Immuno-oncolytic modified vaccinia Tiantan virus and method for treating cancer

Abstract

The present invention relates to a method for treating a subject's cancer by administering to the subject a combination of an oncolytic virus and a therapy, wherein the therapy induces the consumption of tumor-induced polymorphonuclear bone marrow myeloid-derived suppressor cells (PMN-MDSC). In certain preferred embodiments, the oncolytic virus is a modified vaccinia temple of heaven (MVTT) virus with a replication defect having a viral M1L-K2L gene deletion. In other preferred embodiments, the therapy for inducing the consumption of tumor-induced PMN-MDSC includes administering an antibody to Ly6G, such as 1A8. The cancer therapy of the present invention can be administered in combination with one or more other anticancer therapies. Preferred additional anticancer therapies are immunotherapy, such as the administration of checkpoint inhibitors.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 660,546, filed on April 20, 2018, and U.S. Provisional Patent Application Serial No. 62 / 687,531, filed on June 20, 2018, which are hereby incorporated by reference in their entirety, including any tables, figures, or drawings. Background of the Invention

[0004] Mesothelioma is an asbestos-related malignant form of tumor that generally has a poor prognosis in humans. For this life-threatening malignancy, the current standard of care can only achieve suboptimal improvements in patient survival. Harnessing the host immune system to eradicate malignant cells has become a clinical strategy for cancer immunotherapy. Although immune checkpoint inhibitors have improved the treatment of certain cancers, their effects are not satisfactory in mesothelioma patients. Therefore, new strategies are needed to treat mesothelioma. Recently, oncolytic virotherapy has emerged as a promising cancer immunotherapy for the treatment of solid tumors including malignant mesothelioma. However, the mechanisms of limited viral therapeutic efficacy remain elusive.

[0005] Direct virus-mediated oncolysis of cancer cells is one of the main mechanisms of oncolytic virotherapy. During oncolysis, danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) are released into the tumor microenvironment (TME), which can modulate the immunogenicity of released tumor antigens by creating an immune-activating environment and subsequently eliciting or enhancing tumor-reactive T cell responses. Both preclinical and clinical studies have demonstrated the key role of adaptive T cell immunity in oncolytic virotherapy. However, the TME is typically an immunosuppressive environment that inducing tolerogenic dendritic cells (DCs) and CD25 + Foxp3 + Regulatory T lymphocytes (Tregs) are used to inhibit the activation of tumor-reactive T cells. Bone marrow myeloid-derived suppressor cells (MDSCs) in the TME can suppress the responsiveness of cytotoxic T lymphocytes (CTLs), resulting in limited efficacy in patients, especially when the TME is highly immunosuppressive. Since T cell immunity is essential for the efficacy of oncolytic virotherapy, a better understanding of the restrictive mechanisms in the TME is particularly important for improving the clinical efficacy of oncolytic virotherapy.

[0006] MDSC is one of the main immunosuppressive groups in TME and a major obstacle to the effectiveness of cancer immunotherapy. In a malignant mesothelioma model, MDSC rapidly proliferates with the development of tumor lesions and helps to suppress tumor-reactive CTL responses. Consistently, the reduction in the number of MDSC in TME may be related to the generation and therapeutic effect of antigen-specific CTL responses during oncolytic virus therapy in patients. MDSC can be monocytes (M) or multinuclear cells (PMN). Targeting the COX-2-PGE2 pathway during vaccinia virus therapy can reduce PMN-MDSC levels while increasing anti-tumor CTL responses. In addition, early studies using the COX-2 inhibitor celecoxib improved DC-based immunotherapy for mesothelioma by reducing PMN-MDSC frequency. Although these studies have shown the key role of PMN-MDSC in cancer immunotherapy, cure of established tumors has rarely been observed. To date, the underlying mechanisms of MDSC accumulation in TME, the functional differences between MDSC subpopulations, and their effects on eliciting anti-tumor CTLs during oncolytic virus therapy are not fully understood. SUMMARY OF THE INVENTION

[0008] In certain embodiments, the present invention provides a method for treating a subject's cancer by administering a combination of an oncolytic virus and therapy to a subject, wherein the therapy induces the consumption of tumor-induced PMN-MDSC. In a preferred embodiment, the oncolytic virus is a modified vaccinia temple of heaven (MVTT) virus with a replication defect of the M1L-K2L gene deletion of the virus. In other preferred embodiments, the therapy for inducing the consumption of tumor-induced PMN-MDSC includes administering antibodies to Ly6G, such as 1A8. The cancer therapy of the present invention can be administered in combination with one or more other anticancer therapies. Preferred additional anticancer therapies are immunotherapy, such as the administration of checkpoint inhibitors.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 A-1E shows the production of recombinant MVTT virus encoding two detection markers, HIV-1p24 and RFP. (A) Schematic diagram of the vaccinia shuttle vector pZCxz encoding both HIV-1p24 and HcRed. The expression of each protein is driven by a different promoter. (B) AB1 cells were infected with recombinant MVTT for 24 hours. The HcRed signal was obtained using a fluorescence microscope. BF, bright field. (C) Western blot analysis of viral protein expression in AB1 cells after recombinant MVTT infection. As indicated by the arrow, an anti-p24 antibody (clone: ​​183-H12-5C) was used to detect the presence of exogenous protein. GAPDH is an internal control, indicating that equal amounts of protein were loaded in each lane. (D) AB1 cells were plated at 2×10 5Cells were seeded at a density of 10 cells / well in a 24-well plate. After 24 hours, cells were infected with recombinant MVTT virus at a multiplicity of infection (MOI) of 0.2. Cells were harvested at three indicated time points and analyzed for HcRed using flow cytometry. + (E) Culture supernatants from AB1 cells infected with recombinant MVTT virus were collected at different time points, and viral particles released into the supernatant were measured.

[0011] Figure 2 A-2E shows MVTT-mediated oncolysis of AB1 cells, resulting in CRT exposure and release of ATP and HMGB1. (A) AB1 cell viability after infection with 0.2 MOI of recombinant MVTT. CRT expression on AB1 cells was detected by anti-CRT antibody and analyzed by flow cytometry analysis (B) or western blotting (C). β-actin is an internal control, indicating that the same amount of protein was used in the analysis. (D) Western blot analysis of HMGB1 released in the culture supernatant after MVTT virus infection. (E) ATP levels released in the culture supernatant.

[0012] Figure 3 A-3F shows that the oncolytic effect of recombinant MVTT virus on AB1 mesothelioma does not induce immunogenic death of tumor cells. (A) Schematic diagram of the treatment study of AB1 tumor-bearing mice using different doses of MVTT. 5×10 5 AB1 cells were used to establish solid AB1 mesothelioma. In the high-dose group, 1×10 cells were delivered intrathecally (IT) every 2 days. 8 PFU MVTT virus 5 times, while in the medium-dose group, 1 × 10 7 PFU 4 times and 2 times in the low-dose group. (B) Tumor volume was measured over time with a caliper. (C) Growth curves of individual tumors in each group. Each line represents one mouse. (D) Survival curves were determined by caliper measurements and taken as the time until tumor length was >15 mm. (E) T cell responses in splenocytes from tumor-free or AB1 tumor-bearing mice. Secreted IFN-γ was quantified by ELIspot assay after in vitro stimulation of splenocytes with gp70-AH1, TWIST1, or the irrelevant antigen OVA. As indicated by the arrow, only one tumor-free mouse responded strongly to the gp70-AH1 epitope. (F) CD3 isolated from tumor-free mice + CTL assay of T cells. Gray line indicates CD3 T cells in mice with strong AH1 response. + CTL activity of T cells: Compared with the PBS group, P = 0.08.

[0013] Figure 4A-4F shows the accumulation of PMN-MDSC in tumors after intratumoral MVTT treatment. (A) The percentage of total MDSC in spleen and tumor (left panel) and the absolute number of MDSC in tumor (right panel). The number of MDSC per mg of tumor was calculated at the indicated time points. (B) Representative dot plots show the CD11b + Intracellular PMN-MDSC and M-MDSC populations. Numbers indicate cell ratios. (C) The percentages of MDSC subsets were calculated for M-MDSC (left panel) and PMN-MDSC (right panel). (D) Absolute cell numbers of M-MDSC and PMN-MDSC in tumors. At the indicated time points, the number of MDSC subsets was calculated per mg of tumor. (E) CD4 + The percentage of Treg (left) and CD4 + Absolute number of Treg cells (right panel). (F) Percentage of NK cells in spleen and tumor (left panel) and absolute number of NK cells in tumor (right panel).

[0014] Figure 5 A-5F shows the transportation (trafficking) of PMN-MDSC to the tumor site after intratumoral MVTT treatment. (A) Flow cytometric analysis of chemokine receptor expression on different MDSC subpopulations from AB1 tumor-bearing mice. Representative histograms are shown. The shaded area represents isotype control. Expression of CXC chemokines (B) and CC chemokines (C) in tumors after MVTT treatment. (D) Frequency (left figure) and absolute number (right figure) of CFSE-labeled MDSC in spleen and tumor 24 hours after MVTT treatment. (E) M-MDSC and PMN-MDSC cell subpopulations in tumors 24 hours after MVTT treatment. Representative dot plots are shown with numbers, indicating the ratio of gated cells to total single peaks. (F) The ratio change of PMN-MDSC ratio relative to M-MDSC ratio was analyzed (left figure). The PMN- / M-MDSC ratio measured before adoptive transfer is shown as baseline. Changes in the absolute numbers of M-MDSCs and PMN-MDSCs in tumors are shown (right panel).

[0015] Figure 6 A-6D shows that MVTT treatment disrupts PMN-MDSC tumor trafficking. (A) In CD11b +Representative dot plots of gated cells show the number of PMN-MDSC and M-MDSC in spleen and tumor 2 and 4 days after intrathecal injection of 100 μg 1A8 or anti-rat IgG2a (clone: ​​2A3) isotype control. Numbers in the dot plots indicate the proportion of cells in the gate. (B) Percentages of MDSC subsets were calculated using PMN-MDSC (left panel) and M-MDSC (right panel). (C) Representative dot plots show PMN-MDSC and M-MDSC populations in spleen and tumor 2 and 4 days after combined treatment. 100 μg 1A8 or isotype 2A3 were combined with 1×10 7 PFUMVTT was combined and injected intrathecally into AB1 mesothelioma. (D) Analysis of the changes in PMN-MDSC (left) and M-MDSC (right) in MDSC subsets.

[0016] Figure 7 A-7K shows that the combination of oncolysis and PMN-MDSC depletion restored anti-tumor T cell immunity for tumor elimination. (A) Schematic diagram of the treatment regimen. 5×10 5 AB1 cells were inoculated subcutaneously (sc) into Balb / c mice and allowed to grow for 7 days, followed by intrathecal administration of MVTT, 1A8 antibody, MVTT+1A8 combination, or PBS control. Each group was scheduled for additional treatment on day 9. Tumor growth (B) and survival curves (C) of mice were calculated. 40 days after tumor ablation, protected mice in the combined treatment group were challenged again, and tumor growth (D) was measured with representative bioluminescent images of AB1-Luc tumors (E). (F) T cell responses in splenocytes measured by ELISpot assay. (G) CD3 + CD4 T cells or MVTT+1A8 treated groups + and CD8 + T cells have in vitro cytotoxic activity against AB1 cells at different effector:target (E:T) ratios. (H) Schematic diagram of T cell depletion by 2-fold MVTT+1A8 combination treatment. + T cells (YTS191.1), CD8 + AB1-Luc tumor growth (I) and survival curves (J) of MVTT+1A8-treated mice treated with T cells (YTS169.4) or AB1-Luc tumor-bearing mice receiving isotype control (LTF-2). (K) Representative bioluminescent images of AB1-Luc tumors in T cell depleted groups.

[0017] Figure 8 A-8F shows that PMN-MDSCs prevent the induction of anti-tumor T cell immunity by limiting DC activation. (A) CD3 +Cytokine production after incubation of T cells with antigen-pulsed BMDCs. After washing with medium, BMDCs were pulsed with supernatant of AB1 cells treated with rMVTT overnight. Then, purified CD3 + T cells, and culture supernatants were collected for analysis of cytokine production. Anti-CRT antibodies or isotype controls were present in several cultures during antigen pulsing. BALB / c mouse naive, purified CD3 + T cells. (B) After co-culture with antigen-pulsed BMDC, CFSE-labeled CD3 + Proliferation of T cells. Representative histograms are shown in each figure with numbers indicating the proliferating population. (C) Expression of CD80 and CD86 on BMDC pulsed with medium (unstimulated) or LPS. Purified PMN-MDSC or M-MDSC were labeled with CFSE and present in culture at a 2:1 ratio with BMDC. Figures from (A) to (C) show cumulative data from two independent experiments. (D) IL-10 + and TGF-β1 + Frequency of PMN-MDSC and M-MDSC. Representative dot plots from 3 independent experiments are shown, with numbers indicating the positive cell population in each gate. (E) Crosstalk between PMN-MDSC and BMDC enhanced IL-10 production. 5 In the case of BMDC (BMDC:MDSC=1:2), 5×10 4 Purified PMN-MDSC or M-MDSC. Supernatants were collected 4 days after incubation and cytokine production was measured. (F) Expression of CD80 and CD86 on LPS-activated BMDC in the presence of IL-10 receptor blocking antibody or isotype control. Purified PMN-MDSC or M-MDSC were labeled with CFSE and present in culture with BMDC at a ratio of 2:1. The IL-10 receptor was blocked by anti-IL-10R antibody (5 μg / ml) before stimulating BMDC with 100 ng / ml LPS. Figures from (E) to (F) show representative data from two independent experiments.

[0018] Fig. 9 A-9C shows that the combined treatment significantly inhibited the growth of B16F10 melanoma in C57BL / 6 mice. Seven days before treatment, C57BL / 6 mice were implanted subcutaneously with 5 x 10 5 B16F10-Luc cells. Tumor growth (A), survival curve (B) and T cell response of splenocytes (C) at their endpoints are shown.

[0019] Fig.10 A-10E shows that MVTT treatment recruits PMN-MDSCs into the TME. (A) Expression of HcRed in established AB1 mesothelioma tumors after rMVTT treatment. Overlay of representative light and fluorescence images of HcRed in tumors with or without rMVTT injection (left). Fluorescence images were acquired using an IVIS Spectrum instrument. Color bars represent the fluorescence radiant efficiency multiplied by 10 7 . Representative images are shown. HcRed fluorescence signals from tumors were calculated (right panel). (B) Immunohistochemistry of vaccinia virus proteins in AB1 tumors 2 days after rMVTT injection. AB1 tumor sections were stained with hematoxylin and eosin (H&E) (left panel) or stained for vaccinia virus proteins (green) (right panel) using a commercially available rabbit anti-vaccinia virus antibody (WR, Access Biomedical) and Hoechst 33258 staining (blue). Representative images are shown. The dotted line shows the boundary between infected and uninfected tumor tissue. (C) Gating strategy for flow cytometry dot plots showing gating for MDSC subsets, NK cells, and CD4 + Treg and PD1 + / Tim3 + CD3 + Identification of T cells. (D) CD3 + The frequency (left) and absolute number (right) of T cells. (E) PD1 + CD3 + T cells (left) and Tim3 + CD3 + The frequency of T cells (right panel).

[0020] Fig.11 Flow cytometric analysis of CFSE-labeled MDSCs is shown. Adoptively transferred MDSCs accumulated at tumor sites in representative mice 24 h after rMVTT treatment. Numbers in dot plots indicate CFSE + The proportion of cells relative to total singlets.

[0021] Fig.12 A-12H shows preferential depletion of MDSC subsets by antibody and peptibody treatment. (A) Schematic representation of the H6 / G3-pep encoding plasmid. IL2ss, IL2 secretion signal. Binding affinity of H6-pep, G3-pep, or peptibody without the 12-mer specific sequence (control-pep) was measured by flow cytometry. Splenocytes from AB1 tumor-bearing mice were incubated with 2 μg of peptibody after detection with anti-mouse IgG2b AF568. (B) Shows the expression of peptibody in CD11b +Representative dot plots of cells gated on CD11b + Representative histograms of gating on cells stained with pep-H6 (dashed lines), G3-pep (solid lines), or control pep (shaded histograms). (D) Percentage of total MDSCs in spleen and tumor after intrathecal administration of 100 μg 1A8, H6-pep, or 2A3 isotype control. Representative dot plots are shown for changes in PMN-MDSC and M-MDSC frequencies after intrathecal (it) H6-pep treatment (E) and analyzed (F). Intrathecal co-administration of 1×10 7 After PFUrMVTT and 100 μg H6-pep, the changes in PMN-MDSC and M-MDSC frequencies were shown (G) and analyzed (H).

[0022] Fig.13 A-13I shows that depletion of PMN-MDSCs enhances MVTT therapeutic efficacy by inducing anti-tumor T cell immunity. (A) Schematic diagram of the treatment regimen, in which PBS, 1A8 alone, rMVTT and 1A8 in combination, or rMVTT and H6-pep in combination were administered once 7 days after AB1 cell inoculation. Tumor growth (B) and survival curves (C) of mice receiving one round of treatment. Tumor growth (D), survival curves (E), and T cell responses of splenocytes (F) of mice receiving 2 injections of PBS, H6-pep, or rMVTT and H6-pep in combination. Seven days before treatment, 5×10 5 B16F10-Luc cells were implanted subcutaneously in C57BL / 6 mice. rMVTT, 1A8 antibody, combination rMVTT and 1A8 or PBS control were administered intrathecally on days 7 and 9. Tumor growth (G), survival curves (H) and T cell responses of splenocytes (I) at their endpoints are shown.

[0023] Fig.14 A-14E shows that PMN-MDSCs prevent the induction of anti-tumor T cell immunity by limiting DC activation. (A) In CD3 + IL-6, IL-17A, and IL-22 secretion in co-cultures of T cells and antigen-pulsed BMDC. Naive, purified CD3 +T cells. (B) Secreted cytokines in co-culture supernatants were collected after 48 h of incubation. (C) Secretion of IL-6 and TNF-α in antigen-pulsed BMDC cultures in the presence of PMN-MDSC or M-MDSC at MDSC:BMDC ratios of 1:1 and 3:1. BMDCs were pulsed with supernatant of AB1 cells treated with rMVTT. The data shown are representative of two independent experiments. (D) IL-10 in tumor homogenates after rMVTT treatment. (E) Production of TNF-α and IL-12p70 in culture supernatants in the presence of IL-10 receptor blocking antibody or isotype control. Culture supernatants were collected after 48 h of incubation and cytokine secretion was measured. DETAILED DESCRIPTION OF THE INVENTION

[0025] Cancer virotherapy using oncolytic viruses is a promising treatment strategy with demonstrated clinical benefit. Following the approval of T-vec (also known as Imlygic), a recombinant herpes simplex virus expressing the immune-activating cytokine GM-CSF, for the treatment of skin and lymph node melanoma in the United States and Europe, multiple oncolytic viruses have advanced to clinical development. Among them, the use of ONCOS-102 adenovirus in the treatment of malignant mesothelioma in a clinical setting was able to induce CD8 + T cells, systemic anti-tumor CD8 + Infiltration of tumors by T cells and Th1-type polarization. Although the therapeutic effects of T-vec and ONCOS-102 are promising, only a small fraction of treated patients experienced a clinical response in these studies. Therefore, studying how to induce an effective anti-tumor immune response is critical to enhancing the therapeutic efficacy of viral therapy for patients. Most viruses currently being tested in clinical trials are designed to acquire the ability to trigger an immune response. To this end, understanding the underlying mechanisms of blocking and regulating systemic anti-tumor immunity is critical to further improving oncolytic viral therapy.

[0026] The replication of oncolytic viruses in tumors releases danger signals CRT, HMGB1 and ATP, as well as tumor antigens of DCs, thereby triggering anti-tumor immune responses. Therefore, combined immunotherapy has become a useful strategy to improve the efficacy of oncolytic virus therapy against various types of tumors, including malignant mesothelioma and melanoma. Immunotherapy includes enhancing host anti-tumor responses by incorporating immune activation molecules (e.g., GM-CSF), immunomodulatory drugs (e.g., cyclophosphamide), or immune checkpoint inhibitors.

[0027] In addition to the rapid increase in the use of immune checkpoint inhibitors, herpes simplex virus (T-vec) spiked with GM-CSF has also received regulatory approval for the treatment of patients with advanced melanoma. Clinical trials have shown that reducing the immunosuppressive effects of MDSCs and Tregs with sunitinib can enhance the immune response against renal cell carcinoma during oncolytic reovirus therapy. In the case of malignant mesothelioma, the use of first-line chemotherapeutic agents (cisplatin or pemetrexed) during oncolytic adenoviral therapy has been shown to enhance virus-mediated cytotoxicity in mice.

[0028] MVTT virotherapy alone is not sufficient to effectively eliminate tumors. The replication of oncolytic viruses in tumors releases the danger signals CRT, HMGB1, ATP, and tumor antigens of DCs, thereby triggering an antitumor immune response. However, complete eradication of mesothelioma by intratumoral administration of extremely high doses of MVTT alone in multiple sites of solid tumors rarely elicited antitumor T cell responses, even in protected mice.

[0029] The present invention describes that viral therapy significantly expands MDSCs in mesothelioma TME. The expansion of MDSCs is a key immune escape mechanism in various human cancers (e.g., renal cell carcinoma, squamous cell carcinoma, breast cancer, and non-small cell lung cancer). In mice with mesothelioma, tumors induced a rapid increase in MDSCs as early as 7 days after AB1 cell inoculation, and the elimination of MDSCs during immunotherapy was closely related to tumor rejection. The expansion of PMN-MDSCs in mesothelioma TME during MVTT viral therapy was due to the production of CXC chemokines associated with viral infection of tumor cells. CXC chemokines then preferentially chemotactically target CXCR2 + PMN-MDSCs are recruited to tumor sites from peripheral lymphoid organs. These results emphasize the role of the CC and CXC axes in the trafficking of M-MDSCs and PMN-MDSCs, respectively.

[0030] It was found that PMN-MDSCs recruited by viral infection were responsible for inhibiting NK cells through reactive oxygen species (ROS) production, or enhancing local immunosuppression through PD-L1 expression. The present invention demonstrates that PMN-MDSCs exhibit effective immunosuppressive function on DC activation. M-MDSCs did not find similar immunosuppressive effects on DCs, indicating functional differences between these two MDSC subsets in mesothelioma TME.

[0031] Depletion of PMN-MDSC alone is also insufficient to effectively eliminate tumors. Targeted depletion of PMN-MDSC allows modest CTL responses in models of pancreatic ductal adenocarcinoma and lung cancer. However, AB1 mesothelioma in mice has been considered a poor immunogenic model. AB1 mesothelioma in immunodeficient SCID mice shows similar growth kinetics compared to immunocompetent BALB / c mice.

[0032] Moreover, T cells purified from mesothelioma-bearing mice did not contain antigen-specific T cells with potent cytotoxic activity. To better define the functions of PMN-MDSC and M-MDSC in regulating antitumor immunity, depletion experiments using anti-Ly6G or H6-pep monotherapy were performed, respectively. Depletion of either PMN-MDSC or M-MDSC did not induce any inhibitory effect on mesothelioma growth. In addition, no measurable antitumor CTLs were detected. Therefore, depletion of MDSC subsets alone does not promote the exposure of mesothelioma antigens to trigger DC activation. Therefore, oncolytic virus therapy is necessary to promote tumor antigen exposure and subsequent induction of systemic antitumor T cell responses.

[0033] Therefore, the present invention proves that the combination of oncolytic virus therapy (such as MVTT virus therapy) and PMN-MDSC depletion is required to cure established mesothelioma, and despite the increase of intratumoral M-MDSC and enhanced DC to induce effective anti-tumor CTL, they can still overcome immunosuppression. PMN-MDSC plays a key role in regulating anti-tumor CTL response. Using the antibody 1A8 that consumes PMN-MDSC and the peptide antibody H6-pep that consumes M-MDSC, it is shown that PMN-MDSC rather than M-MDSC during oncolytic virus therapy (such as MVTT virus therapy) is essential for the induction of TME-restricted tumor-reactive CTL responses.

[0034] In addition, the combination of oncolytic virus therapy (e.g., MVTT virus therapy) and PMN-MDSC depletion activated endogenous T cells to elicit antitumor CTLs with broad-spectrum reactivity, cytolytic activity, and protective long-term memory responses. In this process, the increased intratumoral M-MDSCs could not block T cell activation and antitumor CTLs.

[0035] Mechanistically, PMN-MDSCs, but not M-MDSCs, within tumors inhibit DC activation by preventing upregulation of CD80 and CD86 and secretion of IL-6, TNF-α, and IL-12p70. Thus, in addition to the inhibitory effects of MDSCs on T cells, the present invention also describes a mechanism by which mesothelioma-derived PMN-MDSCs exhibit immunosuppressive activity on DCs. The crosstalk between PMN-MDSCs and DCs undermines antitumor immunity by increasing IL-10 production and reducing DC activation.

[0036] In mouse models, tumor-derived MDSCs upregulated IL-10 production, and neutralization of IL-10 abolished the suppressive effects of MDSCs. Considering the plasticity of the immunosuppressive myeloid compartment under various tumors and infectious agents, acute phase responders can induce the expansion and polarization of IL-10-secreting tumor-associated neutrophils, thereby suppressing antigen-specific T cell responses in melanoma patients. Therefore, IL-10-secreting PMN-MDSCs can serve as a barrier to protect tumors from immune surveillance. Chemotactic recruitment of IL-10-secreting PMN-MDSCs is a key DC suppressor for aborting T cell activation during MVTT virus therapy.

[0037] Inhibition of cell cycle-related kinase (CCRK) signaling reduced PMN-MDSC-mediated immunosuppression and suppressed the tumorigenicity of hepatocellular carcinoma. Therefore, epigenetic regulatory approaches targeting CCRK to specifically disrupt PMN-MDSC accumulation are particularly important in developing combination therapies with MVTT for the treatment of various human cancers such as mesothelioma.

[0038] Thus, the present invention describes that intratumoral PMN-MDSCs are key suppressors of DCs in the mesothelioma TME, which limits the induction of antitumor CTLs and impairs the efficacy of MVTT-based virotherapy.

[0039] Accordingly, certain embodiments of the invention provide methods of treating cancer (eg, mesothelioma) by administering an oncolytic virus in combination with a therapy that induces tumor-induced PMN-MDSC depletion.

[0040] Oncolytic viruses and the therapy of inducing tumor-induced PMN-MDSC consumption can be administered simultaneously or continuously. Oncolytic viruses can be administered before or after the therapy of inducing tumor-induced PMN-MDSC consumption. The co-administration of oncolytic viruses and the therapy of inducing tumor-induced PMN-MDSC consumption can be carried out in the same or separate compositions. The separate administration of these therapies can be carried out together with one or more other agents.

[0041] When administered alone, the oncolytic virus can be administered within about one day to about seven days of the therapy for inducing tumor-induced PMN-MDSC consumption, preferably within about two days to about six days, more preferably within about three to five days, and even more preferably within about four days. In other embodiments, when administered alone, the oncolytic virus can be administered within about 20 to 40 hours of the therapy for inducing tumor-induced PMN-MDSC consumption, preferably within about 25 to 35 hours, even more preferably within about 30 hours, and most preferably within about 24 hours.

[0042] In a preferred embodiment, the oncolytic virus is administered prior to administration of a therapy that induces tumor-induced PMN-MDSC depletion.

[0043] Oncolytic viruses and therapies that induce tumor-induced PMN-MDSC depletion can be administered multiple times over a period of several days, for example, during 2 to 14 days, more preferably during 4 to 12 days, more preferably during 6 to 10 days, and even more preferably during about 7 days.

[0044] In some embodiments, the oncolytic virus is an adenovirus, a reovirus, a herpes virus, a picornavirus (including coxsackievirus, poliovirus and Seneca Valley virus), a paramyxovirus (including measles virus and Newcastle disease virus (NDV)), a parvovirus, a rhabdovirus (eg, vesicular stomatitis virus (VSV), or vaccinia virus). The oncolytic virus may be replication-competent or replication-deficient. Methods for producing replication-deficient viruses are known in the art and are within the scope of the present invention.

[0045] In certain embodiments, the oncolytic virus is a modified vaccinia virus. Preferably, the modified vaccinia virus is a live attenuated vaccinia virus, such as a vaccinia virus that cannot replicate. In some embodiments, the modified vaccinia virus is a genetically modified vaccinia virus with one or more gene deletions required for replication. For example, the deletion of the M1L-K2L gene renders the vaccinia virus unable to replicate.

[0046] Examples of modified vaccinia viruses suitable for use in the present invention, particularly modified vaccinia Tiantan virus (MVTT), are described in Zhu et al. (2007), J Virol Methods; 144(1-2): 17-26. The Zhu et al. reference is incorporated by reference in its entirety.

[0047] In certain embodiments, the modified vaccinia virus is an MVTT generated from vaccinia Tiantan (VTT) by deleting the viral M1L-K2L gene. In other embodiments, the modified vaccinia virus is an MVTT generated from VTT by replacing the viral M1L-K2L gene with a heterologous gene (e.g., a gene encoding a marker fluorescent protein). Compared to the parent VTT, MVTT is 100 times less toxic. Therefore, MVTT is an attenuated vaccinia Tiantan vaccine vector with improved safety.

[0048] Thus, in certain embodiments, the oncolytic virus is MVTT.

[0049] In a further embodiment, the oncolytic virus is a recombinant MVTT (rMVTT). The rMVTT comprises a deletion of the viral M1L-K2L gene from VTT, and further comprises two or more heterologous genes that replace the deleted viral M1L-K2L gene. One of the two or more heterologous genes can be a gene encoding a protein tag, such as a fluorescent protein or an enzyme. The fluorescent protein can be a green fluorescent protein or a red fluorescent protein. The red fluorescent protein can be HcRed or a green fluorescent protein (GFP). Other examples of fluorescent proteins are known to those of ordinary skill in the art, and such embodiments are within the scope of the present invention. For example, the fluorescent protein database (fpbase) is well known in the art and can be found on the World Wide Web site: fpbase.org.

[0050] In a further embodiment, one of the two or more heterologous genes is a gene encoding a heterologous viral capsid protein, preferably a gene for the p24 protein of the human immunodeficiency virus (HIV).As used herein, the term "heterologous virus" refers to a virus other than VTT.

[0051] In a specific embodiment, one of the two or more heterologous genes is a gene encoding a fluorescent protein, and another of the two or more heterologous genes is a gene encoding a capsid protein of a heterologous virus. Preferably, one of the two or more heterologous genes is a gene encoding HcRed, and another of the two or more heterologous genes is a gene encoding HIVp24.

[0052] In a further embodiment, one of the two or more heterologous genes is under the control of the synaptic protein promoter (pSYN), and another of the two or more heterologous genes is under the control of the H5 promoter (pH5). Preferably, one of the two or more heterologous genes is a gene encoding HcRed under the control of pH5, and another of the two or more heterologous genes is a gene encoding HIV p24 under the control of pSYN. MVTT is prone to induce DAMPs (including calreticulin (CRT) exposure, HMGB1 and ATP release), and the oncolytic effect of AB1 mesothelioma cells. MVTT triggers tumor reactive CTLs, which is essential for the treatment of malignant mesothelioma. MVTT virus therapy also induces chemotaxis, which recruits PMN-MDSCs producing IL-10 into TME, where they inhibit DC, thus blocking the induction of anti-tumor CTLs. During MVTT virus therapy, the consumption of PMN-MDSCs instead of M-MDSCs releases tumor reactive CTLs, thereby leading to the treatment and cure of cancer (e.g., mesothelioma). The present invention provides for the depletion of MDSCs, in particular PMN-MDSCs, in combination with oncolytic MVTT therapy, which can be achieved, for example, by inducing cytotoxic CD8 +T cell responses to restore effective anti-tumor T cell immunity.

[0053] Thus, specific embodiments of the invention provide methods of treating cancer (eg, malignant mesothelioma or melanoma) by administering a combination of oncolytic MVTT and a therapy that induces tumor-induced PMN-MDSC depletion.

[0054] Examples of certain therapies that induce tumor-induced PMN-MDSC depletion include gemcitabine, fluorouracil, bindali, PDE5 inhibitors, tadalafil, nitroaspirin, COX-2 inhibitors, ipilimumab, bevacizumab, celecoxib, sildenafil and tadalafil, N-hydroxy-L-arginine, N-acetylcysteine ​​(NAC), CpG oligodeoxynucleotides (ODN), bardoxolone methyl (CDDO-Me), withaferin A, monoclonal anti-Gr1 antibodies, IL4Rα aptamers, and peptibodies targeting MDSC membrane proteins (S100 family).

[0055] In certain embodiments, the therapy that induces depletion of tumor-induced PMN-MDSCs specifically induces depletion of only tumor-induced PMN-MDSCs without affecting tumor-induced M-MDSCs.

[0056] In a preferred embodiment, the therapy for inducing tumor-induced PMN-MDSC depletion is an antibody against lymphocyte antigen 6 complex locus G6D (Ly6G), such as antibody 1A8. Antibodies against Ly6G (e.g., 1A8) can specifically induce tumor-induced PMN-MDSC depletion without affecting tumor-induced M-MDSC.

[0057] In certain embodiments, the methods comprise administering a chemotherapeutic agent before, during, or after administering the combination of an oncolytic virus and a therapy that induces tumor-induced PMN-MDSC depletion.

[0058] In a further embodiment, radiation therapy is administered before or after the combination of an oncolytic virus and a therapy that induces tumor-induced PMN-MDSC depletion is administered to the subject. Radiation therapy can also be administered between the administration of an oncolytic virus and the administration of a therapy that induces tumor-induced PMN-MDSC depletion.

[0059] In certain embodiments, the method includes administering a checkpoint inhibitor before, during, or after administering a combination of an oncolytic virus and a therapy that induces tumor-induced PMN-MDSC depletion to a subject. Checkpoint inhibitor therapy can be administered between administering an oncolytic virus and administering a therapy that induces tumor-induced PMN-MDSC depletion.

[0060] Certain checkpoint inhibitors have been used in cancer therapy. Checkpoints refer to inhibitory pathways in the immune system that are responsible for maintaining self-tolerance and regulating the extent of the immune system response to minimize damage to surrounding tissues. Tumor cells can activate immune system checkpoints to reduce the efficacy of immune responses against tumor tissues. Administration of checkpoint inhibitors can release the inhibitory effects on the immune system and allow the immune system to be active against tumor cells. Exemplary checkpoint inhibitors include inhibitors, such as antibodies, for cytotoxic T lymphocyte antigen 4 (CTLA4, also known as CD152), programmed cell death protein 1 (PD-1, also known as CD279), and programmed cell death 1 ligand 1 (PD-L1, also known as CD274). Exemplary anti-PD-1 antibodies are commercially available and include pembrolizumab, lambrolizumab, nivolumab, AMP-224 (MERCK), and pidilizumab. Exemplary anti-PD-L1 antibodies are also commercially available, including atezolizumab, MDX-1105 (MEDAREX), MEDI4736 (MEDIMMUNE) MPDL3280A (GENENTECH), BMS-936559 (BRISTOL-MYERSSQUIBB) and AFFYMETRIX EBIOSCIENCE (MIH1). Exemplary anti-CTLA4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (PFIZER). Ipilimumab has recently been approved by the FDA for the treatment of metastatic melanoma (Wada et al., 2013, J Transl Med 11: 89). Other checkpoint inhibitors are well known to technicians, and such embodiments are within the scope of the present invention.

[0061] Examples of cancers that can be treated according to the materials and methods disclosed herein include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, cervical cancer, ovarian cancer, peritoneal cancer, liver cancer. For example, liver cancer, bladder cancer, colorectal cancer, endometrial cancer, kidney cancer, and thyroid cancer. In some embodiments, the cancer is melanoma, MDS, ovarian cancer, breast cancer, or multiple myeloma.

[0062] In some embodiments, the cancer is malignant mesothelioma or melanoma.

[0063] Other non-limiting examples of cancer are basal cell carcinoma, biliary tract cancer; bone cancer; brain and CNS cancer; choriocarcinoma; connective tissue cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer; intraepithelial neoplasia; laryngeal cancer; lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (e.g., lip, tongue, mouth and pharynx); retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; sarcoma; skin cancer; stomach cancer; testicular cancer; uterine cancer; urinary system cancer, as well as other cancers and sarcomas. Table 1 lists examples of cancer types that can be treated with the compositions and methods of the present invention.

[0064] Table 1. Examples of cancer types

[0065]

[0066]

[0067]

[0068]

[0069] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. For example, the characteristics of a specific cancer may be a solid tumor or a non-solid tumor. A solid tumor mass (if present) may be a primary tumor mass. A primary tumor mass refers to the growth of cancer cells in a tissue due to the transformation of normal cells of the tissue. In most cases, a primary tumor mass is identified by the presence of a cyst, which can be found by visual or palpation methods, or by the irregularity of the shape, the texture or weight of the tissue. However, some primary tumors are not palpable and can only be detected by medical imaging techniques (such as X-rays (e.g., mammography)) or magnetic resonance imaging (MRI), or by needle biopsy. The use of these latter techniques is more common in early detection. Molecular and phenotypic analysis of cancer cells in a tissue can usually be used to confirm whether the cancer is endogenous to the tissue, or whether the lesion is due to metastasis to other parts. Some tumors are unresectable (due to, for example, the number of metastatic lesions or because it is located in a surgical danger zone and cannot be surgically removed). The therapeutic and prognostic methods of the invention can be used in early, intermediate or advanced stages of disease and in acute or chronic conditions.

[0070] Composition and treatment

[0071] A variety of methods can be used to deliver the therapy of oncolytic virus and / or PMN-MDSC consumption induced by tumor to the subject.Both the therapy of oncolytic virus and PMN-MDSC consumption induced by tumor induction can be administered by the same approach.Or, oncolytic virus can be administered by a way, and the therapy of PMN-MDSC consumption induced by tumor induction can be administered by different approaches.In a preferred embodiment, the therapy of oncolytic virus and PMN-MDSC consumption induced by tumor induction is administered intrathecally (it).

[0072] Oncolytic viruses and the therapy of inducing tumor-induced PMN-MDSC consumption can be applied in one or more compositions. Pharmaceutical compositions may include various other components. Examples of acceptable components or adjuvants that may be used in pharmaceutical compositions include antioxidants, free radical scavengers, peptides, growth factors, antibiotics, antibacterial agents, immunosuppressants, anticoagulants, buffers, anti-inflammatory agents, anti-angiogenic agents, antipyretics, slow-release adhesives, anesthetics, steroids and corticosteroids. Such components can provide additional therapeutic benefits, enhance the therapeutic effect of anticancer therapy or prevent any potential side effects of the anticancer therapy.

[0073] Other agents can be co-administered in the same or independent preparation to the subject or the subject's cancer cell. Such other agents include agents that change a given biological response, such as immunomodulators. Other agents can be, for example, small molecules, polypeptides (proteins, peptides or antibodies or antibody fragments) or nucleic acids (encoding polypeptides or inhibitory nucleic acids, such as antisense oligonucleotides or interfering RNA). For example, proteins such as tumor necrosis factor (TNF), interferons (such as α-interferon and β-interferon), nerve growth factor (NGF), platelet-derived growth factor (PDGF) and tissue plasminogen activator can be administered. Biological response regulators, such as lymphokines, interleukins (such as interleukin-1 (IL-1), interleukin-2 (IL-2) and interleukin-6 (IL-6)), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF) or other growth factors. In one embodiment, the methods and compositions of the present invention include one or more anticancer agents, such as cytotoxic agents, chemotherapeutic agents, anti-signal transducers and anti-angiogenic agents.

[0074] In some embodiments, the composition of the present invention includes at least one additional anticancer agent (e.g., chemotherapeutic agent). In some embodiments of the present method, at least one additional anticancer agent is administered together with the composition of the present invention. In some embodiments, the anticancer agent is selected from suberoylanilide hydroxamic acid (SAHA) or other histone deacetylase inhibitors, arsenic trioxide, doxorubicin or other anthracycline DNA intercalating agents, and etoposide or other topoisomerase II inhibitors.

[0075] In some embodiments, the composition may include, and the method may include administering one or more proteasome inhibitors (e.g., bortezomib), autophagy inhibitors (e.g., chloroquine), alkylating agents (e.g., melphalan, cyclophosphamide), MEK inhibitors (e.g., PD98509), FAK / PYK2 inhibitors (e.g., PF562271), or EGFR inhibitors (e.g., erlotinib, gefitinib, cetuximab, panitumumab, zalutumumab, nimotuzumab, and matuzumab), or a combination of two or more of the foregoing.

[0076] Therefore, the therapy of oncolytic virus or PMN-MDSC consumption induced by tumor induction, whether it is applied alone or as a pharmaceutical composition, can include various other ingredients as additives. Examples of acceptable components or adjuvants that can be used in relevant situations include antioxidants, free radical scavengers, peptides, growth factors, antibiotics, antibacterial agents, immunosuppressants, anticoagulants, buffers, anti-inflammatory agents, anti-angiogenic agents, antipyretics, slow-release adhesives, anesthetics, steroids and corticosteroids. Such components can provide additional therapeutic benefits, play a therapeutic effect that affects the compounds of the present invention, or play a role in preventing any potential side effects that may be caused by the administration of compounds. Immunotherapeutics can also be conjugated with therapeutic agents or other agents.

[0077] As used herein, the term "immunotherapy" refers to the treatment of disease by stimulating, inducing, subverting, simulating, enhancing, increasing or modulating the subject's immune system in any other way to induce or enhance adaptive or innate immunity (active or passive) against cancerous or other harmful proteins, cells or tissues. Immunotherapy (i.e., immunotherapeutic agents) include cancer vaccines, immunomodulators, monoclonal antibodies (e.g., humanized monoclonal antibodies), immunostimulants, dendritic cells and virotherapy, whether designed to treat existing cancers or prevent the development of cancer, or used in an adjuvant environment to reduce the likelihood of cancer recurrence. Examples of cancer vaccines include GVAX, Stimuvax, DCVax and other vaccines designed to induce immune responses to tumors and other antigens (including MUC1, NY-ESO-1, MAGE, p53, etc.). Examples of immunomodulators include 1MT, Ipilimumab, Tremelimumab, and / or any drug intended to inhibit or otherwise modulate cytotoxicity or other T cell activity against tumors or other antigens, including but not limited to treatments that modulate T-Reg cell control pathways through CTLA-4, CD80, CD86, MHC, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, CD28, other TCRs, PD-1, PDL-1, CD80, ICOS, and their ligands, whether by blockers, agonists, or antagonists. Examples of immunostimulants include corticosteroids and any other anti-inflammatory or pro-inflammatory agent, steroidal or non-steroidal, including but not limited to GM-CSF, interleukins (e.g., IL-2, IL-7, IL-12), cytokines (e.g., interferons), and the like. Examples of dendritic cell (DC) therapy include modified dendritic cells and any other autologous, allogeneic or xenogeneic antigen presenting cells, whether modified by multiple antigens, whole cancer cells, single antigens, by mRNA, phage display or any other modification, including but not limited to ex vivo generated, antigen-loaded dendritic cells (DC) to induce antigen-specific T cell immunity, ex vivo gene-loaded DC to induce humoral immunity, ex vivo generated antigen-loaded DC to induce tumor-specific immunity, ex vivo generated immature DC to induce tolerance, including but not limited to Previx, etc. Examples of virotherapy include oncolytic viruses or virus-derived genetic or other materials that can induce anti-tumor immunity and infectious virus inhibitors associated with tumor development, such as the Prophage series of drugs. Examples of monoclonal antibodies include Alemtuzumab, Bevacizumab, Cetuximab, Gemtuzumab, Rituximab, Trastuzumab, Radioimmunotherapy, Ibritumomab Tiuxetan, Tositumomab / Iodine Tositumomab regimens. Immunotherapy can be used as a monotherapy or in combination with one or more other therapies (one or more other immunotherapies or non-immunotherapy).

[0078] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and / or causes cell destruction in vitro and / or in vivo. The term is intended to include radioactive isotopes (e.g., At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 and radioactive isotopes of Lu), chemotherapeutic agents, toxins (e.g., small molecule toxins) or enzymatically active toxins of bacterial, fungal, plant or animal origin and antibodies, including fragments and / or variants thereof.

[0079] As used herein, the term "chemotherapeutic agent" is a compound useful for treating cancer, such as taxanes, such as paclitaxel (TAXOL, BRISTOL-MYERS SQUIBB Oncology, Princeton, NJ) and doxetaxel (TAXOTERE, Rhone-Poulenc Rorer, Antony, France), chlorambucil, vincristine, vinblastine, antiestrogens including, for example, tamoxifen, raloxifene, 4 (5) -imidazoles that inhibit aromatase, 4-hydroxytamoxifen, troxifene, keoxifene, LY117018, onapristone and toremifene (Fareston, GTx, Memphis, TN) and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin, etc. Examples of anticancer agents, including chemotherapeutic agents that can be used in conjunction with the compounds of the present invention, are listed in Table 2. In a preferred embodiment, the chemotherapeutic agent is one or more anthracyclines. Anthracyclines are a class of chemotherapy drugs that are also antibiotics. Anthracyclines prevent cell division by destroying the structure of DNA and terminate its function by: (1) inserting into the base pairs of the minor groove of DNA; and (2) causing free radical damage to the ribose in DNA. Anthracyclines are commonly used in the treatment of leukemia. Examples of anthracyclines include daunorubicin (CERUBIDINE), doxorubicin (ADRIAMYCIN, RUBEX), epirubicin (ELLENCE, PHARMORUBICIN) and idarubicin (IDAMYCIN).

[0080] Table 2. Examples of anticancer drugs

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Although the oncolytic virus of the present invention and / or the therapy of inducing tumor-induced PMN-MDSC consumption can be applied to the subject as a separated reagent, it is preferred that these viruses or therapies are applied as part of a pharmaceutical composition. Therefore, the present invention further provides a composition comprising an oncolytic virus, a compound for inducing tumor-induced PMN-MDSC consumption, and a combination of at least one pharmaceutically acceptable carrier. The pharmaceutical composition may be suitable for various routes of administration, such as enteral, parenteral, intravenous, intramuscular, topical, subcutaneous, etc. As can be determined by a person of ordinary skill in the art, administration may be continuous or at different intervals. "Pharmaceutically acceptable carrier" refers to ingredients other than active ingredients in a pharmaceutical preparation, and includes but is not limited to buffers, excipients, stabilizers, or preservatives.

[0087] The compositions for use according to the methods of the invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations are described in a number of sources, which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science (Martin, EW, 1995, Easton Pennsylvania, Mack Publishing Company, 19 th ed.) describes formulations that can be used in the present invention. Formulations suitable for administration include, for example, sterile water injections, which may contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. Before use, the formulations may be present in unit dose or multi-dose containers, such as sealed ampoules and vials, and may be stored under freeze-dried (lyophilized) conditions that require only sterile liquid carriers (e.g., water for injection). Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, tablets, and the like. It should be understood that, in view of the type of formulation being discussed, in addition to the ingredients specifically mentioned above, the compositions of the present invention may include other agents conventional in the art.

[0088] The compositions of the present invention, oncolytic viruses, therapies for inducing tumor-induced PMN-MDSC consumption, and other agents for the methods of the present invention, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent, can be locally administered at one or more anatomical sites, such as unwanted cell growth sites (e.g., tumor sites, such as injection or topical administration to tumors). The compositions of the present invention and other agents used in the methods of the present invention can be systemically administered, such as intravenously or orally, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent or an absorbable edible carrier for oral delivery. They can be loaded into hard or soft shell gelatin capsules, can be pressed into tablets, or can be directly mixed with food in the patient's diet. For oral therapeutic administration, the agent can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, sprays, etc.

[0089] Tablets, lozenges, pills, capsules, etc. may also contain the following substances: binders, such as tragacanth, gum arabic, corn starch or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; sweeteners, such as sucrose, fructose, lactose or aspartame, or flavorings, such as mint, wintergreen oil or cherry flavoring, may be added. When the unit dosage form is a capsule, it may contain a liquid carrier, such as a vegetable oil or polyethylene glycol, in addition to the above-mentioned types of materials. Various other materials may exist as coatings or otherwise change the physical form of the solid unit dosage form. For example, tablets, pills or capsules may be coated with gelatin, wax, shellac or sugar, etc. Syrups or elixirs may contain active compounds, sucrose or fructose as sweeteners, methylparabens and propylparabens as preservatives, dyes and flavorings (e.g., cherry or orange flavoring). Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. Additionally, the compositions and agents can be incorporated into sustained-release preparations and devices.

[0090] Oncolytic viruses and / or therapies inducing tumor-induced PMN-MDSC consumption can be administered to tumors (intratumor) or lymph nodes, such as inguinal lymph nodes of subjects. Oncolytic viruses and / or therapies inducing tumor-induced PMN-MDSC consumption can also be administered intradermally, intravenously or intraperitoneally by infusion or injection.

[0091] Solutions of the active agent can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin and mixtures thereof, and in oils. Under conventional conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0092] The pharmaceutical dosage form suitable for injection or infusion may include a sterile aqueous solution or dispersion or a sterile powder suitable for the extemporaneous preparation of a sterile injectable or infusible solution or dispersion, optionally encapsulated in a liposome. The final dosage form should be sterile, fluid and stable under production and storage conditions. The liquid carrier or vehicle may be a solvent or liquid dispersion medium, which comprises, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), a vegetable oil, a non-toxic glyceride and a suitable mixture thereof. Appropriate fluidity can be maintained, for example, by forming liposomes, in the case of a dispersion by maintaining the desired particle size or by using a surfactant. Optionally, the action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is best to include an isotonic agent, such as sugar, buffer or sodium chloride. By including an agent that delays absorption, such as aluminum monostearate and gelatin, the injectable composition can be prolonged for absorption.

[0093] Sterile injectable solutions are prepared by incorporating the oncolytic virus and / or the therapy that induces tumor-induced PMN-MDSC depletion in the desired amount in an appropriate solvent and, as required, various other ingredients as described above, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques that produce a powder of the active ingredient plus any other desired ingredients present in a previously sterile-filtered solution.

[0094] For topical administration, the compositions and agents may be applied in pure form, ie, when they are liquids. However, it will generally be desirable to apply them topically to the skin as compositions in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

[0095] Useful solid carriers include finely divided solids, such as talc, clay, microcrystalline cellulose, silicon dioxide, aluminum oxide, etc. Useful liquid carriers include water, ethanol or ethylene glycol or water-ethanol / ethylene glycol blends, in which the peptides can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Additives such as fragrances and other antimicrobial agents can be added to optimize the performance for a given use. The resulting liquid composition can be applied from an absorbent pad, used to impregnate bandages and other dressings, or sprayed onto the affected area using, for example, a pump or aerosol sprayer.

[0096] Thickeners, such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials, may also be used with the liquid carrier to form a spreadable paste, gel, ointment, soap, etc., for direct application to the user's skin. Examples of useful dermatological compositions that can be used to deliver peptides to the skin are disclosed in Jacquet et al. (US Patent No. 4,608,392), Geria (US Patent No. 4,992,478), Smith et al. (US Patent No. 4,559,157) and Woltzman (US Patent No. 4,820,508).

[0097] Useful dosages of the pharmaceutical compositions of the invention can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known in the art; for example, see U.S. Pat. No. 4,938,949.

[0098] Accordingly, the present invention includes a pharmaceutical composition, which includes oncolytic viruses and / or the therapy of PMN-MDSC consumption induced by tumor induction, optionally in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions suitable for oral, topical or parenteral administration, including oncolytic viruses and / or the therapy of PMN-MDSC consumption induced by tumor induction, constitute a preferred embodiment of the present invention. In the context of the present invention, the dosage applied to the patient, particularly the dosage of the person should be sufficient to achieve a therapeutic response in the patient within a reasonable time frame, without fatal toxicity, and preferably cause side effects or morbidity not exceeding an acceptable level. It will be appreciated by those skilled in the art that dosage will depend on a variety of factors, including the condition (health) of the subject, the weight of the subject, the type of simultaneous treatment (if any), the frequency of treatment, the treatment ratio, and the severity and stage as a pathological condition. Advantageously, in some embodiments, the administration of the compounds of the present invention does not cause weight loss or obvious signs of toxicity in the subject.

[0099] A suitable dose results in a concentration of the active agent in a cancer tissue (e.g., a malignant tumor) that is known to achieve the desired response. A preferred dose is an amount that results in maximum inhibition of cancer cell growth without unmanageable side effects. Therapies of oncolytic viruses and induction of tumor-induced PMN-MDSC depletion, and optional administration of other agents can be continuous or at different intervals.

[0100] In order to provide such dosages for the desired treatment, in some embodiments, the pharmaceutical compositions of the present invention may contain between about 0.1% and 45%, especially 1 to 15%, based on the weight of the total composition including the carrier or diluent, based on the total weight of one or more agents of the present invention. Illustratively, the dosage level of the active ingredient administered may be: intravenous, 0.01 to about 20 mg / kg; intraperitoneal, 0.01 to about 100 mg / kg; subcutaneous, 0.01 to about 100 mg / kg; intramuscular, 0.01 to about 100 mg / kg; oral, 0.01 to about 200 mg / kg, preferably about 1 to 100 mg / kg; intranasal instillation, 0.01 to about 20 mg / kg; and aerosol, 0.01 to about 20 mg / kg animal (body weight)

[0101] definition

[0102] To facilitate understanding of the subject matter disclosed herein, many of the terms, abbreviations, or other shorthand used herein are defined below. Any term, abbreviation, or shorthand not defined should be understood to have the ordinary meaning used by a skilled artisan at the time of filing of this application.

[0103] As used herein, the term "subject" describes a mammal, including, but not limited to, humans, apes, chimpanzees, orangutans, monkeys, dogs, cats, horses, pigs, sheep, goats, mice, rats, and guinea pigs.

[0104] As used herein, the term "treat" or any grammatical variation thereof (e.g., treat, treating, treatment, etc.) includes, but is not limited to, ameliorating or alleviating the symptoms of a disease or condition; reducing or delaying the recurrence of a condition; reducing, suppressing, inhibiting, alleviating or affecting the progression and / or severity of an adverse physiological change or disease condition. For example, treatment includes, for example, preventing, inhibiting or slowing the development of cancer or the rate at which a benign cancer is transformed into a malignant cancer; slowing the growth and / or spread of cancer; and reducing the size or spread of cancer.

[0105] As used herein, the term "effective amount" refers to an amount that can treat or improve cancer or can otherwise produce a desired therapeutic effect. In certain embodiments, an effective amount can reduce the rate of tumor formation or cancer spread by 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, 99% or 100%. In certain embodiments, an effective amount can reduce tumor size or cancer spread by 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40%.

[0106] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" includes more than one such compound. In addition, to the extent that the terms "including," "comprising," "having," or variations thereof are used in the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising." The transitional terms / phrases (and any grammatical variations thereof) "comprising," "consisting essentially of," and "consisting of" may be used interchangeably.

[0107] The phrase "consisting essentially of" means that a claim covers embodiments that include the specified materials or steps, as well as embodiments that do not materially affect the basic and novel characteristics of the claim.

[0108] With regard to oncolytic viruses with gene deletions, the term "deletion" refers to genetic modifications to a gene, including any open reading frame, upstream regulatory region, and downstream regulatory region that result in downregulation or complete inhibition of transcription of the open reading frame (ORF) of the gene. Deletion can be achieved by deleting the entire ORF or a portion of the ORF, for example, by introducing: a frameshift mutation, a missense mutation, a sequence that destroys the activity of the protein encoded by the gene, a stop codon, or any combination thereof.

[0109] With respect to viruses containing a heterologous gene, the term "heterologous gene" includes an open reading frame and may further optionally comprise one or more other elements of a gene, such as an upstream regulatory region, a downstream regulatory region and / or a terminator.

[0110] Materials and methods

[0111] Mouse

[0112] All mice were maintained according to approved procedures. Female BALB / c and C57BL / 6N mice, 6–8 weeks old, were used.

[0113] Cell culture

[0114] Vero cells purchased from ATCC, and B16F10 cells (as a kind gift) were maintained in complete Dulbecco's modified Eagle medium (DMEM, Gibco; supplemented with 10% FBS and antibiotics). The AB1 cell line purchased from the European Collection of Cell Cultures was maintained in complete Roswell Park Memorial Institute-1640 medium (RPMI, Gibco; supplemented with 10% FBS, 2mM L-glutamine and antibiotics). Cells expressing luciferase were maintained in complete RPMI supplemented with 1 μg / ml puromycin (Invitrogen). T cells and splenocytes were cultured in complete RPMI supplemented with 50 μM 2-mercaptoethanol (Sigma).

[0115] Viral and in vitro infections

[0116] A highly attenuated MVTT virus encoding a dual reporter gene for HcRed and HIV-1p24 was prepared. MVTT virus stocks were prepared and viral titers were determined by plaque formation assay in Vero cells using serial dilutions of the virus. In vitro infections were performed in 24-well plates with 2x10 5 AB1 mesothelioma cells. 0.2MOI recombinant MVTT was added to the culture medium to allow 1 hour of attachment, and then the cells were washed and incubated with 1ml of fresh culture medium. Culture supernatants were collected 24, 48 and 72 hours after infection, and viral titers were measured by serial dilution and plaque formation assay in Vero cells. Anti-HMGB1 antibodies (Abcam, ab79823) were used to check the released HMGB1 by Western blotting. According to the manufacturer's instructions, the ATP released in the supernatant and cell viability were determined by CellTiter-Glo luminescent cell viability assay (Promega). Relative cell viability was calculated by the luminescence ratio between infected cells and uninfected cells. Cells were also separated and incubated with anti-CRT antibodies (Abcam, ab92516) for surface labeling and flow cytometry analysis. CRT expression in cell lysates was also determined by Western blotting. AB1-MVTT viral supernatants for antigen presentation analysis were collected 48 hours after infection. Cell debris was removed by centrifugation, passed through a 0.2 μm low protein binding membrane (Millipore), and heat inactivated for 1 hour at 60° C. Successful elimination of live virus was confirmed by plaque formation assay in Vero cells.

[0117] Tumor models and intratumoral treatments

[0118] Mesothelioma AB1 cells or melanoma B16F10 cells were harvested and injected subcutaneously with 5 × 10 5The cells were suspended in 100 μl PBS and then inoculated into the right hind flank of BALB / c or C57BL / 6N mice. The tumor volume was measured with a caliper and calculated using the formula: tumor volume = 1 / 2 (length × width 2 Luciferase-expressing tumors were also measured by bioluminescence imaging using the IVIS Spectrum (PerkinElmer) as described previously, and signal intensity was expressed as photons / s / cm in the region of interest (ROI) using Living Image software (version 4.0, PerkinElmer). 2 / sr. Intratumoral treatment of established tumors began 7 days after tumor inoculation. Tumors were injected with 100 μl of recombinant MVTT, anti-Ly6G antibody (clone 1A8, BioXCell), or a combination of both. 1A8 was administered at 100 μg per dose, and rat IgG2a (clone 2A3, BioXcell) was injected alone or in combination with recombinant MVTT as an isotype control. Mice that rejected tumors were treated with 2 × 10 6 All animals were euthanized when the tumor length exceeded 15 mm.

[0119] In vitro cell preparation

[0120] Splenocytes were isolated as described above. Tumors were cut into small pieces and digested with 1 mg / ml collagenase IV (Sigma) and 0.5 U / ml DNase I (Roche) for 1.5 hours at 37°C. Cells were passed through a 70 μm filter and then subjected to a 40% / 80% Percoll gradient analysis (Sigma). After centrifugation at 800 g for 20 minutes, interphase leukocytes were recovered. Bone marrow leukocytes were flushed out from the tibia and femur. The cells were then passed through a 70 μm filter and erythrocyte lysis buffer (BD Biosciences) was used to remove erythrocytes.

[0121] Isolation of T cells and MDSCs

[0122] Single cell suspensions of splenocytes were used for cell isolation. CD3 + T cells. CD4 + and CD8 + T cells. Total MDSCs or MDSC subsets were isolated using an MDSC isolation kit (Miltenyi) according to the manufacturer's instructions.

[0123] Adoptive MDSC transfer

[0124] Purified MDSCs were labeled with CFSE (Thermo Scientific). 4 × 10 6 Labeled MDSCs were detected 24 hours after transfer.

[0125] Cell consumption in the body

[0126] CD4 was depleted during treatment by intraperitoneal injection of 250 μg of anti-CD4 (YTS191.1, BioXcell) or anti-CD8 (YTS169.4, BioXcell) every 5 days, starting 1 day before treatment. + and CD8 + T cells. Successful T cell depletion was confirmed by flow cytometric analysis of peripheral blood mononuclear cells (PBMCs). Anti-Ly6G (clone 1A8) and the corresponding isotype (clone 2A3) were also purchased from BioXcell.

[0127] Measuring cytokine and chemokine production

[0128] Cytokine concentrations in culture supernatants were measured by LEGENDplex T Helper Cytokine Panel (BioLegend). Tumors were cut into small pieces and homogenized in T-PER Tissue Protein Extraction Reagent (Thermo Scientific) supplemented with Protease Inhibitor Cocktail (Roche). Chemokine concentrations were determined by LEGENDplex Proinflammatory Chemokine Panel (BioLegend) and normalized to total protein determined by BCA protein assay (Thermo Scientific). BMDC culture, in vitro antigen presentation and inhibition assays

[0129] According to standard protocols, isolated bone marrow cells were cultured at 3 × 10 6 Cells / well were seeded in 6-well plates. Half of the differentiation medium was changed every two days. On day 9, loosely adherent cells were resuspended by repeated pipetting and collected together with non-adherent cells in the supernatant for flow cytometry analysis and surface staining with anti-CD3, anti-CD11c, and anti-MHC II, resulting in >90% CD11c + MHC II +BMDCs. For BMDCs-T cell co-culture, BMDCs were pooled and cultured at 2 × 10 per well in the presence of 100 μl of inactivated AB1-MVTT viral supernatant or culture medium. 4 Cells were seeded into 96-well V-bottom plates. In some cultures, anti-CRT antibody (Abcam, ab92516) or rabbit IgG was added at 100 ng / ml. After overnight incubation, BMDCs were washed thoroughly with culture medium, and CFSE-labeled CD3 + T cells were cultured for another 10 days, with half of the culture medium replaced every 4 days. The culture supernatant collected on day 7 and the cells collected on day 10 were analyzed for cytokine secretion and T cell proliferation, respectively. For BMDC-MDSC co-culture, BMDCs were plated at 5 × 10 per well. 4 Cells were seeded in 96-well U-bottom plates and stimulated with 100 ng / ml LPS (Sigma) or 100 μl inactivated AB1-MVTT viral supernatant in the presence of purified PMN-MDSC or M-MDSC. In order to clearly distinguish BMDC from MDSC by flow cytometry, the purified MDSC subpopulation was labeled with CFSE before incubation with BMDC. BMDC maturation was assessed by flow cytometry 48 hours after LPS stimulation. When cells were stimulated with AB1-MVTT viral supernatant, half of the culture medium was replaced with fresh culture medium on day 4, and the supernatant was collected on day 7 to assess cytokine secretion.

[0130] IL-10 receptor blockade test

[0131] BMDCs were plated at 5 × 10 per well. 4 1×10 cells were seeded into 96-well U-bottom plates and incubated with 5 μg / ml anti-mouse CD210 (IL-10R, clone 1B1.3a, BioLegend) antibody at 37°C for 30 min. Then, 1×10 cells were stimulated with 100 ng / ml LPS in an incubator for 48 h. 5 CFSE-labeled PMN-MDSCs or M-MDSCs were added to the culture medium with BMDCs at a ratio of 2: 1. The volume of the culture was kept at 100 μl per well, and rat IgG1 (eBioscience) was used as an isotype control.

[0132] Flow cytometry

[0133] Cell surface and intracellular immunostaining were performed as described previously. The following antibodies were purchased from eBioscience: anti-CD11b (clone M1 / 70), anti-Ly6C (clone HK1.4), anti-Ly6G (clone 1A8-Ly6g), anti-CD3 (clone 17A2), anti-CD4 (clone GK1.5), anti-CD8 (clone 53-6.7), anti-PD1 (clone J43), anti-Tim3 (clone RMT3-23), anti-CD11c (clone N418), anti-MHC II (clone M5 / 114.15.2), anti-CD80 (clone 16-10A1), and anti-CD49b (clone DX5). The following antibodies were purchased from BioLegend: anti-CD25 (clone 3C7), anti-Foxp3 (clone 150D), anti-CXCR2 (clone SA045E1), and anti-CXCR3 (clone CXCR3-173). Anti-CCR2 (clone REA538) antibody was purchased from Miltenyi. Samples were run on a BD FACSAria II cell sorter (BD Biosciences) and analyzed using FlowJo (Tree Star, v10).

[0134] ELISpot and T cell cytotoxicity assays

[0135] IFN-γ-producing T cells in isolated splenocytes were evaluated by ELISpot assay. gp70-AH1 (SPSYVYHQF), OVA257-264 (SIINFEKL), GP100 (EGPRNQDWL), TRP2 (SVYDFFVWL), and TWIST1 peptides (15-mer, spanning the entire amino acid sequence with 11 amino acid overlaps) were synthesized by GL Biochem (Shanghai). The cytotoxic effect of purified T cells on AB1 cells was determined using the LIVE / DEAD Viability / Cytotoxicity Kit (Thermo Scientific) as described previously.

[0136] Statistical analysis

[0137] All data are presented as mean ± standard error. Significance was determined by two-tailed Student's t-test, and p value < 0.05 was considered statistically significant. The survival rates of all animals were plotted on Kaplan-Meier survival curves, and log-rank tests were performed to analyze differences in GraphPad Prism 5 software.

[0138] All patents, patent applications, provisional applications, and publications mentioned or cited herein, including all figures and tables, are incorporated by reference in their entirety to the extent they do not contradict the explicit teachings of this specification.

[0139] The following are examples illustrating the methods of implementing the present invention. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are by weight and all solvent mixture ratios are by volume.

[0140] Example 1 - Oncolytic effect of MVTT on mesothelioma cells triggers exposure of CRT and release of HMGB1 and ATP

[0141] To determine the oncolytic effect of MVTT, recombinant MVTT (rMVTT) was generated to simultaneously express two detection markers, HIV-1p24 and the far-red fluorescent mutant HcRed ( Figure 1 A). Expression of both markers facilitates detection of viral replication as well as encoded gene expression. MVTT has a broad mammalian cell infection range. AB1 mesothelioma cells are susceptible to rMVTT infection and show the presence of red fluorescent syncytia ( Figure 1 B) and expression of virally encoded p24 protein ( Figure 1 C). The increase in HcRed signal and the free virus released over time indicate that rMVTT virus can infect AB1 cells and replicate in them ( Figure 1 D-1E). The oncolytic capacity of rMVTT was subsequently determined, showing that viral infection significantly reduced AB1 cell viability ( Figure 2 A). Calreticulin (CRT) is a DAMP that is normally located in the lumen of the endoplasmic reticulum and is transferred to the surface of dying cells after the induction of immunogenic apoptosis, where it acts as an "eat-me" signal for professional phagocytes. Therefore, the expression of CRT protein in AB1 cells after MVTT infection was determined by flow cytometric analysis. When infected with 0.2 MOI rMVTT, less than 5% of AB1 cells exposed CRT on their surface after 24 h. However, due to active viral replication, this percentage increased to 70% and 90% at 48 and 72 h after infection, respectively ( Figure 2 Importantly, all CRT-positive cells showed HcRed expression, indicating that rMVTT infection was responsible for CRT protein exposure ( Figure 2 B, right). In addition, Western blot analysis also showed that rMVTT infection led to upregulated expression of CRT protein in AB1 cells ( Figure 2C). In addition to CRT protein, the release of other DAMPs such as high mobility group box 1 (HMGB1) and ATP from dying cells may activate antigen presenting cells (APCs) to enhance antitumor immunity. Therefore, the expression of CRT and HMGB1 proteins was measured to test the possibility that oncolysis may lead to immunogenic cell death. HMGB1 protein was readily detected in the culture supernatant 72 h after rMVTT infection but not in uninfected AB1 cell controls ( Figure 2 D). In addition, after rMVTT infection, the ATP released in the supernatant also increased significantly over time ( Figure 2 E). Thus, oncolytic effect of rMVTT on AB1 mesothelioma cells induced upregulated expression and exposure of CRT as well as release of ATP and HMGB1 from dying cells, which are recognized as three major hallmarks of immunogenic cell death that stimulate adaptive antitumor immune responses.

[0142] Example 2 - rMVTT treatment dose-dependently eliminated established AB1 tumors but failed to enhance anti-tumor T cell immunity

[0143] To investigate the therapeutic capacity of rMVTT in established AB1 mesothelioma in Balb / c mice, it injection was explored as a means to determine its direct antitumor efficacy. Mice were inoculated with AB1 mesothelioma cells ( Figure 3 A). AB1 mesothelioma growth was significantly inhibited in all mice treated with rMVTT ( Figure 3 B). In addition, observation of tumor growth in individual mice showed that high-dose virus treatment completely abolished tumor growth ( Figure 3 C), resulting in 100% survival ( Figure 3 D), while the medium-dose and low-dose groups showed reduced antitumor efficacy, with only 37.5% and 50% of the mice remaining tumor-free, respectively ( Figure 3 B-3D), indicating that rMVTT treatment eliminated established AB1 mesothelioma in a dose-dependent manner. The oncolytic effect of rMVTT can create an immunostimulatory environment to induce an immune response against AB1 tumor antigens. Therefore, two tumor antigens were tested by immunological methods, namely the immunodominant AH1 (gp70 423–431) and twist-related protein 1 (TWIST1) peptides. The peptide gp70-AH1 is a well-characterized immunodominant CTL epitope derived from endogenous murine leukemia virus glycoprotein 70 (gp70). The expression of the transcription factor TWIST1 is essential for the metastatic process of tumors and their resistance to drug treatment. Since both gp70-AH1 and TWIST1 were detected simultaneously in AB1 cells, the presence of antitumor T cell responses could be explored by ELIspot and the differences between tumor-bearing and tumor-free mice could be compared. Splenocytes from only one treated and tumor-free mouse showed an AH1-specific ELIspot response ( Figure 3 E) and cytotoxic effects against AB1 cells ( Figure 3 F). There was no statistically significant difference in the induction of antitumor T cell responses between tumor-bearing mice and tumor-free mice ( Figure 3 E-3F). Thus, although rMVTT treatment dose-dependently eliminated established AB1 mesothelioma, tumor oncolysis did not readily induce antitumor T cell immunity.

[0144] Example 3 - rMVTT treatment leads to accumulation of PMN-MDSCs in the TME

[0145] Since the initiation of adaptive antitumor immunity after oncolysis occurs primarily within the tumor, the TME after rMVTT treatment was examined. At two time points after intratumoral rMVTT treatment (day 2 and day 4), different tumor-resident immune cells, including CD3 + T cells, natural killer (NK) cells, CD4 + Treg (CD4 + CD25 + Foxp3 + ) and MDSC subsets (PMN-MDSC, CD11b + 6G + Ly6C low / int ;M-MDSC, CD11b + 6G - Ly6C hi ) ratio, and in CD3 + Expression of PD-1 and Tim-3, exhaustion surface markers on T cells. MDSC and Treg are major components of the tumor suppressor microenvironment. After rMVTT treatment, the overall level of MDSC found in the spleen appeared to decrease over time, while the frequency of tumor-infiltrating MDSC remained at a similar level ( Figure 4A). We then examined the two main subpopulations of MDSCs, PMN-MDSCs and M-MDSCs, as these two subpopulations exhibit significant differences in morphology and suppressive properties. While PMN-MDSCs are abundantly expanded in peripheral lymphoid organs, M-MDSCs preferentially accumulate within tumors ( Figure 4 B). In addition, rMVTT treatment did not affect the frequency of M-MDSCs in the spleen or tumors, but PMN-MDSCs were significantly reduced in the spleen and increased in tumors during rMVTT treatment ( Figure 4 B-4C). Consistently, in response to rMVTT treatment, the absolute PMN-MDSC cell number in tumors also increased significantly ( Figure 4 D) For comparison, although rMVTT treatment reduced CD4 + The frequency of Treg cells was 2.3 fold, but no significant differences were found in their frequency or cell number in tumors ( Figure 4 E). Interestingly, PMN-MDSCs accumulated significantly in tumors as early as day 2 after rMVTT treatment. In contrast, the frequency and number of NK cells were significantly reduced ( Figure 4 F), suggesting a possible counter-action between these two cell types. The inflammatory response induced by viral infection may increase the infiltration of lymphocytes into the tumor. Indeed, intratumoral CD3 + The infiltration of T cells increased significantly ( Fig.10 D). However, the increased T cell infiltration was accompanied by significantly elevated expression of the exhaustion markers PD-1 and Tim-3 ( Fig.10 E) Thus, rMVTT treatment altered the local and systemic distribution of a group of immune cells, specifically leading to a significant accumulation of PMN-MDSCs in the TME.

[0146] Example 4 - PMN-MDSC trafficking to tumor sites after intratumoral rMVTT treatment

[0147] To understand how PMN-MDSCs are recruited to tumors, the effects of chemokines induced by rMVTT treatment were determined. Flow cytometric analysis of chemokine receptors showed that CXCR2 was expressed only on PMN-MDSCs but not on M-MDSCs. In contrast, high levels of CCR2 expression were found on M-MDSCs but not on PMN-MDSCs ( Figure 5 A). Levels of various chemokines were measured after rMVTT treatment. As early as 2 days after treatment, a panel of CXC chemokines (including CXCL5, CXCL9, and CXCL13) were significantly upregulated in tumors ( Figure 5 B), whereas upregulated CC chemokine production was observed only at 4 days ( Figure 5C). These results suggest that CXCR2-expressing PMN-MDSCs may migrate and adhere to the tumor bed, primarily in response to increased CXC chemokines. To support this notion, CFSE-labeled MDSCs derived from mesothelioma-bearing mice were subcultured and transferred into recipient mice bearing the same tumors but treated with rMVTT or PBS after transfer. CFSE-labeled MDSCs were quantified in spleens and tumors by flow cytometry 24 hours after rMVTT treatment. CFSE was observed in tumors of rMVTT-treated recipients compared to PBS-treated recipients. + Both the percentage and absolute number of MDSCs increased significantly ( Figure 5 D) Ly6G expression can be used to distinguish PMN-MDSCs from M-MDSCs migrating in tumors ( Figure 5 E). In addition, spleens showed a slightly decreased PMN- / M-MDSC ratio in rMVTT-treated recipients, whereas tumors showed a significantly increased PMN- / M-MDSC ratio and the absolute number of PMN-MDSCs ( Figure 5 F) Thus, in response to chemotaxis induced by rMVTT treatment, PMN-MDSCs preferentially migrate from the peripheral lymphatic system to the TME.

[0148] Example 5 - Disruption of PMN-MDSC tumor trafficking after rMVTT treatment

[0149] To prevent MDSC migration into tumors, the efficacy of the MDSC-depleting antibody anti-Ly6G monoclonal antibody 1A8 was tested. + MDSCs, therefore AB1 tumor-bearing mice were treated with 1A8 or isotype control by the it route. The frequency of splenic MDSCs was significantly reduced in mice treated with 1A8 compared to the isotype control, but the antibody did not show efficacy in reducing total MDSC accumulation in tumors. However, as expected, 1A8 selectively reduced Ly6G in the spleen and tumors on day 2 after injection. + PMN-MDSCs Figure 6 A). Although this effect was maintained in the tumor at day 4, splenic PMN-MDSCs began to reappear ( Figure 6 A-6B). Unlike PMN-MDSCs, the frequency of M-MDSCs in tumors was not affected by 1A8, as a significant increase in splenic M-MDSCs was observed ( Figure 6 A-6B), which may be due to the continuous generation of PMN-MDSC from the bone marrow. Subsequently, the effect of 1A8 combined with rMVTT was studied. rMVTT treatment resulted in an expanded population of PMN-MDSC in the tumor. However, on day 2, this expanded population was almost eliminated by the 1A8 antibody ( Figure 6C). Despite a significant increase in the frequency of splenic PMN-MDSCs, 1A8 continued to prevent tumor trafficking of PMN-MDSCs at day 4 ( Figure 6 D) Therefore, administration of anti-Ly6G 1A8 can specifically disrupt MVTT-induced tumor trafficking of PMN-MDSCs.

[0150] Example 6 - Combination of MVTT-based oncolysis and PMN-MDSC depletion restores anti-tumor T cell immunity

[0151] Given that MDSCs are one of the main immunosuppressive cell types that inhibit antitumor T cell responses, it was examined whether preventing MVTT-induced tumor trafficking of PMN-MDSCs would enhance the efficacy of oncolytic virus treatment. In a setting similar to that described above, Balb / c mice bearing 7-day-old AB1 tumors were co-injected with rMVTT plus 1A8 or isotype control. To improve the antitumor effect, an additional combination treatment was performed two days later ( Figure 7 A). A single combined treatment slowed tumor growth and resulted in tumor regression in 1 / 7 mice, whereas depletion of PMN-MDSCs by 1A8 alone did not affect tumor growth ( Figure 7 B-7C). Crucially, the second combined treatment effectively controlled tumor growth and ultimately led to complete elimination of established AB1 mesothelioma ( Figure 7 To determine whether prolonged antitumor T cell immunity was generated in these controller mice, a higher dose (2 × 10 6 cells) were challenged with AB1 cells (AB1-Luc) that stably expressed firefly luciferase Figure 7 A). After 11 days, complete rejection of the AB1-Luc tumors was observed in these control mice, resulting in tumor-free survival for >30 weeks, whereas all control mice developed tumors ( Figure 7 D-7E).

[0152] Therefore, the depletion of PMN-MDSCs can greatly improve the effect of rMVTT treatment by inducing prolonged anti-tumor immunity. To test this, tumor-specific T cell responses were measured. Mouse splenocytes were harvested and tested against the tumor antigen gp70-AH1 or TWIST1 peptide. In mice treated with the rMVTT+1A8 combination, a significantly increased T cell response against gp70-AH1 and TWIST1 was induced ( Figure 7 F). In vitro CTL assays also demonstrated enhanced CD8 + Cytotoxic T cells ( Figure 7G) In addition, CD4+ T cells were depleted using monoclonal antibodies before AB1 tumor-bearing mice were treated with rMVTT+1A8 combination. + or CD8 + T cells ( Figure 7 H). It is noteworthy that CD8 + Depletion of YTS169.4 T cells completely abolished the antitumor activity of the combination treatment, resulting in rapid tumor growth and death of all mice within 21 days. + Depletion of T cells (YTS191.1) still retained the therapeutic effect and resulted in tumor regression in 3 / 5 mice ( Figure 7 I-7K). Therefore, CD8 + T cells are essential for this MVTT-based immuno-oncolytic approach. Moreover, depletion of PMN-MDSCs during local rMVTT treatment can restore effective systemic anti-tumor T cell immunity.

[0153] Example 7 - PMN-MDSC induce anti-tumor T cell immunity by limiting the activation tissue of dendritic cells

[0154] As described above, MVTT-induced tumor oncolysis establishes an immune activation environment with CRT, HMGB1, and ATP production. However, dendritic cells (DCs) are unable to recognize and integrate these signals to drive T cell activation. The presence of PMN-MDSCs during MVTT-induced tumor oncolysis may inhibit DC function. To test this, the direct effects of PMN-MDSCs on DCs were determined. The ability of bone marrow-derived DCs (BMDCs) to process and present antigens to activate CD3 T cells from control mice treated with the MVTT+1A8 combination was determined. + T cells. The supernatant of AB1 cells infected with MVTT was used as a source of tumor antigen pool to prime BMDC. CD3 + When co-cultured with T cells, antigen-loaded BMDCs significantly increased the production of TNF-α and IFN-γ ( Figure 8 A), indicating T cell activation in response to tumor antigens. At the same time, it was tested whether surface-exposed CRT protein would chaperone multiple tumor antigens to promote DC uptake. Indeed, inhibition of this process by anti-CR-sensitized T antibodies significantly reduced the production of TNF-α and IFN-γ ( Figure 8 A) To confirm this, T cell proliferation was measured. Antigen-sensitized BMDCs can effectively induce CD4 + and CD8 + T cell proliferation ( Figure 8B), thus demonstrating the activation of tumor antigen-specific T cells. Again, the presence of anti-CRT antibodies can inhibit T cell proliferation ( Figure 8 B), indicating the role of CRT in activating the DC-T cell axis. Therefore, in the absence of an immunosuppressive environment, the oncolytic effect of rMVTT on tumor cells can effectively induce the activation and antigen presentation of BMDCs.

[0155] Subsequently, direct interactions between AB1-induced MDSCs and BMDCs were measured using either medium alone or LPS as a maturation signal. As expected, LPS alone significantly increased the levels of CD80 expression on BMDCs (P < 0.0001, Med vs. LPS) ( Figure 8 C). Notably, when MDSCs were present in the co-culture, only PMN-MDSCs, but not M-MDSCs, significantly suppressed CD80 expression on both unstimulated and LPS-stimulated BMDCs ( Figure 8 C). Whether similar inhibitory effects from PMN-MDSCs could be observed was tested in a more relevant model in which BMDCs were primed with supernatant of AB1 cells infected with MVTT instead of LPS. Cytokine secretion was measured in co-cultures as a probe of BMDC activation. BMDCs were more sensitive to PMN-MDSC-mediated inhibition, with reduced production of IL-6 and TNF-α compared to co-cultures of M-MDSCs and BMDCs. The immunosuppressive cytokine IL-10 is well known to block the DC maturation process and limit the ability of DCs to elicit a Th1 response. Indeed, only PMN-MDSCs showed a subpopulation that produced IL-10 ( Figure 8 D), and release relatively high levels of IL-10 in culture. Therefore, PMN-MDSC can directly inhibit DC activation induced by tumor oncolysis. Therefore, removal of PMN-MDSC can rescue DC function, thereby initiating adoptive antitumor immunity.

[0156] Furthermore, the efficacy of the combination therapy was also confirmed in a different syngeneic C57BL / 6 melanoma model, where enhanced B16F10 tumor regression, prolonged survival, and antitumor T cell responses were observed ( Fig. 9 AC), further demonstrating the efficacy of the MVTT-based immuno-oncolytic approach.

[0157] Example 8 - MVTT treatment recruits PMN-MDSCs into the TME

[0158] Since the initiation of adaptive antitumor immunity after oncolysis occurs primarily within the tumor, the TME was examined after rMVTT treatment. Analysis of AB1 mesothelioma injected with rMVTT showed that expression of virally encoded HcRed was readily detectable 2 days after intratumoral injection and then rapidly declined ( Fig.10 A). Consistently, immunohistochemical staining for vaccinia virus proteins was only found in tumor tissues 2 days but not 4 days after rMVTT treatment, with visible necrotic areas within and adjacent to the infected areas ( Fig.10 B) These results demonstrate rapid but limited replication of rMVTT in the TME. Different tumor-resident immune cells were then measured, including CD3 + T cells, natural killer (NK) cells, CD4 + Tregs (CD4 + CD25 + Foxp3 + ) and MDSC subsets (PMN-MDSC, CD11b + 6G + Ly6C low / int ;M-MDSC, CD11b + Ly6G-Ly6C hi ) and CD3 + Expression of PD-1 and Tim-3, exhaustion markers on T cells ( Fig.10 C). The overall level of MDSCs in the spleen appears to decrease during rMVTT treatment, while the frequency of tumor-infiltrating MDSCs remains at a similar level ( Figure 4 A). The two major subpopulations of MDSCs, PMN-MDSCs and M-MDSCs, were examined because they differ significantly in morphology and suppressive activity. PMN-MDSCs expanded abundantly in peripheral lymphoid organs, whereas M-MDSCs preferentially accumulated within tumors of untreated control mice ( Figure 4 B). In addition, rMVTT treatment did not affect the frequency of M-MDSCs in the spleen or tumor. However, PMN-MDSCs were significantly reduced in the spleen and increased in the TME ( Figure 4 B and 4C). After rMVTT treatment, the absolute number of PMN-MDSCs in the tumor also increased significantly ( Figure 4 D) For comparison, although rMVTT treatment reduced CD4 + The frequency of Tregs was 2.3 fold higher than that of controls, but no significant differences were found in their frequency or cell number in tumors ( Figure 4 E). In contrast to the significant accumulation of PMN-MDSCs in tumors as early as day 2 after rMVTT treatment, the frequency and cell number of NK cells were significantly reduced ( Figure 4 F), suggesting a possible counter-action between these two cell types. Infection-induced inflammatory responses have been shown to increase lymphocyte infiltration into the TME. Indeed, CD3 + T cells increased significantly ( Fig.10 D). However, the increased T cell infiltration was coupled with significantly elevated expression of the exhaustion markers PD-1 and Tim-3 ( Fig.10 E). Overall, rMVTT treatment altered the local and systemic distribution of immune cells, specifically the accumulation of PMN-MDSCs in the TME.

[0159] Example 9 - Trafficking of PMN-MDSCs into the TME via MVTT-induced chemotaxis

[0160] To determine whether PMN-MDSCs could be preferentially recruited to the TME after rMVTT treatment, the expression of chemokine receptors and the levels of chemokines on both MDSC subsets in rMVTT-treated tumors were determined. Flow cytometric analysis of chemokine receptor expression showed that CXCR2 was expressed only on PMN-MDSCs but not on M-MDSCs. In contrast, high levels of CCR2 expression were found on M-MDSCs but not on PMN-MDSCs ( Figure 5 A). The levels of various chemokines were then measured in tumor homogenates after rMVTT treatment. As early as 2 days after treatment, a panel of CXC chemokines (including CXCL5, CXCL9, and CXCL13) were significantly upregulated in AB1 mesothelioma ( Figure 5 B), whereas upregulation of CC chemokine production was observed only 4 days after treatment ( Figure 5 C). Therefore, PMN-MDSC expressing CXCR2 may migrate and adhere to the tumor bed, mainly in response to the rapidly increasing CXC chemokines in the TME. To test this hypothesis, CFSE-labeled MDSC derived from mesothelioma-bearing mice were adoptively transferred into recipient mice also bearing mesothelioma tumors, but were treated with rMVTT or PBS after MDSC transfer. CFSE-labeled MDSC in spleen and mesothelioma were then quantified by flow cytometry 24 hours after rMVTT treatment ( Fig.11 CFSE was observed in tumors of rMVTT-treated recipients compared with PBS-treated recipients. + Both the percentage and absolute number of MDSCs increased significantly ( Figure 5 D) Ly6G expression can be used to distinguish PMN-MDSCs from M-MDSCs migrating in tumors ( Figure 5E). In rMVTT-treated recipients, spleens showed a slightly decreased PMN- / M-MDSC ratio, whereas their tumors exhibited a significantly increased PMN- / M-MDSC ratio and the absolute number of PMN-MDSCs ( Figure 5 E and 5F). Overall, PMN-MDSCs preferentially migrated from the peripheral lymphatic system to the TME in response to rMVTT treatment-induced chemotaxis.

[0161] Example 10 - Preferential depletion of MDSC subsets by antibodies and peptibodies

[0162] To investigate the role of MDSCs in rMVTT treatment, two MDSC depleting agents, the anti-Ly6G monoclonal antibody 1A8 and the specific depleting peptibody H6-pep, were explored in our mesothelioma model. 1A8 is commonly used to deplete Ly6G + cells, mainly PMN-MDSCs, while H6-pep and G3-pep are two peptide bodies with binding specificity to PMN-MDSCs and M-MDSCs. Therefore, these two peptide antibodies were prepared in 293F cells by transient expression system using expression plasmids ( Fig.12 A). H6-pep showed higher binding affinity than G3-pep to total MDSCs derived from AB1 mesothelioma-bearing mice ( Fig.12 B and 12C). Therefore, H6-pep was used in the elimination experiments. When tumor-bearing mice were treated with intratumoral injections of 1A8 or H6-pep, only the frequency of splenic MDSCs was significantly reduced in 1A8-treated mice, whereas neither 1A8 nor H6-pep appeared to reduce total MDSC accumulation in the tumor ( Fig.12 D) However, 1A8 selectively reduced Ly6G in spleen and tumor at day 2 post-injection + PMN-MDSCs Figure 6 A and 6B). Although this effect was maintained in tumors at day 4, spleen but not TME PMN-MDSCs reappeared. Unlike PMN-MDSCs, tumor M-MDSCs were not affected by 1A8, whereas a significant increase in spleen M-MDSCs was observed compared with isotype controls, likely due to the continuous generation of MDSCs from the bone marrow. In contrast, due to the higher binding affinity of H6-pep for M-MDSCs, H6-pep treatment significantly depleted M-MDSCs but not PMN-MDSCs, particularly in the TME; this effect persisted until day 4 ( Fig.12 E and 12F). After M-MDSC depletion, a clear compensatory increase in the frequency of splenic PMN-MDSC was observed.

[0163] The efficacy of 1A8 and H6-pep during rMVTT treatment was then investigated. rMVTT treatment resulted in increased recruitment of PMN-MDSCs in tumors ( Figure 6 A and 6C). However, this increased population was almost eliminated by 1A8 antibody treatment on day 2 ( Figure 6 C and 6D). 1A8 also prevented tumor recruitment of PMN-MDSCs at day 4, although the frequency of splenic PMN-MDSCs was significantly increased. In contrast, H6-pep treatment decreased M-MDSCs in both spleen and tumor, while increasing PMN-MDSCs ( Fig.12 G and 12H). Thus, administration of 1A8 and H6-pep, respectively, preferentially depleted PMN-MDSCs and M-MDSCs and maintained their depletion effects even after rMVTT administration, allowing us to investigate the impact of PMN-MDSCs and M-MDSCs on the induction of antitumor immunity during MVTT-based oncolytic virus therapy.

[0164] Example 11 - Depletion of PMN-MDSCs enhances MVTT therapeutic efficacy by inducing anti-tumor T cell immunity

[0165] Considering that MDSCs are one of the main immunosuppressive cells that inhibit antitumor T cell responses, we investigated whether the depletion of PMN-MDSCs enhances the efficacy of MVTT-based oncolytic virus therapy. In a similar situation as above, BALB / c mice bearing 7-day-old wild-type AB1 mesothelioma were simultaneously injected with low-dose rMVTT (1×10 7 PFU) and 100 μg 1A8 or H6-pep for specific depletion of PMN-MDSC and M-MDSC ( Fig.13 A). A single delivery of low-dose rMVTT failed to control tumor growth. However, incorporating MDSC depletion in this setting did not slow tumor progression or prolong survival ( Fig.13 B and 13C). Considering the known dose-dependent effects of rMVTT treatment, it would be reasonable to supplement the antitumor effect by an additional low dose 2 days later ( Figure 7 A). Two rMVTT treatments alone slowed tumor growth and resulted in tumor regression in 1 / 7 mice, whereas 1A8 alone had no effect on tumor growth at all ( Figure 7 B and 7C). However, strikingly, a second low-dose rMVTT combined with 1A8 effectively controlled tumor growth and ultimately led to complete elimination of established AB1 mesothelioma ( Figure 7 B and 7C). In contrast, combined rMVTT and H6-pep treatment showed no significant antitumor activity or synergistic effect in mesothelioma elimination ( Fig.13To determine whether prolonged antitumor T cell immunity was generated in these controller mice, their contralateral flanks were treated with a higher dose (2 × 10 6 cells) of AB1-Luc cells to attack these mice ( Figure 7 A). Complete rejection of the AB1-Luc mesothelioma was observed in these control mice after 11 days, resulting in tumor-free survival for >30 weeks, whereas all mice from the control group developed tumors ( Figure 7 D and 7E). These results suggest that depletion of PMN-MDSCs but not M-MDSCs significantly improves the efficacy of rMVTT treatment, possibly by inducing prolonged antitumor immunity.

[0166] To further test this hypothesis, tumor-reactive T cell responses were measured. Mouse splenocytes were harvested and tested against gp70-AH1 or TWIST1 peptides ( Figure 7 A). T cell responses against gp70-AH1 and TWIST1 were significantly increased in mice treated twice with a combination of low-dose rMVTT and 1A8 ( Figure 7 F). Depletion of M-MDSCs with a dual rMVTT and H6-pep combination did not reveal this enhancement ( Fig.13 F) In addition, in vitro cytotoxicity assays demonstrated enhanced CD8 + CTL( Figure 7 G) In addition, CD4 T cells were depleted using monoclonal antibodies YTS191.1 and YTS169.4, respectively, before AB1 tumor-bearing mice were treated with rMVTT and 1A8. + or CD8 + T cells ( Figure 7 H). Notably, YTS169.4 depletes CD8 + T cells completely reduced the antitumor activity of the combination therapy, resulting in uncontrolled tumor growth and death of all mice within 21 days. In contrast, depletion of CD4+ T cells by YTS191.1 retained partial therapeutic efficacy and resulted in tumor regression in 3 / 5 mice ( Figure 7 I-7K). To determine whether this finding could be applied to other malignancies, the efficacy of rMVTT and 1A8 combination therapy was tested in a different syngeneic C57BL / 6 melanoma model. Similarly, this combination therapy resulted in enhanced B16F10 tumor regression, prolonged survival, and enhanced anti-tumor T cell responses ( Fig.13 G-13I). Collectively, PMN-MDSC depletion during local MVTT-based oncolytic virus therapy elicited robust systemic and persistent antitumor T cell immunity.

[0167] Example 12 - PMN-MDSCs prevent the induction of anti-tumor T cell immunity by limiting the activation of dendritic cells

[0168] Although rMVTT-induced oncolysis established an immune activation environment with CRT, HMGB1, and ATP production, anti-mesothelioma-specific T cell responses were not easily induced ( Figure 3 E and 3F). However, this situation completely changed when PMN-MDSCs were depleted during rMVTT treatment ( Figure 7 F and 7G). Therefore, PMN-MDSC may have an inhibitory effect on DCs through direct crosstalk in the TME of our model. To test this possibility, the direct effects of PMN-MDSC on DCs were examined. First, the ability of bone marrow-derived DCs (BMDCs) to process and present activated CD3 + T cell antigen capacity, the CD3 + T cells were derived from control mice treated with a combination of rMVTT and 1A8. The supernatant of AB1 cells treated with rMVTT was used as a tumor antigen supply for sensitizing BMDCs. When BMDCs were sensitized with antigens, a significant increase in the production of the proinflammatory cytokine IL-6 was observed in the co-culture ( Fig.14 A) At the same time, CD3 + When co-cultured with T cells, antigen-loaded BMDCs significantly increased the expression of TNF-α and IFN-γ ( Figure 8 A) and Th17 cytokines IL-17A and IL-22 ( Fig.14 A) production, suggesting T cell activation in response to tumor antigens. It has been previously shown that surface-exposed CRT protein accompanies tumor antigens to promote their uptake by DCs. Indeed, anti-CRT antibodies significantly reduced the production of TNF-α and IFN-γ ( Figure 8 A). To confirm these findings, T cell proliferation was measured. Antigen-primed BMDCs efficiently induced + and CD8 + T cell proliferation ( Figure 8 B), demonstrating the activation of tumor antigen-specific T cells. Again, the presence of anti-CRT antibodies inhibited T cell proliferation ( Figure 8 B), indicating a role for CRT in activating the DC-T cell axis. Thus, in the absence of PMN-MDSCs, rMVTT-induced CRT exposure enhances BMDC activation, thereby eliciting potent anti-tumor T cell immunity.

[0169] Subsequently, the direct interaction between AB1-induced MDSCs and BMDCs was measured. BMDCs were co-cultured with AB1-induced MDSCs in the presence or absence of LPS. LPS stimulation significantly upregulated the expression of CD80 and CD86 on BMDCs (P < 0.001 for CD80 and P < 0.05 for CD86, unstimulated vs LPS), indicating that BMDCs matured ( Figure 8 C). Notably, when MDSCs were present in the co-culture, PMN-MDSCs, but not M-MDSCs, significantly suppressed the expression of CD80 and CD86 on both unstimulated and LPS-stimulated BMDCs ( Figure 8 C). Changes in LPS-induced cytokine production were also analyzed. Supernatants collected from BMDCs without LPS consistently showed very low levels of cytokines. In contrast, culture supernatants with LPS resulted in a significant increase in the proinflammatory cytokines IL-6 and TNF-α, as well as the type 1 cytokine IL-12p70 ( Fig.14 B). Consistent with the ability of PMN-MDSC to downregulate BMDC activation, the presence of PMN-MDSC in co-cultures significantly inhibited the induction of IL-6, TNF-α, and IL-12p70, further supporting a role for PMN-MDSC in inhibiting BMDC activation ( Fig.14 B). We then tested whether PMN-MDSCs had a similar inhibitory effect when BMDCs were sensitized with supernatant of AB1 cells treated with rMVTT instead of LPS. By measuring cytokines associated with BMDC activation, PMN-MDSCs, but not M-MDSCs, significantly inhibited the production of IL-6 and TNF-α in co-culture, and the inhibitory effect of PMN-MDSCs on TNF-α was dose-dependent ( Fig.14 C).

[0170] To understand the potential mechanisms of PMN-MDSC-mediated immunosuppression, we examined the production of IL-10 and TGF-β in MDSC subsets. MDSCs do not produce TGF-β, and only PMN-MDSCs exhibit an IL-10-producing subset ( Figure 8 D). In addition, when PMN-MDSCs were co-cultured with BMDCs in vitro ( Figure 8 E), and after MVTT treatment in vivo ( Fig.14D), IL-10 production is enhanced. It is well known that the immunosuppressive cytokine IL-10 inhibits DC maturation and prevents DC from initiating a Th1 response. Crosstalk between MDSC and macrophages has been reported to reduce IL-12 production by macrophages and increase IL-10 production by MDSC to promote tumor progression. Therefore, the suppressive capacity of PMN-MDSC may depend on their IL-10 production. To test this, purified PMN-MDSC or M-MDSC derived from AB1-bearing mice were co-cultured with LPS-activated BMDC in the presence of IL-10 receptor blocking antibodies or isotype controls. Comparing the expression of activation markers on BMDC, the presence of PMN-MDSC consistently downregulated CD80 and CD86 expression on BMDC ( Figure 8 F). However, PMN-MDSC-mediated suppression can be partially alleviated by blocking IL-10 receptors ( Figure 8 F). In addition, the secreted cytokines in the supernatant were detected, and blocking the IL-10 receptor also significantly increased the production of TNF-α and IL-12p70 ( Fig.14 E), indicating that IL-10 production by PMN-MDSCs appears to be a direct inhibitory means in our in vitro inhibitory assay. Overall, although rMVTT treatment promotes CRT-dependent antigen uptake and activation and antigen presentation of BMDCs, PMN-MDSCs may directly inhibit DC activation and lead to reduced efficacy or failure of oncolytic virus treatment.

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Claims

1. Use of a combination of an oncolytic virus and a therapeutic agent in the preparation of a medicament for treating cancer in a subject, wherein the therapeutic agent is a therapeutic agent that induces the consumption of tumor-induced polymorphonuclear bone marrow myeloid-derived suppressor cells, wherein the oncolytic virus is a modified MVTT virus having a viral M1L-K2L gene deletion; wherein the therapeutic agent that induces the consumption of tumor-induced polymorphonuclear bone marrow myeloid-derived suppressor cells is an antibody against Ly6G; wherein the cancer is mesothelioma or melanoma.

2. The use according to claim 1, wherein the MVTT comprises a heterologous polynucleotide replacing the M1L-K2L of the deleted virus.

3. The use according to claim 1 or 2, wherein the oncolytic virus and / or the therapeutic agent that induces depletion of tumor-induced polymorphonuclear bone marrow myeloid-derived suppressor cells is administered multiple times over a period of two to fourteen days.

4. The use according to claim 1 or 2, wherein the oncolytic virus and / or the therapeutic agent that induces the depletion of tumor-induced polymorphonuclear bone marrow myeloid-derived suppressor cells is administered by intratumoral injection.

5. The use according to claim 1 or 2, wherein the medicament is combined with one or more other anti-cancer therapeutic agents.

6. The use of claim 5, wherein the one or more other anti-cancer therapeutic agents include chemotherapeutic drugs, checkpoint inhibitors, adjuvants, anemia drugs, radiotherapeutic agents, stem cells, T cells expressing chimeric antigen receptors (CAR) (CAR T cells), or a combination of two or more of the foregoing.

7. The use according to claim 6, wherein the checkpoint inhibitor is an inhibitor of: CTLA4, PD-1 and PD-L1.

8. The use according to claim 7, wherein the inhibitor of CTLA4 is an antibody that binds to CTLA4, the inhibitor of PD-1 is an antibody that binds to PD-1, and the inhibitor of PD-L1 is an antibody that binds to PD-L1.

9. The use of claim 1 or 2, wherein the subject is a human.

10. A composition comprising an oncolytic virus, a therapeutic agent that induces the depletion of tumor-induced polymorphonuclear bone marrow myeloid-derived suppressor cells, and a pharmaceutically acceptable carrier, wherein the oncolytic virus is a modified MVTT virus having a viral M1L-K2L gene deletion; wherein the therapeutic agent that induces the depletion of tumor-induced polymorphonuclear bone marrow myeloid-derived suppressor cells is an antibody against Ly6G.

11. The composition of claim 10, wherein the MVTT comprises a heterologous polynucleotide that replaces M1L-K2L of the deleted virus.

12. The composition of claim 10 or 11, wherein the therapeutic agent that induces depletion of tumor-induced polymorphonuclear bone marrow myeloid-derived suppressor cells specifically induces depletion of tumor-induced polymorphonuclear bone marrow myeloid-derived suppressor cells without affecting tumor-induced M-MDSCs.

13. The composition of claim 10 or 11, wherein the antibody against Ly6G is 1A8.

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