Pharmaceutical composition for tumor therapy

Abstract

The present invention provides: a pharmaceutical composition for tumor therapy, comprising an oncolytic genetically modified measles virus, wherein the tumor contains a tumor cell that expresses PVRL4 / Nectin4, the oncolytic genetically modified measles virus is rMV-SLAMblind or rMV-V(-)-SLAMblind that is obtained by genetically modifying a wild-type measles virus, and the oncolytic genetically modified measles virus and an immune checkpoint inhibitor are administered in combination; and a method for tumor therapy, comprising administering, to a patient with a tumor, an oncolytic genetically modified measles virus and an immune checkpoint inhibitor in combination, wherein the tumor contains a tumor cell that expresses PVRL4 / Nectin4, and the oncolytic genetically modified measles virus is rMV-SLAMblind or rMV-V(-)-SLAMblind that is obtained by genetically modifying a wild-type measles virus.

Description

Pharmaceutical composition for tumor treatment

[0001] The present invention relates to a new use for enhancing the efficacy of an oncolytic gene-modified measles virus. Specifically, the present invention relates to a new use for an oncolytic gene-modified measles virus, a pharmaceutical composition utilizing the new use, and a new method for treating tumors. This application claims priority to Patent Application No. 63 / 683246 filed in the United States on August 15, 2024, and Patent Application No. 2024-164339 filed in Japan on September 20, 2024, the contents of which are incorporated herein by reference.

[0002] Oncolytic virus therapy is a novel cancer treatment method that utilizes two mechanisms: direct tumor cell death through infection and indirect tumor cell death through immune cell-mediated tumor cell death. Measles virus (MV) is a known oncolytic virus. It belongs to the genus Morbillivirus in the family Paramyxoviridae. It infects humans as a natural host, replicates entirely in the cytoplasm (i.e., viral sequences are not integrated into the host's chromosomal DNA), and is a pathogenic virus that causes immunosuppression and respiratory symptoms.

[0003] Our research group discovered that a recombinant measles virus (rMV-SLAMblind), based on the wild-type measles virus HL strain, which does not recognize the signaling lymphocyte activation molecule (SLAM), the receptor for the pathogenicity of wild-type measles virus, is a promising oncolytic virus targeting NECTIN4+ tumor cells (Non-Patent Document 1). In addition to having a stronger antitumor activity against breast cancer than conventional measles virus vaccine strains, rMV-SLAMblind is also fully attenuated and does not induce typical clinical symptoms of measles when administered subcutaneously to monkeys, making it highly safe (Patent Document 1, Non-Patent Document 1).

[0004] The inventors' research group further created rMV-V(-)-SLAMblind, a genetically modified measles virus that does not recognize SLAM, and found that this rMV-V(-)-SLAMblind can also be used to treat breast cancer and metastatic cancer (Patent Document 2). Furthermore, it has been reported that when genetically modified measles viruses that do not recognize SLAM (rMV-SLAMblind and rMV-V(-)-SLAMblind) are directly administered to tumor masses, not only does it induce cell death in tumor cells into which the virus has been introduced, but it also induces strong systemic cellular immunity against tumor cells, thereby suppressing tumor growth even in tumor cells that have not been directly infected with the virus, such as metastatic tumor cells and deep tumor cells (Patent Document 3).

[0005] JP 2013-216609 A International Publication No. 2016 / 047645 International Publication No. 2023 / 176938

[0006] Sugiyama et al., Gene Therapy, 2013, vol.20, p.338-347.Moritoh et al., Cancer Science. 2023, vol.114, p.2158-2168.Tamura et al., Scientific Reports, 2023, vol.13, id:18168.

[0007] In tumor treatment, the combined use of multiple therapeutic methods is widely practiced. It is expected that oncolytic virus therapy will also achieve a more effective therapeutic effect when combined with other therapeutic methods. Therefore, a primary object of the present invention is to provide a pharmaceutical composition for achieving a more effective therapeutic effect in oncolytic virus therapy, and a tumor treatment method using the pharmaceutical composition.

[0008] As a result of extensive research, the inventors discovered that combining antitumor therapy using a genetically modified measles virus that does not recognize SLAM with immune checkpoint inhibitor therapy can produce a synergistic effect, resulting in excellent antitumor effects, and thus completed the present invention.

[0009] The present invention includes the following pharmaceutical compositions, etc.: [1] A pharmaceutical composition for treating a tumor, comprising an oncolytic gene-modified measles virus, wherein the tumor comprises tumor cells expressing PVRL4 / Nectin4, and the oncolytic gene-modified measles virus is rMV-SLAMblind or rMV-V(-)-SLAMblind, which is a wild-type measles virus genetically modified, and the pharmaceutical composition is administered in combination with an immune checkpoint inhibitor. [2] The pharmaceutical composition of [1] above, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. [3] The pharmaceutical composition of [2] above, wherein the PD-1 inhibitor is a small molecule compound with PD-1 inhibitory activity or an anti-PD-1 antibody, and the PD-L1 inhibitor is a small molecule compound with PD-L1 inhibitory activity or an anti-PD-L1 antibody. [4] The pharmaceutical composition of [2] or [3] above, wherein the PD-1 inhibitor is an anti-PD-1 antibody having PD-1 inhibitory activity, and the PD-L1 inhibitor is an anti-PD-L1 antibody having PD-L1 inhibitory activity. [5] The pharmaceutical composition of [3] or [4] above, wherein the anti-PD-1 antibody is one or more selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab, MEDI0608, and tislelizumab, and the anti-PD-L1 antibody is one or more selected from the group consisting of avelumab, atezolizumab, durvalumab, and BMS-936559. [6] The pharmaceutical composition of any of [1] to [5] above, wherein the immune checkpoint inhibitor is one or more anti-PD-1 antibodies selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab, MEDI0608, and tislelizumab. [7] The pharmaceutical composition of any of [1] to [5] above, wherein the immune checkpoint inhibitor is one or more anti-PD-L1 antibodies selected from the group consisting of avelumab, atezolizumab, durvalumab, and BMS-936559. [8] The pharmaceutical composition of any of [1] to [7] above, which is administered in combination with the immune checkpoint inhibitor and a CD4 inhibitor. [9] The pharmaceutical composition of [8] above, wherein the CD4 inhibitor is a small molecule compound having CD4 inhibitory activity or an anti-CD4 antibody.

[10] The pharmaceutical composition of any of [1] to [9] above, wherein the tumor is breast cancer, lung cancer, colon cancer, gastric cancer, pancreatic cancer, or bladder cancer.

[11] The pharmaceutical composition of

[10] above, wherein the breast cancer is triple-negative breast cancer.

[12] The pharmaceutical composition of any of [1] to

[11] above, wherein the tumor is selected from the group consisting of a primary tumor, a deep tumor, a recurrent tumor, a metastatic tumor, and a peritoneally disseminated tumor.

[13] A method for treating a tumor, comprising administering to a tumor patient a combination of an oncolytic gene-modified measles virus and an immune checkpoint inhibitor, wherein the tumor comprises tumor cells expressing PVRL4 / Nectin4, and the oncolytic gene-modified measles virus is rMV-SLAMblind or rMV-V(-)-SLAMblind, which is a wild-type measles virus genetically modified.

[14] The method of

[13] , wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

[15] The method of

[14] , wherein the PD-1 inhibitor is a low-molecular-weight compound having PD-1 inhibitory activity or an anti-PD-1 antibody, and the PD-L1 inhibitor is a low-molecular-weight compound having PD-L1 inhibitory activity or an anti-PD-L1 antibody.

[16] The method of

[14] or

[15] , wherein the PD-1 inhibitor is an anti-PD-1 antibody having PD-1 inhibitory activity, and the PD-L1 inhibitor is an anti-PD-L1 antibody having PD-L1 inhibitory activity.

[17] The method of

[15] or

[16] above, wherein the anti-PD-1 antibody is one or more selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab, MEDI0608, and tislelizumab, and the anti-PD-L1 antibody is one or more selected from the group consisting of avelumab, atezolizumab, durvalumab, and BMS-936559.

[18] The method of any of

[13] to

[17] above, wherein the immune checkpoint inhibitor is one or more anti-PD-1 antibodies selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab, MEDI0608, and tislelizumab.

[19] The method according to any one of

[13] to

[17] , wherein the immune checkpoint inhibitor is one or more anti-PD-L1 antibodies selected from the group consisting of avelumab, atezolizumab, durvalumab, and BMS-936559.

[20] Any of the methods according to any one of

[13] to

[19] , wherein the oncolytic gene-modified measles virus is administered to the tumor patient, followed by administration of the immune checkpoint inhibitor.

[21] Any of the methods according to any one of

[13] to

[19] , wherein the period during which the oncolytic gene-modified measles virus is administered to the tumor patient and the period during which the immune checkpoint inhibitor is administered to the tumor patient at least partially overlap.

[22] Any of the methods according to any one of

[13] to

[19] , wherein the oncolytic gene-modified measles virus, the immune checkpoint inhibitor, and a CD4 inhibitor are administered concomitantly to the tumor patient.

[23] The method of

[22] , wherein the oncolytic gene-modified measles virus is administered to the tumor patient, followed by administration of the immune checkpoint inhibitor, and then administration of the CD4 inhibitor.

[24] The method of

[22] or

[23] , wherein the period during which the oncolytic gene-modified measles virus is administered to the tumor patient and the period during which the immune checkpoint inhibitor is administered to the tumor patient overlap at least partially.

[25] Any of the methods of

[22] to

[24] , wherein the period during which the immune checkpoint inhibitor is administered to the tumor patient and the period during which the CD4 inhibitor is administered to the tumor patient overlap at least partially.

[26] Any of the methods of

[22] to

[25] , wherein the period during which the oncolytic gene-modified measles virus is administered to the tumor patient and the period during which the CD4 inhibitor is administered to the tumor patient overlap at least partially.

[27] The method according to any one of

[22] to

[26] above, wherein the CD4 inhibitor is a low-molecular-weight compound having CD4 inhibitory activity or an anti-CD4 antibody.

[28] The method according to any one of

[13] to

[27] above, wherein the tumor is breast cancer, lung cancer, colon cancer, gastric cancer, pancreatic cancer, or bladder cancer.

[29] The method according to

[28] above, wherein the breast cancer is triple-negative breast cancer.

[30] Any of the methods

[13] to

[29] above, wherein the tumor is selected from the group consisting of a primary tumor, a deep tumor, a recurrent tumor, a metastatic tumor, and a peritoneally disseminated tumor.

[31] Any of the methods

[13] to

[30] above, wherein the oncolytic gene-modified measles virus is administered directly to the tumor in the body of the tumor patient.

[32] Any of the methods

[13] to

[30] above, wherein the oncolytic gene-modified measles virus is administered intraperitoneally to the tumor patient.

[33] Any of the methods

[13] to

[32] above, wherein the immune checkpoint inhibitor is administered intravenously or intraperitoneally to the tumor patient.

[34] Any of the methods

[22] to

[33] above, wherein the CD4 inhibitor is administered intravenously to the tumor patient.

[35] A kit for use in treating a tumor, comprising an oncolytic genetically modified measles virus and an immune checkpoint inhibitor, wherein the tumor comprises tumor cells expressing PVRL4 / Nectin4, and the oncolytic genetically modified measles virus is rMV-SLAMblind or rMV-V(-)-SLAMblind, which is a wild-type measles virus genetically modified.

[36] The kit of

[35] , wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

[37] The kit of

[36] , wherein the PD-1 inhibitor is a small molecule compound with PD-1 inhibitory activity or an anti-PD-1 antibody, and the PD-L1 inhibitor is a small molecule compound with PD-L1 inhibitory activity or an anti-PD-L1 antibody.

[38] The kit according to

[36] or

[37] , wherein the PD-1 inhibitor is an anti-PD-1 antibody having PD-1 inhibitory activity, and the PD-L1 inhibitor is an anti-PD-L1 antibody having PD-L1 inhibitory activity.

[39] The kit according to

[37] or

[38] , wherein the anti-PD-1 antibody is one or more selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab, MEDI0608, and tislelizumab, and the anti-PD-L1 antibody is one or more selected from the group consisting of avelumab, atezolizumab, durvalumab, and BMS-936559.

[40] Any of the kits according to

[35] to

[39] , wherein the immune checkpoint inhibitor is one or more anti-PD-1 antibodies selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab, MEDI0608, and tislelizumab.

[41] Any of the kits according to

[35] to

[39] , wherein the immune checkpoint inhibitor is one or more anti-PD-L1 antibodies selected from the group consisting of avelumab, atezolizumab, durvalumab, and BMS-936559.

[42] Any of the kits according to

[35] to

[41] , further comprising a CD4 inhibitor.

[43] The kit according to

[42] , wherein the CD4 inhibitor is a small molecule compound having CD4 inhibitory activity or an anti-CD4 antibody.

[0010] The pharmaceutical composition and tumor treatment method of the present invention provide a significantly higher antitumor effect than treatment using a genetically modified measles virus that does not recognize SLAM alone. Furthermore, the kit of the present invention is useful for combined treatment using a genetically modified measles virus that does not recognize SLAM and an immune checkpoint inhibitor.

[0011] 1(A) shows the results of examining the expression of checkpoint molecules (Pdl1 and Pdl2) in tumor-bearing mice inoculated with B16F10N4 cells, after administration of rMV or saline. FIG. 1(A) is a diagram schematically illustrating the experimental protocol. FIG. 1(B) shows the results of measuring the relative expression levels of Pdl1 (left panel) and Pdl2 (right panel) in tumor tissues of tumor-bearing mice administered rMV or saline. FIG. 2(A) shows the results of examining the expression levels of Pdl1 and Pdl2 genes in B16F10N4 cells infected with rMV, after administration of rMV or saline. FIG. 2(A) shows the results of measuring the relative expression levels of Pdl1 (left panel) and Pdl2 (right panel) in tumor-bearing mice administered rMV and control tumor-bearing mice. Figure 2(B) is a histogram showing PDL1 expression in B16F10N4 cells infected with rMV at an MOI of 0.1 or 1. Figure 2(C) is a histogram showing PDL1 expression in B16F10N4 cells infected with rMV-EGFP. The left panel shows the results of flow cytometry separation of infected (virus+EGFP+) and uninfected (virus+EGFP-) cells, and the right panel shows the results of analyzing PDL1 expression in each type of cell. In Example 1, bone marrow-derived dendritic cells (BMDCs) were cocultured with rMV-infected or uninfected B16F10N4 cells and magnetically selected CD11c+ cells, and the expression levels of Pdl1 and Pdl2 were examined. Figure 3(A) is a schematic diagram of the experimental protocol. Figure 3(B) shows the results of measuring the relative expression levels of Pdl1 (left panel) and Pdl2 (right panel) in BMDCs after coculture with rMV-infected or uninfected B16F10N4 cells. Figure 3(B) shows the results of examining the expression levels of Pdl1 and Pdl2 genes in rMV-infected LL2N4 cells and 3T3N4 cells in Example 1. Figure 4(A) shows the results of measuring the relative expression levels of Pdl1 in rMV-administered tumor-bearing mice and control tumor-bearing mice. Figure 4(B) shows the results of measuring the relative expression levels of Pdl2 in rMV-administered tumor-bearing mice and control tumor-bearing mice.Figure 4(C) is a histogram showing PDL1 expression in LL2N4 cells (left panel) and 3T3N4 cells (right panel) infected with rMV at an MOI of 0.1 or 1. Figure 4(C) shows the results of measuring the expression levels of Pdl1 and Pdl2 in BMDCs cocultured with rMV-infected or uninfected LL2N4 and 3T3N4 cells and magnetically selected CD11c+ cells in Example 1. Figure 5(A) is a schematic diagram of the experimental protocol. Figure 5(B) shows the results of measuring the relative expression levels of Pdl1 in BMDCs after coculture with rMV-infected or uninfected cells, and Figure 5(C) shows the results of measuring the relative expression levels of Pdl2 in BMDCs after coculture with rMV-infected or uninfected cells. 7A and 7B are diagrams schematically illustrating the experimental protocol for measuring the expression of PDL1 and PDL2 in tumor cells (N4+ cells) in B16F10N4 tumor tissue and TDLN in Example 1.

[0034] Figure 7A is a diagram illustrating the results of examining the expression of PDL1 and PDL2 in tumor cells (N4+ cells) and DCs in B16F10N4 tumor tissue and TDLN in Example 1.

[0035] Figure 7A is a Coulter plot showing PDL1 expression in N4+ cells, Figure 7B is the PDL1 expression frequency in N4+ cells, Figure 7C is a Coulter plot showing PDL2 expression in N4+ cells, Figure 7D is the PDL2 expression frequency in N4+ cells, Figure 7E is a Coulter plot showing PDL1 expression in DCs in the tumor, Figure 7F is the number of PDL1-expressing DCs (PDL1+DCs) in the tumor, and Figure 7G is a diagram illustrating the expression of PDL1 and PDL2 in DCs in the tumor. Figure 7(H) shows the number of PDL1-expressing DCs in the TDLN. Figure 7(I) shows the Coulter plot showing PDL2 expression in DCs in the tumor. Figure 7(J) shows the number of PDL2-expressing DCs (PDL2 + DCs) in the tumor. Figure 7(K) shows the Coulter plot showing PDL2 expression in DCs in the TDLN. Figure 7(L) shows the number of PDL2-expressing DCs in the TDLN. This figure shows a schematic diagram of the experimental protocol used to measure the antitumor effect of coadministration of rMV and anti-PD1 antibody (aPD1) to B16F10N4 tumor-bearing mice in Example 2.9 shows the results of investigating the antitumor effect of combined administration of rMV and anti-PD1 antibody (aPD1) in B16F10N4 tumor-bearing mice in Example 2. For the group administered saline and IgG2B antibody (iso) (saline + iso), the group administered saline and anti-PD1 antibody (saline + aPD1), the group administered rMV and IgG2B antibody (iso) (rMV + iso), and the group administered rMV and anti-PD1 antibody (rMV + aPD1), Figure 9(A) shows the time course of tumor volume in individual mice in each group, Figure 9(B) shows tumor growth curves from B16F10N4 cell inoculation to day 13 (D13) for each group, and Figure 9(C) shows Kaplan-Meier survival curves for each group. [Example 2] Figure 9 shows the results of investigating T cell profiling after combined therapy with rMV and a PDL1 inhibitor. Figure 10(A) shows a schematic diagram of the experimental protocol. Figure 10(B) shows the number of IFNγ+CD8+ T cells, Figure 10(C) shows the number of granulocyte B (GrB)+CD8+ T cells, Figure 10(D) shows the number of IFNγ+CD4+ T cells, Figure 10(E) shows the number of IL4+CD4+ T cells, and Figure 10(F) shows the number of FoxP3+CD4+ T cells in TDLN and tumor tissues of tumor-bearing mice administered rMV or saline with anti-PD1 antibody (aPD1) or rat IgG2B antibody (iso). In Example 2, the antitumor effect of administering rMV, anti-PD1 antibody (aPD1), and anti-CD4 antibody (aCD4) in combination to B16F10N4 tumor-bearing mice was investigated. Figure 11(A) shows a schematic diagram of the experimental protocol. Figure 11(B) shows the time course of tumor volume in individual mice in each group for the group administered saline and IgG2B antibody (iso) (Saline+iso), the group administered rMV, anti-PD1 antibody, and IgG2B antibody (iso) (Combo+iso), and the group administered rMV, anti-PD1 antibody, and anti-CD4 antibody (aCD4) (Combo+aCD4). Figure 11(C) shows the results of PD1+CD8+ cell analysis of blood samples obtained on day 18 using the experimental protocol shown in Figure 11(A) in Example 2.Figure 12(A) shows a Coulter plot showing PD1 expression in CD8+ T cells. Figure 12(B) and (C) show the frequency of PD1 expression in CD8+ T cells (the percentage of PD1-expressing CD8+ T cells (PD1+CD8+ T cells) among all CD8+ T cells) and the cell number, respectively.

[0012] <Pharmaceutical Composition> The pharmaceutical composition according to this embodiment is a pharmaceutical composition for treating tumors, comprising an oncolytic genetically modified measles virus, and is administered in combination with an immune checkpoint inhibitor and / or a CD4 inhibitor. When administered in combination with an immune checkpoint inhibitor, the pharmaceutical composition according to this embodiment exhibits a stronger antitumor effect than when used alone.

[0013] The term "oncolytic gene-modified measles virus" used in the present invention refers to a measles virus that has been genetically modified to enhance safety and have oncolytic activity. For example, the oncolytic gene-modified measles virus used in the present invention may be a wild-type measles virus (MV HL strain), which has been found to efficiently infect various tumor cells that express PVRL4 / Nectin4, proliferate within the cells, and induce efficient cell death.

[0014] The genetic modifications used to create the oncolytic viruses used in the present invention are preferably genetic modifications that render them infective to tumor cells but not to normal cells, including genetic modifications that eliminate the ability to bind to SLAM, thereby eliminating infectivity to SLAM-positive cells. By eliminating the ability to bind to SLAM, the oncolytic viruses of the present invention do not infect SLAM-positive immune system cells, which are the normal target cells of measles virus. Furthermore, SLAM-deficient viruses do not cause measles pathology in monkey infection experiments (Patent Document 1, Non-Patent Document 1). Furthermore, the loss of infectivity to immune cells can improve efficacy and safety during treatment.

[0015] The oncolytic gene-modified measles virus used in the present invention is not particularly limited, and any virus having the above-mentioned characteristics of an oncolytic gene-modified measles virus can be used. The oncolytic gene-modified measles virus used in the present invention is particularly preferably rMV-SLAMblind or rMV-V(-)-SLAMblind, which were previously produced by the inventors of the present invention and have actually been shown to have a direct antitumor effect.

[0016] The oncolytic genetically modified measles virus used in the present invention, "rMV-SLAMblind," is an example of an oncolytic genetically modified measles virus described in Patent Documents 1 and 2. This virus strain was created using reverse genetics techniques as a recombinant MV that is unable to selectively utilize SLAM. This genetically modified measles virus strain has lost its infectivity for SLAM-positive cells and does not originally infect or induce cytotoxicity in CD46-positive cells. However, because it uses PVRL4 / Nectin4 as a receptor for cell infection, it specifically infects PVRL4-positive tumor cells and induces their cell death. Therefore, it has specific oncolytic activity against SLAM-negative tumor cells (e.g., breast cancer cells).

[0017] Another example of an oncolytic genetically modified measles virus used in the present invention is "rMV-V(-)-SLAMblind," which is an oncolytic genetically modified measles virus described in Patent Document 2. This virus strain is a genetically modified measles virus strain that has oncolytic activity similar to that of "rMV-SLAMblind," although it has an additional modification in the P gene compared to "rMV-SLAMblind." This genetically modified measles virus strain has similar characteristics to those of the rMV-SLAMblind strain (infection specificity, oncolytic activity), and is attenuated by being genetically modified so as not to express the V gene involved in pathogenicity.

[0018] Specifically, rMV-SLAMblind is a variant in which the arginine at position 533 in the H protein of a measles virus strain has been replaced with alanine, whereas rMV-V(-)-SLAMblind is a variant in which the arginine at position 533 in the H protein has been replaced with alanine, and bases 687 and 690 in the P gene have been replaced with U to C and C to U, respectively.

[0019] rMV-SLAMblind can be prepared by reverse genetics using, for example, the plasmid pMV-HL(7+) encoding the full-length antigenome cDNA of the wild-type measles virus HL strain to prepare a vector (pMV-SLAMblind) in which the arginine at amino acid residue 533 of the H protein has been substituted with alanine. The full-length antigenome cDNA of the wild-type measles virus HL strain in which the arginine at amino acid residue 533 has been substituted with alanine contains the nucleotide sequence shown in SEQ ID NO: 1. Among the proteins encoded by this nucleotide sequence, the N protein consists of the amino acid sequence shown in SEQ ID NO: 2, the P protein consists of the amino acid sequence shown in SEQ ID NO: 3, the M protein consists of the amino acid sequence shown in SEQ ID NO: 4, the F protein consists of the amino acid sequence shown in SEQ ID NO: 5, the H protein consists of the amino acid sequence shown in SEQ ID NO: 6, and the L protein consists of the amino acid sequence shown in SEQ ID NO: 7. These proteins constitute the virus.

[0020] On the other hand, rMV-V(-)-SLAMblind can be prepared by reverse genetics using the plasmid pMV-HL(7+), which encodes the full-length antigenome cDNA of the wild-type measles virus HL strain. The arginine at amino acid residue 533 of the H protein of the vector (pMV-SLAMblind) is then substituted with alanine to create a vector (pMV-SLAMblind) in which two additional mutations (U at residue 687 and C at residue 690) are made in the P gene. The amino acid sequences of the viral proteins encoded by pMV-V(-)SLAMblind are identical to those of rMV-SLAMblind.

[0021] The original virus strain used to prepare MV-SLAMblind or rMV-V(-)-SLAMblind may be a strain other than the wild-type MV-HL strain. Therefore, the rMV-SLAMblind or rMV-V(-)-SLAMblind used as the oncolytic gene-modified measles virus in the present invention is not limited to those using the above-mentioned pMV-SLAMblind vector or pMV-V(-)SLAMblind.

[0022] The tumor cells to be treated with the pharmaceutical composition of this embodiment are not particularly limited as long as they express PVRL4 / Nectin4. Because the oncolytic genetically modified measles virus, which is the active ingredient, can efficiently infect tumors containing tumor cells that highly express PVRL4 / Nectin4, the pharmaceutical composition of this embodiment is preferably used to treat tumors containing tumor cells that highly express PVRL4 / Nectin4. Examples of tumor cells that highly express PVRL4 / Nectin4 include epithelial cancer cells such as breast cancer (including triple-negative breast cancer (TNBC)), lung cancer, colorectal cancer (e.g., colorectal cancer), pancreatic cancer, gastric cancer (including scirrhous gastric cancer), bladder cancer, and ovarian cancer.

[0023] Oncolytic genetically modified measles viruses (e.g., rMV-SLAMblind or rMV-V(-)-SLAMblind) have both an antitumor effect (direct antitumor effect) that directly induces significant cell death in tumor cells upon intratumoral administration, and an antitumor effect (secondary antitumor effect) that indirectly induces cellular immunity and suppresses tumor growth even in tumor cells not directly infected with the virus (e.g., metastatic tumor cells and deep tumor cells that have spread to sites that cannot be directly infected with the virus). Therefore, the pharmaceutical composition of this embodiment can be used to treat primary tumors, and can also treat tumor cells that remain in the body after tumor treatment using an oncolytic genetically modified measles virus due to the direct antitumor effect (e.g., tumor cells constituting deep tumors, recurrent tumors, metastatic tumors, peritoneal dissemination tumors, etc.). Tumors that are difficult to treat by direct administration into the tumor mass are more likely to be refractory or highly malignant than tumors that can be administered directly into the tumor mass, and therefore there is a greater need for treatment. The pharmaceutical composition of this embodiment can be a very effective therapeutic agent against these diseases.

[0024] The pharmaceutical composition according to this embodiment may contain only the oncolytic genetically modified measles virus (e.g., rMV-SLAMblind or rMV-V(-)-SLAMblind) as the active ingredient, or may contain it in combination with one or more other active ingredients. Examples of other active ingredients include oncolytic viruses other than oncolytic genetically modified measles viruses, anticancer agents other than oncolytic genetically modified measles viruses, or auxiliary ingredients for supporting the function of anticancer agents. Examples of such auxiliary ingredients include immune checkpoint inhibitors and immunostimulants. Examples of immune checkpoint inhibitors that can be used include those described below. Examples of immunostimulants include GM-CSF.

[0025] The pharmaceutical composition of this embodiment may contain one unit dose of the oncolytic genetically modified measles virus as the active ingredient, or two or more unit doses. Here, "unit dose" refers to the amount of oncolytic genetically modified measles virus administered in a single administration. "Single administration" includes not only a single infusion treatment but also continuous infusion treatment over a set period of time. The specific unit dose may be a unit dose that has already been investigated for its direct antitumor effect (Patent Document 2).

[0026] The pharmaceutical composition of this embodiment can be obtained by appropriately incorporating an oncolytic genetically modified measles virus (e.g., rMV-SLAMblind or rMV-V(-)-SLAMblind) and a pharmaceutically acceptable carrier. The pharmaceutical composition containing an oncolytic genetically modified measles virus and a pharmaceutically acceptable carrier can be produced by methods commonly used in the field of pharmaceutical production, using appropriate additives as needed. The pharmaceutically acceptable carrier to be incorporated into the pharmaceutical composition of this embodiment can be appropriately selected from known carriers depending on the dosage form of the pharmaceutical composition. The dosage form of the pharmaceutical composition of this embodiment may be any suitable form for administering an oncolytic virus, and can be used, for example, as a liquid formulation.

[0027] Pharmaceutically acceptable carriers are diluents, excipients, binders, solvents, etc. that do not induce adverse physiological reactions in the recipient and do not adversely interact with other components, such as active ingredients. Specific examples of such carriers include water, physiological saline, various buffer solutions, etc. Usable additives include diluents, excipients, binders, stabilizers, isotonicity agents, buffers, solubilizers, suspending agents, preservatives, cryoprotectants, cryoprotectants, lyoprotectants, bacteriostatic agents, etc. Specific examples include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange resins, methylcellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, Veegum, titanium oxide, sorbitan fatty acid esters, sodium lauryl sulfate, glycerin, fatty acid glycerin esters, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oils, wax, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, and water.

[0028] The pharmaceutical composition according to this embodiment may be in any of the following dosage forms: liquid, frozen, and lyophilized. A culture solution obtained by culturing a suitable medium or cultured cells can be collected, and additives such as stabilizers can be added. The culture solution can be dispensed into small bottles, ampoules, or the like, and then sealed. In the case of frozen or lyophilized compositions, a dissolving solution is used for dissolving the composition at the time of use. The dissolving solution can be any of various buffer solutions or physiological saline.

[0029] When the pharmaceutical composition according to this embodiment is an injection, the oncolytic genetically modified measles virus may be dissolved in distilled water for injection, optionally with a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc., and an isotonic agent such as sodium chloride or glucose, and then filled into the solution. Alternatively, the active ingredient may be further added with mannitol, dextrin, cyclodextrin, gelatin, etc., followed by vacuum freeze-drying to prepare an injection that can be dissolved immediately before use. Alternatively, the active ingredient may be emulsified in water with lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, etc., to prepare an emulsion for injection.

[0030] The method of administration of the pharmaceutical composition according to this embodiment is not particularly limited, and can be various administration methods such as direct administration to a tumor, intraperitoneal administration, intravenous administration, subcutaneous administration, intramuscular administration, and intravesical administration. Direct administration to a tumor mass is preferred because it reliably achieves a direct antitumor effect and is also likely to achieve a secondary antitumor effect. Intraperitoneal administration is also preferred for tumors in the peritoneal cavity and nearby tissues. In particular, intraperitoneal administration of the pharmaceutical composition according to this embodiment is preferred for the treatment or prevention of gastric scirrhous cancer. Intravesical administration of the pharmaceutical composition according to this embodiment is also preferred for the treatment of bladder cancer.

[0031] The pharmaceutical composition according to this embodiment is administered in combination with an immune checkpoint inhibitor. An "immune checkpoint" is a molecule that functions to suppress excessive immune responses during the immune response process. The "immune checkpoint inhibitor" may be any substance that can inhibit the function of any immune checkpoint. For example, it may be a substance that directly binds to an immune checkpoint and inhibits its function, or it may be a substance that binds to an immune checkpoint via another molecule and inhibits its function.

[0032] The immune checkpoint inhibitor to be administered in combination with the pharmaceutical composition according to this embodiment may be any substance having immune checkpoint inhibitory activity, and various substances such as low molecular weight compounds, antibodies, peptides, antisense molecules used in RNA interference such as siRNA, and nucleic acid aptamers may be used. The antibody may be any substance capable of specifically binding to the target immune checkpoint, and may be a monoclonal antibody or a polyclonal antibody. It may also be a chimeric antibody, a humanized antibody, or a fully humanized antibody. rIgG, Fab, F(ab') ２ The antibody may be an antibody fragment such as a bispecific antibody, a trispecific antibody, an antibody-drug conjugate (ADC), or a Nanobody (registered trademark).

[0033] The immune checkpoint inhibitor to be administered in combination with the pharmaceutical composition of this embodiment is preferably an inhibitor of one or more immune checkpoints selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4 (cytotoxic T-lymphocyte-associated protein-4), LAG-3 (lymphocyte activation gene 3), TIM-3 (mucin domain-containing protein 3), TIGIT (T cell immunoglobulin and ITIM domain), ICOS (inducible costimulatory molecule), VISTA (V domain Ig suppressor of T cell activation), B7-H3 (CD276), and BTLA (B and T lymphocyte inhibitory costimulatory molecule), more preferably an inhibitor of one or more immune checkpoints selected from the group consisting of PD-1, PD-L1, and CTLA-4, and particularly preferably a PD-1 inhibitor or PD-L1 inhibitor.

[0034] The PD-1 inhibitor may be any substance that has PD-1 inhibitory activity, and various substances can be used, such as low molecular weight compounds, antibodies, peptides, antisense molecules used in RNA interference such as siRNA, nucleic acid aptamers, etc. Similarly, the PD-L1 inhibitor may be any substance that has PD-L1 inhibitory activity, and various substances can be used, such as low molecular weight compounds, antibodies, peptides, antisense molecules used in RNA interference such as siRNA, nucleic acid aptamers, etc.

[0035] Examples of low molecular weight compounds having PD-L1 inhibitory activity as PD-L1 inhibitors to be administered in combination with the pharmaceutical composition of this embodiment include BMS-202 (CAS RN: 1422185-22-5), BMS-1327 (Bristol Myers Squibb), SA-49 ((6S,6aR,13S,13aS)-1-((1-Methyl-1H-imidazol-4-yl)sulfonyl)-1,3,4,6,6a,7,8,9,10,12,13,13a-dodecahydro-2H-6,13-methanodipyrido[1,2-a:3',2'-e]azocine), JQ-1 (CAS RN: 1268524-70-4), BMS-8 (CAS RN: 1675201-90-7), and BMS-1166 (CAS Preferably, one or more selected from the group consisting of CA-170 (CAS RN: 1818314-88-3) and CA-170 (CAS RN: 1673534-76-3).

[0036] The PD-1 inhibitor to be administered in combination with the pharmaceutical composition according to this embodiment is preferably an anti-PD-1 antibody having PD-1 inhibitory activity, and examples thereof include nivolumab (CAS RN: 946414-94-4), pembrolizumab (lambrolizumab, MK-3475) (CAS RN: 1374853-91-4), pidilizumab (CT-011) (CAS RN: 1036730-42-3), MEDI0608 (Naing et al. Journal for ImmunoTherapy of Cancer, 2019, 7:225), cemiplimab (CAS RN: 1801342-60-8), spartalizumab (CAS RN: 1935694-88-4), tislelizumab (BGB-A317) (CAS RN: 1418628-81-5), Camrelizumab (CAS RN: 1798286-48-2), Serplulimab (CAS RN: 2231029-82-4), Toripalimab (CAS RN: 1924598-82-2), Retifanlimab (CAS RN: 2079108-44-2), Dostarlimab (TSR-042) (CAS RN: 2022215-59-2), Sintilimab (CAS RN: 2072873-06-2), Penpulimab (CAS more preferred are one or more anti-PD-1 antibodies selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, MEDI0608, cemiplimab, spartalizumab, and tislelizumab, with nivolumab or pembrolizumab being particularly preferred.

[0037] The PD-L1 inhibitor to be administered in combination with the pharmaceutical composition of this embodiment is preferably an anti-PD-L1 antibody that has PD-L1 inhibitory activity. The anti-PD-L1 antibody used as the PD-L1 inhibitor is preferably a monoclonal antibody or a bispecific antibody. The anti-PD-L1 antibodies include avelumab (CAS RN: 1537032-82-8), atezolizumab (CAS RN: 1380723-44-3), durvalumab (CAS RN: 1428935-60-7), BMS-936559 (CAS RN: 1422185-22-5), cosibelimab (CAS RN: 2216751-26-5), envafolimab (ASC 22; KN 035) (CAS RN: 2102192-68-5), sugemalimab (CAS RN: 2256084-03-2), BNT327 (BioNTech), and FAZ053 (CB No. CB115668405), SGN-PDL1V (PF-08046054) (MedChemExpress), AB003 (AKSO Biopharmaceuticals), and Anti-PD-L1 ISAC (SGN-PDL1iT) (PF-08046037). The PD-L1 inhibitor to be co-administered with the pharmaceutical composition of this embodiment is preferably one or more anti-PD-L1 antibodies selected from the group consisting of avelumab, atezolizumab, durvalumab, and BMS-936559, and particularly preferably one or more anti-PD-L1 antibodies selected from the group consisting of avelumab, atezolizumab, and durvalumab.

[0038] The immune checkpoint inhibitor administered in combination with the pharmaceutical composition of this embodiment may be a single type, or a combination of two or more types. The pharmaceutical composition of this embodiment may be administered in combination with an anti-PD-1 antibody and an anti-CTLA4 antibody, or an anti-PD-1 antibody and an anti-LAG-3 antibody. Examples of combinations of the pharmaceutical composition of this embodiment with two or more immune checkpoint inhibitors include a combination of the pharmaceutical composition of this embodiment with at least one of a PD-1 inhibitor and a PD-L1 inhibitor and an anti-CTLA4 antibody; a combination of the pharmaceutical composition of this embodiment with at least one of a PD-1 inhibitor and a PD-L1 inhibitor and an anti-LAG-3 antibody; or a combination of the pharmaceutical composition of this embodiment with at least one of a PD-1 inhibitor and a PD-L1 inhibitor, an anti-CTLA4 antibody, and an anti-LAG-3 antibody. Ipilimumab (CAS RN: 477202-00-9) and the like are preferred anti-CTLA4 antibodies. As the anti-LAG-3 antibody, Relatlimab (CAS RN: 1673516-98-7) and the like are preferably used. More specifically, examples include a combined use of the pharmaceutical composition according to this embodiment, nivolumab, and ipilimumab; a combined use of the pharmaceutical composition according to this embodiment, nivolumab, and Relatlimab; and the like.

[0039] The pharmaceutical composition according to this embodiment is also preferably administered in combination with an immune checkpoint inhibitor and a CD4 inhibitor. The CD4 inhibitor administered in combination with the pharmaceutical composition according to this embodiment may be any substance having CD4 inhibitory activity, and various substances such as low molecular weight compounds, antibodies, peptides, antisense molecules used in RNA interference such as siRNA, and nucleic acid aptamers can be used. Specifically, IT1208 (manufactured by IDAC Theranostics, Inc.), which has CD4 inhibitory activity, is preferred as the CD4 inhibitor to be administered in combination.

[0040] The pharmaceutical composition of this embodiment is preferably used in combination with other drugs in addition to immune checkpoint inhibitors. The other drugs are not particularly limited, as long as they are drugs other than immune checkpoint inhibitors and CD4 inhibitors that are expected to have anti-cancer effects and reduce side effects. Examples include anti-cancer drugs used in chemotherapy, molecularly targeted drugs (antibodies or small molecules), and hormones. Examples of anti-cancer drugs include the platinum complex carboplatin (CAS RN: 41575-94-4). Examples of small molecular targeted drugs include the angiogenesis inhibitor bevacizumab (CAS RN: 216974-75-3) and the tyrosine kinase inhibitor axitinib (CAS RN: 319460-85-0). Examples of hormones include the anti-estrogen drug fulvestrant (CAS RN: 129453-61-8). More specifically, examples of such combinations include a combination of the pharmaceutical composition according to this embodiment, pembrolizumab, and carboplatin; a combination of the pharmaceutical composition according to this embodiment, atezolizumab, and bevacizumab; and a combination of the pharmaceutical composition according to this embodiment, pembrolizumab, and axitinib.

[0041] Here, "co-administration" of two or three types of drugs means that the period in which one drug exhibits its efficacy and the period in which the other drug exhibits its efficacy overlap at least partially in the body of the administered animal. The same applies to "co-administration" of four or more types of drugs. The multiple drugs administered in combination may be administered simultaneously via the same administration route, simultaneously via different administration routes, at different times via the same administration route, or at different times via different administration routes.

[0042] "Administering the pharmaceutical composition according to this embodiment in combination with an immune checkpoint inhibitor" means administering the compositions in such a manner that the period during which the oncolytic genetically modified measles virus, which is the active ingredient of the pharmaceutical composition according to this embodiment, exhibits its therapeutic effect in the body of the administered animal and the period during which the administered immune checkpoint inhibitor exhibits its therapeutic effect at least partially overlap. The pharmaceutical composition according to this embodiment may be administered simultaneously with the immune checkpoint inhibitor, or the administration times may be staggered by several hours to several days. For example, the pharmaceutical composition according to this embodiment and the immune checkpoint inhibitor may be administered, for example, every other day, once a week, or at longer intervals.

[0043] "Administering the pharmaceutical composition of this embodiment in combination with an immune checkpoint inhibitor and a CD4 inhibitor" means that the pharmaceutical composition is administered in such a manner that the period during which the oncolytic genetically modified measles virus, which is the active ingredient of the pharmaceutical composition of this embodiment, exhibits its therapeutic effect, the period during which the administered immune checkpoint inhibitor exhibits its therapeutic effect, and the period during which the administered CD4 inhibitor exhibits its therapeutic effect at least partially overlap in the body of the administered animal. The pharmaceutical composition of this embodiment, the immune checkpoint inhibitor, and the CD4 inhibitor may be administered simultaneously, or the pharmaceutical composition of this embodiment and the immune checkpoint inhibitor may be administered followed by the CD4 inhibitor, or the pharmaceutical composition of this embodiment may be administered followed by the immune checkpoint inhibitor, and then the CD4 inhibitor.

[0044] The pharmaceutical composition of this embodiment is administered to animals with confirmed or suspected tumor formation to treat tumors. The pharmaceutical composition of this embodiment is also administered to animals at risk of tumor recurrence to prevent tumor recurrence. The animal to which the pharmaceutical composition of this embodiment is administered may be any animal that can be infected with an oncolytic genetically modified measles virus (e.g., rMV-SLAMblind or rMV-V(-)-SLAMblind), preferably a mammal. The term "mammal" refers to any animal classified as a mammal, including, but not limited to, humans, pet animals such as dogs, cats, and rabbits, and livestock animals such as cows, pigs, sheep, and horses. Preferably, the animal is a human or a dog.

[0045] <Tumor Treatment Method> The tumor treatment method of this embodiment is characterized by administering to a tumor patient a combination of an oncolytic gene-modified measles virus and an immune checkpoint inhibitor. By administering the oncolytic gene-modified measles virus and the immune checkpoint inhibitor in combination, a synergistic therapeutic effect can be achieved.

[0046] In the tumor treatment method of this embodiment, the tumor patient to be treated is a patient with a tumor containing tumor cells expressing PVRL4 / Nectin4. The tumor is not particularly limited as long as it contains tumor cells expressing PVRL4 / Nectin4, but is preferably breast cancer (including triple-negative breast cancer (TNBC)), lung cancer, colon cancer, gastric cancer, pancreatic cancer, or bladder cancer, as this provides a more satisfactory therapeutic effect. Furthermore, the tumor may be any tumor selected from the group consisting of primary tumors, deep tumors, recurrent tumors, metastatic tumors, and peritoneally disseminated tumors.

[0047] In the tumor treatment method of this embodiment, the malignancy of the tumor to be treated is not particularly limited, and it may be an early stage tumor of stage 0 to 1, or an advanced or recurrent tumor of stage 2 to 4. Furthermore, tumor patients to be treated with the tumor treatment method of this embodiment may be tumor patients who have been treated with other cancer treatments such as drug therapy, radiation therapy, or surgical therapy, or may be tumor patients who have not yet been treated with other cancer treatments.

[0048] The oncolytic gene-modified measles virus used in the tumor treatment method of this embodiment is the same as the oncolytic gene-modified measles virus used as the active ingredient in the pharmaceutical composition of this embodiment. For the tumor treatment method of this embodiment, rMV-SLAMblind or rMV-V(-)-SLAMblind, which are wild-type measles viruses that have been genetically modified, are preferred. In the tumor treatment method of this embodiment, the pharmaceutical composition of this embodiment can be administered as the oncolytic gene-modified measles virus.

[0049] The immune checkpoint inhibitor used in the tumor treatment method of this embodiment can be the same as those listed above as those administered in combination with the pharmaceutical composition of this embodiment. The immune checkpoint inhibitor used in the tumor treatment method of this embodiment is preferably a PD-1 inhibitor or a PD-L1 inhibitor, more preferably a small molecule compound with PD-1 inhibitory activity, an anti-PD-1 antibody with PD-1 inhibitory activity, a small molecule compound with PD-L1 inhibitory activity, or an anti-PD-L1 antibody with PD-L1 inhibitory activity, and even more preferably an anti-PD-1 antibody with PD-1 inhibitory activity or an anti-PD-L1 antibody with PD-L1 inhibitory activity.

[0050] In the tumor treatment method of this embodiment, the oncolytic gene-modified measles virus and the immune checkpoint inhibitor are administered to a tumor patient so that the period during which the secondary antitumor effect of the oncolytic gene-modified measles virus administered to the tumor patient is exerted at least partially overlaps with the period during which the therapeutic effect of the immune checkpoint inhibitor administered to the tumor patient is exerted. The oncolytic gene-modified measles virus and the immune checkpoint inhibitor may be administered simultaneously to the tumor patient, or the oncolytic gene-modified measles virus may be administered first and then the immune checkpoint inhibitor, or the immune checkpoint inhibitor may be administered first and then the oncolytic gene-modified measles virus. Because the synergistic effect of the immune checkpoint inhibitor is exerted more on the secondary antitumor effect than on the direct antitumor effect of the oncolytic gene-modified measles virus, it is preferable to administer the immune checkpoint inhibitor to the tumor patient after administering the oncolytic gene-modified measles virus.

[0051] Like many other anticancer agents, the oncolytic gene-modified measles virus is preferably administered multiple times consecutively at predetermined intervals. For example, the oncolytic gene-modified measles virus is administered 3 to 8 times consecutively at 1 to 4 week intervals. Similarly, the immune checkpoint inhibitor may also be administered multiple times consecutively at predetermined intervals. The administration intervals of the oncolytic gene-modified measles virus and the immune checkpoint inhibitor may be the same or different. Furthermore, it is preferable that the period during which the oncolytic gene-modified measles virus is administered to a tumor patient overlaps at least partially with the period during which the immune checkpoint inhibitor is administered to the tumor patient (the time of administration in the case of a single administration). For example, when the oncolytic gene-modified measles virus is administered 3 to 8 times consecutively at 1 to 4 week intervals to a tumor patient, the immune checkpoint inhibitor can be administered at 2 to 4 week intervals for 2 years from the second administration onward after the start of administration of the oncolytic gene-modified measles virus.

[0052] In the tumor treatment method of this embodiment, it is also preferable to administer an oncolytic gene-modified measles virus, an immune checkpoint inhibitor, and a CD4 inhibitor in combination. The CD4 inhibitor used in the tumor treatment method of this embodiment can be the same as those listed above as those administered in combination with the pharmaceutical composition of this embodiment. The CD4 inhibitor used in the tumor treatment method of this embodiment is preferably a low-molecular-weight compound having CD4 inhibitory activity or an anti-CD4 antibody.

[0053] When a CD4 inhibitor is used, the oncolytic gene-modified measles virus, the immune checkpoint inhibitor, and the CD4 inhibitor are administered so that the period during which the secondary antitumor effect of the oncolytic gene-modified measles virus administered to the tumor patient is exerted, the period during which the therapeutic effect of the immune checkpoint inhibitor administered to the patient is exerted, and the period during which the therapeutic effect of the CD4 inhibitor administered to the patient is exerted at least partially overlap. The oncolytic gene-modified measles virus, the immune checkpoint inhibitor, and the CD4 inhibitor may be administered simultaneously to the tumor patient, or the immune checkpoint inhibitor and CD4 inhibitor may be administered after the oncolytic gene-modified measles virus is administered, or the immune checkpoint inhibitor and CD4 inhibitor may be administered after the oncolytic gene-modified measles virus is administered. Since the synergistic effect of an immune checkpoint inhibitor and a CD4 inhibitor is exerted on the secondary antitumor effect rather than the direct antitumor effect of an oncolytic gene-modified measles virus, it is preferable to administer an immune checkpoint inhibitor and a CD4 inhibitor to a tumor patient after administering an oncolytic gene-modified measles virus, and it is more preferable to administer an immune checkpoint inhibitor and then a CD4 inhibitor to a tumor patient after administering an oncolytic gene-modified measles virus.

[0054] When an oncolytic gene-modified measles virus is administered multiple times, it is preferable that the period during which the oncolytic gene-modified measles virus is administered to a tumor patient overlaps at least partially with the period during which an immune checkpoint inhibitor is administered to the tumor patient (or the time of administration in the case of a single administration).It is also preferable that the period during which the oncolytic gene-modified measles virus is administered to a tumor patient overlaps at least partially with the period during which a CD4 inhibitor is administered to the tumor patient (or the time of administration in the case of a single administration).It is more preferable that the period during which an immune checkpoint inhibitor is administered to a tumor patient (or the time of administration in the case of a single administration) overlaps at least partially with the period during which a CD4 inhibitor is administered to the tumor patient (or the time of administration in the case of a single administration).It is also preferable that the period during which an immune checkpoint inhibitor is administered to a tumor patient (or the time of administration in the case of a single administration) overlaps at least partially with the period during which a CD4 inhibitor is administered to the tumor patient (or the time of administration in the case of a single administration). For example, when an oncolytic gene-modified measles virus is administered to a tumor patient three to eight consecutive times at one- to four-week intervals, from the second administration of the oncolytic gene-modified measles virus onwards, an immune checkpoint inhibitor can be administered at two- to four-week intervals for two years, and after one or two administrations of the immune checkpoint inhibitor, a CD4 inhibitor can be administered two to three times at one-day to two-week intervals.

[0055] In the tumor treatment method of this embodiment, the administration routes of the oncolytic gene-modified measles virus, immune checkpoint inhibitor, and CD4 inhibitor, or the administration route of the combined use of other drugs (drugs expected to have anti-cancer effects or side effect reduction effects, etc.), may be the same or different from each other. In the tumor treatment method of this embodiment, the oncolytic gene-modified measles virus is preferably administered directly to the tumor mass in order to fully obtain a direct anti-tumor effect. When the tumor is a peritoneally disseminated tumor or a tumor formed in tissue near the peritoneum, such as gastric cancer, the oncolytic gene-modified measles virus is also preferably administered intraperitoneally. Meanwhile, the immune checkpoint inhibitor is preferably administered intravenously or intraperitoneally, which are commonly used routes. The CD4 inhibitor is preferably administered intravenously.

[0056] <Kit used for tumor treatment> The kit of this embodiment is a kit used for treating tumors containing tumor cells expressing PVRL4 / Nectin4, and includes an oncolytic gene-modified measles virus and an immune checkpoint inhibitor. Use of the kit including the oncolytic gene-modified measles virus and the immune checkpoint inhibitor makes it easier to carry out the tumor treatment method of this embodiment.

[0057] The oncolytic genetically modified measles virus contained in the kit of this embodiment is the same as the oncolytic genetically modified measles virus used as the active ingredient in the pharmaceutical composition of this embodiment. The kit of this embodiment preferably uses rMV-SLAMblind or rMV-V(-)-SLAMblind, which are wild-type measles viruses genetically modified, with rMV-SLAMblind being particularly preferred. The kit of this embodiment may contain the pharmaceutical composition of this embodiment as the oncolytic genetically modified measles virus.

[0058] The immune checkpoint inhibitor contained in the kit of this embodiment can be the same as those listed above as those to be administered in combination with the pharmaceutical composition of this embodiment. The immune checkpoint inhibitor contained in the kit of this embodiment is preferably a PD-1 inhibitor or a PD-L1 inhibitor, more preferably a small molecule compound with PD-1 inhibitory activity, an anti-PD-1 antibody with PD-1 inhibitory activity, a small molecule compound with PD-L1 inhibitory activity, or an anti-PD-L1 antibody with PD-L1 inhibitory activity, and even more preferably an anti-PD-1 antibody with PD-1 inhibitory activity or an anti-PD-L1 antibody with PD-L1 inhibitory activity.

[0059] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0060] <Cells> The B16F10N4 cell line was established in the inventors' laboratory. The MC38 cell line was purchased from Kerafast and maintained in DMEM containing 10% FCS, 10 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were cultured at 37°C and 5% CO2, and routine mycoplasma testing was performed using the e-Myco® plus Mycoplasma PCR Detection Kit (iNtRON Biotechnology).

[0061] Viruses: rMV-SLAMblind and rMV-EGFP-SLAMblind were prepared in the VeroN4 cell line and used for in vitro studies. For in vivo studies, rMV-infected VeroN4 cells were cultured for 3 days in VP-SFM (Invitrogen) supplemented with 4 mM L-glutamine, 50 U / mL penicillin, and 50 μg / mL streptomycin. The culture medium was collected and centrifuged at 2000 g for 10 min at 4°C. The supernatant was incubated with 10 U / mL benzonase (Millipore) at 37°C for 40 min and then centrifuged at 5000 g for 20 min at room temperature. The resulting supernatant was filtered through a 450 nm filter and subsequently centrifuged at 53,900 g for 3 h at 4°C. The resulting pellet was resuspended in saline.

[0062] In the following examples, unless otherwise specified, "rMV" refers to rMV-SLAMblind, and "rMV-EGFP" refers to rMV-EGFP-SLAMblind.

[0063] <Animal Experiments> C57BL / 6J mice were purchased from CLEA, and 7-week-old female mice were used in the experiments (n = 5-8 per group). B16F10N4 cells were injected subcutaneously into the flank of the mice. Unless otherwise specified, 1 × 10 cells were injected into each mouse. ６ 1 × 10 cells were inoculated. ６ TCID 50 rMV-SLAMblind (1 x 10 in 100 µL saline) ６The tumor volume was calculated by measuring the long and short diameters with a caliper and calculating the volume based on the formula (width x width x length) / 2. In the survival analysis, the survival rate was 1500 mm ３ Mice exceeding this threshold or exhibiting severe bleeding were euthanized and declared dead. Axillary lymph nodes (LN) and inguinal lymph nodes were harvested as tumor-draining lymph nodes (TDLN). For PD-1 studies, mice were intraperitoneally injected with 100 μg of rat IgG2a (2A3) antibody or anti-PD-1 antibody (29F.1A12). For depletion studies, mice were intraperitoneally injected with 200 μg of rat IgG2b (LTF-2) antibody or anti-CD4 antibody (GK1.5). All antibodies were purchased from BioXcell.

[0064] <Processing of tumor tissue and TDLN> Tumor tissue was digested as described by Morito et al. (Non-Patent Document 2). The tissue was incubated in RPMI 1640 medium with 200 U / mL type I collagenase (Wako), 100 U / mL hyaluronidase (Nacalai Tesque), and 200 U / mL DNase I (Worthington Biochemical) at 37°C for 30 minutes, and then filtered through a 70 μm cell strainer. The immune cell fraction was isolated using Lympholyte-M (Cedarlane). A single-cell suspension of TDLN was prepared using a 70 μm cell strainer.

[0065] Flow cytometry: Dead cells, surface markers, and intracellular staining were assessed as follows. For FoxP3 analysis, cells were fixed and permeabilized with True-Nucleart® Transcription Factor Buffer Set (Biolegend) according to the manufacturer's instructions. All antibodies and reagents are listed in Table 1. Data were acquired on an Attune NxT acoustic cytometer (Invitrogen) and analyzed using Flowjo (TreeStar).

[0066]

[0067] <In vitro infection and sample preparation for gene expression analysis> B16F10N4, LL2N4, or 3T3N4 cells (2 × 10 ６ pcs), 0.2 x 10 ６ TCID 50 (MOI = 0.1) or 2 x 10 ６ TCID 50 (MOI = 1) rMV, or 0.4 x 10 ６ TCID 50 The cells were infected with rMV-EGFPSLAMblind at an MOI of 0.2. A pellet of uninfected VeroN4 cell lysate was used as a control. After overnight incubation, the cells were washed twice with PBS and then lysed with TRIZOL LS (Invitrogen) or collected with a cell scraper and filtered through a 70 μm cell strainer for flow cytometry. Bone marrow-derived dendritic cells (BMDCs) were prepared as described by Morito et al. (Non-Patent Document 2). One day before co-culture, 5 × 10 ５ 1 x 10 B16F10N4, LL2N4, or 3T3N4 cells ６ TCID 50 The cells were infected with rMV at 37°C for 1 hour, washed with medium, and seeded into 6-well plates. After overnight incubation, 2 x 10 ６ BMDCs were added to control or rMV-treated cells, and plates were incubated overnight at 37°C. Adherent and nonadherent cells were harvested with a cell scraper, and CD11c+ cells were positively fractionated using biotin-conjugated anti-CD11c and Mojosort streptavidin nanobeads (BioLegend) according to the manufacturer's instructions. Cell purity (>96%) was determined by staining with streptavidin-APC-efluor780 conjugate (eBioscience). Cells were solubilized with TRIZOL LS reagent (Invitrogen), and RNA was extracted according to the manufacturer's instructions.

[0068] <RT-qPCR> RNA extraction, reverse transcription, and qPCR analysis were performed in the same manner as in the method of Tamura et al. (Non-Patent Document 3). The primers used were those listed in Table 2.

[0069]

[0070] Statistical analysis: Statistical analysis was performed using Welch's t-test, Mann-Whitney U test, Kruskal-Wallis test with Steele-Dwas test, and log-rank test using Igor Pro 7 (WaveMetrics, Inc.), Excel, or EZR. A P value of <0.05 was considered statistically significant.

[0071] Example 1: Oncolytic virus therapy (OVT) induces inflammation in tumor tissue, creating a "hot" tumor microenvironment. Therefore, we administered rMV to mice and investigated its effect on the expression of checkpoint molecules in the tumor microenvironment (TME).

[0072] To analyze whether the expression of checkpoint molecules (Pdl1 and Pdl2) was increased in tumor tissue, we tested the B16F10N4 model. Figure 1(A) shows a schematic of the experimental protocol. Mice inoculated with B16F10N4 cells were administered rMV or saline on days 8 and 11 after inoculation. Three days after the last administration (day 14 after tumor cell inoculation), tumor tissues were harvested and gene expression in the tumor tissue was analyzed by RT-qPCR. Gene expression levels were normalized by Gapdh. Figure 1(B) shows the relative expression levels of Pdl1 (left panel) and Pdl2 (right panel), respectively (n = 5 for both panels). Data are representative of two independent experiments. Statistical significance was calculated using the Mann-Whitney U test. In the figure, "**" indicates p < 0.01, and "ns" indicates not significant. As shown in Figure 1(B), OVT using rMV (rMV-OVT) was found to induce an increase in the expression levels of Pdl1 and Pdl2 genes in tumor tissues.

[0073] To elucidate the mechanism, we first examined the expression levels of Pdl1 and Pdl2 genes in B16F10N4 cells infected in vitro. First, B16F10N4 cells were infected with rMV at an MOI of 0.1. After overnight incubation, cells were harvested and RNA was isolated. Gene expression was analyzed by RT-qPCR. Uninfected B16F10N4 cells served as a control. Figure 2(A) shows the relative expression levels of Pdl1 (left panel) and Pdl2 (right panel), respectively. Figure 2(B) shows a histogram showing PDL1 expression in B16F10N4 cells infected with rMV at an MOI of 0.1 or 1. Cells were harvested one day after rMV infection, stained with anti-PD-L1 antibody or IgG2B antibody (iso), and analyzed by flow cytometry. Figure 2(C) shows a histogram of PDL1 expression in B16F10N4 cells infected with rMV-EGFP. Cells were first infected with rMV or rMV-EGFP at an MOI of 0.2 and cultured overnight. Infected (virus + EGFP +) and uninfected (virus + EGFP -) cells were separated by flow cytometry (left panel). PDL1 expression was then analyzed for each type of cell (right panel). Increased Pdl1 expression was detected in rMV-infected B16F10N4 cells by qPCR and flow cytometry (Figures 2(A) and 2(B)). Meanwhile, Pdl2 expression in rMV-infected cells was comparable to that in the control group (Figure 2(A)). Using rMV-EGFP, we found that increased PDL1 protein expression was not limited to infected cells but was also observed in uninfected cells (Figure 2(C)).

[0074] Dendritic cells (DCs) are one of the cell subsets that express PDL1 and PDL2 proteins in the tumor microenvironment. Next, we analyzed gene expression levels in DCs. To mimic the rMV-treated tumor microenvironment, bone marrow-derived dendritic cells (BMDCs) were cocultured with rMV-infected or uninfected B16F10N4 cells and magnetically selected CD11c+ cells, and qPCR was performed to examine the expression of Pdl1 and Pdl2 (Figure 3(A)). Specifically, for the tumor cell-DC coculture assay, B16F10N4 cells were first infected with or without rMV at an MOI of 2 and incubated overnight, and then BMDCs were added to the B16F10N4 cells. After overnight coculture, CD11c+ cells were magnetically selected with biotin-conjugated anti-CD11c+ antibody and streptavidin nanobeads, followed by RT-qPCR. Gene expression levels were normalized by Gapdh. The expression levels of Pdl1 and Pdl2 in BMDCs cocultured with tumor cells were measured and the results are shown in Figure 3(B). Data are shown as mean ± SD. Statistical significance was analyzed by Welch's t-test. In the figure, "**" indicates p<0.01. Similar results were obtained from at least two independent experiments. As shown in Figure 3(B), coculture with rMV-infected cells increased the gene expression of Pdl1 and Pdl2 in BMDCs.

[0075] Instead of B16F10N4 cells, a mouse melanoma-derived cell line, we used LL2N4 cells, a mouse lung cancer cell line, or 3T3N4 cells, a mouse non-tumor cell line, to examine the expression of checkpoint molecules (Pdl1 and Pdl2) in a similar manner. LL2N4 and 3T3N4 cells were infected with rMV at an MOI of 0.1 and cultured, and then the expression levels of Pdl1 and Pdl2 were analyzed by RT-qPCR. The relative expression levels of Pdl1 and Pdl2 are shown in Figure 4(A) and Figure 4(B), respectively. LL2N4 and 3T3N4 cells infected with rMV at an MOI of 0.1 or 1 were harvested on day 1 after rMV infection, stained with anti-PD-L1 antibody, and analyzed by flow cytometry. Gene expression levels were normalized by Gapdh. Figure 4(C) shows the analysis results for LL2N4 cells (left panel) and 3T3N4 cells (right panel). Data are shown as mean ± SD. Statistical significance was analyzed by Welch's t-test. In the figure, "**" indicates p<0.01. In all experiments, similar results were obtained from at least two independent experiments. These results demonstrate that rMV infection induced the expression of PDL1, but not PDL2, in both LL2N4 and 3T3N4 cells, as in B16F10N4 cells.

[0076] Furthermore, as shown in Figure 5(A), we performed coculture assays of rMV-infected and uninfected LL2N4 and 3T3N4 cells with DCs to examine the expression of Pdl1 and Pdl2. The coculture assays and expression measurements were performed as in the case of B16F10N4 cells. Figure 5(B) shows the results of Pdl1 expression in BMDCs cocultured with LL2N4 or 3T3N4 cells, and Figure 5(C) shows the results of Pdl2 expression in BMDCs cocultured with LL2N4 and 3T3N4 cells. As shown in Figure 5(A), rMV infection and coculture with BMDCs increased Pdl1 and Pdl2 gene expression in both LL2N4 and 3T3N4 cells, as in B16F10N4 cells.

[0077] Finally, we examined the expression of PDL1 and PDL2 in tumor cells (NECTIN4+ cells, hereafter referred to as "N4+ cells") and DCs in B16F10N4 tumor tissue and TDLN. Figure 6 shows a schematic diagram of the experimental protocol. B16F10N4 tumor-bearing mice were treated twice or three times with rMV or saline (n = 6 / group). Two or three days after the final injection, tumor tissue and TDLN were dissected and isolated, and flow cytometry was performed. The results are shown in Figures 7(A)–7(L). Data are representative values ​​from two independent experiments. Statistical significance was calculated using the Mann-Whitney U test. In the figures, "*" indicates p<0.05, "**" indicates p<0.01, and "ns" indicates not significant.

[0078] Figure 7(A) shows a Coulter plot of PDL1 expression in N4+ cells, and Figure 7(B) shows the frequency of PDL1 expression in N4+ cells (the percentage of N4+ cells expressing PDL1). Figure 7(C) shows a Coulter plot of PDL2 expression in N4+ cells, and Figure 7(D) shows the frequency of PDL2 expression in N4+ cells (the percentage of N4+ cells expressing PDL2). As shown in Figures 7(A) to 7(D), consistent with the in vitro results, rMV-OVT increased PDL1 expression but not PDL2 expression on N4+ cells (PDL1, p<0.05; PDL2, ns, p=0.329).

[0079] Figure 7(E) shows a Coulter plot indicating PDL1 expression in DCs in the tumor, and Figure 7(F) shows the number of PDL1-expressing DCs (PDL1+DCs) in the tumor. Figure 7(G) shows a Coulter plot indicating PDL1 expression in DCs in the TDLN, and Figure 7(H) shows the number of PDL1-expressing DCs in the TDLN. Figure 7(I) shows a Coulter plot indicating PDL2 expression in DCs in the tumor, and Figure 7(J) shows the number of PDL2-expressing DCs (PDL2+DCs) in the tumor. Figure 7(K) shows a Coulter plot indicating PDL2 expression in DCs in the TDLN, and Figure 7(L) shows the number of PDL2-expressing DCs in the TDLN. The numbers of PDL1+DCs and PDL2+DCs increased in tumor tissues (Fig. 7(E), (F), (I), (J); PDL1+DCs, p<0.01; PDL2+DCs, p<0.05) and TDLNs (Fig. 7(G), (H), (K), (L); PDL1+DCs, p<0.05; PDL2+DCs, ns, p=0.067). Taken together, these results suggest that rMV-OVT induces immunosuppression by increasing PDL1 and PDL2 in tumor tissues.

[0080] Example 2: In Example 1, rMV-OVT was shown to induce the expression of PDL1 and PDL2 in tumor tissue. Therefore, the effect of combining rMV-OVT with immune checkpoint inhibitor therapy was investigated. Anti-PD1 antibody was used as the immune checkpoint inhibitor.

[0081] Figure 8 shows a schematic representation of the experimental protocol. To evaluate the therapeutic efficacy of the combination therapy, saline or rMV was administered directly into tumors in tumor-bearing mice inoculated with B16F10N4 cells. At the indicated time points, 100 μg of anti-PD1 antibody (aPD1) or rat IgG2b antibody (iso) was then administered intraperitoneally (n = 8 / group). Figure 9(A) shows the time course of tumor volume in individual mice in the saline and IgG2B antibody (iso) group (saline + iso), saline and anti-PD1 antibody (saline + aPD1), rMV and IgG2b antibody (iso) group (rMV + iso), and rMV and anti-PD1 antibody (rMV + aPD1) groups. Figure 9(B) also shows tumor growth curves for each group from B16F10N4 cell inoculation to day 13 (D13). Data are presented as mean ± SEM. Statistical analysis was performed by the Kruskal-Wallis test with Steele-Dwas post-hoc test. Kaplan-Meier survival curves for each group are shown in Figure 9(C).

[0082] As shown in Figure 9(A), treatment with rMV or aPD1 alone significantly suppressed tumor growth, whereas treatment with rMV and aPD1 combined significantly enhanced tumor growth suppression compared with treatment with aPD1 or rMV alone (Saline + iso vs. Saline + aPD1, p = 0.152; Saline + iso vs. rMV + iso, p < 0.05; Saline + iso vs. rMV + aPD1, p < 0.01). As shown in Figure 9(C), the survival rates were 14.0% for the Saline + iso group, 17.0% for the Saline + aPD1 group, 17.5% for the rMV + iso group, and 22.5% for the rMV + aPD1 group. The survival time of mice treated with the combination therapy of anti-PD1 antibody and rMV was also significantly longer than that of untreated or monotreated mice (Saline + iso group vs. Saline + aPD1 group, p < 0.05; Saline + iso group vs. rMV + iso group, p < 0.05; Saline + iso group vs. rMV + aPD1 group, p < 0.01).

[0083] Next, we investigated T cell profiling after combination therapy with rMV and a PDL1 inhibitor. Figure 10(A) shows a schematic diagram of the experimental protocol. Mice bearing B16F10N4 cells were administered rMV or saline, along with anti-PD1 antibody (aPD1) or rat IgG2b antibody (iso) at the indicated times (n = 7 / group). On day 15 (D15) after tumor cell inoculation, T cells in the TDLN and tumor tissues were analyzed by flow cytometry. The results are shown in Figures 10(B)–(F). Figure 10(B) shows the number of IFNγ+CD8+ T cells, Figure 10(C) shows the number of granulocyte B (GrB)+CD8+ T cells, Figure 10(D) shows the number of IFNγ+CD4+ T cells, Figure 10(E) shows the number of IL4+CD4+ T cells, and Figure 10(F) shows the number of FoxP3+CD4+ T cells. Statistical significance was determined by the Kruskal-Wallis test with a Steele-Dwas post-hoc test. "*" indicates p<0.05, and "**" indicates p<0.01. Analysis of these effector CD8+ T cells revealed that the combination of rMV-OVT and aPD1 significantly increased IFNγ+CD8+ T cells and GrB+CD8+ T cells in the TDLN (IFNγ+CD8, p<0.01; GrB+CD8, p<0.01), but the increase in the tumor did not reach significance (Figures 10(B) and 10(C)).

[0084] Based on these results, we further investigated the effect of CD4+ cell depletion on the antitumor effect of rMV-OVT. Figure 11(A) shows a schematic diagram of the experimental protocol. Mice bearing B16F10N4 cells were administered rMV or saline, anti-PD1 antibody (aPD1) or rat IgG2b antibody (iso), and 200 μg of anti-CD4 antibody (aCD4) or rat IgG2b antibody (iso) at the times indicated. On day 18 after B16F10N4 cell inoculation, blood was collected to examine the status of CD4+ and CD8+ T cells. Figure 11(B) shows the time course of tumor volume in individual mice in the saline and IgG2b antibody (iso) group (Saline+iso), the rMV, anti-PD1, and IgG2b antibody (iso) group (Combo+iso), and the rMV, anti-PD1, and anti-CD4 antibody (aCD4) group (Combo+aCD4). Kaplan-Meier survival curves for each group are also shown in Figure 11(C). Data are representative values ​​from two independent experiments. Statistical significance of survival analysis was determined by the log-rank test using Holm's correction. In the figure, "*" indicates p<0.05, "**" indicates p<0.01, and "n.s." indicates not significant.

[0085] As shown in Figure 11(B), depletion of CD4+ T cells suppressed tumor growth in mice treated with rMV and anti-PD1 antibodies. Furthermore, as shown in Figure 11(C), the survival rates were 18.0% in the Saline+iso group, 23.5% in the Combo+iso group, and 34.0% in the Combo+aCD4 group. The survival time in the group receiving anti-CD4 (aCD4) antibodies in addition to the combination therapy was extended compared with control mice or mice receiving only the combination therapy (p<0.01 for Saline+iso vs. Combo+iso, p<0.05 for Combo+iso vs. Combo+aCD4). These results confirmed that depletion of CD4+ T cells by the combination therapy of rMV-OVT and immune checkpoint inhibitors with anti-CD4 antibodies produced a synergistic antitumor effect.

[0086] PD1+CD8+ cell analysis was performed on blood samples on day 18. Figure 12(A) shows a Coulter plot of PD1 expression on CD8+ T cells, and Figure 12(B) shows the frequency of PD1 expression on CD8+ T cells (the percentage of PD1-expressing CD8+ T cells (PD1+CD8+ T cells) among all CD8+ T cells). Data are representative of two independent experiments. Statistical significance was calculated using the Mann-Whitney test. In the figure, "**" indicates p<0.01, and "ns" indicates not significant. These results demonstrate that depletion of CD4+ T cells enhances the antitumor effect of rMV and anti-PD1 antibody combination therapy and increases activated CD8+ T cells in the blood.

Claims

1. A pharmaceutical composition for treating a tumor, comprising an oncolytic genetically modified measles virus, wherein the tumor comprises tumor cells expressing PVRL4 / Nectin4, and the oncolytic genetically modified measles virus is rMV-SLAMblind or rMV-V(-)-SLAMblind, which is a wild-type measles virus genetically modified, and the pharmaceutical composition is administered in combination with an immune checkpoint inhibitor.

2. The pharmaceutical composition according to claim 1, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

3. The pharmaceutical composition according to claim 2, wherein the PD-1 inhibitor is a low molecular weight compound with PD-1 inhibitory activity or an anti-PD-1 antibody, and the PD-L1 inhibitor is a low molecular weight compound with PD-L1 inhibitory activity or an anti-PD-L1 antibody.

4. The pharmaceutical composition according to claim 2 or 3, wherein the PD-1 inhibitor is an anti-PD-1 antibody having PD-1 inhibitory activity, and the PD-L1 inhibitor is an anti-PD-L1 antibody having PD-L1 inhibitory activity.

5. The pharmaceutical composition of claim 3 or 4, wherein the anti-PD-1 antibody is one or more selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab, MEDI0608, and tislelizumab, and the anti-PD-L1 antibody is one or more selected from the group consisting of avelumab, atezolizumab, durvalumab, and BMS-936559.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the immune checkpoint inhibitor is one or more anti-PD-1 antibodies selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab, MEDI0608, and tislelizumab.

7. The pharmaceutical composition of any one of claims 1 to 5, wherein the immune checkpoint inhibitor is one or more anti-PD-L1 antibodies selected from the group consisting of avelumab, atezolizumab, durvalumab, and BMS-936559.

8. The pharmaceutical composition according to any one of claims 1 to 7, which is administered in combination with the immune checkpoint inhibitor and the CD4 inhibitor.

9. The pharmaceutical composition according to claim 8, wherein the CD4 inhibitor is a low molecular weight compound or an anti-CD4 antibody having CD4 inhibitory activity.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the tumor is breast cancer, lung cancer, colon cancer, stomach cancer, pancreatic cancer, or bladder cancer.

11. The pharmaceutical composition of claim 10, wherein the breast cancer is triple-negative breast cancer.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the tumor is selected from the group consisting of a primary tumor, a deep tumor, a recurrent tumor, a metastatic tumor, and a peritoneally disseminated tumor.

13. A method for treating a tumor, comprising administering to a tumor patient a combination of an oncolytic genetically modified measles virus and an immune checkpoint inhibitor, wherein the tumor contains tumor cells expressing PVRL4 / Nectin4, and the oncolytic genetically modified measles virus is rMV-SLAMblind or rMV-V(-)-SLAMblind, which is a wild-type measles virus genetically modified.

14. The method of claim 13, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

15. The method according to claim 14, wherein the PD-1 inhibitor is a small molecule compound having PD-1 inhibitory activity or an anti-PD-1 antibody, and the PD-L1 inhibitor is a small molecule compound having PD-L1 inhibitory activity or an anti-PD-L1 antibody.

16. The method according to claim 14 or 15, wherein the PD-1 inhibitor is an anti-PD-1 antibody having PD-1 inhibitory activity, and the PD-L1 inhibitor is an anti-PD-L1 antibody having PD-L1 inhibitory activity.

17. The method of claim 15 or 16, wherein the anti-PD-1 antibody is one or more selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab, MEDI0608, and tislelizumab, and the anti-PD-L1 antibody is one or more selected from the group consisting of avelumab, atezolizumab, durvalumab, and BMS-936559.

18. The method of any one of claims 13 to 17, wherein the immune checkpoint inhibitor is one or more anti-PD-1 antibodies selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab, MEDI0608, and tislelizumab.

19. The method of any one of claims 13 to 17, wherein the immune checkpoint inhibitor is one or more anti-PD-L1 antibodies selected from the group consisting of avelumab, atezolizumab, durvalumab, and BMS-936559.

20. The method of any one of claims 13 to 19, wherein the tumor patient is administered the oncolytic genetically modified measles virus followed by administration of the immune checkpoint inhibitor.

21. The method of any one of claims 13 to 19, wherein the period during which the oncolytic genetically modified measles virus is administered to the tumor patient and the period during which the immune checkpoint inhibitor is administered to the tumor patient overlap at least partially.

22. The method of any one of claims 13 to 19, wherein the oncolytic genetically modified measles virus, the immune checkpoint inhibitor, and a CD4 inhibitor are administered in combination to the tumor patient.

23. The method of claim 22, wherein the tumor patient is administered the oncolytic genetically modified measles virus, followed by the immune checkpoint inhibitor, and then the CD4 inhibitor.

24. The method of claim 22 or 23, wherein the period during which the oncolytic genetically modified measles virus is administered to the tumor patient and the period during which the immune checkpoint inhibitor is administered to the tumor patient overlap at least in part.

25. The method of any one of claims 22 to 24, wherein the period during which the immune checkpoint inhibitor is administered to the tumor patient and the period during which the CD4 inhibitor is administered to the tumor patient overlap at least in part.

26. The method of any one of claims 22 to 25, wherein the period during which the oncolytic genetically modified measles virus is administered to the tumor patient and the period during which the CD4 inhibitor is administered to the tumor patient overlap at least partially.

27. The method according to any one of claims 22 to 26, wherein the CD4 inhibitor is a low molecular weight compound having CD4 inhibitory activity or an anti-CD4 antibody.

28. The method of any one of claims 13 to 27, wherein the tumor is breast cancer, lung cancer, colon cancer, gastric cancer, pancreatic cancer, or bladder cancer.

29. The method of claim 28, wherein the breast cancer is triple-negative breast cancer.

30. The method according to any one of claims 13 to 29, wherein the tumor is selected from the group consisting of a primary tumor, a deep tumor, a recurrent tumor, a metastatic tumor, and a peritoneally disseminated tumor.

31. The method of any one of claims 13 to 30, wherein the oncolytic genetically modified measles virus is administered directly to the tumor in the tumor patient.

32. The method of any one of claims 13 to 30, wherein the oncolytic genetically modified measles virus is administered intraperitoneally to the tumor patient.

33. The method of any one of claims 13 to 32, wherein the immune checkpoint inhibitor is administered intravenously or intraperitoneally to the tumor patient.

34. The method of any one of claims 22 to 33, wherein the CD4 inhibitor is administered intravenously to the tumor patient.

35. A kit for use in treating a tumor, comprising an oncolytic genetically modified measles virus and an immune checkpoint inhibitor, wherein the tumor contains tumor cells expressing PVRL4 / Nectin4, and the oncolytic genetically modified measles virus is rMV-SLAMblind or rMV-V(-)-SLAMblind, which is a wild-type measles virus genetically modified.

36. The kit of claim 35, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

37. The kit according to claim 36, wherein the PD-1 inhibitor is a low molecular weight compound having PD-1 inhibitory activity or an anti-PD-1 antibody, and the PD-L1 inhibitor is a low molecular weight compound having PD-L1 inhibitory activity or an anti-PD-L1 antibody.

38. The kit described in claim 36 or 37, wherein the PD-1 inhibitor is an anti-PD-1 antibody having PD-1 inhibitory activity, and the PD-L1 inhibitor is an anti-PD-L1 antibody having PD-L1 inhibitory activity.

39. The kit of claim 37 or 38, wherein the anti-PD-1 antibody is one or more selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab, MEDI0608, and tislelizumab, and the anti-PD-L1 antibody is one or more selected from the group consisting of avelumab, atezolizumab, durvalumab, and BMS-936559.

40. The kit of any one of claims 35 to 39, wherein the immune checkpoint inhibitor is one or more anti-PD-1 antibodies selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab, MEDI0608, and tislelizumab.

41. The kit of any one of claims 35 to 39, wherein the immune checkpoint inhibitor is one or more anti-PD-L1 antibodies selected from the group consisting of avelumab, atezolizumab, durvalumab, and BMS-936559.

42. The kit of any one of claims 35 to 41, further comprising a CD4 inhibitor.

43. The kit according to claim 42, wherein the CD4 inhibitor is a low molecular weight compound having CD4 inhibitory activity or an anti-CD4 antibody.

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