VIRUS AND TUMOR THERAPEUTIC DRUG FOR SPECIFICALLY KILLING TUMOR CELLS

Abstract

Provided are a virus and a tumor therapeutic drug for specifically killing tumor cells. The virus is a recombinant oncolytic virus, and the genome thereof has an exogenous promoter inserted which is located upstream of an essential gene of the virus to replace the exogenous promoter of the essential gene, and to drive the expression of the essential gene in tumor cells but not in normal cells. The virus can kill a variety of tumor cells with an efficacy similar to that of the wild-type virus while it is safe to non-tumor cells. In vivo studies indicate that the oncolytic viruses provided in this disclosure can significantly inhibit tumor growth in various tumor animal models.

Claims

1-16. (canceled)

17. A virus for specifically killing tumor cells, wherein the virus is a recombinant oncolytic virus comprising an exogenous promoter inserted into the viral genome, wherein the exogenous promoter is located upstream the regulated essential gene of the virus to drive expression of the regulated essential gene in tumor cells but not in normal cells.

18. The virus for specifically killing tumor cells of claim 17, wherein the exogenous promoter is a tumor cell-specific promoter.

19. The virus for specifically killing tumor cells of claim 17, wherein the virus further comprises an enhancer inserted, and the enhancer is located between the exogenous promoter and the regulated essential gene to enhance the expression of the regulated essential gene in tumor cells.

20. The virus for specifically killing tumor cells of claim 19, wherein an additional copy of the regulated essential gene is further inserted into the genome or more than one, gene is regulated. The exogenous promoter and the enhancer are accordingly inserted into the genome and located upstream each regulated essential gene.

21. The virus for specifically killing tumor cells of claim 19, wherein the genome of the virus further comprises an immunostimulatory factor expression sequence and a viral late gene promoter which drives expression of the immunostimulatory factor from the sequence, wherein the viral late gene promoter is activated by the gene product of the regulated essential gene.

22. The virus for specifically killing tumor cells of claim 21, wherein an immunostimulatory factor expressed from the immunostimulatory factor expression sequence is either interleukin 12 or granulocyte-macrophage colony stimulating factor (GMCSF).

23. The virus for specifically killing tumor cells of claim 21, wherein the virus late gene promoter is glycoprotein D promoter when the oncolytic virus is derived from HSV- or adenovirus late gene promoter is E3 promoter when the oncolytic virus is derived from adenovirus.

24. The virus for specifically killing tumor cells of claim 17, wherein the recombinant oncolytic virus is selected from the group consisting of herpes simplex virus, coxsackie virus, influenza virus, vaccinia virus, measles virus, poliovirus, mumps virus, vesicular stomatitis virus, Newcastle disease virus and adenovirus.

25. The virus for specifically killing tumor cells of claim 22, wherein the recombinant oncolytic virus is derived from herpes simplex virus type 1, the essential gene is ICP27, and the viral late gene promoter is glycoprotein D promoter; or when the recombinant oncolytic virus is derived from adenovirus, the essential gene is E1A, and the viral late gene promoter is adenovirus late gene E3 promoter.

26. A tumor therapeutic drug, comprising the virus for specifically killing tumor cells of claim 17.

27. The virus for specifically killing tumor cells of claim 17, wherein the tumor specific promoter is any one selected from the group consisting of telomerase reverse transcriptase promoter, human epidermal growth factor receptor-2 promoter, E2F1 promoter, osteocalcin promoter, carcinoembryonic antigen promoter, survivin promoter and ceruloplasmin promoter.

28. A nucleic acid fragment for preparing the virus of claim 17, wherein the nucleic acid fragment consists of the 5′ UTR of the regulated essential gene without the endogenous promoter, the tumor specific promoter and enhancer, the open reading frame of the regulated essential gene, a poly(A) sequence, and a second copy of the regulated gene or the immunostimulatory factor expression sequence followed by 3′ UTR of the regulated essential gene. The 5′ and 3′ UTRs serve as the sequence basis for homologous recombination between the fragment-comprising plasmid DNA and parental virus genome for generation of the oncolytic viruses provided in this disclosure.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0051] Exemplary features of the present disclosure, its nature and various advantages will be apparent from the accompanying drawings and the following detailed description of various embodiments. Non-limiting and non-exhaustive embodiments are described with reference to the accompanying drawings. The sizes and relative positions of elements in the drawings are not necessarily drawn to scale. For example, the shapes of various elements are selected, enlarged, and positioned to improve drawing legibility. The particular shapes of the elements as drawn have been selected for ease of recognition in the drawings. One or more embodiments are described hereinafter with reference to the accompanying drawings in which

[0052] FIG. 1 Schematic showing of the genome structure of the recombinant oncolytic virus provided by the examples of the present disclosure. A: Schematic showing of the genome structure of the recombinant oncolytic virus 1 of Example 1, wherein an additional copy of a regulated essential gene is inserted. The regulatory element contains an exogenous tumor cell-specific promoter and an enhancer located upstream of the reading frame of each copy of the regulated essential gene. A1 Schematic showing of the genome structure of the recombinant oncolytic virus 1 (oHSV-BJTT), wherein the virus is HSV-1, the essential gene was ICP27, tumor-specific promoter was hTERT, and the enhancer is the CMV enhancer. B Schematic showing of the genome structure of the recombinant oncolytic virus 2 provided by Example 2, the first expression cassette contains tumor specific promoter, an enhancer, the open reading frame of the regulated essential gene, followed by a ploy (A) sequence. and the second expression cassette contains a viral late gene promoter, and the open reading frame of an immunostimulatory factor followed by a ploy (A) sequence. B1 Schematic showing of the genome structure of the recombinant oncolytic virus 2 (oHSV-BJGMCSF), wherein the virus is HSV-1, the essential gene is HSV-1 ICP27, the tumor cell-specific promoter is hTERT, the enhancer is the CMV enhancer, the viral late gene promoter is HSV-1 gD promoter, and the immunostimulatory factor was GM-CSF.

[0053] FIG. 2 Schematic showing of the parental plasmid pcDNA3.1-EGFP unitized for constructing a plasmid expressing HSV-1 ICP27. In the plasmid, EGFP is constitutively expressed under the control of the CMV promoter and the plasmid contains neomycin-resistant gene expression sequence.

[0054] FIG. 3 The expression of ICP27 from the recombinant virus oHSV-BJTT or oHSV-BJGMCSF in African green monkey kidney cells (Vero) (normal cells) is lower than the detection limit. Vero cells were infected with 3 multiplicities of infection (MOIs, virus/cell) oHSV-BJTT, oHSV-BJGMCSF or HSV-1 wild-type virus KOS. One day later, the cells were harvested, RNA was isolated, and protein extracted, and ICP27 mRNA (A) was detected by reverse transcription combined with semi-quantitative PCR, and HSV-1 ICP27 protein (B) was detected by Western blotting assays. (i): oHSV-BJTT; and (ii): oHSV-BJGMCSF.

[0055] FIG. 4 No significant difference was observed in ICP27 protein expression of in tumor cells from the recombinant virus oHSV-BJTT, oHSV-BJGMCSF or wild-type virus KOS. The tumor cells Hela, siHA, SK-BR3 and ME-180 were respectively infected with 3 MOI oHSV-BJTT, oHSV-BJGMCSF or wild-type virus KOS. One day later, the cells were collected, the protein was extracted, and the ICP27 protein was detected by Western blotting assays. A: oHSV-BJTT; B: oHSV-BJGMCSF.

[0056] FIG. 5 Basically identical replication kinetics was observed among oncolytic viruses oHSV-BJTT, oHSV-BJGMCSF and wild-type virus KOS. Various tumor cells were infected with 0.1 MOI oHSV-BJTT, oHSV-BJGMCSF or KOS. At different days after infection, the cells and culture medium were collected, and the virus remaining in the cells was released into the culture medium through three freeze-thawing cycles. Complementing cells were infected with the virus, and virus titer (plaque forming unit/ml, PFU/ml) was determined by the plaque method. (i): oHSV-BJTT; and (ii): oHSV-BJGMCSF. In (i) and (ii): A: cervical tumor Hela cell; B: cervical squamous cancer siHa cell; C: breast cancer SK-BR3 cell; D: breast cancer ME-180 cell.

[0057] FIG. 6 Recombinant oncolytic viruses oHSV-BJTT and oHSV-BJGMCSF significantly inhibit the proliferation of lung cancer, gastric cancer, liver cancer and rectal cancer in animal tumor models by recombinant oncolytic viruses oHSV-BJTT and oHSV-BJGMCSF. A tumor animal model was established. After the tumor grew to 50-80 mm.sup.3, the oncolytic virus was injected into the tumor every 3 days for a total of 3 times. PBS (without oncolytic virus) was injected as a negative control. After the oncolytic virus was injected, the tumor volume was measured twice a week. When the negative control animals needed to be euthanized, the experiment ended. A tumor growth curve was plotted based on the tumor volume (in the FIG., A: lung cancer; B: gastric cancer; C: liver cancer; D: rectal cancer), and the relative inhibition rate (E) was calculated by comparing the tumor volume in the test group at the end of the test with that observed in the negative control.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0058] In order to demonstrate the features of the present disclosure, its nature and various advantages, exemplary embodiments were executed and are described in details below. All experiments were conducted using standard methods as described in literature. Reagents were purchased from commercial providers and used according to the instructions of the manufacturers.

[0059] The features and performance of the oncolytic virus provided in the present disclosure will be further described in detail below in conjunction with examples.

Example 1

[0060] The recombinant oncolytic virus 1 provided in this example was constructed with wild-type HSV type 1 KOS as the starting material. The genome of the recombinant oncolytic virus 1 has the following structural features.

[0061] Referring to A in FIG. 1, in the genome of the recombinant oncolytic virus 1 of this example, an additional copy of an essential gene is inserted so that the number of copies of the essential gene was two, and the regulatory element is composed of a tumor cell-specific promoter and an enhancer is inserted into upstream of the reading frame of each copy of the essential gene to replace the endogenous promoter of the essential gene, thereby driving the expression of the essential gene in tumor cells. There is an exogenous terminator 1 downstream of the reading frame of the first copy of the essential gene, and an exogenous terminator 2 downstream of the reading frame of the second copy of the essential gene, and the essential gene is driven by the above-mentioned regulatory element to be specifically expressed in tumor cells.

[0062] Referring to A1 in FIG. 1, specifically, in this example, the regulated essential gene is ICP27, and an additional copy of ICP27 is inserted. The tumor cell-specific promoter is hTERT, the enhancer is the CMV enhancer, the exogenous terminator 1 is SV40 Poly (A), and the exogenous terminator 2 is BGH Poly (A).

[0063] In the following text, the virus is named as oHSV-BJTT.

Example 2

[0064] The recombinant oncolytic virus 2 provided in this example contains a regulated essential gene and a late viral promoter-driven immunostimulatory factor expression cassette. The genome of the recombinant oncolytic virus 2 possesses the following structural features.

[0065] Referring to B in FIG. 1, a first regulatory element is composed of a tumor-specific promoter and an enhancer is located upstream of the reading frame of the regulatory essential gene in the genome of the recombinant oncolytic virus 2 of this example; a second regulatory element set composed of a viral late gene promoter and an immunostimulatory factor reading frame is located downstream of the reading frame of the essential gene. Herein, the viral late gene promoter is regulated by essential gene expression product; there are an exogenous terminator 1 located downstream of essential gene reading frame and an exogenous terminator 2 located downstream of the reading frame of the immunostimulatory factor.

[0066] Referring to B1 in FIG. 1, specifically, in this example, the essential gene is ICP27, the tumor cell-specific promoter is hTERT, the enhancer is the CMV enhancer, and the exogenous terminator 1 is SV40 Poly(A), the viral late gene promoter is the gD promoter, the immunostimulatory factor reading frame is the GMCSF reading frame, and the exogenous terminator 2 is BGH Ploy(A).

[0067] In the following text, the recombinant oncolytic virus is named as oHSV-BJGMCSF.

Example 3

[0068] This example provides the methods for preparing the recombinant oncolytic virus provided in the foregoing Example 1 or 2, and the specific manipulations were as follows.

(1) Preparation of Complementing Cells Expressing HSV-1 ICP27

[0069] (a) plasmid construction: Using the DNA of wild-type HSV-1 KOS as a template, the encoding region of ICP27 was amplified by PCR, and inserted into HindIII and XbaI sites of plasmid pcDNA3.1-EGFP expressing the neomycin-resistant gene (see FIG. 2 for the structure) to replace EGFP. The recombinant plasmid was named as ICP27 expression plasmid, and the ICP27 gene was expressed under the control of the CMV promoter.

[0070] (b) G418 dose determination for selection: Vero cells were treated with G418 of different concentrations, the culture medium containing G418 was refreshed every three days with media containing G418 of different concentrations, and cell death was monitored every day. The minimal concentration of G418 required for all cells to die after 6 days of G418 treatment was determined. Such a concentration of G418 (500 μg/ml) was utilized for complementing cell establishment.

[0071] (c) Cell line establishment: Totally 3.5×10.sup.5 Vero cells were seeded into each well of a 6-well cell culture plate and cultured overnight in an antibiotic-free culture medium, and 4 μg of the ICP27 expression plasmid obtained in step (a) were transfected into cells in each well with Lipofectamine 2000. After 24 hours of culture, cells in each well were harvested, and diluted by 1:20, 1:40, and 1:60, respectively. Cells were cultured in the culture medium containing G418, and the medium was refreshed with medium containing G418 every 3 days. After 6-7 changes of the culture medium, the clones were collected and propagated step by step from the 24-well plate to T150 tissue culture flasks. Subsequently, the protein was separated and the expression of ICP27 detected by Western blotting assays. The cells with the highest level of ICP27 expression were selected as the complementing cells to support the growth and replication of replication-defective viruses in which ICP27 was not expressed; the cells were named as C.sub.ICP27

[0072] The complementing cell has been preserved at China Center for Type Culture Collection (CCTCC), Wuhan University, Luojiashan, Wuchang, Wuhan City on Apr. 24, 2019 with a preservation number of CCTCC NO. C201974.

(2) Preparation of the Parental Virus rHSV-EGFP

[0073] The wild-type HSV type 1 was used as the starting material, the recombinant parental virus HSV-EGFP was obtained by homologous recombination between plasmid and KOS genome. In HSV-EGFP, HSV-ICP27 was replaced by EGFP. HSV-EGFP will serve as the parental virus for generating the oncolytic viruses provided in this disclosure. The manipulations are detailed as follows:

[0074] (a) the first nucleic acid fragment was synthesized and its nucleotide sequence is shown in SEQ ID NO. 1:

[0075] From 5′ to 3′ end, the fragment includes the following elements: ICP27 5′ sequence, CMV promoter, Kozak sequence, EGFP encoding frame, BGH Poly(A) and ICP27 3′ sequence.

[0076] Sequence seen in SEQ ID NO. 1:

[0077] site 1-6: irrelevant sequence, increasing the end length to facilitate enzyme digestion;

[0078] site 7-12: Xho1 site, C/TCGAG;

[0079] site 13-575: ICP27 5′ end sequence;

[0080] site 576-1163: CMV promoter;

[0081] site 1164-1174: interval sequence;

[0082] site 1175-1180: Kozak sequence, increasing protein expression;

[0083] site 1181-1900: EGFP encoding frame;

[0084] site 1901-2145: BGH Poly(A);

[0085] site 2146-2667: ICP27 3′ end sequence;

[0086] site 2668-2673: HindIII site, A/AGCTT; and

[0087] site 2674-2679: irrelevant sequence, increasing the end length to facilitate enzyme digestion.

[0088] (b) the first fragment was cleaved and ligated to the HindIII and Xho1 sites of the pcDNA3.1-EGFP plasmid, and the resulting recombinant plasmid was named as EGFP expression plasmid.

[0089] (c) 3.5×10.sup.5 above-mentioned complementing C.sub.ICP27 cells were seeded into each well in 6-well cell culture plate and cultured overnight in an antibiotic-free culture medium.

[0090] (d) the cells were infected with wild-type virus KOS of 0.1, 0.5, 1, 3 MOI (virus/cell), respectively. After 1 hour, the above-mentioned EGFP expression plasmid (4 μg DNA/well) was transfected into the cells using Lipofectamine 2000. Four hours later, the transfection mixture was replaced with complete medium. After all the cells became spherical, the cells and culture medium were collected, and after three cycles of freeze-thawing, the mixture was centrifuged and the supernatant collected by centrifugation, diluted, and infected by the above-mentioned complementing cell C.sub.ICP27. The viruses were isolated by using plaque separation method. Four to 5 days later, the green virus plaque was selected under a fluorescence microscope, and then the obtained virus plaques were subjected to 2 or 3 rounds of screening to obtain pure virus plaques, and the virus was propagated. The recombinant virus with ICP27 replaced by EGFP was named as HSV-EGFP. HSV-EGFP served as the parental virus for generation of the oncolytic viruses provided in this disclosure

(3) Construction of Recombinant Plasmid

[0091] (a) the TA cloning plasmid was modified such that the multiple cloning site in the plasmid only contains a XhoI site. The resulting plasmid was named as TA-XhoI plasmid, for subsequent use.

[0092] (b) the second nucleic acid fragment was synthesized, and its nucleotide sequence was shown in SEQ ID NO. 2.

[0093] The nucleic acid fragment from 5′ to 3′ end contains the following elements: ICP27 5′ end sequence (excluding the natural promoter), hTERT promoter, CMV enhancer sequence, ICP27 open reading frame, SV40 Poly(A) sequence and ICP27 3′ end sequence. In addition, the 5′ and 3′ ends of the second nucleic acid fragment each contain one XhoI site, and there is one HindIII site between the SV40 Poly(A) and ICP27 3′ end sequence.

[0094] In SEQ ID NO. 2:

[0095] site 1-6: Xho1 site;

[0096] site 7-517: ICP27 5′ end non-coding region;

[0097] site 518-973: hTERT promoter;

[0098] site 974-1039: CMV enhancer;

[0099] site 1040-2578: ICP27 open reading frame;

[0100] site 2579-3050: SV40 Poly(A);

[0101] site 3051-3056: HindIII site;

[0102] site 3057-3576: ICP27 3′ end non-coding region; and

[0103] site 3577-3582: Xho1 site.

[0104] (c) the second nucleic acid fragment was cleaved and inserted into the XhoI site of the plasmid TA-XhoI, and the resulting recombinant plasmid was named as: TA-XhoI-hTERT-CMVminimal-ICP27 plasmid, for subsequent use.

[0105] (d) the third nucleic acid fragment was artificially synthesized, and its nucleotide sequence is shown in SEQ ID NO. 3:

[0106] The nucleic acid fragment from 5′ to 3′ end contains the following elements: hTERT promoter plus CMV enhancer sequence, ICP27 open reading frame and BGH Poly(A) sequence; The 5′ and 3′ ends each contain one HindIII site.

[0107] In SEQ ID NO. 3:

[0108] site 1-6: HindIII site;

[0109] site 7-462: hTERT promoter;

[0110] site 463-528: CMV enhancer;

[0111] site 529-2067: ICP27 open reading frame;

[0112] site 2068-2304: BGH Poly(A); and

[0113] site 2305-2310: HindIII site.

[0114] (e) the fourth nucleic acid fragment was artificially synthesized, and its nucleotide sequence is shown in SEQ ID NO. 4:

[0115] The nucleic acid fragment from 5′ to 3′ end contains the following elements: gD promoter, Kozak sequence, GMCSF open reading frame and BGH Poly(A) sequence; each of the 5′ and 3′ ends contains one HindIII site.

[0116] In SEQ ID NO. 4:

[0117] site 1-6: HindIII site;

[0118] site 7-439: gD promoter;

[0119] site 440-448: Kozak sequence;

[0120] site 449-883: GMCSF open reading frame;

[0121] site 884-1100: BGH Poly(A); and

[0122] site 1100-1115: HindIII site.

[0123] Herein, the amino acid sequence (SEQ ID NO. 5) of GMCSF is

TABLE-US-00001 MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTA AEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASH YKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE.

[0124] (f) the third nucleic acid fragment was digested with HindIII and inserted into the HindIII site of the TA-XhoI-hTERT-CMVminimal-ICP27 plasmid. The resulting recombinant plasmid was named as ICP27-TT.

[0125] The fourth nucleic acid fragment was digested with HindIII and inserted into the HindIII site of the TA-XhoI-hTERT-CMVminimal-ICP27 plasmid. The resulting recombinant plasmid was named as ICP27-GMCSF.

(4) Construction of Recombinant Oncolytic Viruses oHSV-BJTT and oHSV-BJGMCSF

[0126] (a) the above-mentioned complementing C.sub.ICP27 cells were seeded into a 6-well cell culture plate with 3.5×10.sup.5 cells per well, and cultured overnight in an antibiotic-free culture medium.

[0127] (b) the complementing C.sub.ICP27 cells were respectively infected at an MOI of 0.1, 0.5, 1, and 3 of the parental virus HSV-EGFP; 1 hour later, plasmid ICP27-TT DNA (4 μg DNA/well) was transfected to cells by using Lipofectamine 2000. After 4 hours, the transduction solution was replaced with complete medium. After all the cells were infected, the cells and culture solution were collected, through three times of freeze-thawing, the supernatant was collected by centrifugation, diluted, and infected by the above-mentioned complementing C.sub.ICP27 cells.

[0128] (c) the viruses were separated using plaque separation method. Four to 5 days later, virus plaques without green fluorescence were selected under a fluorescence microscope, and then the obtained virus plaques were subjected to 2 or 3 rounds of screening to obtain pure viruses, which were then propagated. DNA was isolated from the infected cells, and was amplified by PCR using specific primers and confirmed by sequencing. The recombinant oncolytic virus obtained was named as oHSV-BJTT.

[0129] (d) Repeated steps from a to d to obtain oncolytic oHSV-BJGMCSF.

[0130] These two viruses have been both preserved in the Chinese Center of Type Culture Collection (CCTCC), Wuhan University, Luojiashan, Wuchang, Wuhan on Apr. 24, 2019. The preservation number of the oHSV-BJTT virus is CCTCC NO: V201922, and the preservation number of the oHSV-BJGMCSF virus is CCTCC NO: V201921.

Experimental Example 1

Detection of the Expression of ICP27 in Normal Cells Infected with Recombinant Oncolytic Virus

[0131] Detection method: Vero cells were infected with HSV-1 wild-type virus KOS, oHSV-BJTT or oHSV-BJGMCSF at 3 MOI (virus/cell). One day later, the cells were collected, and RNA and protein were isolated. The expression level of ICP27 mRNA was detected using semi-quantitative PCR, and the expression level of HSV-1 ICP27 protein detected by Western blotting assays. Both mRNA and protein analysis used β-actin as loading control.

[0132] In the cells infected with KOS, ICP27 mRNA and protein were expressed to an easily detectable level. In the cells infected with oHSV-BJTT or oHSV-BJGMCSF, ICP27 mRNA and protein were below detectable level (FIG. 3: A: mRNA; B: protein). It indicates that in normal cells, the expression of ICP27 mRNA and protein were expressed at a very low level or not expressed from oHSV-BJTT or oHSV-BJGMCSF.

Experimental Example 2

Detection of the Expression of GMCSF and ICP27 in Tumor Cells Infected with Recombinant Oncolytic Virus

[0133] Detection method: 3 MOI oHSV-BJTT, oHSV-BJGMCSF or wild-type virus KOS was used to infect tumor cells, respectively: cervical tumor cells Hela, cervical squamous tumor cells siHA, breast tumor cells SK-BR3 and breast tumor cells ME-180. After 6 hours, a small portion of infected cell culture medium was collected. The content of GMCSF in the cell culture medium was analyzed by ELISA. One day later, all infected cells were collected, proteins isolated, and the ICP27 protein was detected by Western blotting assays. For protein analysis, β-actin was used as the loading control.

[0134] The GMCSF content in oHSV-BJGMCSF-infected cell culture medium reached a detectable level, but in oHSV-BJTT- or KOS-infected cell culture medium, the GMCSF was not detectable (Table 1). In different cells infected with oHSV-BJTT and oHSV-BJGMCSF, ICP27 protein level in one cell type is different from that observed in another cell type, but for a given cell type, there was not much difference in the levels of ICP27 protein in the cells infected by oHSV-BJTT, oHSV-BJGMCSF or by KOS (FIG. 4). GMCSF can be expressed from oHSV-BJGMCSF in tumor cells; meanwhile, ICP27 can be expressed specifically from oHSV-BJTT and oHSV-BJGMCSF, and the expression level was not much different from that of wild-type virus.

TABLE-US-00002 TABLE 1 GMCSF content in different tumor cell culture media with cells infected by oHSV-BJGMCSF (ng/ml) Tumor cell type Hela siHA BR-SK3 ME-180 oHSV-BJGMCSF 4.5 5.6 9.5 7.2 oHSV-BJTT 0 0 0 0 KOS 0 0 0 0

Experimental Example 3

Detection of the Replication Kinetics of Recombinant Oncolytic Viruses oHSV-BJTT and oHSV-BJGMCSF in Tumor Cells

[0135] Detection method: Tumor cells were infected with 0.1 MOI of oHSV-BJTT, oHSV-BJGMCSF or KOS. After different days, the cells and culture medium were collected, separately, and the viruses remaining in the cells were released into the culture medium through three −80/37° C. freeze-thawing cycles. The complementing C.sub.ICP27 cells were infected with the virus, and the virus titer (plaque forming unit/ml, PFU/ml) was determined by the plaque assay.

[0136] oHSV-BJTT (FIG. 5i) and oHSV-BJGMCSF (FIG. 5.ii) like KOS can replicate in all the four tumor cell types tested. For a given day after infection, the replication ability of oHSV-BJTT or oHSV-BJGMCSF was slightly lower than that of KOS (A: Hela cells; B: siHA cells; C: SK-BR3 cells and D: ME-180 cells), but there was no significant difference between oHSV-BJTT and KOS or between oHSV-BJGMCSF and KOS. The results demonstrates that genetic engineering of the virus whereby the oncolytic viruses are generated basically maintains the replicative ability of the virus.

Experimental Example 4

Detection of the Ability of Recombinant Oncolytic Viruses oHSV-BJTT and oHSV-BJGMCSF to Kill Tumor Cells

[0137] Detection method: Tumor cells including Hela cells, siHA cells, SK-BR3 cells and ME-180 cells, were respectively infected with 0.25 or 0.5 MOI oHSV-BJTT, oHSV-BJGMCSF or wild-type KOS. Cell survival rate was analyzed at different days after infection. The results are shown in Table 2-5 (for the recombinant oncolytic virus oHSV-BJTT) and Table 6-9 (for the recombinant oncolytic virus oHSV-BJGMCSF).

TABLE-US-00003 TABLE 2 Survival rate (%) of Hela cells infected with oHSV-BJTT at different days after infection oHSV-BJTT KOS Virus (MOI 0.25) the first day 56 ± 3.0 47 ± 2.8 the second day 37 ± 1.5 25 ± 1.3 the third day 15 ± 0.8  8 ± 0.5 the fourth day  5 ± 0.2 0 Virus (MOI 0.5) the first day 49 ± 3.0 36 ± 1.9 the second day 32 ± 1.8 19 ± 1.4 the third day  8 ± 0.5  4 ± 0.2 the fourth day 0 0

TABLE-US-00004 TABLE 3 Survival rate (%) of siHA cells infected with oHSV-BJTT at different days after infection oHSV-BJTT KOS Virus (MOI 0.25) the first day 25 ± 2   15 ± 0.9 the second day 5 ± 0.5 0 the third day 0 0 Virus (MOI 0.5) the first day 11 ± 0.4   5 ± 0.4 the second day 1 ± 0.6 0 the third day 0 0

TABLE-US-00005 TABLE 4 Survival rate (%) of SK-BR3 cells infected with oHSV-BJTT at different days after infection oHSV-BJTT KOS Virus (MOI 0.25) the first day 25 ± 3   10 ± 0.8 the second day 0 0 the third day 0 0 Virus (MOI 0.5) the first day 6 ± 0.5  4 ± 0.3 the second day 1 ± 0.6 0 the third day 0 0

TABLE-US-00006 TABLE 5 Survival rate (%) of ME-180 cells infected with oHSV-BJTT at different days after infection oHSV-BJTT KOS Virus (MOI 0.25) the first day 18 ± 1   9 ± 0.5 the second day 7.4 ± 0.4.sup.  0 the third day 0 0 Virus (MOI 0.5) the first day 9 ± 0.6 7 ± 0.4 the second day 1 ± 0.6 0 the third day 0 0

[0138] From Table 2-5, it can be seen that oHSV-BJTT can kill different cell types, and the ability is similar to that observed for KOS.

TABLE-US-00007 TABLE 6 Survival rate (%) of Hela cell infected with oHSV- BJGMCSF at different days after infection oHSV-BJGMCSF KOS Virus (MOI 0.25) the first day 62 ± 3.5 42 ± 2.6 the second day 41 ± 2.4 22 ± 1.3 the third day 18 ± 1.0  7 ± 0.4 the fourth day  5 ± 0.3 0 virus (MOI 0.5) the first day 52 ± 3.5 33 ± 2.0 the second day 29 ± 1.6 19 ± 1.1 the third day 11 ± 0.7  3 ± 0.2 the fourth day 0 0

TABLE-US-00008 TABLE 7 Survival rate (%) of siHA cells infected with oHSV-BJGMCSF at different days after infection oHSV-BJGMCSF KOS Virus (MOI 0.25) the first day 27 ± 1.5 18 ± 1.2 the second day  8 ± 0.5 0 the third day 0 0 Virus (MOI 0.5) the first day 14 ± 0.8  6 ± 0.5 the second day  1 ± 0.08 0 the third day 0 0

TABLE-US-00009 TABLE 8 Survival rate (%) of SK-BR3 cells infected with oHSV-BJGMCSF at different MOIs. oHSV-BJGMCSF KOS Virus (MOI 0.25) the first day 24 ± 1.2  11 ± 0.8 the second day 0 0 the third day 0 0 Virus (MOI 0.5) the first day 8 ± 0.5  5 ± 0.3 the second day  1 ± 0.06 0 the third day 0 0

TABLE-US-00010 TABLE 9 Survival rate (%) of ME-180 cells infected with oHSV-BJGMCSF at different days after infection. oHSV-BJGMCSF KOS Virus (MOI 0.25) the first day 15 ± 0.9  9 ± 0.6 the second day 3 ± 0.2 0 the third day 0 0 Virus (MOI 0.5) the first day 4 ± 0.3 3 ± 0.2 the second day 1 ± 0.1 0 the third day 0 0

[0139] Tables 6-9 indicates that oHSV-BJGMCSF kills four tumor cells with a similar ability to that seen with KOS. In summary, the results demonstrate that the genetic engineering used to generate the oncolytic viruses mentioned in Example 3 does not significantly affect the ability of the virus and kill tumor cells, and oncolytic viruses oHSV-BJTT and oHSV-BJGMCSF oncolytic viruses provided in the examples of the present disclosure possesses the capacity to kill a variety of tumor cells.

Experimental Example 5

Effect of Recombinant Oncolytic Herpesvirus oHSV-BJTT and oHSV-BJGMCSF on the Viability of Normal Cells

[0140] Detection method: Vero cells or primary human corneal epidermal cells were infected with oncolytic viruses oHSV-BJTT, oHSV-BJGMCSF (2 MOI) and wild virus KOS (0.5 MOI). Untreated cells were used as negative control. The viability of the cells infected with oHSV-BJTT and oHSV-BJGMCSF and untreated cells were assayed 3 days after infection, and the viability of the cells infected with wild virus KOS were analyzed 2 days after infection.

[0141] Two days after KOS infection, all Vero cells or primary human corneal epithelial cells died, but the viability of the cells infected with oncolytic viruses oHSV-BJTT and oHSV-BJGMCSF was basically the same as that observed for untreated cells (Table 10). This result indicates that the oncolytic viruses oHSV-BJTT and oHSV-BJGMCSF do not affect the viability of normal cells, i.e. the oncolytic viruses are safe to normal cells.

TABLE-US-00011 TABLE 10 Survival rate (%) of normal cells 3 days after oHSV- BJS or oHSV-BJGMCSF, or 2 days after KOS infection. oHSV- virus oHSV-BJTT BJGMCSF KOS untreated Vero cells 96 ± 2  95 ± 2  0 98 ± 2 corneal 97 ± 12 96 ± 12 0 97 ± 1 epithelial cells

Experimental Example 6

[0142] In order to evaluate the effectiveness and the broad spectrum of oncolytic viruses oHSV-BJTT and oHSV-BJGMCSF in tumor treatment, mouse tumor models for human lung, gastric, liver and colon tumors were established. In vitro cultured human non-small cell tumor A549, gastric tumor NCI-N87, and liver cancer SK-HEP-1 cells were subcutaneously injected into BALB/c (lung and gastric tumors) or NPG (liver tumor) mice. When the tumors grew to 800-1000 mm.sup.3, they were dissected, cut into 30 mm.sup.3 pieces, and then implanted into mice. When the tumors grew to 40-120 mm.sup.3, intratumoral injection of oncolytic virus oHSV-BJR was initiated.

[0143] The rectal tumor model was established by direct subcutaneous injection of cultured rectal adenocarcinoma HCT-8 cells into a BALB/c mouse. When the tumor grew to 40-120 mm.sup.3, intratumoral injection of oncolytic virus oHSV-BJR started.

[0144] Intratumoral injection of oncolytic virus oHSV-BJTT or oHSV-BJGMCSF was performed by multiple-point injection once every three days for a total of three times with 2×10.sup.7 infectious units suspended in 40 μl PBS injected each time. Each group in each model included 8 animals and injection of 40 μl PBS served as a negative control. Tumor volume was measured twice a week after the first virus injection, and the study lasted for 25 to 32 days after the first virus injection depending on when animals in the control group needed to be euthanized. A tumor growth curve of tumor volume over days after the first virus injection was made and the relative inhibition rate calculated by comparing the tumor volume in the test group to the tumor volume in the control group.

[0145] The tumor volume of animals in the test group was smaller than that observed in the control group starting from the 8.sup.th day or so after the first virus injection (FIG. 6. A: lung tumor; B: gastric tumor C: liver tumor; and D: rectal tumor). The difference in tumor volumes between the virus-injected and control groups increased with the days after the first virus injection. Increased with the days after the first virus injection increasing, the inhibition rates of oncolytic virus oHSV-BJTT on the growth of lung, liver, gastric and rectal tumors at the end of study were 44.5, 44.6, 29.7, 57.5%, respectively. And the inhibition rates of oncolytic virus oHSV-BJGMCSF on the growth of lung, liver, gastric and rectal tumors at the end of study were 36.3, 42.2, 24.5, 43.1%, respectively.

[0146] The results demonstrate that in various tumor models, the tumor volume in animals injected with oHSV-BJTT or oHSV-BJGMCSF is smaller than that observed for negative control group, indicating that oHSV-BJTT and oHSV-BJGMCSF significantly inhibit the proliferation of various tumors (FIG. 6E). The foregoing descriptions are only preferred examples of the present disclosure, and are not intended to limit the present disclosure. Any modification, equivalent replacement, and improvement, etc. made within the concept and principle of the present disclosure shall be included in the protection scope of the present disclosure.

INDUSTRIAL APPLICABILITY

[0147] The present disclosure provides a virus and a tumor therapeutic drug for specifically killing tumor cells which can kill a variety of tumor cells effectively while they are safe to non-tumor cells, and have been demonstrated to inhibit tumor growth in animals. Most importantly, the oncolytic viruses retain an intact genome, a basic feature that is strikingly different for currently available oncolytic viruses.