Application of oncolytic viruses as immunostimulants for treatment of tumors and/or cancers

Abstract

Provided is the use of selectively replicating oncolytic viruses in the preparation of immunostimulants for treatment of tumors and/or cancers, wherein the oncolytic viruses do not carry exogenous immunoregulatory genes.

Claims

1. A method for treating a tumor and/or cancer, said method comprising the steps of: administering to a tumor and/or cancer patient an immunostimulating effective amount of a selectively replicating oncolytic virus as an immunostimulant in the treatment of the tumor and/or cancer, wherein the oncolytic virus does not carry an exogenous immunoregulatory gene, and wherein the immune function of the patient is sufficient to cause an anti-tumor cellular immune response, and the total number of leukocytes in whole blood of the patient is ≥4.0×10.sup.9/L; wherein, 7-10 days after completion of the first course of consecutive administration, the immunostimulant is administered again to carry out an immune-boosting course, wherein the immune-boosting course is performed via intratumoral injection at a dose of 0.5×10.sup.12 to 1.5×10.sup.12 vp/day of the oncolytic virus, once daily, consecutively for 1 to 2 days, and wherein, the oncolytic virus is H101, wherein said method does not include any steps of either of chemotherapy and/or radiotherapy.

2. The method according to claim 1, wherein, in addition to directly killing tumor and/or cancer cells, the oncolytic virus also effectively induces activation and amplification of dendritic cells and T cells, induces up-regulation of the expression of chemokines at tumor and/or cancer cell sites, promotes the infiltration of dendritic cells and lymphocytes at tumor and/or cancer cell sites, and induces up-regulation of the expression of MHC-I and/or MHC-II molecules on tumor and/or cancer cells, thereby comprehensively stimulating the anti-tumor immune response of the patient.

3. The method according to claim 1, wherein the first course of consecutive administration of the immunostimulant is administered via intratumoral injection at a dose of 0.5×10.sup.12 to 1.5×10.sup.12 vp/day of the oncolytic virus, once daily, consecutively for 4 to 5 days.

4. The method according to claim 1, wherein the immunostimulant is administered in combination with one or more other tumor immunological agents.

5. The method according to claim 1, wherein the immune-boosting course is further repeated 2 to 3 times, at an interval of 7-10 days from the end of one immune-boosting course to the beginning of the next immune-boosting course.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the results of MTT assay of in vitro infection of human and murine tumor cell lines with H101 in Experimental example 1 according to the present disclosure, wherein FIG. 1A shows the results of A549 cells infected with H101, and FIG. 1B shows the results of B16-F0 cells infected with H101.

(2) FIG. 2 shows the results of H101 promoting apoptosis and tumor antigen release in Experimental example 2 according to the present disclosure, wherein FIG. 2A shows the immunohistochemical results of tumor antigen release, in which the arrows indicate the positive sites of the immunohistochemistry of Caspase-3 protein; and FIG. 2B shows the HE staining results of cell lysis, in which the upper and lower images on the left were taken from different parts of the PBS group sample, while the upper and lower images on the right were taken from different parts of the H101 group sample, with the arrows indicating the cell lysis sites.

(3) FIG. 3 shows the results of H101 activating dendritic cells in Experimental example 3 according to the present disclosure, wherein FIG. 3A shows the flow cytometry results of immature DC cells on Days 1, 3 and 7 after administration of H101, in which the ordinate represents the percentage (%) of CD11b.sup.+CD11c.sup.+ double positive cells in the spleen, and the abscissa represents Days after H101 injection; FIG. 3B shows the flow cytometry results of immature DC cells on Day 12 after administration of H101, in which the ordinate represents the percentage (%) of CD11b.sup.+CD11c.sup.+ double positive cells in the spleen; and FIGS. 3C-3D show the flow cytometry results of mature DC cells on Day 12 after administration of H101, in which the ordinate of FIG. 3C represents the percentage (%) of CD11c.sup.+CD80.sup.+ double positive cells in the spleen, and the ordinate of FIG. 3D represents the percentage (%) of CD11c.sup.+CD86.sup.+ double positive cells in the spleen.

(4) FIG. 4 shows the results of H101 activating T cells in Experimental example 4 according to the present disclosure, wherein FIGS. 4A-4C show the flow cytometry results of CD3.sup.+, CD4.sup.+ and CD8.sup.+ T cells on Day 7 after administration of H101, respectively, in which the ordinate of FIG. 4A represents the percentage (%) of CD3.sup.+ positive cells in the spleen, the ordinate of FIG. 4B represents the percentage (%) of CD3.sup.+CD4.sup.+CD8.sup.− cells in the spleen, and the ordinate of FIG. 4C represents the percentage (%) of CD3.sup.+CD4.sup.−CD8.sup.+ cells in the spleen; and FIG. 4D shows the results of T lymphocytes typing by flow cytometry on Day 10 after administration of H101, in which the ordinate represents the percentage (%) of CD3.sup.+ T cells in the spleen.

(5) FIG. 5 shows the results of expression of the chemokine CXCL12 in Experimental example 5 according to the present disclosure, in which the arrows indicate the positive sites of immunohistochemistry of CXCL12 protein.

(6) FIG. 6 shows the results of H101 promoting infiltration of NKs, DCs and CTLs into tumor entities in Experimental example 6 according to the present disclosure, wherein FIGS. 6A and 6B show the flow cytometry results of CD4.sup.+ T cells and the CD4.sup.+ T cells in secretory activation state after administration of H101, respectively, in which the ordinate of FIG. 6A represents the percentage (%) of CD3.sup.+CD4.sup.+ double positive cells in the tumor, the ordinate of FIG. 6B represents the percentage (%) of CD4.sup.+CD107.sup.+ double positive cells in the tumor, and the abscissas thereof both represent Days after H101 injection; FIGS. 6C and 6D show the flow cytometry results of CD8.sup.+ T cells and the CD8.sup.+ T cells in secretory activation state after administration of H101, respectively, in which the ordinate of FIG. 6C represents the percentage (%) of CD3.sup.+CD8.sup.+ double positive cells in the tumor, the ordinate of FIG. 6D represents the percentage (%) of CD8.sup.+CD107.sup.+ double positive cells in the tumor, and the abscissas thereof both represent Days after H101 injection; FIG. 6E shows the flow cytometry results of NK cells after administration of H101, in which the ordinate represents the percentage (%) of CD3.sup.−NK1.1.sup.+NKG2D.sup.+ cells in the tumor, and the abscissa represents Days after H101 injection; FIG. 6F shows the flow cytometry results of immature DC cells after administration of H101, in which the ordinate represents the percentage (%) of CD11b.sup.+CD11c.sup.+ double positive cells in the tumor, and the abscissa represents Days after H101 injection; and FIGS. 6G and 6H show the flow cytometry results of mature DC cells after administration of H101, in which the ordinate of FIG. 6G represents the percentage (%) of CD11c.sup.+MHCI.sup.+ double positive cells in the tumor, the ordinate of FIG. 6H represents the percentage (%) of CD11c.sup.+MHCII.sup.+ double positive cells in the tumor, and the abscissas thereof both represent days after H101 injection.

(7) FIG. 7 shows the results of H101 stimulating the MHCI/II expression of tumor cells in Experimental example 7 according to the present disclosure, wherein FIG. 7A shows the flow cytometry results of MHC-I molecule-expressing cells, in which the ordinate represents the percentage (%) of MHCI.sup.+ positive cells in the tumor, and the abscissa represents Days after H101 injection; and FIG. 7B shows the flow cytometry results of MHC-II molecule-expressing cells, in which the ordinate represents the percentage (%) of MHCII.sup.+ positive cells in the tumor, and the abscissa represents Days after H101 injection.

(8) FIG. 8 shows the results of relative inhibition of tumor growth by H101 in Experimental example 8 according to the present disclosure, wherein FIG. 8A shows the results of tumor weight over time; FIG. 8B shows the results of tumor volume over time; and FIG. 8C is a photograph showing tumor size over time.

(9) FIG. 9 shows the results of H101 improving the overall survival rate of laboratory animals in Experimental example 9 according to the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

(10) The present disclosure is further explained by the following description of the embodiments and with reference to the accompanied drawings, but it is not intended to limit the invention, and those skilled in the art can make various modifications or improvements according to the spirit of the disclosure. The modifications and improvements are within the scope of the disclosure, without departing from the spirit of the disclosure.

(11) Current treatments of tumors and/or cancers by oncolytic viruses primarily utilize the mechanism that oncolytic viruses can specifically replicate in tumor cells and cause lysis and death of the cells. U.S. Pat. No. 5,677,178 discloses an oncolytic adenovirus, ONYX-015, which is a specific oncolytic adenovirus constructed by deleting E1B-55KD fragment in the DNA sequence of human wild-type adenovirus type 5. ONYX-015 has no significant effect on normal cells, but can selectively replicate and proliferate in tumor cells, eventually leading to lysis of the tumor cells. The clinical trials of ONYX-015 for the treatment of malignant tumors were started in 1996 in subjects with tumors such as head and neck tumor, brain glioma, pancreatic cancer, primary liver and gallbladder tumor, hepatic metastases from colorectal cancer, non-small cell lung cancer, cervical cancer and the like, and the results indicated that the treatment is effective and safe to a certain extent. Also, Chinese Patent No. CN1110553C discloses an oncolytic adenovirus, H101 (CCTCC Deposit No.: V98003), which is an oncolytic adenovirus obtained by genetic recombination of human adenovirus type 5 (Ad5) using genetic engineering techniques. H101 cannot express E1B-55KD polypeptide and lacks the E3 region. Since H101 can specifically replicate in tumor cells, eventually leading to lysis of the cells, oncolytic efficacy can be achieved. H101 was approved by CFDA for sale in China in 2006, with the New Drug Application (NDA) No. Guo Yao Zhun Zi S20060027, under the trade name “Oncorine”.

(12) The therapeutic concept and the purpose of application of the above-mentioned oncolytic viral agents are identical to many other specific targeting anti-tumor drugs that have been developed so far, and they are all used as drugs which are expected to directly act on tumors per se and thereby inhibit the same. In other words, these agents take advantage of the function that oncolytic viruses can selectively replicate in tumor cells to cause lysis and death of the cells.

(13) However, based on long-term research and experiments, the present inventors have discovered a novel use of oncolytic viruses. Specifically, the inventors have found that the oncolytic viruses according to the present disclosure can be used as immunostimulants in the treatment of tumors and/or cancers to individualizedly induce the release of tumor antigens in vivo and act on all the seven steps of the anti-tumor immune system, thereby comprehensively activating the anti-tumor immune function, achieving secondary attack on tumors, and producing sustained efficacy.

(14) The inventors have found that the oncolytic viruses according to the present disclosure can function to stimulate all the steps of anti-tumor immune responses and comprehensively activate the anti-tumor immune function of the body, without the need to undergo more genetic modification to carry an exogenous immunoregulatory gene. Therefore, unlike T-Vec, the inventors have found that the selectively replicating oncolytic viruses per se (without carrying an exogenous immunoregulatory gene) can be used as immunostimulants in the treatment of tumors and/or cancers.

(15) In addition, in order to better exert the function of the immunostimulants, the inventors have further found that the subject to whom the immunostimulant is to be administered should be a tumor and/or cancer patient whose immune function is sufficient to cause an anti-tumor cellular immune response, and the total number of leukocytes in whole blood of the tumor and/or cancer patient should be greater than or equal to 4.0×10.sup.9/L.

(16) Based on the above findings, the present disclosure provides the use of a selectively replicating oncolytic virus in the preparation of an immunostimulant for treatment of tumors and/or cancers, wherein the oncolytic virus does not carry an exogenous immunoregulatory gene, and wherein a subject to whom the immunostimulant is to be administered is a tumor and/or cancer patient whose immune function is sufficient to cause an anti-tumor cellular immune response, wherein the total number of leukocytes in whole blood of the tumor and/or cancer patient is 4.0×10.sup.9/L.

(17) In addition to directly killing tumor and/or cancer cells, the oncolytic virus can also effectively induce activation and amplification of dendritic cells and T cells, induce up-regulation of the expression of chemokines at tumor and/or cancer cell sites, promote the infiltration of dendritic cells and lymphocytes at tumor and/or cancer cell sites, and induce up-regulation of the expression of MHC-I and/or MHC-II molecules on tumor and/or cancer cells, thereby comprehensively stimulating the anti-tumor immune response of the patient.

(18) According to the present disclosure, the oncolytic virus is preferably a human adenovirus type 5, a human 5/3 chimeric adenovirus or a human 5/2 chimeric adenovirus. Preferably, the virus cannot express E1B-55KD polypeptide or the E1A protein of the virus is deleted partially. More preferably, the oncolytic virus lacks at least a portion of genes in the E3 region. Still more preferably, the oncolytic virus is H101.

(19) Preferably, the immunostimulant comprises the oncolytic virus at a clinically applied dosage, e.g., 0.5×10.sup.12 to 1.5×10.sup.12 vp/day.

(20) The oncolytic virus as the immunostimulant may be administered via intratumoral or intravenous injection at a dose of 0.5×10.sup.12 to 1.5×10.sup.12 vp/day of the oncolytic virus, once daily, consecutively for 4 to 5 days. Alternatively, the oncolytic virus may be loaded into a cell carrier and administered by infusion. For example, the oncolytic virus may be loaded into autologous or allogeneic immune cells that are cultured in vitro, and then administered by intravenous infusion or the like (see Ilett et al., 2009, Dendritic cells and T cells deliver oncolytic reovirus for tumour killing despite pre-existing anti-viral immunity, Gene therapy, 16, 689-699).

(21) Also, the immunostimulant according to the present disclosure may be administered in combination with one or more other tumor immunological agents including, but not limited to, some interleukin cytokines having immunostimulatory effect, such as IL-1, IL-2, IL-7, IL-12, IL-15, and IL-21; interferons, such as IFN-alpha; colony-stimulating factors, such as G-CSF, and GM-CSF; tumor necrosis factors, such as TNF-alpha; immune cytokines, such as CD40L/CD40; chemokines, such as CCL-3, CCL-5, CCL-26, CX3CL1, CXCL9, and CXCL10; Toll-like receptor (TLR) signal-related molecules; and immune checkpoint related agents, such as anti-PD-1 antibodies and anti-CTLA4 antibodies.

(22) In another aspect, the present disclosure provides a method for treating a tumor and/or cancer, including administering to a tumor and/or cancer patient an immunostimulating effective amount of the selectively replicating oncolytic virus according to the present disclosure as an immunostimulant in the treatment of the tumor and/or cancer, wherein the oncolytic virus does not carry an exogenous immunoregulatory gene, and wherein the immune function of the patient is sufficient to cause an anti-tumor cellular immune response, and the total number of leukocytes in whole blood of the patient is ≥4.0×10.sup.9/L.

(23) In the method according to the present disclosure, in addition to directly killing tumor and/or cancer cells, the oncolytic virus can also effectively induce activation and amplification of dendritic cells and T cells, induce up-regulation of the expression of chemokines at tumor and/or cancer cell sites, promote the infiltration of the dendritic cells and lymphocytes at tumor and/or cancer cell sites, and induce up-regulation of the expression of MHC-I and/or MHC-II molecules on tumor and/or cancer cells, thereby comprehensively stimulating the anti-tumor immune response of the patient.

(24) In the method, the immunostimulant is preferably administered via intratumoral injection at a dose of 0.5×10.sup.12 to 1.5×10.sup.12 vp/day of the oncolytic virus, once daily, consecutively for 4 to 5 days.

(25) In an embodiment according to the present disclosure, the immunostimulant may be administered in combination with one or more other tumor immunological agents including, but not limited to, some interleukin cytokines having immunostimulatory effect, such as IL-1, IL-2, IL-7, IL-12, IL-15, and IL-21; interferons, such as IFN-alpha; colony-stimulating factors, such as G-CSF, and GM-CSF; tumor necrosis factors, such as TNF-alpha; immune cytokines, such as CD40L/CD40; chemokines, such as CCL-3, CCL-5, CCL-26, CX3CL1, CXCL9, and CXCL10; Toll-like receptor (TLR) signal-related molecules; and immune checkpoint related agents, such as anti-PD-1 antibodies and anti-CTLA4 antibodies.

(26) According to the present disclosure, DC proliferation and tumor infiltration can be induced significantly on Day 7 after administration of H101, and enhancing immunity at this time can synergistically promote the immune effect. Therefore, in another embodiment according to the present disclosure, 7-10 days (more preferably, 7 days) after completion of the first course of consecutive administration, the immunostimulant is administered again to carry out an immune-boosting course. The immune-boosting course is performed via intratumoral injection at a dose of 0.5×10.sup.12 to 1.5×10.sup.12 vp/day of the oncolytic virus, once daily, consecutively for 1 to 2 days. At an interval of 7-10 days after completion of the immune-boosting course, a following-up immune-boosting course may be performed at the same dose and in the same manner as described above. In this way, the immune-boosting course may be performed 3-4 times.

(27) Hereinafter, the present disclosure will be further explained or described by way of examples, but these examples are not intended to limit the scope of protection of the present disclosure.

EXAMPLES

(28) Unless otherwise specified, all the methods and techniques used in the following examples were carried out in accordance with the conventional operations and conditions in the art.

(29) The materials used in the following examples are described below.

(30) A549 (human non-small cell lung cancer cells) and B16-F0 (mouse melanoma cells) were obtained from the Cell Bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences.

(31) Oncolytic adenovirus, H101, was obtained from Shanghai Sunway Biotech Co., Ltd.

(32) C57BL/6 mice (male, 6-8 weeks old) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. The flow cytometer, D2060R, was obtained from ACEA Biosciences (Hangzhou) Co., Ltd.

(33) Experiments in vitro were repeated at least 3 times, with at least 3 duplicate wells per group for each repetition, and statistical analysis was performed.

(34) Experiments in vivo were performed on at least 5 laboratory animals per group, unless otherwise specified.

Experimental Example 1: Infection of Human and Murine Tumor Cell Lines In Vitro

(35) A549 cells (3×10.sup.3 cells/well) or B16-F0 cells (4×10.sup.3 cells/well) were seeded in a 96-well cell culture plate, and incubated in high-glucose DMEM containing 10% FBS at 37° C. in 5% CO.sub.2. After incubation for 12 hours when the cells completely adhered to the wells, H101 (10 MOI for A549 cell line, or 100 MOI for B16-F0 cell line) was added, and the cells were further incubated for 12 hours. Then, the medium was replaced with a fresh one. The time point at which the virus was added was taken as 0 hour. MTT assay was performed every 24 hours (MTT reagent was purchased from Sigma-Aldrich; Item No. M5655-1G). The control group was carried out in complete medium without the addition of any oncolytic virus.

(36) From Day 3 after infection of A549 cells by H101, the MTT results showed that the amount of viable cells was drastically reduced and it was observed under the microscope that a large number of cells were dead and disrupted, indicating that H101 infection in vitro can directly kill human tumor cell lines and promote the release of tumor antigens (as shown in FIG. 1A). However, direct inhibition of the proliferation of murine B16-F0 cells was not observed, even though the infective dose MOI was increased 10-fold (as shown in FIG. 1B).

Experimental Example 2: Effective Promotion of the Release of Tumor Antigens from Murine Tumor Cell Line In Vivo

(37) To study the immunological effects of H101, the present inventors developed an animal model bearing murine melanoma subcutaneously and verified the in vitro cytotoxicity of H101 to the melanoma cell line (B16-F0) in advance. The results demonstrated that the direct killing effect of H101 on the murine B16-F0 cell line was extremely weak due to the species specificity of the human adenovirus type 5 infection (see FIG. 1B of Experimental example 1). The model was in line with the research purpose of studying the immunostimulant effect of H101 while ruling out the direct oncolytic effect thereof as far as possible. Thereafter, the promoting effect of H101 on apoptosis and complete antigen release of the cell line (B16-F0) in the animal model was investigated.

(38) For tumor bearing, B16-F0 cells (2×10.sup.6 cells/100 μl DMEM) were injected into the right back of C57BL/6 male mice at a dose of 100 μl per animal. On the 7.sup.th day of tumor-bearing when the tumor volume reached about 150 mm.sup.3, for the experimental group, H101 at a dose of 2×10.sup.10 vp/50 μl pH7.4 PBS (PBS reagent was purchased from Sangon Biotech (Shanghai) Co., Ltd., Item No. B548117) was intratumorally injected, once daily for 4 consecutive days; meanwhile for the control group, 50 μl PBS was intratumorally injected, once daily for 4 consecutive days. The first day of the administration was taken as Day 0. Then, on Days 3, 7, and 13, the mice were sacrificed and tumors were taken to perform immunohistochemistry (IHC) detection of Caspase-3 (using anti-mouse Caspase-3 antibody, purchased from R&D Systems, Item No. AF-605-NA) and HE staining (using Hematoxylin-Eosin (HE) staining kit, purchased from Sangon Biotech (Shanghai) Co., Ltd., Item No. E607318).

(39) The IHC results demonstrated that Caspase-3 was mainly expressed in the nucleus. In the H101 group, the expression amounts of Caspase-3 on Day 7 and Day 10 were significantly higher than that on Day 3. Also, on Day 7 and Day 10, the counts of Caspase-3 protein-positive cells in the H101 experimental group were significantly higher than that in the PBS control group (as shown in FIG. 2A), and significant cell lysis was observed only on Day 13 (as shown in FIG. 2B). Caspase-3 is the main indicator of apoptosis, and cell lysis is an important manifestation of immunogenic cell death. Therefore, this experiment indicates that the oncolytic virus can induce apoptosis and further induce immunogenic cell death, thereby effectively promoting the release of tumor antigens.

Experimental Example 3: Stimulation of the Immune System and Activation of Dendritic Cells (DCs)

(40) For tumor bearing, B16-F0 cells (2×10.sup.6 cells/100 μl DMEM) were injected into the right back of C57BL/6 male mice at a dose of 100 μl per animal. On the 7th day of tumor-bearing, for the experimental group, H101 (1×10.sup.10 vp/60 μl PBS) was intratumorally injected once for one day; meanwhile for the control group, 60 μl PBS was intratumorally injected once for one day. The first day of the administration was taken as Day 0. Then, on Days 1, 3 and 7, the mice were sacrificed and spleens were taken to perform flow cytometry detection of DCs (using anti-mouse CD11b-FITC and CD11c-APC antibodies which were purchased from eBioscience, Item Nos. 11-0112-85 and 17-0114-81). The results are shown in FIG. 3A.

(41) For tumor bearing, B16-F0 cells (2×10.sup.6 cells/100 μl DMEM) were injected into the right back of C57BL/6 male mice at a dose of 100 μl per animal. On the 7.sup.th day of tumor-bearing, for the experimental group, H101 (5×10.sup.9 vp/50 μl PBS) was intratumorally injected, once daily for 4 consecutive days; meanwhile for the control group, 50 μl PBS was intratumorally injected, once daily for 4 consecutive days. The first day of the administration was taken as Day 0. Then, on Day 12, the mice were sacrificed and spleens were taken to perform flow cytometry detection of DCs (anti-mouse CD80-APC, CD11b-FITC and CD11c-APC antibodies were purchased from eBioscience, Item Nos. 17-0801-82, 11-0112-85 and 17-0114-81; anti-mouse CD86-FITC antibody was purchased from BD Pharmingen, Item No. 561962). The results are shown in FIGS. 3B, 3C and 3D, respectively.

(42) The laboratory animals after administration of H101 were monitored continuously, and it was found that immature DCs in the peripheral blood represented by the spleen were significantly up-regulated from Day 7 after the administration (FIG. 3A). On Day 12 after the administration, immature DCs (CD11b.sup.+CD11c.sup.+) and mature DCs (CD11c.sup.+CD80.sup.+ or CD11c.sup.+CD86.sup.+) in the spleens of the animals still showed an up-regulated trend (FIGS. 3B-3D).

Experimental Example 4: Stimulation of the Immune System and Activation of T Cells

(43) For tumor bearing, B16-F0 cells (1.5×10.sup.6 cells/100 μl DMEM) were injected into the right back of C57BL/6 male mice at a dose of 100 μl per animal. On the 7.sup.th day of tumor-bearing, for the experimental group, H101 (4×10.sup.9 vp/50 μl PBS) was intratumorally injected, once daily for 4 consecutive days; meanwhile for the control group, 50 μl PBS was intratumorally injected, once daily for 4 consecutive days. The first day of the administration was taken as Day 0. Then, on Day 7, the mice were sacrificed and spleens were taken to perform flow cytometry detection of T lymphocytes (anti-mouse CD3e PE-Cyanine7 antibody was purchased from eBioscience, Item No. 35-0031-82; anti-mouse CD4-FITC and CD8-APC antibodies were purchased from BD Pharmingen, Item Nos. 553729 and 553035). The results are shown in FIGS. 4A, 4B and 4C, respectively.

(44) For tumor bearing, B16-F0 cells (2×10.sup.6 cells/100 μl DMEM) were injected into the right back of C57BL/6 male mice at a dose of 100 μl per animal. On the 7th day of tumor-bearing, for the experimental group, H101 (1×10.sup.10 vp/60 μl PBS) was intratumorally injected once for one day; meanwhile for the control group, 60 μl PBS was intratumorally injected once for one day. The first day of the administration was taken as Day 0. Then, on Day 10, the mice were sacrificed and spleens were taken to perform T lymphocytes typing by the flow cytometric detection (using anti-mouse CD3e PE-Cyanine7 antibody, purchased from eBioscience, Item No. 35-0031-82). The results are shown in FIG. 4D.

(45) On Day 7 after administration of H101, the CD3.sup.+, CD4.sup.+ and CD8.sup.+ T cells in the spleens were significantly up-regulated (FIGS. 4A-4C). On Day 10, T cell subtype analysis (anti-mouse CD4-FITC and CD8-APC antibodies were purchased from BD Pharmingen, Item Nos. 553729 and 553035; anti-mouse CD44-PE and CD62L-APC antibodies were purchased from eBioscience, Item Nos. 12-0441-83 and 17-0621-82) indicated that in the CD3.sup.+ T cell population, Naive T (CD44.sup.low CD62L.sup.hi) cells and Tcm (central memory T cells, CD44.sup.hi CD62L.sup.hi) of the laboratory animals in the H101-administered group were down-regulated, while Tem (effector memory T cells, CD44.sup.hi CD62L.sup.low) was up-regulated, indicating that T cell immunity was mobilized and activated in the body (FIG. 4D).

Experimental Example 5: Intratumoral Administration Stimulates the Expression of Chemokine CXCL12 at Tumor Site, which Facilitates the Homing and Aggregation of Immune Cells at the Tumor Site

(46) For tumor bearing, B16-F0 cells (1×10.sup.6 cells/100 μl DMEM) were injected into the right back of C57BL/6 male mice at a dose of 100 μl per animal. On the 7.sup.th day of tumor-bearing, for the experimental group, H101 (4×10.sup.9 vp/50 μl PBS) was intratumorally injected, once daily for 4 consecutive days; meanwhile for the control group, 50 μl PBS was intratumorally injected, once daily for 4 consecutive days. The first day of the administration was taken as Day 0. Then, on Day 13, the mice were sacrificed and tumors were taken to perform IHC detection of the chemokine CXCL12 (using anti-mouse CXCL12 antibody, purchased from Abcam, Item No. AB25117). The results are shown in FIG. 5.

(47) The IHC results demonstrated that the expression of the chemokine CXCL12 in tumor site was up-regulated on Day 13 after administration of H101. The up-regulation of the chemokine can promote vascular proliferation, improve the permeability of tumor entities, and facilitate the homing and aggregation of immune cells at the tumor site.

Experimental Example 6: Intratumoral Injection of Oncolytic Virus Promotes the Infiltration of NKs, DCs and CTLs into Tumor Entities

(48) For tumor bearing, B16-F0 cells (2×10.sup.6 cells/100 μl DMEM) were injected into the right back of C57BL/6 male mice at a dose of 100 μl per animal. On the 7th day of tumor-bearing when the tumor volume reached about 150 mm.sup.3, for the experimental group, H101 (2×10.sup.10 vp/50 μl PBS) was intratumorally injected, once daily for 4 consecutive days; meanwhile for the control group, 50 μl PBS was intratumorally injected, once daily for 4 consecutive days. The first day of the administration was taken as Day 0. Then, on Days 3, 7 and 10, the mice were sacrificed and tumors were taken to perform flow cytometric analysis (anti-mouse CD314 (NKG2D)-APC, NK1.1-FITC, CD107a-PE, CD3e PE-Cyanine7, MHC Class I (H-2Kd)-PE and MHC Class II (I-A/I-E)-PE-Cyanine7 antibodies were purchased from eBioscience, Item Nos. 17-5882-82, 11-5941-85, 12-1071-83, 35-0031-82, 12-5957-82 and 25-5321-82; anti-mouse CD4-FITC and CD8-APC antibodies were purchased from BD Pharmingen, Item Nos. 553729 and 553035; anti-mouse CD11b-FITC and CD11c-APC antibodies were purchased from eBioscience, Item Nos. 11-0112-85 and 17-0114-81). The results are shown in FIG. 6.

(49) On Day 3 and Day 7 after administration of H101, CD4.sup.+ T cells and the CD4.sup.+ T cells in secretory activation state (CD4.sup.+CD107a.sup.+) in tumor tissues were significantly up-regulated (FIGS. 6A and 6B), while CD8.sup.+ T cells were significantly up-regulated on Day 7. Also, on Day 7 and Day 10, CD8.sup.+ T cells and the CD8.sup.+ T cells in secretory activation state (CD8.sup.+CD107a.sup.+) showed an up-regulated trend (FIGS. 6C and 6D). On Day 7 after the administration, NK cells were significantly up-regulated (FIG. 6E). Meanwhile, immature DCs (CD11b.sup.+CD11c.sup.+) in the tumors were significantly up-regulated on Day 7, while mature DCs (CD11c.sup.+MHCI.sup.+ or CD11c.sup.+MHCII.sup.+) were significantly up-regulated on Day 3 and Day 7 (FIGS. 6F-6H).

Experimental Example 7: Injection Stimulates MHCI/II Expression of Tumor Cells and Enhances Immune Recognition

(50) For tumor bearing, B16-F0 cells (2×10.sup.6 cells/100 μl DMEM) were injected into the right back of C57BL/6 male mice at a dose of 100 μl per animal. From the 7th day of tumor-bearing when the tumor volume reached about 150 mm.sup.3, for the experimental group, H101 (2×10.sup.10 vp/50 μl PBS) was intratumorally injected for 4 consecutive days; meanwhile for the control group, 50 μl PBS was intratumorally injected for 4 consecutive days. The first day of the administration was taken as Day 0. Then, on Days 3, 7 and 10, the mice were sacrificed and tumors were taken to perform flow cytometric analysis (anti-mouse MHC Class I (H-2Kd)-PE and MHC Class II (I-A/I-E)-PE-Cyanine7 antibodies were purchased from eBioscience, Item Nos. 12-5957-82 and 25-5321-82). The results are shown in FIGS. 7A and 7B.

(51) On Day 3 and Day 7 after administration of H101, the percentages of MHC-I molecule-expressing cells in the tumor site were significantly up-regulated. On Day 7 and Day 10, the percentages of MHC-II molecule-expressing cells were significantly up-regulated. The up-regulation of the expression of the MHC molecules strongly promotes the tumor immune recognition response and improves the killing efficiency of immune cells.

Experimental Example 8: Relative Inhibition of Tumor Growth in Laboratory Animals

(52) For tumor bearing, B16-F0 cells (2×10.sup.6 cells/100 μl DMEM) were injected into the right back of C57BL/6 male mice at a dose of 100 μl per animal. On the 7th day of tumor-bearing when the tumor volume reached about 150 mm.sup.3, for the experimental group, H101 (2×10.sup.10 vp/50 μl PBS) was intratumorally injected, once daily for 4 consecutive days; meanwhile for the control group, 50 μl PBS was intratumorally injected, once daily for 4 consecutive days. The first day of the administration was taken as Day 0. Tumor sizes were measured every 2 days, and on Days 3, 7 and 10, the mice were sacrificed and tumors were taken to weigh. The results are shown in FIG. 8.

(53) Compared with the control group, from Day 7 after the administration of H101, the tumor volumes of the animals were significantly smaller than those of the PBS-injected control group, and the difference was significant (FIGS. 8A-8C).

Experimental Example 9: Increasing of the Overall Survival Rate of Laboratory Animals

(54) For tumor bearing, B16-F0 cells (2×10.sup.6 cells/100 μl DMEM) were injected into the right back of C57BL/6 male mice at a dose of 100 μl per animal. On the 7.sup.th day of tumor-bearing, for the experimental group, H101 (4×10.sup.9 vp/50 μl PBS) was intratumorally injected, once daily for 4 consecutive days; meanwhile for the control group, 50 μl PBS was intratumorally injected, once daily for 4 consecutive days. The survival status of the mice was observed every day. The results are shown in FIG. 9.

(55) The experiment of survival rate demonstrated that administering H101 can significantly prolong the overall survival rate of laboratory animals.

(56) Although in the in vitro killing experiment the proliferation of the murine B16-F0 cell line was almost unaffected by H101, the results of the in vivo administration demonstrated that administering H101 can significantly inhibit the tumor growth in laboratory animals relative to the control group. By combining the results of the above experiments, it is believed that H101 as an immunostimulant can effectively improve the tumor-inhibitive effect on the immunocompetent murine tumor model. Its efficacy is achieved not only by the direct oncolytic effect of the oncolytic virus, but further by promoting all the seven steps of anti-tumor immunity and effectively stimulating the anti-tumor immunity.