene sequence of key protein EP0 of wild-type PRV (of which the DNA sequence was shown in SEQ ID NO: 2) was replaced by the gene sequence of green fluorescent protein (GFP) (of which the DNA sequence was shown in SEQ ID NO: 3) to obtain the genome (of which the DNA sequence was shown in SEQ ID NO: 4) of the recombinant virus (which was named as PRV-del-EP0, and also called BK61-dEP0).

[0161] (3) The sgRNA constructed above was transferred into 293T cells to form a stable cell strain, then the target fragment was transferred into the cells, followed by infection with PRV virus. And a new recombinant virus was formed by using intracellular homologous recombination technology.

[0162] (4) The progeny virus formed after infection with 293T was used to infect PK-15 cells. The successfully recombined progeny virus carried a fluorescent signal and could be used for the screening and isolation of the progeny virus.

Example 2: In Vitro Evaluation of Anti-Tumor Activity of Wild-Type PRV

[0163] 2.1 Virus and Cell Lines as Used

[0164] (1) Virus: In this example, the PRV-WT (SEQ ID NO: 1) provided in Example 1 was used.

[0165] (2) Cell lines: human rhabdomyosarcoma cell RD (ATCC® Number: CCL-136™); human colorectal cancer cell lines SW1116 (ATCC® Number: CCL-233™), SW480 (ATCC® Number: CCL-228™) and HT-29 (ATCC® Number: HTB-38™); human gastric cancer cell lines AGS (ATCC® Number: CRL-1739™), SGC7901 (CCTCC deposit number: GDC150), BGC823 (CCTCC deposit number: GDC151) and NCI-N87 (ATCC® Number: CRL-5822™); human esophageal cancer cell line TE-1 (purchased from the Cell Resource Center, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, No. 3131C0001000700089); human small cell lung cancer cell line DMS114 (ATCC® Number: CRL-2066™); human non-small cell lung cancer cell lines SPC-A-1 (CCTCC deposit number: GDC050), NCI-H1975 (ATCC® Number: CRL-5908™), NCI-H1299 (ATCC® Number: CRL-5803™), A549 (ATCC® Number: CCL-185™), NCI-H661 (ATCC® Number: HTB-183™), EBC-1 (Thermo Fisher Scientific, Catalog #: 11875101) and NCI-H1703 (ATCC® Number: CRL-5889™); human liver cancer cell lines C3A (ATCC® Number: CRL-10741™), HepG2 (ATCC® Number: HB-8065™), SMMC7721 (purchased from the Basic Medical Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Number: 3111C0001CCC000087), BEL7402 (CCTCC deposit number: GDC035), BEL7404 (purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, number: 3131C0001000700064), Huh? (CCTCC deposit number: GDC134) and PLC/PRF/5 (ATCC® Number: CRL-8024™); human ovarian cancer cell lines SKOV3 (ATCC® Number: HTB-77™) and Caov3 (ATCC® Number: HTB-75™); human endometrial cancer cell lines Hec-1-A (ATCC® Number: HTB-112™), Hec-1-B (ATCC® Number: HTB-113™) and Ishikawa (ECACC No. 99040201); human cervical cancer cell lines Hela (ATCC® Number: CCL-2™), Caski (ATCC® Number: CRL-1550™) and C-33A (ATCC® Number: HTB-31™); human melanoma cell lines SK-MEL-1 (ATCC® Number: HTB-67™) and MeWo (ATCC® Number: HTB-65™); human breast cancer cell lines BcaP37 (CCTCC deposit number: GDC206), BT-474 (ATCC® Number: HTB-20™) and MDA-MB-231 (ATCC® Number: HTB-26™); human kidney cancer cell lines A-498 (ATCC® Number: HTB-44™) and 786-0 (ATCC® Number: CRL-1932™); human pancreatic cancer cell lines Capan-2 (ATCC® Number: HTB-80™) and HPAF-2 (ATCC® Number: CRL-1997™); human osteosarcoma cell line U2OS (ATCC® Number: HTB-96™); human prostate cancer cell lines DU145 (ATCC® Number: HTB-81™) and LNCap (ATCC® Number: CRL-1740™); human neuroglioma cell line GBM (primary tumor cell line isolated from a patient tumor tissue); human neuroblastoma cell line SH-SY5Y (ATCC® Number: CRL-2266™); human nasopharyngeal carcinoma cell line CNE (purchased from the Center for Basic Medical Cells, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, No.: 3131C0001000700013); human nasal septum squamous cell carcinoma cell line RPMI 2650 (ATCC® Number: CCL-30™); human laryngeal carcinoma cell line HEp-2 (ATCC® Number: CCL-23™); human thyroid cancer cell line SW579 (preserved by the National Engineering Research Center for Diagnostic Reagents and Vaccines for Infectious Disease) and human ductal carcinoma of thyroid cell line TT (ATCC® Number: CRL-1803™); human bladder cancer cell lines J82 (ATCC® Number: HTB-1™) and 5637 (ATCC® Number: HTB-9™); human Burkitt's lymphoma cell lines Daudi (ATCC® Number: CCL-213™) and Raji (ATCC® Number: CCL-86™); human normal cell lines including: human skin keratinocyte cell line HaCat (CCTCC, deposit number: GDC106), human embryonic lung fibroblast cell line MRC-5 (ATCC® Number: CCL-171™), human foreskin fibroblast cell line HFF-1 (ATCC® Number: SCRC-1041™), human prostate stromal cell line WPMY-1 (ATCC® Number: CRL-2854™), human umbilical vein endothelial cell line HUVEC (Thermo Fisher Scientific, Catalog #: C01510C) and the differentiated human liver progenitor cell line HepaRG (with the characteristics of primary hepatocytes; Thermo Fisher Scientific, Catalog #: HPRGC10). The above cells were all preserved by the National Engineering Research Center for Diagnostic Reagents and Vaccines for Infectious Disease, Xiamen University, China. HepaRG cells were cultured in WME medium (added with 1.5% DMSO); AGS and TT were cultured in F-12K medium; SH-SY5Y was cultured in DMEM:F12 (1:1) medium; RD, C-33A, EBC-1, SK-MEL-1, J82 and DU145 were cultured in MEM medium; Raji, Daudi, 5637, 786-0, TE-1, Caski, NCI-H1299, NCI-H1703, NCI-H1975, NCI-H661, SGC7901, BGC823, SW1116, HEp-2 and LNCap were cultured in RPMI-1640 medium; and other cells were cultured in DMEM medium. These mediums were all supplemented with 10% fetal bovine serum, glutamine and penicillin-streptomycin. All the above cells were cultured under standard conditions of 37° C. and 5% CO.sub.2.

[0166] 2.2 Cultivation of Virus

[0167] The RD cells were evenly plated on 10 cm cell culture plates, and the culturing conditions were DMEM medium containing 10% fetal bovine serum, glutamine, penicillin and streptomycin, 37° C., 5% CO.sub.2, and saturated humidity; when the cell confluence reached 90% or more, the cell culture medium was replaced with DMEM medium containing 2% serum, and each plate was inoculated with 10.sup.6 PFU of PRV-WT.

[0168] After 24 hours of continuous culture, the PRV-WT proliferated in RD cells and caused CPE in the cells. When more than 90% of the cells turned contracted and rounded, showed increased graininess, and became detached and lysed, the cells and culture supernatant thereof were harvested. After freezing and thawing for 3 cycles, the culture supernatant was collected and centrifuged to remove cell debris, in which the centrifugation conditions were 4000 rpm, 10 min, and 4° C. Finally, the supernatant was filtered with 0.22 μm disposable filter (Millipore) to remove impurities such as cell debris.

[0169] 2.3 Determination of Virus Titer

[0170] The RD cells were plated on 6-well plates with cell density of 10.sup.5 cells/well. After the cells adhered, the virus was diluted 10-fold. 100 μl of the dilution of virus was added to each well to infect the cells, followed by shaking and mixing once every 15 minutes. After shaking and mixing 5 times, the supernatant was removed. 2% agarose solution prepared with pbs was dissolved by heating and then mixed with DMEM medium containing 10% serum at a volume ratio of 1:1, and was added to the cells. When it was cooled and solidified, it was inverted and placed in an incubator. After cultivating for three days, 10% formaldehyde solution was added for fixation for 1 hour, and then the gel was inverted and taken out, followed by staining with crystal violet staining solution for 15 minutes. The titer of the virus was determined by counting the number of plaques formed.

[0171] 2.4 In Vitro Anti-Tumor Experiments of Virus

[0172] The human tumor cells and normal cells were inoculated into 96-well plates at 10.sup.4 per well. After the cells adhered, each well was replaced with corresponding cell culture medium without serum, and inoculated with viruses with MOIs of 10, 1, 0.1 and 0.01, respectively. Subsequently, CPE of the cells were monitored daily by a microscope.

[0173] The micrographs are shown in FIGS. 1A to 1C. The results show that after 72 hours of infection with a multiplicity of infection (MOI) of 1, a significant reduction in the number of the tumor cells, marked shrinking and lysis and the like, were detected in the virus-infected groups; while the non-tumor cells infected with the virus had less change in cell morphology as compared to the non-tumor cells in the Mock group. The above results indicate that the PRV has a significant oncolytic effect on a variety of human and murine tumor cell lines, but has little effect on the non-tumor cell human embryonic lung fibroblast MRCS. In addition, after 24 hours of infection with a MOI of 1, the CPE of Renca cells was very obvious, and almost all of the cells were lysed to death by 48 hours (FIG. 2).

[0174] Cell Counting Kit-8 (CCK-8 kit; Shanghai Biyuntian Biotechnology Co., Ltd.) was used to detect cell survival rate after 72 hours of virus infection and culture. The specific methods were as follows:

[0175] For adherent cells, the original medium in a 96-well cell culture plate was directly discarded; for suspension cells, the original medium in a 96-well cell culture plate was carefully discarded after centrifugation; and then 100 μl of fresh serum-free medium was added per well. 10 μl of CCK-8 solution was added to each of the wells inoculated with cells, and an equal amount of CCK-8 solution was also added to the blank culture medium as a negative control, followed by incubation at 37° C. in a cell culture incubator for 0.5-3 hours. The absorbance was detected at 450 nm using a microplate reader at 0.5, 1, 2, 3 hours, respectively, and the time point where the absorbance was within a suitable range was selected as a reference for cell survival rate. The CCK-8 detection results of the cells against PRV-WT are shown in Table 2, where “−” indicated that the cell survival rate after virus treatment was not significantly different from that of the MOCK group; “+” indicated that after virus treatment, the cell number was reduced, the survival rate was still greater than 50% but was significantly different from that of the MOCK group; “++” indicated that the cell survival rate after virus treatment was less than 50%, and was significantly different from that of the MOCK group.

[0176] The calculation method of cell survival rate is:

[00001]$\mathrm{survival\_rate}\left(\%\right)=\frac{\begin{array}{c}(\mathrm{read\_of}\mathrm{\_test}\mathrm{\_group}-\\ \mathrm{read\_of}\mathrm{\_negative}\mathrm{\_control}\mathrm{\_group})\end{array}}{\begin{array}{c}(\mathrm{read\_of}\mathrm{\_positive}\mathrm{\_control}\mathrm{\_group}-\\ \mathrm{read\_of}\mathrm{\_negative}\mathrm{\_control}\mathrm{\_group})\end{array}}\times 100\%$

TABLE-US-00002 TABLE 2 Results of in vitro anti-tumor test of PRV-WT MOI Cell line 10 1 0.1 0.01 A549 ++ ++ ++ + H661 ++ ++ ++ + H1299 ++ ++ ++ ++ LLC ++ + − − BEL7704 ++ ++ ++ ++ BEL7402 ++ ++ ++ ++ GSQ7701 ++ ++ ++ + HEP1-6 ++ ++ ++ + HUH7 ++ ++ ++ ++ QGY7703 ++ + + − SMMC7721 ++ ++ ++ + AGS ++ ++ ++ ++ BGC823 ++ + + + SGC7901 ++ ++ + + CT26 ++ ++ + + HCT116 + − − − SW1116 ++ + + + MCF7 ++ ++ + + 4T1 ++ + + + MEWO ++ ++ ++ ++ B16 ++ ++ + − Renca ++ ++ ++ + SKOV3 ++ ++ ++ ++ A2780 ++ ++ ++ ++ CNE1 ++ ++ ++ ++ CNE2 ++ ++ + + GBM ++ ++ ++ ++ Hep-2 ++ ++ ++ + Panc-1 ++ ++ ++ ++ Raji ++ ++ + + A20 ++ ++ ++ ++ Tramp C2 ++ ++ ++ ++ MRC5 ++ + − − Note: “−” indicated that there was no significant difference in cell survival rate between virus treatment group and MOCK group; “+” indicated that after virus treatment, the number of cells was reduced, the survival rate was greater than 50% but was significantly different from that of MOCK group; “++” indicated that the cell survival rate after virus treatment was less than 50%, and was significantly different from that of the MOCK group.

[0177] It can be seen from Table 2 that the PRV-WT had a good killing effect on most of the detected tumor cells. In particular, the virus had a very significant killing effect on lung cancer, liver cancer, ovarian cancer, neuroblastoma, cervical cancer, lymphoma, and kidney cancer. On the other hand, the PRV-WT had a certain killing effect on non-tumor cell lines including human embryonic lung fibroblast cell line MRC-5.

Example 3: In Vivo Anti-Tumor Experiment of Wild-Type PRV

[0178] 3.1 Virus, Cell Lines and Experimental Animals

[0179] (1) Virus: In this example, the PRV-WT provided in Example 1 was used. For the virus cultivation and virus titer determination methods, see Examples 2.2 and 2.3, respectively.

[0180] (2) Cell lines: human nasopharyngeal carcinoma cell line CNE1, human Burkitt's lymphoma cell Raji (ATCC® Number: CCL-86™), human neuroglioma cell line GBM (primary tumor cell line isolated from patient tumor tissue), mouse colon cancer cell CT26, mouse liver cancer cell Hep1-6, mouse kidney cancer cell Renca and mouse breast cancer cell 4T1. Except Raji, the above cells were cultured in RPMI-1640 medium, and all other cells were cultured in DMEM medium; and the above mediums were all added with 10% fetal bovine serum, glutamine and penicillin-streptomycin. All the above cells were cultured under standard conditions of 37° C. and 5% CO.sub.2.

[0181] (3) Experimental animals: 6-8 week-old female C.B17 SCID mice or Bab/c mice were from Shanghai Silaike Experimental Animal Co., Ltd.; according to the plan approved by Experimental Animal Center and Ethics Committee, Xiamen University, the mice were raised under SPF conditions.

[0182] 3.2 In Vivo Anti-Tumor Experiments of Virus

[0183] For the human tumor transplantation model, SCID mice were used. The tumor cells used for subcutaneous tumor formation were digested with 0.01% trypsin, and then resuspended into a single cell suspension using cell culture medium containing 10% fetal bovine serum. The cell density of the suspension was counted. The cells were precipitated by centrifugation under 1000 g for 3 min, and then the cells were resuspended with an appropriate volume of PBS to reach a concentration of about 10.sup.6-10.sup.7 cells/100 μl PBS. The tumor cells were subcutaneously inoculated in the back of SCID mice at 10.sup.6-10.sup.7 cells/100 μl PBS/site with a syringe. When the tumor cells grew into a tumor mass of about 100 mm.sup.3 under the skin of SCID mice after about 14-21 days, the tumor-bearing SCID mice were randomly divided into experimental groups treated with PRV-WT (BK61) and the negative control group (Mock). For the mouse tumor model, Bab/c mice were used, and were subcutaneously inoculated with tumor cells. After 7-14 days, the mice with tumor mass of about 100 mm.sup.3 were selected for treatment.

[0184] The oncolytic virus PRV-WT at a dose of 10.sup.6 TCID50/100 μl serum-free medium/tumor mass and an equal amount of serum-free medium were intratumorally injected respectively, once every two days for a total of 5 times of treatment.

[0185] The tumor size was measured with a vernier caliper and recorded every two days. The calculation method of tumor size is:

[0186] Tumor size (mm.sup.3)=tumor length value×(tumor width value)/2.

[0187] The treatment results of PRV-WT on human tumor transplantation model and mouse tumor model are shown in FIG. 3 and FIG. 4, respectively. The results show that after treatment with PRV-WT, the growth of the tumors of CNE1 (FIG. 3A), GBM (FIG. 3B), Raji (FIG. 3C) and CT26 (FIG. 4A), Hep1-6 (FIG. 4B), Renca (FIG. 4C) and 4T1 (FIG. 4D) gradually slowed down and arrested, and the tumors were even lysed and disappeared; in contrast, the tumors in the negative group (Mock) maintained normal growth, and the tumor sizes were significantly larger than those in the experimental groups.

[0188] The above results indicate that PRV-WT exhibited significantly favorable antitumor activity in vivo.

Example 4: Safety Evaluation of Oncolytic Virus

[0189] 4.1 Virus and Experimental Animals as Used

[0190] (1) Virus: In this example, the PRV-WT provided in Example 1 was used. For virus cultivation and virus titer determination methods, Examples 2.2 and 2.3 were referred to, respectively.

[0191] (2) Experimental animals: 6-8 weeks old Bab/c mice were from Shanghai Silaike Experimental Animal Co., Ltd.; according to the protocol approved by the Experimental Animal Center and Ethics Committee, Xiamen University, the mice were raised under clean-grade conditions, and subsequently used for in vivo virulence assessment of pseudorabies virus.

[0192] 4.2 Safety Evaluation of Virus in Mice

[0193] (1) Bab/c mice were selected and subjected to single intravenous injection of PRV-WT or PBS, the challenge titer dose was 10.sup.7 TCID50/mouse (6 mice per group), and then the survival rate and body weight of the Bab/c mice of the challenge group were monitored and recorded every day. The statistical results of body weight of the mice after injection of PBS or PRV-WT are shown in FIGS. 5A-B, which showed that within 15 days after challenge, none of the mice in the challenge group died, and the trend of animal body weight growth of the challenge group was consistent with that of the control group, i.e., there was no statistical difference (P>0.05). This result indicates that PRV-WT had very good safety in the mouse intravenous model.

[0194] (2) Bab/c mice were selected and intracranially injected with different doses of PRV-WT with challenge titers of 2*10.sup.6, 2*10.sup.5, 2*10.sup.4 PFU/mouse (4 mice per group). Subsequently, the survival rates of Bab/c mice in different dose challenge groups were recorded every day. The results are shown in FIG. 6, which showed that the death of mice occurred one after another, and this phenomenon was dose-dependent, indicating that PRV-WT had certain neurotoxicity.

Example 5: Anti-Tumor Activity and Safety Evaluation of Modified Form of PRV

[0195] 5.1 Virus as Used:

[0196] In this example, the PRV-del-EP0 (SEQ ID NO: 4) provided in Example 1 was used. For virus cultivation and virus titer determination methods, Examples 2.2 and 2.3 were referred to, respectively.

[0197] 5.2 In Vitro Oncolytic Activity Evaluation of PRV-Del-EP0

[0198] The target cells were treated with PRV-del-EP0 (BK61-dEP0) with MOI=1 according to the method described in Example 2, and the survival rate of cells after treatment with PRV-del-EP0 (BK61-dEP0) was detected using CCK8 method. FIG. 7A shows the killing results of PRV-del-EP0 (BK61-dEP0) on various tumor cell lines, and the results indicate that it substantially maintained a killing effect comparable to that of wild-type PRV of parental strain. FIG. 7B shows the killing results of PRV-del-EP0 (BK61-dEP0) on a variety of non-tumor cell lines, and the results indicate that PRV-del-EP0 showed a significantly reduced killing activity to diploid cell lines (similar to normal cell line) as compared to the wild-type PRV of parent strain. The above results indicate that PRV-del-EP0 not only retained the significant tumor-killing activity of wild-type PRV, but also had an improved safety to some extent.

[0199] 5.3 In Vivo Safety Evaluation of PRV-Del-EP0

[0200] ICR mice were selected and intracranially injected with different doses of PRV-WT and PRV-del-EP0 with challenge titer doses of 1*10.sup.6, 1*10.sup.5, 1*10.sup.4, 1*10.sup.3, 1*10.sup.2, 1*10.sup.1 PFU/mouse (4 mice per group), and then the survival rates of ICR mice in different dose challenge groups were recorded every day. The results of the PRV-WT (BK61-WT) group are shown in FIG. 8A, and the results of the PRV-del-EP0 (BK61-dEP0) group are shown in FIG. 8B. The results show that the mice in the PRV-WT group died one after another, all the mice in the 1*10.sup.6 and 1*10.sup.5 groups died, and the survival rate of the mice in the 1*10.sup.4 PFU group was only 25%; in contrast, among the PRV-del-EP0 groups, the death of mice only occurred in the 1*10.sup.6 group, and the survival rate was 50%. The above results indicate that the PRV-del-EP0 had significantly improved in vivo safety as compared to the wild-type PRV.

[0201] 5.4 Evaluation of in vivo therapeutic effect of PRV-del-EP0

[0202] The tumor cells Hep1-6 used for subcutaneous tumor formation in C57/B6 immune mice were digested with 0.01% trypsin, and then resuspended into a single cell suspension using cell culture medium containing 10% fetal bovine serum. The cell density of the suspension was counted. The cells were precipitated by centrifugation under 1000 g for 3 min, and then the cells were resuspended with an appropriate volume of PBS to reach a concentration of about 10.sup.6-10.sup.7 cells/100 μl PBS. The tumor cells were subcutaneously inoculated in the back of C57/B6 mice at 10.sup.6-10.sup.7 cells/100 μl PBS/site with a syringe. When the tumor cells grew into a tumor mass of about 100 mm.sup.3 under the skin of SCID mice after about 7-14 days, the tumor-bearing SCID mice were randomly divided into 3 groups, which were intratumorally injected with PRV-WT (BK61-WT), PRV-del-EP0 (BK61-dEP0) and PBS, respectively, once every two days, for a total of 3 treatments. The tumor size was measured with a vernier caliper and recorded every two days, and the method for calculating the tumor size was:

Tumor size (mm.sup.3)=tumor length value×(tumor width value).sup.2/2.

[0203] The results are shown in FIG. 9. The tumors were completely cleared in the mice of the PRV-WT group and the PRV-del-EP0 group, indicating that the PRV-del-EP0 exhibited the same significant in vivo therapeutic effect as the wild strain.

[0204] At the same time, the survival rates of the mice were determined. According to animal ethics, the mice were killed when the tumor size reached 2000 mm.sup.3. The results are shown in FIG. 10, which indicate that both PRV-WT and PRV-del-EP0 treatments could significantly improve the survival rate of the mice.

[0205] Further, the tumor recurrence rate in the mice was evaluated. Specifically, the mice that were cured in the above-mentioned PRV-WT group and PRV-del-EP0 group were inoculated again with tumors, and the number of inoculated cells was 10 times the number of the initially inoculated cells; and, the mice that were not treated with PRV-WT and PRV-del-EP0 were used as the control group (NC) and inoculated with the same amount of tumor cells. The tumor growth of the mice was monitored, the number of the mice with recurring tumors was counted, and the tumor recurrence rate was calculated as the following: tumor recurrence rate=(number of mice with recurring tumor/total number of mice inoculated with tumor)×100%. The results are shown in FIG. 11, which showed that none of the mice in the PRV-WT group and the PRV-del-EP0 group had recurring tumors, while tumors were observed in all of the mice of the control group that had not been treated with PRV-WT and PRV-del-EP0. This result suggests that mice cured by PRV-WT and PRV-del-EP0 had good anti-tumor immunity and tumor recurrence could be prevented.

[0206] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings that have been published, and these changes are within the protection scope of the present invention. The protection scope of the invention is given by the appended claims and any equivalents thereof.