Echovirus for treatment of tumors

Abstract

Provided are use of an Echovirus 25 (ECHO25) or a modified form thereof, or a nucleic acid molecule comprising a genomic sequence or cDNA sequence of the ECHO25 or a modified form thereof, or a complementary sequence of the genomic sequence or cDNA sequence, in treatment of a tumor in a subject, and in the manufacture of a medicament for treatment a tumor in a subject.

Claims

1. A method of treating a tumor, the method comprising administering, to a subject in need thereof, an effective amount of a modified Echovirus 25 (ECHO25) or a medicament comprising the modified ECHO25, wherein as compared to a genome of a wild-type ECHO25, a genome of the modified ECHO25 has an insertion of one or more exogenous nucleic acids, and wherein the one or more exogenous nucleic acids are selected from the group consisting of a nucleic acid sequence encoding a cytokine, a nucleic acid sequence encoding an antitumor protein or polypeptide, and a target sequence of microRNA.

2. The method of claim 1, wherein as compared to the genome of the wild-type ECHO25, the genome of the modified ECHO25 has a substitution of an internal ribosome entry site (IRES) sequence in a 5′ untranslated region (5′UTR) with an exogenous IRES sequence.

3. The method of claim 2, wherein the exogenous IRES sequence is an internal ribosome entry site sequence of human rhinovirus 2 (HRV2).

4. The method of claim 1, wherein the modified ECHO25 has one of the following characteristics: (1) a genomic sequence of the modified ECHO25 has a sequence identity of at least 80% to a nucleotide sequence as shown in SEQ ID NOs: 13-16; and (2) a cDNA sequence of the modified ECHO25 has a sequence identity of at least 80% to a nucleotide sequence as shown in SEQ ID NOs: 8-11.

5. The method of claim 1, wherein the modified ECHO25is administered in combination with an additional pharmaceutically active agent having antitumor activity.

6. A modified ECHO25, comprising: a substitution of an internal ribosome entry site (IRES) sequence in a 5′ untranslated region (5′UTR) with an internal ribosome entry site sequence of human rhinovirus 2 (HRV2) as compared to a wild-type ECHO25, and an exogenous nucleic acid which is selected from the group consisting of a nucleic acid sequence encoding a cytokine, a nucleic acid sequence encoding an antitumor protein or polypeptide, and a target sequence of microRNA.

7. The modified ECHO25 of claim 6, wherein the modified ECHO25 has one of the following characteristics: (1) a genomic sequence of the modified ECHO25 has a sequence identity of at least 80% to a nucleotide sequence as shown in SEQ ID NO: 13; (2) a cDNA sequence of the modified ECHO25 has a sequence identity of at least 80% to a nucleotide sequence as shown in SEQ ID NO: 8.

8. An isolated nucleic acid molecule, comprising a genomic sequence or a cDNA sequence of the modified ECHO25 of claim 6.

9. The isolated nucleic acid molecule of claim 8, which consists of the genomic sequence of the modified ECHO25.

10. The isolated nucleic acid molecule of claim 8, which is a vector comprising the cDNA sequence of the modified ECHO25.

11. A method of treating a tumor, the method comprising administering, to a subject in need thereof, an effective amount of the modified ECHO25 of claim 6, or a medicament comprising the modified ECHO25.

12. The method of claim 1, wherein at least one of the following conditions is satisfied: (i) the cytokine is GM-CSF; (ii) the antitumor protein or polypeptide is a scFv against PD-1 or PD-L1; (iii) the microRNA is miR-133 and/or miR-206.

13. The method of claim 1, wherein at least one of the following conditions is satisfied: (i) the tumor is selected from the group consisting of gastric cancer, liver cancer, ovarian cancer, endometrial cancer, melanoma, prostate cancer, glioma, esophageal cancer, bladder cancer, lymphoma, leukemia, rhabdomyosarcoma, colorectal cancer, non-small cell lung cancer, cervical cancer, breast cancer, kidney cancer, and pancreatic cancer; (ii) the subject is a human.

14. The modified ECHO25 of claim 6, wherein at least one of the following conditions is satisfied: (i) the cytokine is GM-CSF; (ii) the antitumor protein or polypeptide is a scFv against PD-1 or PD-L1; (iii) the microRNA is miR-133 and/or miR-206.

15. The method of claim 11, wherein at least one of the following conditions is satisfied: (i) the tumor is selected from the group consisting of gastric cancer, liver cancer, ovarian cancer, endometrial cancer, melanoma, prostate cancer, glioma, esophageal cancer, bladder cancer, lymphoma, leukemia, pharyngeal squamous cell carcinoma, thyroid cancer, rhabdomyosarcoma, colorectal cancer, non-small cell lung cancer, cervical cancer, breast cancer, kidney cancer, and pancreatic cancer; (ii) the subject is a human.

16. A method of treating a tumor, the method comprising administering, to a subject in need thereof, an effective amount of an isolated nucleic acid molecule comprising a genomic sequence or a cDNA sequence of a modified ECHO25, or a medicament comprising the isolated nucleic acid molecule, wherein as compared to a genome of a wild-type ECHO25, a genome of the modified ECHO25 has an insertion of one or more exogenous nucleic acids, and wherein the one or more exogenous nucleic acids are selected from the group consisting of a nucleic acid sequence encoding a cytokine, a nucleic acid sequence encoding an antitumor protein or polypeptide, and a target sequence of microRNA.

17. The method of claim 16, wherein the isolated nucleic acid molecule consists of the genomic sequence of the modified ECHO25, or is a vector comprising the cDNA sequence of the modified ECHO25.

18. A method of treating a tumor, the method comprising administering, to a subject in need thereof, an effective amount of an isolated nucleic acid molecule comprising a genomic sequence or a cDNA sequence of the modified ECHO25 of claim 6, or a medicament comprising the isolated nucleic acid molecule.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows photomicrographs of the in vitro killing tests of the wild-type ECHO25 on human umbilical vein endothelial cell line HUVEC, human esophageal cancer cell line TE-1, human endometrial cancer cell lines HEC-1-A and HEC-1-B in Example 2, in which MOCK represents cells that are not infected with the virus. The results showed that the ECHO25 had significant oncolytic effects on human tumor cell lines TE-1, HEC-1-A, and HEC-1-B after 72 hours of infection at a multiplicity of infection (MOI) of 1, but had no effect on HUVEC as human non-tumor cells.

(2) FIG. 2 shows the photos of crystal violet staining of the in vitro killing tests of the wild-type ECHO25 on human non-small cell lung cancer cell lines A549 and NCI-H661, human ovarian cancer cell line Caov3, human pancreatic cancer cell line HPAF-2, human gastric cancer cell lines AGS, SGC7901 and BGC823, human foreskin fibroblast cell line HFF-1 and human skin keratinocyte cell line HaCat in Example 2, wherein MOCK represents cells that are not infected with the virus. The results showed that the ECHO25 had significant oncolytic effects on A549, NCI-H661, Caov3, HPAF-2, AGS, SGC7901 and BGC823, after 72 hours of infection at MOIs of 10, 1, and 0.1, but had no effect on HFF-1 and HaCat of human non-tumor cells.

(3) FIG. 3 shows an electrophoresis image of two samples of wild-type ECHO25 virus genomic RNA of the same batch obtained by the in vitro transcription method in Example 2.

(4) FIG. 4 shows the killing effect of the wild-type ECHO25 virus genomic RNA on human colorectal cancer cell line SW480 in Example 2. The results showed that SW480 cells showed obvious CPE after 24 hours of transfection with ECHO25 genomic RNA, and were almost all lysed to death by 48 hours.

(5) FIGS. 5A to 5D show the results of in vivo antitumor experiment of the wild-type ECHO25 on human glioma cell line GBM (A), human endometrial cancer cell line Ishikawa (B), human prostate cancer cell line PC-3 (C) and human breast cancer cell line BcaP37 (D) in Example 3. The results showed that, in the challenge experimental groups, 10.sup.6 TCID50 per tumor mass of ECHO25 were injected intratumorally every third day. After 5 treatments in total, the growth of the tumors formed by subcutaneous inoculation of GBM, Ishikawa, PC-3 or BcaP37 cells in SCID mice significantly slowed down and arrested, and the tumors were even lysed and disappeared. In contrast, the tumors of the negative group (CTRL) without treatment of oncolytic virus maintained the normal growth, and their tumor volumes were significantly larger than those in the challenge groups.

(6) FIG. 6 shows the results of in vivo antitumor experiment of ECHO25-WT, ECHO25-HRV2, ECHO25-miR133&206T, ECHO25-GM-CSF and ECHO25-Anti-PD-1 on human glioma cell line GBM in Example 3. The results showed that, in the challenge experimental groups, 10.sup.6 TCID50 per tumor mass of ECHO25 were injected intratumorally every third day. After 5 treatments in total for 10 days, the growth of the tumors formed by subcutaneous inoculation of GBM cells in SCID mice arrested, and the tumors were even lysed and disappeared. In contrast, the tumors of the negative group (CTRL) without treatment of oncolytic virus maintained the normal growth, and their tumor volumes were significantly larger than those in the challenge groups.

(7) FIG. 7 shows the results of toxicity detection of ECHO25-WT in BALB/c mice in Example 4. 1-Day-old BALB/c mice were subjected to intraperitoneal injection of ECHO25 at different doses (10.sup.4, 10.sup.3, 10.sup.6, and 10.sup.7 TCID50/mouse), and then survival rates and health scores of the mice after challenge were obtained. The results showed that ECHO25 had very limited toxicity to BALB/c mice and did not cause disease or death at high doses, indicating that ECHO25 had good safety in vivo.

SEQUENCE INFORMATION

(8) Information of a part of sequences involved in the present invention is provided in Table 1 as below.

(9) TABLE-US-00001 TABLE 1 Sequence description SEQ ID NO: Description 1 cDNA sequence of wild type ECHO25 (ECHO25-WT) 2 RNA sequence of the internal ribosome entry site of human rhinovirus 2 (HRV2) 3 RNA sequence of miR-133 target sequence 4 RNA sequence of miR-206 target sequence 5 RNA sequence of tandem sequence of miR-133 target sequence and miR-206 target sequence 6 DNA sequence of human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene 7 DNA sequence of single chain antibody against human programmed death receptor 1 (Anti-PD-1 scFv) 8 cDNA sequence of one modified form of ECHO25 (ECHO25-HRV2) 9 cDNA sequence of one modified form of ECHO25 (ECHO25-miR133&206T) 10 cDNA sequence of one modified form of ECHO25 (ECHO25-GM-CSF) 11 cDNA sequence of one modified form of ECHO25 (ECHO25-Anti-PD1) 12 Genomic sequence of wild-type ECHO25 (ECHO25-WT) 13 Genomic sequence of one modified form of ECHO25 (ECHO25-HRV2) 14 Genomic sequence of one modified form of ECHO25 (ECHO25-miR133 & 206T) 15 Genomic sequence of one modified form of ECHO25 (ECHO25-GM-CSF) 16 Genomic sequence of one modified form of ECHO25 (ECHO25-Anti-PD1) 17 DNA sequence of miR-133 target sequence 18 DNA sequence of miR-206 target sequence 19 DNA sequence of tandem sequence of miR-133 target sequence and miR-206 target sequence 20 DNA sequence of the internal ribosome entry site sequence of human rhinovirus 2 (HRV2)
Specific Models for Carrying Out the Invention

(10) The present invention is now described with reference to the following examples which are intended to illustrate the present invention (rather than to limit the present invention).

(11) Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention were carried out substantially by referring to the methods of J. Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubelet a., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; restriction enzymes were used under conditions recommended by the product manufacturer. If the specific conditions were not indicated in the examples, the conventional conditions or the conditions recommended by the manufacturer were used. If the reagents or instruments used were not specified by the manufacturer, they were all conventional products that were commercially available. Those skilled in the art will understand that the examples describe the present invention by way of example, and are not intended to limit the scope of protection claimed by the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1: Obtainment and Preparation of ECHO25 and Modified Forms Thereof

(12) 1.1 Isolation of ECHO025 from Patient Clinical Samples

(13) (1) A throat swab and anal swab of patient were gained from the Center for Disease Control and Prevention of Xiamen City, China; African green monkey kidney cells (Vero cells; ATCC® Number: CCL-81™) were was kept by the National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, China, and cultured in MEM medium containing 10% fetal bovine serum, glutamine, penicillin and streptomycin.

(14) (2) Sample processing: the throat swab and anal swab of patient were sufficiently agitated in a sample preservation solution to wash off the virus and virus-containing cells adhering to the swabs, and then the sample preservation solution was subjected to a high speed centrifugation at 4000 rpm and 4° C. for 30 min;

(15) (3) Inoculation and observation:

(16) A) The Vero cells were plated in a 24-well plate with 1×10.sup.5 cells/well. The growth medium (MEM medium, containing 10% fetal bovine serum, as well as glutamine, penicillin and streptomycin) was aspirated, and 1 mL of maintenance medium (MEM medium, containing 2% fetal calf serum, as well as glutamine, penicillin and streptomycin) was added in each well. Then, except the negative control wells, each well was inoculated with 50 μL of the sample supernatant, and cultured in an incubator at 37° C., 5% CO.sub.2.

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

(18) C) If the enterovirus-specific cytopathic effect appeared in the cells in the inoculated wells within 7 days, the cells and supernatant were collected and frozen at −80° C.; if no CPE appeared after 7 days, the cells were subjected to blind passage.

(19) D) If CPE appeared within 6 blind passages, the cells and supernatant were collected and frozen at −80° C.; If CPE did not appear after 6 blind passages, the cells were determined as negative.

(20) (4) Isolation and Cloning of Viruses:

(21) RT-PCR (Hou et al., Virus Res 2015, 205: 41-44) and enzyme-linked immunospot method (ELISPOT) based on specific antibody (Li Shuxuan et al., Biotechnology News (2016) 27 (1): 52-57) were used to identify the viruses isolated from the clinical samples, and echovirus 25-positive culture was selected and subjected to at least 3 cloning experiments. The virus clones obtained by the limiting dilution method in each experiment were also identified by RT-PCR and ELISPOT, and the ECHO25-positive clones were selected for the next round of cloning. A single ECHO25 strain with strong growth viability was selected as a candidate oncolytic virus strain.

(22) 1.2 Obtainment of Rescued Strain of ECHO25 and Modified Forms Thereof by Infectious Cloning and Reverse Genetics Technology

(23) In this example, wild-type ECHO25 (SEQ ID NO: 1) was used as an example to show how to obtain ECHO25 and its modified form for the present invention through reverse genetics technology. The specific method was as follows.

(24) (1) Construction of viral infectious clone: the cDNA sequence of wild-type ECHO25 (named ECHO25-WT) was shown in SEQ ID NO: 1, and its genomic RNA sequence was SEQ ID NO: 12; or gene insertion or replacement based on the cDNA (SEQ ID NO: 1) of ECHO25 was performed, comprising:

(25) Modified form 1: the internal ribosome entry site sequence of wild-type ECHO25 was replaced with the internal ribosome entry site sequence of human rhinovirus 2 (which has a DNA sequence shown in SEQ ID NO: 20) to obtain the cDNA (SEQ ID NO: 8) of the recombinant virus (named as ECHO25-HRV2), which has a genomic RNA sequence shown as SEQ ID NO: 13;

(26) Modified form 2: the tandem sequence (which has a DNA sequence shown in SEQ ID NO: 19) of miR-133 target sequence (which has a DNA sequence shown in SEQ ID NO: 17) and miR-206 target sequence (which has a DNA sequence shown in SEQ ID NO: 18) was inserted between 7337-7338 bp of the 3′ untranslated region of the cDNA (SEQ ID NO: 1) of the wild-type ECHO25, to obtain the cDNA (SEQ D NO: 9) of the recombinant virus (named ECHO25-miR133 & 206T), which has a genomic RNA sequence shown as SEQ ID NO: 14;

(27) Modified form 3: the human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene (SEQ ID NO: 6) was inserted between the VP1 gene and 2A gene of the cDNA (SEQ ID NO: 1) of wild-type ECHO25 to obtain the cDNA (SEQ ID NO: 10) of the recombinant virus (named ECHO25-GM-CSF), which has a genomic RNA sequence shown as SEQ ID NO: 15;

(28) Modified form 4: the sequence (SEQ ID NO: 7) encoding the single chain antibody against human programmed death receptor 1 (Anti-PD-1 scFv) was inserted between the VP1 gene and 2A gene of the cDNA (SEQ ID NO: 1) of wild-type ECHO25 to obtain the cDNA (SEQ ID NO: 11) of the recombinant virus (named ECHO25-Anti-PD-1), which has a genomic RNA sequence shown as SEQ ID NO: 16.

(29) Then, the cDNA sequences (SEQ ID NO: 1, 8-11) of the above five oncolytic viruses were sent to the gene synthesis company (Shanghai Biotech Engineering Co., Ltd.) for full gene synthesis, and ligated into the pSVA plasmids (Hou et al. Virus Res 2015, 205: 41-44) to obtain the infectious cloning plasmids of ECHO25 or modified forms thereof (i.e., ECHO25-WT, ECHO25-HRV2, ECHO25-miR133&206T, ECHO25-GM-CSF and ECHO25-Anti-PD-1).

(30) (2) Plasmid mini-kit and E. coli. DH5a competent cells were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; 293T cells (ATCC® Number: CRL-3216™) and human rhabdomyosarcoma cells (RD cells; ATCC® Number: CCL-136™) were kept by National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, China, and were cultured with DMEM and MEM media respectively, in which 10% fetal bovine serum as well as glutamine, penicillin and streptomycin were added; transfection reagents Lipofactamine2000 and Opti-MEM were purchased from Thermo Fisher Scientific Company.

(31) (3) The infectious cloning plasmids containing the cDNA sequences of the above five oncolytic viruses were transformed into E. coli DH5a competent cells, the monoclonal strains were picked out and shaken after the outgrowth of clones, and the plasmids were extracted using the plasmid mini-kit, and then sent to the company (Shanghai Biotech Engineering Co., Ltd.) for sequencing analysis.

(32) (4) The infectious cloning plasmids with correct sequence and the helper plasmid pAR3126 were co-transfected into the cells to rescue virus (Hou et al. Virus Res 2015, 205: 41-44). 293T cells were first transfected according to the instructions of the transfection reagent; then observed under a microscope. When CPE appeared in 293T cells, the cells and culture supernatant were harvested, and inoculated with RD cells followed by passaging and culturing, thereby obtaining the candidate strain of oncolytic virus.

Example 2: In Vitro Antitumor Experiment of ECHO25 and Modified Forms Thereof

(33) 2.1 Viruses and Cell Lines as Used

(34) (1) Viruses: this example used ECHO25-WT (SEQ ID NO: 12), ECHO25-HRV2 (SEQ ID NO: 13), ECHO25-miR133&206T (SEQ ID NO: 14), ECHO25-GM-CSF (SEQ ID NO: 15) and ECHO25-Anti-PD-1 (SEQ ID NO: 16) as provided in Example 1.

(35) (2) Cell lines: human rhabdomyosarcoma cell RD (ATCC® Number: CCL-136™); human colorectal cancer cell lines SW480 (ATCC® Number: CCL-228™) and HT-29 (ATCC® Number: HTB-38™); humans 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 small cell lung cancer cell line NCI-H1417 (ATCC® Number: CRL-5869™); human non-small cell lung cancer cell lines SPC-A-1 (CCTCC Deposit Number: GDC050), NCT-H1299 (ATCC® Number: CRL-5803™), NCI-H1975 (ATCC® Number: CRL-5908™), 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™), Hep3B (ATCC® Number: HB-8064™), Huh7 (CCTCC Deposit Number: GDC134) and PLC/PRF/5 (ATCC® Number: CRL-8024™); human ovarian cancer cell lines ES-2 (ATCC® Number: CRL-1978™) 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 A-375 (ATCC® Number: CRL-1619™) and SK-MEL-1 (ATCC® Number: HTB-67™); human breast cancer cell lines BT-474 (ATCC® Number: HTB-20™), MDA-MB-231 (ATCC® Number: HTB-26™), MDA-MB-453 (ATCC® Number: HTB-131™), MCF-7 (ATCC® Number: HTB-22™), ZR-75-30 (ATCC® Number: CRL-1504™), SK-BR-3 (ATCC® Number: HTB-30™) and BcaP37 (CCTCC deposit number: GDC206); human kidney cancer cell lines A-498 (ATCC® Number: HTB-44™), 786-O (ATCC® Number: CRL-1932™) and Caki-1 (ATCC® Number: HTB-46™); human pancreatic cancer cell line HPAF-2 (ATCC® Number: CRL-1997™); human prostate cancer cells lines PC-3 (ATCC® Number: CRL-1435™) and DU145 (ATCC® Number: HTB-81™); human glioma cell lines GBM (primary tumor cell line isolated from patient tumor tissue) and U118-MG (ATCC® Number: HTB-15™); human pharyngeal squamous carcinoma cell line FaDu (ATCC® Number: HTB-43™); human tongue squamous cell carcinoma cell line CAL 27 (ATCC® Number: CRL-2095™); human nasopharyngeal carcinoma cell line CNE (purchased from the Cell Center of Basic Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, No. 3131C0001000700013); human nasal septum squamous carcinoma cell line RPMI 2650 (ATCC® Number: CCL-30™); human laryngeal carcinoma cell line HEp-2 (ATCC® Number: CCL-23™); metastatic cells from pleural effusion of human pharyngeal carcinoma Detroit 562 (ATCC® Number: CCL-138™); human submandibular adenocarcinoma cell line A-235 (preserved by National Institute of Diagnostics and Vaccine Development in Infectious Diseases); human thyroid cancer cell lines SW579 (preserved by National Institute of Diagnostics and Vaccine Development in Infectious Diseases) and TT (ATCC® Number: CRL-1803™); human esophageal cancer cell line TE-1 (purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, No. 3131C0001000700089); human bladder cancer cell lines J82 (ATCC® Number: HTB-1™) and 5637 (ATCC® Number: HTB-9™); human leukemia cell lines Jurkat (ATCC® Number: T1B-152™), THP-1 (ATCC® Number: TIB-202™), CCRF-CEM (ATCC® Number: CCL-119™), MOLT-4 (ATCC® Number: CRL-1582™), K562 (ATCC® Number: CCL-243™); human lymphoma cell lines Daudi (ATCC® Number: CCL-213™), Raji (ATCC® Number: CCL-86™) and U937 (ATCC® Number: CRL-1593.2™); human normal cell lines including: human foreskin fibroblast cell line HFF-1 (ATCC® Number: SCRC-1041™), human skin keratinocyte cell line HaCat (CCTCC deposit number: GDC106), human prostate stromal cell line WPMY-1 (ATCC® Number: CRL-2854™) and human umbilical vein endothelial cell line HUVEC (Thermo Fisher Scientific, Catalog #: C01510C). The above cells were all preserved by National Institute of Diagnostics and Vaccine Development in Infectious Diseases, China, Xiamen University. AGS and TT were cultured with F-12K medium; RD, C-33A, SK-MEL-1, J82, FaDu, EBC-1, RPMI2650, Detroit 562 and DU145 were cultured with MEM medium; NCI-H1417, NCI-H1703, Caski, BT-474, ZR-75-30, SK-BR-3, 786-0, Jurkat, THP-1, CCRF-CEM, MOLT-4, Daudi, Raji, K562, U937, 5637, TE-1, Caski, NCI-H1975, NCI-H661, SGC7901 and BGC823 were cultured with RPMI-1640 medium; ES-2, A-235 were cultured with McCoy's 5A medium; MDA-MB-231 and MDA-MB-453 were cultured with Leibovitz's L-15 medium; other cells were cultured with DMEM medium. All of these mediums were supplemented with 10% fetal bovine serum, glutamine and penicillin-streptomycin. All the above cells were cultured under the standard conditions of 37° C. and 5% CO.sub.2.

(36) 2.2 Culture of Viruses

(37) RD cells were evenly plated on 10 cm cell culture plates, and the culture conditions included MEM medium containing 10% fetal bovine serum and 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 serum-free MEM medium, and each plate was inoculated with 10.sup.7 TCID50 of ECHO25-WT, ECHO25-HRV2, ECHO25-miR133&206T, ECHO25-GM-CSF or ECHO25-Anti-PD-1. After continuous culture for 24 hours, the ECHO25 or its modified form proliferated in RD cells and caused CPE in 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 freeze-thawing for three cycles, the culture supernatant was collected and centrifuged to remove cell debris, wherein the centrifuge conditions were 4000 rpm, 10 min, 4° C. Finally, the supernatant was filtered with a 0.22 μm disposable filter (Millipore Company) to remove impurities such as cell debris.

(38) 2.3 Determination of Virus Titer

(39) The RD cells were plated in a 96-well plate with a cell density of 10.sup.4 cells/well. After the cells adhered, the virus solution obtained in Example 2.2 was diluted 10-fold with serum-free MEM medium from the first 10-fold dilution. 50 μl of the dilution of virus was added to the wells with cells. After 7 days, the wells where CPE appeared were monitored and recorded, followed by calculation using Karber method, in which the calculation formula was 1g.sup.TCID50=L−D (S−0.5), L: logarithm of the highest dilution, D: difference between the logarithms of dilutions, S: sum of proportions of positive wells. The unit of TCID50 thus calculated was TCID50/50 μl, which should be converted to TCID50/ml.

(40) 2.4 In Vitro Antitumor Experiments of Viruses

(41) Human tumor cells and normal cells were inoculated into 96-well plates at 10.sup.4 cells/well. After the cells adhered, the medium in each well was replaced with the corresponding cell culture medium without serum, and viruses were inoculated at an MOI of 0.1, 1, 10 or 100. Subsequently, CPE of the cells were monitored daily by a microscope.

(42) FIG. 1 shows micrographs of the human umbilical vein endothelial cell line HUVEC, the human esophageal cancer cell line TE-1, the human endometrial cancer cell lines HEC-1-A and HEC-1-B, which were not infected with viruses (negative control groups, Mock) or treated with ECHO25-WT at MOI=1 for 72 hours. The results showed that after 72 hours of infection at 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 as compared to the non-tumor cells in the Mock groups, the non-tumor cells infected with the viruses showed almost no change in cell morphology. The above results demonstrated that ECHO25 had significant oncolytic effects on human esophageal cancer cell line TE-1, the human endometrial cancer cell lines HEC-1-A and HEC-1-B, but did not have any effect on non-tumor cells HUVEC.

(43) After 72 hours of virus infection and culture, the cell survival rate was detected using Cell Counting Kit-8 (CCK-8 kit; Shanghai Biyuntian Biotechnology Co., Ltd.) and crystal violet staining method (only for adherent cells), and the specific method was as follows:

(44) (1) Cell Survival Rate Detected by CCK8 Method

(45) 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 p 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 test results of ECHO25-WT for each kind of cells were 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.

(46) The calculation of cell survival rate was:

(47) $\mathrm{Cell\_survival}\mathrm{\_rate}\left(\%\right)=\frac{\begin{array}{c}(\mathrm{reading\_of}\mathrm{\_test}\mathrm{\_group}-\\ \mathrm{reading\_of}\mathrm{\_negative}\mathrm{\_group})\end{array}}{\begin{array}{c}(\mathrm{reading\_of}\mathrm{\_positive}\mathrm{\_group}-\\ \mathrm{reading\_of}\mathrm{\_negative}\mathrm{\_group})\end{array}}\times 100\%.$

(48) (2) Cell Survival Rate Detected by Crystal Violet Staining Method (Only for Adherent Cells)

(49) After the cells were infected with viruses for 3 days, the culture supernatant in the 96-well cell culture plate was discarded, 100 s of methanol was added to each well, followed by fixation in the dark for 15 min. Crystal violet powder (Shanghai Biotech Biotechnology Co., Ltd.) was weighed, and formulated as 2% (w/v) crystal violet methanol solution, which was stored at 4° C. An appropriate amount of 2% crystal violet methanol solution was taken and formulated with PBS solution to prepare 0.2% crystal violet working solution. After fixation for 15 minutes, the methanol fixation solution in the 96-well cell culture plate was discarded, and 100 μl of the crystal violet working solution was added to the plate and staining was performed for 30 min. After the crystal violet staining solution was discarded, PBS solution was used for washing for 3 to 5 times, until the excess staining solution was washed off, and air-drying was performed. ImmunSpot @ S5 UV Analyzer (Cellular Technology Limited, USA) was used for photographing. FIG. 2 showed the crystal violet staining results of the human non-small cell lung cancer cell lines A549 and NCI-H661, human ovarian cancer cell line Caov3, human pancreatic cancer cell line HPAF-2, human gastric cancer cell lines AGS, SGC7901, and BGC823, human foreskin fibroblast cell line HFF-1 and human skin keratinocyte line HaCat of human normal cell lines in the control groups (MOCK) and in the experimental groups (infected for 72 hours with ECHO25-WT at MOIs of 0.1, 1, and 10, respectively). As shown in the results, after 72 hours of infection at MOIs of 10, 1, and 0.1, the tumor cells in the experimental groups were significantly reduced as compared to the control group (MOCK) without addition of virus; while the number of non-tumor cells showed no significant change. The above results indicated that the ECHO025-WT had significant oncolytic effects on human tumor cell lines A549. NCI-H661. Caov3. HPAF-2. AGS. SGC7901 and BGC823, but had no significant effect on non-tumor cell lines HFF-1 and HaCat.

(50) TABLE-US-00002 TABLE 2 Results of in vitro antitumor experiments of wild-type enterovirus ECHO25 MOI Cell line 0.1 1 10 100 RD ++ ++ ++ ++ SW480 ++ ++ ++ ++ HT-29 ++ ++ ++ ++ AGS ++ ++ ++ ++ SGC7901 ++ ++ ++ ++ BGC823 ++ ++ ++ ++ NCI-N87 + + ++ ++ SPC-A-1 ++ ++ ++ ++ NCI-H1299 ++ ++ ++ ++ NCI-H1975 − + ++ ++ A549 ++ ++ ++ ++ C3A + ++ ++ ++ Hep3B − + ++ ++ Huh7 − + ++ ++ PLC/PRF/5 − − ++ ++ Caov3 ++ ++ ++ ++ Hcc-1-A ++ ++ ++ ++ Hec-1-B ++ ++ ++ ++ Ishikawa ++ ++ ++ ++ C-33A ++ ++ ++ ++ A-375 − − + ++ SK-MEL-1 + + ++ ++ BcaP37 ++ ++ ++ ++ Caki-1 ++ ++ ++ ++ HPAF-2 ++ ++ ++ ++ PC-3 ++ ++ ++ ++ DU145 − ++ ++ ++ GBM ++ ++ ++ ++ U118-MG ++ ++ ++ ++ FaDu − − + + CAL27 − − + + CNE − − + + Hep2 − − + + TE-1 − ++ ++ ++ J82 − + + ++ 5637 − ++ ++ ++ K562 + + ++ ++ U937 − + ++ ++ EBC-1 − − − − NCI-H1417 − − + + NCI-H1703 − − − − ES-2 − − − − HeLa − − − − CaSki − − − − MCF-7 − − − − BT-474 − − − − MDA-MB-231 − − − − MDA-MB-453 − − − − ZR-75-30 − − − − SK-BR-3 − − − − A498 − − − − 786-O − − − − Jurkat − − − − Daudi − − − − Raji − − − − THP-1 − − − − MOLT-4 − − − − CCRF-CEM − − − − RPMI2650 − − − − Detroit 562 − − − − A-235 − − − − TT − − − − HFF-1 − − − − HaCat − − − − WPMY-1 − − − − HUVEC − − − + 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.

(51) As could be seen from Table 2, ECHO25-WT had good killing effects on specific tumor cell types. In particular, the virus had significant killing effects on colorectal cancer cell lines, gastric cancer cell lines, non-small cell lung adenocarcinoma cell lines, ovarian cancer cell lines, clear cell renal carcinoma cell lines, endometrial cancer cell lines, HPV-negative cervical cancer cell lines, breast medullary carcinoma cell lines, prostate cancer cell lines, glioma cell lines, esophageal cancer cell lines, etc., and had good killing effects on liver cancer cell lines, pancreatic cancer cell lines, bladder cancer cell lines, histiocytic lymphoma cell lines, and chronic myeloid leukemia cell lines; while, ECHO25-WT showed no significant killing activity to non-small cell lung squamous carcinoma cell lines, small cell lung carcinoma cell lines, HPV-positive cervical cancer cell lines, breast non-medullary cancer cell lines, renal adenocarcinoma cell lines, B cell lymphoma cell lines, T cell leukemia cell lines, nasal septum squamous carcinoma cell lines, submandibular adenocarcinoma cell lines, thyroid cancer cell lines, etc. In addition, the virus had substantially no toxicity to non-tumor cell lines including human foreskin fibroblast cell line HFF-1, human skin keratinocyte cell line HaCat and human prostate stromal cell line WPMY-1, except that it showed certain toxicity to human umbilical vein endothelial cell line HUVEC at MOI=100.

(52) In addition, the in vitro antitumor experiments of ECHO25-HRV2, ECHO25-miR133&206T, ECHO25-GM-CSF and ECHO25-Anti-PD-1 showed that the four modified ECHO25 forms all retained the killing effects of the parent wild-type ECHO25 on specific tumor cells, and showed significant killing effects on colorectal cancer cell lines, gastric cancer cell lines, ovarian cancer cell lines, clear cell renal carcinoma cell lines, endometrial cancer cell lines, HPV-negative cervical cancer cell lines, breast medullary carcinoma cell lines, prostate cancer cell lines, glioma cell lines, esophageal cancer cell lines and so on. The CCK-8 detection results of oncolytic activity to human colorectal cancer cell line SW480, human gastric cancer cell line AGS, human endometrial cancer cell line Ishikawa and human glioma cell line U118-MG were shown in Table 3. In addition, the four modified ECHO25 forms showed no significant killing activity to non-small cell lung squamous carcinoma cell lines, small cell lung cancer cell lines, HPV-positive cervical cancer cell lines, breast non-medullary cancer cell lines, renal adenocarcinoma cell lines, B-cell lymphoma cell lines, T-cell leukemia cell lines, nasal septum squamous carcinoma cell lines, submandibular adenocarcinoma carcinoma cell lines, etc. It was worth noting that ECHO25-HRV2 showed significant killing activity on some tumor cells to which ECHO25-WT showed almost no killing activity. The CCK-8 detection results of oncolytic activity to human pharyngeal squamous carcinoma cell line FaDu and human thyroid cancer cell line SW579 were shown in Table 4.

(53) TABLE-US-00003 TABLE 3 In vitro antitumor experimental results of ECHO25-HRV2, ECHO25-miR133&206T, ECHO25-GM-CSF and ECHO25-Anti-PD-1 MOI Cell Line 0.1 1 10 100 ECHO25-HRV2 SW480 ++ ++ ++ ++ AGS ++ ++ ++ ++ Ishikawa ++ ++ ++ ++ U118-MG ++ ++ ++ ++ ECHO25-miR133&206T SW480 ++ ++ ++ ++ AGS ++ ++ ++ ++ Ishikawa ++ ++ ++ ++ U118-MG ++ ++ ++ ++ ECHO25-GM-CSF SW480 ++ ++ ++ ++ AGS ++ ++ ++ ++ Ishikawa ++ ++ ++ ++ U118-MG ++ ++ ++ ++ ECHO25-Anti-PD-1 SW480 ++ ++ ++ ++ AGS ++ ++ ++ ++ Ishikawa ++ ++ ++ ++ U118-MG ++ ++ ++ ++ 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.

(54) TABLE-US-00004 TABLE 4 Comparison of in vitro oncolytic experimental results of ECHO25-WT and ECHO25-HRV2 on human pharyngeal squamous carcinoma cell line FaDu and human thyroid cancer cell line SW579 MOI Cell Line 0.1 1 10 100 ECHO25-WT FaDu − − + + SW579 − − − − ECHO25-HRV2 FaDu ++ ++ ++ ++ SW579 + ++ ++ ++ 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.
2.5 Serial Passaging of ECHO25 for Adaptation

(55) In this example, ECHO25 was serially passaged for adaptation in a certain type of tumor cells to obtain a virus strain with enhanced killing activity to the tumor cell.

(56) The wild-type ECHO25 was serially passaged for adaptation in human liver cancer cell line PLC/PRF/5, human melanoma cell line A-375 or human bladder cancer cell line J82, on which oncolytic effects of wild-type ECHO25 were not very significant, and the specific method was as follows:

(57) One kind of the above tumor cells was evenly plated on a 10 cm cell culture plate, and the culture conditions included a corresponding cell culture media containing 10% fetal bovine serum and 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 serum-free cell culture medium, each plate was inoculated with 10.sup.7 TCID50 of ECHO25, the culture environment was changed to 33° C., 5% CO.sub.2 saturated humidity. When ECHO25 proliferated in tumor cells and caused CPE in the cells (after infection for up to 3 days), the cells and their culture supernatant were harvested. After freeze-thawing for three cycles, centrifugation was performed at 4° C., 4000 rpm for 10 min. The centrifugation supernatant was taken and added onto new tumor cells with a cell confluence of more than 90% to complete one round of virus passage. The passage was repeated for more than 10 times, and a part of the virus solution was taken for virus titer detection in RD cells in each round of passage, and the specific method referred to Example 2.3. Generally, the virus replication ability would increase with the generation, and when a relatively high infectious titer was reached and the virus replication was stable in the tumor cell, the adapted strain of ECHO25 for the tumor cell was obtained.

(58) Subsequently, by the in vitro antitumor experimental method described in Example 2.4, the human tumor cell PLC/PRF/5, A-375 or J82 was inoculated to a 96-well plate at 10.sup.4 cells/well. After the cells adhered, the medium in each well was replaced with the corresponding culture medium free of serum, followed by incubation at 37° C. for 30 min, and then the serially passaged ECHO25 virus strains (viral titers of which were detected on RD cells) adapted for each of the above kinds of cells at MOIs of 0.1, 1, 10, and 100 were inoculated. Subsequently, CPE of the cells were monitored daily by a microscope, and the cell survival rate was detected using CCK-8 method 72 hours after the infection and culture of viruses.

(59) The results were shown in Table 5, in which after serial passaging of the wild-type enterovirus ECHO25 in a certain kind of tumor cells on which ECHO25 had poor oncolytic effect, the killing activity thereof on the tumor cells was significantly enhanced, indicating that the serial passaging method could be used to obtain an ECHO25 adapted strain with enhanced oncolytic effect on the tumor cells.

(60) TABLE-US-00005 TABLE 5 In vitro killing experimental results of ECHO25 on tumor cells after serial passaging for adaptation in tumor cells MOI Cell Line 0.1 1 10 100 PLC/PRF/5 + ++ ++ ++ A-375 − + ++ ++ J82 + ++ ++ ++ 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.
2.6 Evaluation of Oncolytic Effect of Genomic RNA of ECHO25

(61) In this example, a large amount of infectious live viruses of ECHO25 could be produced by transfecting the purified genomic RNA of ECHO25 into a certain kind of tumor cells, and thus kill the tumor cells.

(62) The viral genomic RNA was first obtained by in vitro transcription, and this method could be found in, for example, Hadac E M, Kelly E J and Russell S J. Mol Ther, 2011, 19(6): 1041-1047. Specifically, the infectious cloning plasmid of wild-type ECHO25 obtained in Example 1 was linearized, and the linearized plasmid was used as a template for in vitro transcription using MEGAscript™ T7 Transcription Kit (Thermo Fisher Scientific, AM1333) so as to produce a large amount of viral RNA. And the obtained viral RNA was purified using MEGAclear™ Transcription Clean-Up Kit (Thermo Fisher Scientific, AM1908) for next use. The RNA electropherograms of two parallel samples were shown in FIG. 3.

(63) Subsequently, according to the method of the in vitro antitumor experiment described in Example 2.4, the human colorectal cancer tumor cell line SW480 was inoculated to a 24-well plate at 10.sup.5 cells/well. After the cells adhered, the medium in each well was replaced with a corresponding cell culture medium free of serum, followed by incubation at 37° C. for 30 min. Then, SW480 cells were transfected with purified virus RNA at 1 μg per well using transfection reagent Lipofectamine® 2000 (Thermo Fisher Scientific, 11668019), and the negative control group was transfected with irrelevant RNA nucleic acid molecules. Subsequently, CPE of the cells were monitored daily by a microscope.

(64) The results showed that CPE began to appear in the SW480 cells transfected with genomic RNA of ECHO25 about 8 hours after transfection, and then the cytopathy gradually increased. After 48 hours, the survival rate was measured using the CCK8 method, the SW480 cells had almost all died and lysed, and the micrographs of SW480 cells at 0, 24 and 48 hours after infection were shown in FIG. 4. The culture supernatant was inoculated into new SW480 cells and CPE was quickly produced. The results indicated that the direct administration with the nucleic acid of ECHO25 also had good killing activity and could be used to treat tumors.

Example 3: In Vivo Antitumor Experiments of ECHO25 and Modified Forms Thereof

(65) 3.1 Viruses. Cell Lines and Experimental Animals

(66) (1) Viruses: ECHO25-WT (SEQ ID NO: 12), ECHO25-HRV2 (SEQ ID NO: 13), ECHO25-miR133&206T (SEQ ID NO: 14), ECHO25-GM-CSF (SEQ ID NO: 15) and ECHO25-Anti-PD-1 (SEQ ID NO: 16) as provided in Example 1 were used in this example. The methods of virus culture and virus titer measurement could be seen in Examples 2.2 and 2.3, respectively.

(67) (2) Cell lines: human glioma cell line GBM (primary tumor cell line isolated from patient tumor tissue), human endometrial cancer cell line Ishikawa (ECACC No. 99040201), human prostate cancer cell line PC-3 (ATCC® Number: CRL-1435™) and human breast cancer cell line BcaP37 (CCTCC deposit number: GDC206). The above cells were all cultured in DMEM medium, and the medium was added with 10% fetal bovine serum, glutamine and penicillin-streptomycin. All the above cells were cultured under the standard conditions of 37° C. and 5% CO.sub.2.

(68) (3) Experimental animals: female C.B17 SCID mice aged 6-8 weeks were from Shanghai Slark Experimental Animal Co., Ltd.; according to the protocol approved by the Experimental Animal Center and Ethics Committee of Xiamen University, the mice were raised under SPF conditions.

(69) 3.2 In Vivo Antitumor Experiments of the Viruses

(70) The tumor cells used for subcutaneous tumor formation in SCID mice were digested with 0.01% trypsin, and then resuspended into a single-cell suspension using a 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 (administrated with ECHO25-WT, ECHO25-HRV2, ECHO25-miR133&206T, ECHO25-GM-CSF or ECHO25-Anti-PD-1) and negative control group, with 4 mice (n=4) in each group. Oncolytic virus (ECHO25-WT, ECHO25-HRV2, ECHO25-miR133&206T, ECHO25-GM-CSF or ECHO25-Anti-PD-1) at 10.sup.6 TCID50/100 μl serum-free medium/tumor mass or equivalent amount of serum-free medium were intratumorally injected every two days, for a total of 5 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.

(71) The treatment results of ECHO25-WT for the above four tumors were shown in FIGS. 5A-5D. The results showed that after the challenge of ECHO25-WT, the growth of the four detected tumors of GBM (A), Ishikawa (B), PC-3 (C) and BcaP37 (D) gradually slowed down and arrested, and the tumors were even lysed and disappeared; by contrast, the tumors of the negative group (CTRL) maintained the normal growth, and their tumor sizes were significantly larger than those of the experimental groups.

(72) FIG. 6 showed the results obtained after a treatment of the GBM tumor model with ECHO25-WT, ECHO25-HRV2, ECHO25-miR133&206T, ECHO25-GM-CSF or ECHO25-Anti-PD-1 for 10 days. The results showed that the tumor volumes were significantly reduced after treatment with ECHO25-WT, ECHO25-HRV2, ECHO25-miR133&206T, ECHO25-GM-CSF, and ECHO25-Anti-PD as compared with the negative control group that was not treated with oncolytic virus, and similar reductions in tumor volume were detected after treatment with 5 oncolytic viruses ECHO25-WT, ECHO25-HRV2, ECHO25-miR133&206T, ECHO25-GM-CSF and ECHO25-Anti-PD-1. The above results indicated that all of ECHO25-WT, ECHO25-HRV2, ECHO25-miR133&206T, ECHO25-GM-CSF and ECHO25-Anti-PD-1 showed remarkable and favorable antitumor activity in vivo.

Example 4: Safety Evaluation of Oncolytic Virus

(73) 4.1 Viruses and Laboratory Animals Used

(74) (1) Virus: ECHO25-WT (SEQ ID NO: 12) provided in Example 1 was used in this example. The methods for virus culture and virus titer measurement could refer to Examples 2.2 and 2.3, respectively.

(75) (2) Experimental animals: BALB/c pregnant mice were from Shanghai Slark Experimental Animal Co., Ltd.; according to the protocol approved by the Experimental Animal Center and Ethics Committee of Xiamen University, the mice were raised under clean conditions, and then 1-day-old mice produced by the BALB/c pregnant mice were used for in vivo virulence evaluation of ECHO25.

(76) 4.2 Evaluation of In Vivo Safety of the Virus in Mice

(77) 1-day-old BALB/c suckling mice were selected for challenge with ECHO25-WT by intraperitoneal injection, and the titer doses for challenge were 10.sup.4, 10.sup.5, 10.sup.6, or 10.sup.7 TCID50/mouse. Then, the survival rates and health scores for the BALB/c mice challenged with different doses were recorded daily, wherein the evaluation criteria of the health score were: score of 5 represents dying or died; score of 4 represents severe limb paralysis; score of 3 represents weakness or mild deformity of limb; score of 2 represents wasting; score of 1 represents lethargy, piloerection, and trembling; and score of 0 represents healthy.

(78) The results were shown in FIG. 7. Within 14 days after challenge, no disease or death occurred in all mice in the challenge groups, indicating that ECHO25-WT had limited toxicity to BALB/c mice, and had no effect on the status of mice even at very high doses for challenge. The above results indicate that ECHO25-WT had good safety in vivo.

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