Recombinant rhabdovirus encoding CCL21

Abstract

To provide: in the field of oncolytic viruses, in particular a recombinant rhabdovirus, such as vesicular stomatitis virus encoding a CCL21 protein in its genome; and the use of the recombinant virus in treatment of cancer, and also methods for producing such viruses.SOLUTION: The present invention provides, in the field of oncolytic viruses, in particular a recombinant rhabdovirus, such as vesicular stomatitis virus encoding a CCL21 protein in its genome. The invention further provides the use of the recombinant virus in treatment of cancer, and also methods for producing such viruses.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to the field of oncolytic viruses, in particular to recombinant rhabdoviruses encoding CCL21 protein in their genome.The present invention further relates to the use of recombinant rhabdoviruses in the treatment of cancer, and to methods for producing such viruses.

[0002] Background of the Invention Oncolytic viruses are an emerging class of biologics that can selectively replicate in and kill cancer cells and spread within tumors. Efforts to further improve oncolytic viruses and increase their therapeutic potential have led to the development of so-called armed viruses that encode tumor antigens or immunomodulatory transgenes in their genomes to improve their effectiveness in tumor treatment.

[0003] In many cases, tumors lack T cells, creating what has become known as an "immune desert"—a tumor microenvironment in which T cells from the immune system cannot or will not infiltrate the tumor and kill uncontrolled growing cells. To evade immune surveillance, tumors have been hypothesized to create an immunosuppressive microenvironment by recruiting myeloid-derived suppressor cells or secreting factors, including TGF-β, which plays the dual role of inducing extracellular matrix gene expression and suppressing the expression of chemokines and cytokines required to promote T-cell infiltration into tumors (Pickup M, Novitskiy S, Moses HL. The roles of TGF-beta in the tumor microenvironment. Nat Rev Cancer 2013;13:788-99).Furthermore, studies have found that tumors with high expression of genes corresponding to an immunosuppressive microenvironment are associated with poor outcomes across many cancer types, including ovarian and colorectal cancer (Calon A, Lonardo E, Berenguer-Llergo A, Espinet E, Hernando-Momblona X, Iglesias M, et al. Stromal gene expression defines poor-prognosis subtypes in colorectal cancer. Nat Genet 2015;47:320-9; Ryner L, Guan Y, Firestein R, Xiao Y, Choi Y, Rabe C, et al. Upregulation of periostin and reactive stroma is associated with primary chemoresistance and predicts clinical outcomes in epithelial ovarian cancer. Clin Cancer Res 2015;21:2941-51; Tothill RW, Tinker AV, George J, Brown R, Fox SB, Lade S, et al. Novel molecular subtypes of serous and endometrioid ovarian cancer linked to clinical outcome. Clin Cancer Res 2008;14:5198-208.).

[0004] One recent approach has predicted an oncolytic virus encoding IFN-β protein as cargo in its genome. A further approach has considered the expression of the tumor antigen MAGE-A3. In addition to identifying an appropriate and effective cargo, the expression of additional cargo from the viral backbone always carries the risk of enhancing not only the antitumor effect but also antiviral immunity. It must be noted that the cargo must not limit the oncolytic ability of the virus to such an extent that the benefits obtained by expressing the therapeutic cargo are negated by the loss of oncolytic ability. Thus, there is a need in the art for further improved armed oncolytic viruses that can be used in the effective treatment of tumors. Furthermore, there is a need in the art for selectively improving the infiltration of T cells and / or dendritic cells into the immunosuppressive tumor microenvironment.

[0005] Summary of the Invention The present invention addresses the above needs by providing a recombinant rhabdovirus (such as vesicular stomatitis virus) that encodes a CCL21 protein or a functional variant thereof, preferably human CCL21, in its genome.

[0006] It should be understood that any embodiment relating to a particular aspect may also be combined with other embodiments relating to that particular aspect, even in multiple layers and combinations that include several embodiments for that particular aspect.

[0007] In a first aspect, the present invention relates to a recombinant rhabdovirus encoding in its genome at least one CCL21 protein or a functional variant thereof.

[0008] In one embodiment of the first aspect, the CCL21 protein or functional variant thereof is (i) a plasmin-treated CCL21 protein, (ii) a c-terminally truncated CCL21 protein, (iii) a protein comprising SEQ ID NO: 2 or having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% identity to SEQ ID NO: 2, (iv) a protein comprising SEQ ID NO: 3 or having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% identity to SEQ ID NO: 3, or (v) a protein comprising SEQ ID NO: 4 or having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% identity to SEQ ID NO: 4. (vi) a protein according to any of (i) to (v) further comprising a signal peptide sequence; (vii) a protein comprising SEQ ID NO: 1 or having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% identity to SEQ ID NO: 1; or (viii) a protein comprising SEQ ID NO: 5 or having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% identity to SEQ ID NO: 5.

[0009] In one embodiment of the first aspect, the recombinant rhabdovirus is a vesiculovirus.

[0010] In one embodiment of the first aspect, the vesiculovirus is selected from the group comprising vesicular stomatitis Alagoas virus (VSAV), Karajas virus (CJSV), Chandipura virus (CHPV), Coccoccus virus (COCV), vesicular stomatitis Indiana virus (VSIV), Isfahan virus (ISFV), Maraba virus (MARAV), vesicular stomatitis New Jersey virus (VSNJV), or piri virus (PIRYV), preferably vesicular stomatitis Indiana virus (VSIV) or preferably vesicular stomatitis New Jersey virus (VSNJV).

[0011] In one embodiment of the first aspect, the recombinant rhabdovirus is replication-competent.

[0012] In one embodiment of the first aspect, the CCL21 protein or functional variant thereof is human CCL21.

[0013] In one embodiment of the first aspect, the recombinant rhabdovirus lacks a functional gene encoding glycoprotein G and / or lacks a functional glycoprotein G; alternatively, the gene encoding glycoprotein G is replaced with a gene encoding glycoprotein GP of another virus, and / or glycoprotein G is replaced with glycoprotein GP of another virus; alternatively, the gene encoding glycoprotein G is replaced with a gene encoding glycoprotein GP of an arenavirus, and / or glycoprotein G is replaced with glycoprotein GP of an arenavirus. In a more preferred embodiment, the gene encoding glycoprotein G is replaced with a gene encoding glycoprotein GP of Dandenong virus or Mopeia virus, and / or glycoprotein G is replaced with glycoprotein GP of Dandenong virus or Mopeia virus. Even more preferably, the gene encoding glycoprotein G is replaced with a gene encoding glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), and / or glycoprotein G is replaced with glycoprotein GP of LCMV.

[0014] In a preferred embodiment of the first aspect, the present invention provides a CCL21 protein comprising in its genome: (i) a plasmin-treated CCL21 protein; (ii) a c-terminally truncated CCL21 protein; (iii) a protein comprising SEQ ID NO: 2 or having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% identity to SEQ ID NO: 2; (iv) a protein comprising SEQ ID NO: 3 or having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% identity to SEQ ID NO: 3; (v) a protein comprising SEQ ID NO: 4 or having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% identity to SEQ ID NO: 4; (vi) a protein according to any of (i) to (v), further comprising a signal peptide sequence; The present invention provides a recombinant vesicular stomatitis virus encoding at least one CCL21 protein or functional variant thereof, preferably human CCL21, selected from the group comprising: (i) a protein comprising SEQ ID NO: 1 or having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% identity to SEQ ID NO: 1; or (viii) a protein comprising SEQ ID NO: 5 or having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% identity to SEQ ID NO: 5, wherein the gene encoding glycoprotein G of the recombinant vesicular stomatitis virus is replaced by the gene encoding glycoprotein GP of lymphocytic choriomeningitis virus (LCMV) and / or glycoprotein G is replaced by glycoprotein GP of LCMV.

[0015] In a second aspect, the present invention relates to a recombinant vesicular stomatitis virus encoding in its genome at least the vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M), glycoprotein (G), and at least one CCL21 protein or functional variant thereof, preferably human CCL21.

[0016] In one embodiment of the second aspect, the nucleoprotein (N) comprises the amino acid sequence set forth in SEQ ID NO: 7, or a functional variant that is at least 80%, 85%, 90%, 92%, 94%, 96%, 98% identical to SEQ ID NO: 7.

[0017] In one embodiment of the second aspect, the phosphorylated protein (P) comprises the amino acid sequence set forth in SEQ ID NO: 8, or a functional variant that is at least 80%, 85%, 90%, 92%, 94%, 96%, 98% identical to SEQ ID NO: 8.

[0018] In one embodiment of the second aspect, the large protein (L) comprises the amino acid sequence shown in SEQ ID NO: 9 or a functional variant that is at least 80%, 85%, 90%, 92%, 94%, 96%, 98% identical to SEQ ID NO: 9.

[0019] In one embodiment of the second aspect, the matrix protein (M) comprises the amino acid sequence set forth in SEQ ID NO: 10, or a functional variant that is at least 80%, 85%, 90%, 92%, 94%, 96%, 98% identical to SEQ ID NO: 10.

[0020] In a preferred embodiment of the second aspect, the nucleoprotein (N) comprises the amino acid sequence set forth in SEQ ID NO:7 or a functional variant that is at least 80%, 85%, 90%, 92%, 94%, 96%, 98% identical to SEQ ID NO:7, the phosphoprotein (P) comprises the amino acid sequence set forth in SEQ ID NO:8 or a functional variant that is at least 80%, 85%, 90%, 92%, 94%, 96%, 98% identical to SEQ ID NO:8, the large protein (L) comprises the amino acid sequence set forth in SEQ ID NO:9 or a functional variant that is at least 80%, 85%, 90%, 92%, 94%, 96%, 98% identical to SEQ ID NO:9, and the matrix protein (M) comprises the amino acid sequence set forth in SEQ ID NO:10 or a functional variant that is at least 80%, 85%, 90%, 92%, 94%, 96%, 98% identical to SEQ ID NO:10.

[0021] In one embodiment of the second aspect, the recombinant vesicular stomatitis virus is replication-competent.

[0022] In one embodiment of the second aspect, the recombinant vesicular stomatitis virus lacks a functional gene encoding glycoprotein G and / or lacks a functional glycoprotein G; alternatively, the gene encoding glycoprotein G is replaced by a gene encoding glycoprotein GP of another virus and / or glycoprotein G is replaced by glycoprotein GP of another virus; alternatively, the gene encoding glycoprotein G is replaced by a gene encoding glycoprotein GP of lymphocytic choriomeningitis virus (LCMV) and / or glycoprotein G is replaced by glycoprotein GP of LCMV.

[0023] In one embodiment of the second aspect, the CCL21 protein or functional variant thereof is selected from the group comprising: (i) a plasmin-treated CCL21 protein, (ii) a C-terminally truncated CCL21 protein, (iii) a protein comprising SEQ ID NO: 2 or having at least 80% identity to SEQ ID NO: 2, (iv) a protein comprising SEQ ID NO: 3 or having at least 80% identity to SEQ ID NO: 3, (v) a protein comprising SEQ ID NO: 4 or having at least 80% identity to SEQ ID NO: 4, (vi) a protein according to any of (i) to (v) further comprising a signal peptide sequence, (vii) a protein comprising SEQ ID NO: 1 or having at least 80% identity to SEQ ID NO: 1, or a protein comprising SEQ ID NO: 5 or having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% identity to SEQ ID NO: 5.

[0024] In a preferred embodiment of the second aspect, the present invention provides a recombinant vesicular stomatitis virus encoding in its genome the vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M), glycoprotein (G), and at least one CCL21 protein or functional variant thereof, preferably human CCL21, wherein the CCL21 protein or functional variant thereof is selected from the group consisting of: (i) a plasmin-treated CCL21 protein; (ii) a c-terminally truncated CCL21 protein; (iii) a protein comprising SEQ ID NO:2 or having at least 80% identity to SEQ ID NO:2; (iv) a protein comprising SEQ ID NO:3 or having at least 80% identity to SEQ ID NO:3; (v) a protein comprising SEQ ID NO:4 or having at least 80% identity to SEQ ID NO:4; (vi) a protein according to any of (i) to (v) which further comprises a signal peptide sequence; (vii) a protein comprising SEQ ID NO:1 or having at least 80% identity to SEQ ID NO:1; or (viii) a protein comprising SEQ ID NO:5 or having at least 80% identity to SEQ ID NO:5. a phosphoprotein selected from the group comprising a protein having 80% identity with a gene encoding glycoprotein G of vesicular stomatitis virus (VSV) and / or a gene encoding glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), wherein the gene encoding glycoprotein G of Vesicular Stomatitis Virus (VSV) is replaced by a gene encoding glycoprotein GP of Lymphocytic Choriomeningitis Virus (LCMV), and / or wherein glycoprotein G is replaced by glycoprotein GP of LCMV, and wherein the nucleoprotein (N) comprises the amino acids set forth in SEQ ID NO: 7, or a functional variant at least 80%, 85%, 90%, 92%, 94%, 96%, 98% identical to SEQ ID NO: 7; The protein (P) comprises the amino acids set forth in SEQ ID NO:8 or a functional variant at least 80%, 85%, 90%, 92%, 94%, 96%, 98% identical to SEQ ID NO:8; the large protein (L) comprises the amino acids set forth in SEQ ID NO:9 or a functional variant at least 80%, 85%, 90%, 92%, 94%, 96%, 98% identical to SEQ ID NO:9; and the matrix protein (M) comprises the amino acids set forth in SEQ ID NO:10 or a functional variant at least 80%, 85%, 90%, 92%, 94%, 96%, 98% identical to SEQ ID NO:10.

[0025] In a third aspect, the present invention provides a pharmaceutical composition, characterized in that the composition comprises a recombinant rhabdovirus according to the first aspect or any of its embodiments, or a recombinant vesicular stomatitis virus according to the second aspect or any of its embodiments.

[0026] In a fourth aspect, the present invention provides a recombinant rhabdovirus according to the first aspect or any of its embodiments, or a recombinant vesicular stomatitis virus according to the second aspect or any of its embodiments, or a pharmaceutical composition according to the third aspect or any of its embodiments for use as a medicament.

[0027] In one embodiment of the fourth aspect, the present invention provides a recombinant rhabdovirus, a recombinant vesicular stomatitis virus, or a pharmaceutical composition for use in treating cancer, preferably solid cancer.In a preferred embodiment, the solid cancer is selected from the list comprising reproductive system tumor, ovarian tumor, pancreatic tumor, testicular tumor, endocrine tumor, gastrointestinal tumor, liver tumor, kidney tumor, colon tumor, colorectal tumor, bladder tumor, prostate tumor, skin tumor, melanoma, respiratory system tumor, lung tumor, breast tumor, head and neck tumor, head and neck squamous cell carcinoma (HNSCC) and bone tumor.

[0028] In one embodiment of the fourth aspect, recombinant rhabdovirus, recombinant vesicular stomatitis virus or pharmaceutical compositions should be administered intratumorally or intravenously.In another related embodiment, recombinant rhabdovirus, recombinant vesicular stomatitis virus or pharmaceutical compositions should be administered intratumorally at least once, and then intravenously.In a further related embodiment, the subsequent intravenous administration of recombinant rhabdovirus, recombinant vesicular stomatitis virus or pharmaceutical compositions is given 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or 31 days after the first intratumoral administration.

[0029] In a fifth aspect, the present invention provides a composition comprising a recombinant rhabdovirus according to the first aspect or any of its embodiments, or a recombinant vesicular stomatitis virus according to the second aspect or any of its embodiments, and further an inhibitor, wherein the inhibitor is a PD-1 pathway inhibitor or a SMAC mimetic.

[0030] In one embodiment of the fifth aspect, the PD-1 pathway inhibitor is an antagonist antibody, which is directed against PD-1 or PD-L1. In a further related embodiment, the SMAC mimetic is selected from the group consisting of any of compounds 1 to 26 from Table 2, or a pharmaceutically acceptable salt of one of these compounds. In another related embodiment, the PD-1 pathway inhibitor is an antagonist selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, atezolizumab, avelumab, durvalumab, PDR-001, PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5 (as shown in Table 1).

[0031] In a sixth aspect, the present invention provides a kit of parts comprising: a recombinant rhabdovirus, a recombinant vesicular stomatitis virus, or a pharmaceutical composition as defined in any of the first to third aspects or any of their embodiments, and a PD-1 pathway inhibitor or SMAC mimetic as defined in any of the embodiments relating to the fifth aspect.

[0032] In a seventh aspect, the present invention provides a combination treatment comprising: a) a recombinant rhabdovirus according to the first aspect or any of its embodiments, or a recombinant vesicular stomatitis virus according to the second aspect or any of its embodiments, or a pharmaceutical composition according to the third aspect or any of its embodiments, and b) a PD-1 pathway inhibitor or a SMAC mimetic. In one embodiment related to the seventh aspect, a) and b) may be administered simultaneously, sequentially, or alternately. In a related embodiment, a) and b) are administered via different administration routes. In a further related embodiment, a) is administered intratumorally and b) is administered intravenously.

[0033] In one embodiment of the seventh aspect, the PD-1 pathway inhibitor is an antagonist antibody, which is directed against PD-1 or PD-L1. In a related embodiment, the PD-1 pathway inhibitor is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, atezolizumab, avelumab, durvalumab, PDR-001, PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5 (see Table 1). In a further related embodiment, the SMAC mimetic is selected from the group consisting of any one of compounds 1 to 26 according to Table 2, or a pharmaceutically acceptable salt of one of these compounds.

[0034] In an eighth aspect, the present invention provides a virus-producing cell, characterized in that the cell produces a recombinant rhabdovirus according to the first aspect or any of its embodiments, or a recombinant vesicular stomatitis virus according to the second aspect or any of its embodiments.

[0035] In one embodiment of the eighth aspect, the virus producer cells are Vero cells, HEK cells, HEK293 cells, Chinese hamster ovary cells (CHO), or baby hamster kidney (BHK) cells.

[0036] In a ninth aspect, the present invention provides a method for producing a recombinant rhabdovirus in cell culture: (i) infecting a host cell with a recombinant rhabdovirus, preferably a vesicular stomatitis virus; (ii) culturing the host cells under conditions that allow replication of the recombinant rhabdovirus; (iii) recovering the recombinant rhabdovirus from the cell culture; (iv) optionally, enzymatic treatment of the virus harvest, preferably with benzonase; (v) capturing the rhabdovirus harvest by loading onto a cation exchange monolith membrane sorber or resin followed by elution; (vi) subjecting the eluate of step (v) to size exclusion, multimodal size exclusion / ion exchange, or tangential flow filtration to polish the rhabdovirus; (vii) buffer exchange of polished rhabdovirus by ultrafiltration / diafiltration; (viii) Sterile filtration of rhabdovirus.

[0037] In one embodiment of the ninth aspect, the host cell is a HEK293 cell.

[0038] In one embodiment of the ninth aspect, the host cells are cultured in suspension.

[0039] In one embodiment of the ninth aspect, recombinant rhabdovirus is formulated in pharmaceutical composition.In a preferred embodiment, the recombinant rhabdovirus according to the first aspect or any of its embodiments, or the recombinant vesicular stomatitis virus according to the second aspect or any of its embodiments is formulated in pharmaceutical composition.

[0040] In a further embodiment, the recombinant rhabdovirus encodes in its RNA genome at least one CCL21 protein or functional variant thereof, preferably human CCL21, wherein the RNA genome of the recombinant rhabdovirus comprises or consists of a coding sequence identical to or at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:24. [Brief explanation of the drawings]

[0041] [Figure 1] Expression analysis of LLC1-IFNARKO tumors (total RNA) from control or VSV-GP-treated mice for the indicated genes. [Figure 2]Single tumor growth curves of CT26.CL25-IFNARKO tumor-bearing mice: (A) control mice (mock-treated), (B) mice treated with anti-PD-1, (C) mice treated with VSV-GP iv, or (D) mice treated with a combination of VSV-GP and anti-PD-1. [Figure 3] Tumor (re)challenge analysis of cured mice pretreated with VSV-GP (see Figure 2C) or the combination of VSV-GP and anti-PD-1 (see Figure 2D). (A) Naive mice with CT26.CL25-IFNARKO tumor cells were used as controls. For tumor (re)challenge, CT26Cl25 IFNAR cells were injected into (B) VSV-GP-treated long-term tumor-free mice (cured) from the experiment shown in Figure 2C or (C) long-term tumor-free mice (cured) treated with the combination of VSV-GP and anti-PD-1 from the experiment shown in Figure 2D. [Figure 4] Expression analysis of LLC1-IFNARKO tumors (total RNA) from control or VSV-GP-treated mice for the indicated chemokines. [Figure 5] (A) Diagram illustrating the CCL21 (transgene) insertion site within the VSV-GP genome. (B) Diagram illustrating virus rescue. [Figure 6] (A) Analysis of mouse CCL21 in the supernatant of HEK293 cells infected with VSV-GP-muCCL21 (VSV-GP encoding full-length mouse CCL21). (B) Transwell setup and functional analysis of mouse T cell migration using recombinant mouse CCL21 or supernatant from VSV-GP or VSV-GP-muCCL21-infected HEK293 cells. [Figure 7] Single tumor growth analysis of CT26.CL25-IFNARKO tumor-bearing mice. Tumor-bearing mice were treated with (A) VSV-GP, (B) SMACm (SMAC mimetic), or (C) VSV-GP in combination with SMACm (SMAC mimetic). The thick black line indicates the tumor volume of untreated control mice, while the thin gray line indicates the tumor volume of mice treated with either (A) VSV-GP, (B) SMACm, or (C) VSV-GP + SMACm. [Figure 8]Single tumor growth analysis of CT26.CL25-IFNARKO tumor-bearing mice. Tumor-bearing mice were treated with (A) VSV-GP-CCL21, (B) SMACm (SMAC mimetic), or (C) VSV-GP-CCL21 in combination with SMACm (SMAC mimetic). The thick black line indicates the tumor volume of untreated control mice, while the thin gray line indicates the tumor volume of mice treated with either (A) VSV-GP-CCL21, (B) SMACm, or (C) VSV-GP-CCL21 + SMACm. [Figure 9] Survival analysis of the experiments depicted in Figures 7A-C and 8A-C. [Figure 10] Expression analysis of LLC1-IFNARKO tumors (total RNA) from control, VSV-GP, or VSV-GPmuCCL21-treated mice for CD3 epsilon, CXCL10, and the codon-optimized sequence of murine CCL21 encoded by VSV-GP-muCCL21. [Figure 11] Evaluation of neurotoxicity induced by intracerebral injection of VSV-G DsRed (neurotoxic wild-type VSV), VSV-GP, VSV-GP-muCCL21, or PBS. Panel (A) shows the percentage of weight gain / loss in mice over time after each injection. Panel (B) shows the survival rate of mice over time after each injection. [Figure 12] (A) Analysis of human CCL21 in the supernatant of HEK293 cells infected with VSV-GP-huCCL21 (VSV-GP encoding full-length human CCL21). (B) Functional analysis of human T cell migration using a Transwell setup and supernatants from recombinant human CCL21, or VSV-GP or VSV-GP-huCCL21-infected HEK293 cells. [Figure 13] Functional analysis of mouse (A, left panel) and rat (B, right panel) T cell migration using a Transwell setup and recombinant mouse CCL21 vs. human CCL21 or rat CCL21 vs. human CCL21, respectively, to test species cross-reactivity. [Figure 14]Single tumor growth analysis of CT26.CL25-IFNARKO tumor-bearing control mice or mice treated with VSV-GP or VSV-GP-huCCL21. [Figure 15] Cartoon illustrating CCL21 processing by plasmin to generate short diffusible N-terminal fragments. [Figure 16] Functional analysis of human T cell migration using a Transwell setup and supernatants from plasmid-transfected HEK293 cells expressing recombinant human CCL21 (rec. CCL21) or the indicated c-terminally truncated versions of CCL21. [Figure 17] Functional analysis of human monocyte-derived dendritic cell (moDC) migration using a Transwell setup and supernatants from recombinant human CCL21 or VSV-GP- or VSV-GP-huCCL21- or VSV-GP-huCCL21(1-79)-infected HEK293 cells. [Figure 18] Western blot (WB) analysis of CCL21 in supernatants from plasmid-transfected HEK293 cells expressing the indicated c-terminally truncated or full-length versions of CCL21 or from VSV-GP- or VSV-GP-huCCL21(1-79)- or VSV-GP-huCCL21-infected HEK293 cells. [Figure 19] The viral titers of VSV-GP(GP), VSV-GP-huCCL21(21), or VSV-GP-huCCL21(1-79)(21k) in the supernatants of the indicated cells were measured at the indicated time points after viral infection to determine viral replication capacity. The different panels show, from left to right, (A) Vero cells, (B) BHK21 cells, and (C) HEK293 cells. In each panel, the titers of VSV-GP(GP), VSV-GP-huCCL21(21), or VSV-GP-huCCL21(1-79)(21k) were measured at 0, 24, and 48 hours. [Figure 20] Cumulative tumor growth in CT26.CL25-IFNARKO tumor-bearing control mice or mice treated with VSV-GP-huCCL21 or VSV-GP-huCCL21(1-79). [Figure 21] 30-day survival of mice from FIG. [Figure 22] IHC-based quantification of T cell infiltration (viable, non-necrotic tumor segments) in CT26.CL25-IFNARKO tumor-bearing control mice or mice treated with VSV-GP-huCCL21 or VSV-GP-huCCL21(1-79). [Figure 23] Cartoon illustrating the effects of VSV-GP, VSV-GP-CCL21 (full-length CCL21), and VSV-GP-CCL21(1-79) (c-terminally truncated CCL21) on immune infiltration of virus-infected tumors. [Figure 24] IHC-based quantification of dendritic cell (CD11c positive) infiltration (tumor areas with active viral replication = necrotic margin) in CT26.CL25 tumor-bearing control mice or mice treated with VSV-GP or VSV-GP-muCCL21.

[0042] Detailed Description of the Invention In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the subject technology may be practiced without some of these specific details. In other instances, well-known structures and techniques have not been shown in detail so as not to obscure the present invention. Headings are included merely for convenience to aid in reading and should not be understood to limit the present invention to any particular aspect or embodiment.

[0043] Rhabdovirus

[0044] The Rhabdoviridae family includes 18 genera and 134 species with negative-sense, single-stranded RNA genomes of approximately 10-16 kb (Walke et al., ICTV Virus Taxonomy Profile: Rhabdoviridae, Journal of General Virology, 99:447-448(2018)).

[0045] Characteristic features of members of the Rhabdovirus family include one or more of the following: bullet- or rod-shaped particles 100-430 nm in length and 45-100 nm in diameter, composed of a helical nucleocapsid surrounded by a matrix layer and a lipid envelope, although some rhabdoviruses have non-enveloped filamentous viruses; negative-sense single-stranded RNA of 10.8-16.1 kb, mostly non-segmented; a genome encoding at least five genes encoding the structural proteins nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M), and glycoprotein (G).

[0046] As used herein, a rhabdovirus may belong to the genus Almendravirus, Curiovirus, Cytorhavirus, Dichorhabdovirus, Ephemelovirus, Hapavirus, Redantevirus, Lyssavirus, Nobilhabdovirus, Nucleorhabdovirus, Perhabdovirus, Sigmavirus, Sprivivirus, Thripvirus, Tibrovirus, Tupavirus, Varicosavirus, or Vesiculovirus.

[0047] Within the genera mentioned herein, the rhabdoviruses can belong to any of the listed species: the genus Almendravirus includes Arboretum Almendravirus, Balsa Almendravirus, Coote Bay Almendravirus, Puerto Almendras Almendravirus, and Rio Chico Almendravirus; the genus Curiovirus includes Curionopolis Curiovirus, Iliri Curiovirus, Itacaionus Curiovirus, and Rochambeau Curiovirus; the genus Cytorhabdovirus includes Alfalfa Dwarf Cytorhabdovirus, and the genus Cytorhabdovirus. Virus, Barley yellow streak mosaic cytorhabdovirus, Broccoli necrotic yellow cytorhabdovirus, Colocasia bobourn-associated cytorhabdovirus, Festuca leaf streak cytorhabdovirus, Festuca leaf streak cytorhabdovirus, Lettuce necrotic yellow cytorhabdovirus, Lettuce yellow mottle cytorhabdovirus, Northern cereal mosaic cytorhabdovirus, Sonchus cytorhabdovirus 1, Strawberry crinkle cytorhabdovirus, Small The genera of dichorhaviruses include the American streak mosaic cytorhabdovirus; the genera of dichorhaviruses include the coffee ringspot dichorhavirus and the orchid fleck dichorhavirus; the genera of ephemeroviruses include the Adelaide River ephemerovirus, Berrima ephemerovirus, bovine fever ephemerovirus, Kimberley ephemerovirus, Coolpiña ephemerovirus, Cotoncan ephemerovirus, Obodian ephemerovirus, and Yata ephemerovirus. The genus of hapaviruses includes Flanders hapavirus, Grey Lodge hapavirus, Hart Park hapavirus, Joinjakaka hapavirus, Kamese hapavirus, La Joya hapavirus, Landzia hapavirus, Manitoba hapavirus, Marco hapavirus, Mosqueiro hapavirus, Mosslil hapavirus, Ngaingan hapavirus, Ord River hapavirus, Parry Creek hapavirus, and Wongabel hapavirus;The genus Redantevirus is Baruru Redantevirus, Fikirini Redantevirus, Fukuoka Redantevirus, Kanyawala Redantevirus, Cane Canyon Redantevirus, Keuraliva Redantevirus, Corente Redantevirus, Kumasi Redantevirus, Le Dantek Redantevirus, Mount Elgon Bat Redantevirus, Nishimuro Redantevirus, Ncorbisson Redantevirus, Oita Redantevirus, Wuhan Redantevirus, and Yongjia Redantevirus. The lyssavirus genera include Araban lyssavirus, Australian bat lyssavirus, Boquero bat lyssavirus, Duvenhage lyssavirus, European bat 1 lyssavirus, European bat 2 lyssavirus, Gannoruwa bat lyssavirus, Ikoma lyssavirus, Irkut lyssavirus, Khujand lyssavirus, Lagos bat lyssavirus, Lureida bat lyssavirus, and Mokola lyssavirus. The genus Nobilhabdovirus includes the lyssavirus, rabies lyssavirus, Simoni bat lyssavirus, and West Caucasus bat lyssavirus; the genus Nobilhabdovirus includes the flounder nobilhabdovirus, Piscine nobilhabdovirus, Salmonid nobilhabdovirus, and Snakehead nobilhabdovirus; the genus Nucleorhabdovirus includes the Datura yellow-vein nucleorhabdovirus, eggplant spotted dwarf nucleorhabdovirus, maize fine streak nucleorhabdovirus, and tomato The Perhabdovirus genus includes Sorghum Iranian mosaic nucleorhabdovirus, Maize mosaic nucleorhabdovirus, Potato yellow dwarf nucleorhabdovirus, Rice yellow stunt nucleorhabdovirus, Sonchus yellow net nucleorhabdovirus, Sowbuckthorn yellow vein nucleorhabdovirus, and Taro vein chlorosis nucleorhabdovirus; the Perhabdovirus genus includes Anguillid Perhabdovirus, Perch Perhabdovirus, and Sea Trout Perhabdovirus;The sigmavirus genera include Drosophila affinis sigmavirus, Drosophila ananasae sigmavirus, Drosophila immunograns sigmavirus, Drosophila melanogaster sigmavirus, Drosophila obscura sigmavirus, Drosophila tristis sigmavirus, and Muscina stabulans sigmavirus; the sprivivirus genera include carp sprivivirus and pike fly sprivivirus; the thripvirus genera include Armpiwar thripvirus, Chaco thripvirus, Niaka thripvirus, Sena madureira thripvirus, and thrippool thripvirus; the tibrovirus genera include Bas-Congo thripvirus, Beatrice Hill thripvirus, Coastal Plains thripvirus, Ekpoma 1 thripvirus, and Ekpoma 1 thripvirus. The genus Tupavirus includes the 2-tibulovirus, Sweetwater Branch Tibulovirus, and Tibulogargan Tibulovirus; the genus Tupavirus includes the Durham Tupavirus, Klamath Tupavirus, and Tupaia Tupavirus; the genus Varicosavirus includes the Lettuce Major Vein-Associated Varicosavirus; and the genus Vesiculovirus includes the Alagoas vesiculovirus, American bat vesiculovirus, Carajas vesiculovirus, Chandipura vesiculovirus, Kocal vesiculovirus, Indiana vesiculovirus, Isfahan vesiculovirus, Jurona vesiculovirus, Malpais Spring vesiculovirus, Maraba vesiculovirus, Moreton vesiculovirus, New Jersey vesiculovirus, Perinet vesiculovirus, Pirie vesiculovirus, Raji vesiculovirus, Yug-Bogdanovac vesiculovirus, and Mussavirus.

[0048] Preferably, the recombinant rhabdovirus of the present invention is an oncolytic rhabdovirus.In this respect, oncolytic has its usual meaning known in the art, and refers to the ability of rhabdovirus to infect and dissolve (decompose) cancer cells, but not (to a significant extent) normal cells.Preferably, oncolytic rhabdovirus can replicate in cancer cells.Oncolytic activity can be tested in different assay systems known to those skilled in the art (exemplary in vitro assay is described by Muik et al., Cancer Res., 74(13), 3567-78, 2014).It should be understood that oncolytic rhabdovirus can only infect and dissolve certain types of cancer cells.In addition, oncolytic effect can vary depending on the type of cancer cells.

[0049] In a preferred embodiment, Rhabdovirus belongs to the genus Vesiculovirus.Vesiculovirus species are mainly defined by serological means linked with genome phylogenetic analysis.Biological characteristics (such as host range and transmission mechanism) are also used to distinguish virus species within the genus.As such, the genus Vesiculovirus forms a separate monophyletic group that is well supported by the maximum likelihood tree inferred from complete L sequence.

[0050] Viruses assigned to different species within the genus Vesiculovirus may have one or more of the following characteristics: A) a minimum amino acid sequence divergence of 20% in L; B) a minimum amino acid sequence divergence of 10% in N; C) a minimum amino acid sequence divergence of 15% in G; D) can be distinguished in serological tests; and E) occupy different ecological niches as evidenced by differences in hosts and / or arthropod vectors.

[0051] Preferred is vesicular stomatitis virus (VSV), particularly VSV-GP (recombinant with LCMV GP). Advantageous properties of VSV-GP include one or more of the following: highly potent and fast killer (<8 hours); oncolytic virus; possible systemic application; reduced neurotropism / neurotoxins, thereby replicating lytically and inducing immunogenic cell death; non-replicating in healthy human cells due to the interferon (IFN) response; potent activation of innate immunity; approximately 3 kb space for immunomodulatory cargo and antigens; and combination with arenavirus glycoproteins from lymphocytic choriomeningitis virus (LCMV). recombinant; advantageous safety features compared to wild-type VSV (VSV-G) in terms of reduced neurovirulence and less susceptibility to neutralizing antibody responses and complement destruction; specifically replicate in tumor cells that have lost the ability to mount and respond to antiviral innate immune responses (e.g., type I IFN signaling); abortive replication in "healthy cells" so that they are rapidly cleared from normal tissues; viral replication in tumor cells leads to the induction of immunogenic cell death, release of tumor-associated antigens, local inflammation, and induction of anti-tumor immunity.

[0052] The present invention is further embodied by a recombinant vesicular stomatitis virus encoding in its genome at least the vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M), glycoprotein (G), and at least one CCL21 protein or functional variant thereof, preferably human CCL21.

[0053] In a preferred embodiment, the recombinant vesicular stomatitis virus encodes in its genome at least a vesicular stomatitis virus nucleoprotein (N) comprising the amino acid sequence set forth in SEQ ID NO:7 or a functional variant at least 80%, 85%, 90%, 92%, 94%, 96%, or 98% identical to SEQ ID NO:7; a phosphoprotein (P) comprising the amino acid sequence set forth in SEQ ID NO:8 or a functional variant at least 80%, 85%, 90%, 92%, 94%, 96%, or 98% identical to SEQ ID NO:8; a large protein (L) comprising the amino acid sequence set forth in SEQ ID NO:9 or a functional variant at least 80%, 85%, 90%, 92%, 94%, 96%, or 98% identical to SEQ ID NO:9; and a matrix protein (M) comprising the amino acid sequence set forth in SEQ ID NO:10 or a functional variant at least 80%, 85%, 90%, 92%, 94%, 96%, or 98% identical to SEQ ID NO:10.

[0054] Those skilled in the art will understand that modifications to the vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M), or glycoprotein (G) sequences can be made without losing the basic function of those proteins. As used herein, such functional variants retain all or part of their basic function or activity. Protein L, for example, is a polymerase and has an essential function during viral transcription and replication. Its functional variants must retain at least part of this ability. A good indication of the retention of basic functionality or activity is the successful production of viruses (including these functional variants) that are still capable of replicating and infecting tumor cells. Testing virus production and infection and replication in tumor cells may be tested in different assay systems known to those skilled in the art (an exemplary in vitro assay is described by Muik et al., Cancer Res., 74(13), 3567-78, 2014).

[0055] In a preferred embodiment, the recombinant vesicular stomatitis virus encodes in its genome at least the vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M), glycoprotein (G), and at least one CCL21 protein or functional variant thereof, preferably human CCL21, wherein the large protein (L) comprises an amino acid sequence having ≥80% sequence identity to SEQ ID NO:9.

[0056] In a preferred embodiment, the recombinant vesicular stomatitis virus encodes in its genome at least the vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M), glycoprotein (G), and at least one CCL21 protein or functional variant thereof, preferably human CCL21, wherein the nucleoprotein (N) comprises an amino acid sequence having ≥90% sequence identity to SEQ ID NO:7.

[0057] In a further preferred embodiment, the recombinant vesicular stomatitis virus encodes in its genome at least the vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M), glycoprotein (G), and at least one CCL21 protein or functional variant thereof, preferably human CCL21, wherein the large protein (L) comprises an amino acid sequence having equal to or greater than 80% sequence identity to SEQ ID NO:9, and the nucleoprotein (N) comprises an amino acid sequence having ≥ 90% sequence identity to SEQ ID NO:7.

[0058] In a preferred embodiment of the present invention, the RNA genome of recombinant rhabdovirus of the present invention comprises or consists of the sequence shown in SEQ ID NO: 24.In addition, the RNA genome of recombinant rhabdovirus of the present invention can also consist of or comprise these sequences, but the nucleic acid of RNA genome is exchanged according to the degeneracy of genetic code, without leading to the change of each amino acid sequence.In a more preferred embodiment, the RNA genome of recombinant rhabdovirus of the present invention comprises or consists of the coding sequence identical to SEQ ID NO: 24 or at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

[0059] It should be understood that the recombinant rhabdovirus of the present invention may encode additional cargo in its genome, such as tumor antigens, additional chemokines, cytokines, or other immunomodulatory elements.

[0060] In another embodiment, the recombinant rhabdovirus of the present invention further encodes in its genome the sodium iodide symporter protein (NIS).Expression of NIS and simultaneous incubation with, for example, I, allows NIS to be used as an imaging reporter (Carlson et al., Current Gene Therapy, 12, 33-47, 2012).

[0061] Recombinant rhabdovirus

[0062] It is known that certain wild-type rhabdovirus strains, such as wild-type VSV strains, are considered to be neurovirulent.It has also been reported that infected individuals can quickly launch a strong humoral response, with high antibody titers mainly directed against glycoprotein.The neutralizing antibody that targets the glycoprotein G and VSV of rhabdovirus can generally limit the spread of virus, thereby mediating the protection of individuals from reinfection with virus.However, virus neutralization limits the repeated application of rhabdovirus to cancer patients.

[0063] To eliminate these drawbacks, the rhabdovirus wild-type glycoprotein G may be replaced with a glycoprotein from another virus. In this regard, replacing the glycoprotein refers to (i) replacing the gene encoding the wild-type glycoprotein G with the gene encoding the glycoprotein GP of another virus, and / or (ii) replacing the wild-type glycoprotein G with the glycoprotein GP of another virus.

[0064] In a preferred embodiment, rhabdovirus glycoprotein G is replaced with glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), preferably with WE-HPI strain.In a more preferred embodiment, rhabdovirus is vesicular stomatitis virus with glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), preferably with WE-HPI strain.For example, such VSV is described in WO2010 / 045026, and is named VSV-GP.The advantages provided are (i) the loss of VSV-G-mediated neurovirulence and (ii) the lack of vector neutralization by antibody (as shown in mice).

[0065] The glycoprotein GP of lymphocytic choriomeningitis virus (LCMV) can be GP1 or GP2. The present invention includes glycoproteins from different LCMV strains. In particular, LCMV-GP can be derived from LCMV wild-type or LCMV strains LCMV-WE, LCMV-WE-HPI, LCMV-WE-HPlopt. In a preferred embodiment, the gene encoding the glycoprotein GP of LCMV encodes a protein with the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence with at least 80, 85, 90, 95%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 11, and the functional properties of the recombinant rhabdovirus comprising the glycoprotein GP encoding the amino acid sequence shown in SEQ ID NO: 11 are maintained.

[0066] In another embodiment, recombinant rhabdovirus glycoprotein G is replaced with glycoprotein GP of Dandenong virus (DANDV) or Mopeia virus (MOPV).In a more preferred embodiment, recombinant rhabdovirus is vesicular stomatitis virus, and glycoprotein G is replaced with glycoprotein GP of Dandenong virus (DANDV) or Mopeia virus (MOPV).The advantages provided are (i) loss of VSV-G-mediated neurovirulence and (ii) lack of vector neutralization by antibody (as shown in mice).

[0067] Dandenong virus (DANDV) is an Old World arenavirus.To date, there is only one strain known to those skilled in the art that contains glycoprotein GP and can be used in the present invention as the donor of glycoprotein GP contained in recombinant rhabdovirus of the present invention.The DADV glycoprotein GP contained in recombinant rhabdovirus of the present invention has more than six glycosylation sites, particularly seven glycosylation sites.An exemplary and preferred glycoprotein GP is the glycoprotein GP contained in DANDV, which is available under Genbank number EU136038.In one embodiment, the gene encoding glycoprotein GP of DANDV encodes the amino acid sequence shown in SEQ ID NO:12 or a sequence with at least 80, 85, 90 or 95% sequence identity with the amino acid sequence of SEQ ID NO:12, and the functional properties of the recombinant rhabdovirus that contains the glycoprotein GP that encodes the amino acid sequence shown in SEQ ID NO:12 are maintained.

[0068] Mopeia virus (MOPV) is an Old World arenavirus. There are several strains known to those skilled in the art that contain glycoprotein GP and can be used in the present invention as the donor of glycoprotein GP contained in recombinant rhabdovirus of the present invention. The MOPV glycoprotein GP contained in recombinant rhabdovirus of the present invention has more than six glycosylation sites, particularly seven glycosylation sites. An exemplary and preferred glycoprotein GP is the glycoprotein GP contained in Mopeia virus, available under Genbank number AY772170. In one embodiment, the gene encoding the glycoprotein GP of MOPV encodes the amino acid sequence shown in SEQ ID NO: 13 or a sequence with at least 60, 65, 70, 75, 80, 85, 90 or 95% sequence identity with the amino acid sequence of SEQ ID NO: 13, and the functional properties of the recombinant rhabdovirus that contains the glycoprotein GP that encodes the amino acid sequence shown in SEQ ID NO: 13 are maintained.

[0069] CCL21 and functional variants of CCL21.

[0070] Surprisingly, it has been found that recombinant rhabdoviruses that encode CCL21 protein in their genome can induce tumor cell lysis in tumor microenvironment, combined with immunogenic cell death and the stimulation of innate immune cells.In addition, prolonged survival rates have been observed in established mouse tumor models that are treated with this recombinant rhabdovirus that carries CCL21.

[0071] CCL21 belongs to the CC chemokine family and is also known as secondary lymphoid tissue chemokine (SLC), exodus-2, ckb9, scya21, TCA4, or 6Ckine. CCL21 contains a C-terminal region that binds to the extracellular matrix. CCL21 also binds to the CCR7 cell surface receptor, thereby exerting its functions, such as attracting and activating T cells and dendritic cells. CCR7 is expressed on a wide range of peripheral T cells and dendritic cells in both healthy and cancer patients.

[0072] Thus, in one aspect, a recombinant rhabdovirus encoding at least one CCL21 protein or a functional variant thereof in its genome can enhance the recruitment of T cells and dendritic cells to the tumor environment.

[0073] In another aspect, local expression of the highly potent chemokine CCL21 further enhances immune cell recruitment, preferentially T lymphocytes and dendritic cells, into the tumor microenvironment and improved efficacy of the recombinant rhabdovirus.

[0074] In yet another embodiment, the recombinant rhabdovirus encoding at least one CCL21 protein or a functional variant thereof in its genome acts as an innate immune stimulator.

[0075] In one aspect, a recombinant rhabdovirus encoding at least one CCL21 protein or functional variant thereof in its genome transforms a cold tumor into a hot tumor. In particular, the resulting pro-inflammatory tumor microenvironment transforms a non-T cell infiltrated ("cold") tumor into a T cell inflamed ("hot") tumor, accompanied by the generation of an adaptive anti-tumor immune response in the tumor-draining lymph nodes.

[0076] The human CCL21 protein has been described, for example, by M. Nagira et al., The Journal of Biological Chemistry, 272, 19518-19524 (August 1, 1997), and is a highly basic polypeptide of 134 amino acids in total, with a putative signal peptide of 23 amino acids:

[0077] [ka]

[0078] CCL21 proteins particularly include CCL21 comprising or consisting of the following sequence:

[0079] [ka]

[0080] Preferably, the CCL21 protein comprises or consists of a protein having the following sequence:

[0081] [ka]

[0082] or has at least 70%, 72%, 74%, 76%, 78%, 80%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:4.

[0083] More preferably, the CCL21 protein comprises or consists of a protein having the following sequence:

[0084] [ka]

[0085] or has at least 70%, 72%, 74%, 76%, 78%, 80%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:3.

[0086] The term "signal peptide" or "signal peptide sequence" describes a peptide sequence, usually 10 to 30 amino acids in length, present at the N-terminus of a newly synthesized secretory or membrane polypeptide that directs the polypeptide across or into the cell's cytoplasmic membrane (the plasma membrane in prokaryotes and the endoplasmic reticulum membrane in eukaryotes). It is usually subsequently removed. In particular, a signal peptide may be capable of directing the polypeptide into the cell's secretory pathway.

[0087] It should be understood that, for the purposes of the present invention, other (i.e., non-wild-type) signal peptide sequences may be used with the CCL21 protein. Such other signal peptide sequences may replace the original wild-type signal peptide sequence. Signal peptides include peptides that target newly synthesized proteins in ribosomes to the endoplasmic reticulum and further to the Golgi complex for transport to the plasma membrane or extracellularly. They generally contain a series of hydrophobic amino acids and include immunoglobulin leader sequences as well as others known to those skilled in the art. Signal peptides particularly include peptides that can be acted upon by signal peptidases (specific proteases located on the cisternal surface of the endoplasmic reticulum). Signal peptides are well understood by those skilled in the art and may include any known signal peptide. A signal peptide is incorporated at the N-terminus of a protein, and processing of the CCL21 protein by the signal peptidase produces an active biological form.

[0088] In a preferred embodiment, the signal peptide has the sequence shown in SEQ ID NO: 6. In a related preferred embodiment, the CCL21 protein comprises or consists of a protein having the following sequence:

[0089] [ka]

[0090] or has at least 70%, 72%, 74%, 76%, 78%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:5.

[0091] In related embodiments, the CCL21 protein includes a protein comprising or consisting of, or having at least 70%, 72%, 74%, 76%, 78%, 80%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, respectively, SEQ ID NO: 2, 3, or 4, and further comprising a signal peptide sequence. In a preferred embodiment, the signal peptide sequence comprises or consists of amino acids 1-19 of SEQ ID NO: 5.

[0092] CCL21 proteins also include proteins comprising or consisting of the amino acids of SEQ ID NO:2, 3, or 4, or having at least 70%, 72%, 74%, 76%, 78%, 80%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97, 98%, 99%, or 100% identity to SEQ ID NO:2, 3, or 4, respectively, and further comprising a signal peptide sequence comprising or consisting of amino acids 1 to 23 of SEQ ID NO:1.

[0093] CCL21 protein also includes proteins corresponding to plasmin-processed forms of CCL21. CCL21 contains a unique extended C-terminus (e.g., human CCL21 approximately 30 aa) with a net positive charge that contributes to the binding of extracellular matrix components, such as heparin-like glycosaminoglycans. C-terminal truncation / deletion dramatically reduces binding to glycosaminoglycans such as heparin. CCL21 is processed in the human body by plasmin, which is deficient in some human cancers. It has been shown that the need for plasmin cleavage can be overcome by encoding a biologically active N-terminal fragment of CCL21 similar to the plasmin-processed form of CCL21 into a recombinant rhabdovirus. The plasmin-processed form of CCL21 is characterized by C-terminal truncation / deletion, resulting in a reduced ability to bind to heparin and / or heparan sulfate, which can be measured by methods known to those skilled in the art. In this context, a reduced ability to bind to heparin and / or heparan sulfate refers to a binding ability that is reduced to 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or less of the binding ability of a CCL21 protein with the sequence shown in SEQ ID NO: 1 or 2 (with or without the signal peptide sequence, respectively) when compared to a CCL21 protein having SEQ ID NO: 1 or 2 and tested under the same conditions in the same assay.

[0094] Plasmin is a serine protease that acts to dissolve fibrin clots. Apart from fibrinolysis, plasmin proteolyzes proteins in various other systems, thereby activating collagenase, a mediator of the complement system, which weakens the walls of Graafian follicles and leads to ovulation. It cleaves fibrin, fibronectin, thrombospondin, laminin, and von Willebrand factor. Plasmin belongs to the family of serine proteases. Plasmin is released from the liver into the systemic circulation as a proenzyme called plasminogen. The conversion of plasminogen to active plasmin involves cleavage of the peptide bond between Arg-561 and Val-562 by, for example, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and factor XII (Hageman factor).

[0095] Cleavage of the CCL21 protein by plasmin can occur either on cell surface-bound CCL21 or in vivo (in either case by incubating the CCL21 protein with plasmin). Plasmin-treated CCL21 protein thus includes CCL21 comprising or consisting of a sequence corresponding to amino acids 1-88 of SEQ ID NO:2, or having at least 70%, 72%, 74%, 76%, 78%, 80%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 1-88 of SEQ ID NO:2. Further, the CCL21 protein includes a CCL21 comprising or consisting of a sequence corresponding to amino acids 1 to 91 of SEQ ID NO:2, or having at least 70%, 72%, 74%, 76%, 78%, 80%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 1 to 91 of SEQ ID NO:2. CCL21 proteins also include CCL21 comprising or consisting of a sequence corresponding to amino acids 1-104 of SEQ ID NO:2, or having at least 70%, 72%, 74%, 76%, 78%, 80%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 1-104 of SEQ ID NO:2.

[0096] C-terminally truncated CCL21 proteins are characterized by the deletion and / or mutation of amino acids at the extended C-terminus of the CCL21 protein, which results in reduced binding to glycosaminoglycans such as heparin. In a preferred embodiment, the c-terminally truncated CCL21 protein comprises or consists of SEQ ID NO:2 or has at least 70%, 72%, 74%, 76%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 1 to 79 of SEQ ID NO:2 (wherein the protein has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, In a further preferred embodiment, the C-terminally truncated CCL21 is a CCL21 comprising or consisting of SEQ ID NO:2 or having at least 70%, 72%, 74%, 76%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 1-79 of SEQ ID NO:2, in which all amino acids 80-111 of SEQ ID NO:2 have been deleted (i.e., a deletion of 32).

[0097] In each case, either the plasmin-treated or C-terminally truncated CCL21 may further comprise a signal peptide sequence. Particularly preferred are signal peptide sequences comprising or consisting of amino acids 1 to 23 of SEQ ID NO: 1 or amino acids 1 to 19 of SEQ ID NO: 5. Other signal peptide sequences that replace the native signal peptide sequence may also be used. Thus, in a preferred embodiment, the plasmin-treated or C-terminally truncated CCL21 protein comprises or consists of a protein having the following sequence:

[0098] [ka]

[0099] or has at least 70%, 72%, 74%, 76%, 78%, 80%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:5.

[0100] CCL21 proteins can also include CCL21 with a truncated signal peptide sequence. In this context, "truncated" refers to a signal peptide sequence that is shorter than the native signal peptide sequence but still retains at least part of its functionality of acting as a signal peptide. For example, a human signal peptide sequence comprises or consists of amino acids 1-23 of SEQ ID NO: 1. CCL21 with a truncated signal peptide sequence can have 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of amino acids 1-23 of SEQ ID NO: 1. In a further example, the signal peptide can comprise or consist of the sequence set forth in SEQ ID NO: 6. CCL21 with a cleaved signal peptide sequence can have 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of amino acids 1 to 18 of SEQ ID NO:6.

[0101] A CCL21 protein with a truncated signal peptide sequence can also be a protein comprising any of the sequences of SEQ ID NOs: 2 to 4, and also a signal peptide sequence that is shorter than the native signal peptide sequence. Again, by way of example, the signal peptide sequence can have 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of amino acids 1 to 23 of SEQ ID NO: 1, or in a further example, the signal peptide can comprise or consist of the sequence shown in SEQ ID NO: 6. A CCL21 with a truncated signal peptide sequence can have 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of amino acids 1 to 18 of SEQ ID NO: 6.

[0102] The CCL21 protein can be of any origin, including mouse and rat. Preferably, the CCL21 protein is from human origin.

[0103] Functional variants of CCL21 proteins include biologically active variants and biologically active fragments of the CCL21 proteins described above. Variants can have one or more different amino acids at the specifically described CCL21 protein positions. Variants can share at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more amino acid identity with such CCL21 proteins. Fragments can have the same amino acids as a given specifically described CCL21 protein but lack a particular portion or region of the CCL21 protein.

[0104] As used herein, the terms "identical" or "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same, or that have a specified percentage of nucleotides or amino acid residues that are the same when compared and aligned for maximum correspondence. To determine percent identity, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (e.g., overlapping positions) × 100). In some embodiments, the two sequences being compared are the same length after gaps have been introduced into the sequences, where appropriate (e.g., excluding additional sequences that extend beyond the sequences being compared).

[0105] The determination of percent identity or percent similarity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed using the NBLAST program, score=100, word length=12, to obtain nucleotide sequences homologous to a nucleic acid encoding a protein of interest. BLAST protein searches can be performed using the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein of interest. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art, including ADVANCE and ADAM, described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA, described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. When ktup=2, similar regions in the two sequences being compared are found by examining pairs of aligned residues; when ktup=1, single aligned amino acids are examined. ktup can be set to 2 or 1 for protein sequences, or 1 to 6 for DNA sequences. If ktup is not specified, the default is 2 for proteins and 6 for DNA. Alternatively, protein sequence alignments may be performed using the CLUSTAL W algorithm as described by Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0106] In both cases, functional variants include only biologically active CCL21 variants and fragments.For the present invention, the biological activity of the CCL21 variant or CCL21 fragment encoded in the genome of recombinant rhabdovirus is determined after its expression in respective cells or tumor cells.This means that biological activity is determined in the context of the recombinant rhabdovirus encoding the CCL21 variant or CCL21 fragment (for example, in a Transwell assay or an in vitro tumor model).Preferably, biological activity is determined using a vesiculovirus encoding a CCL21 variant or CCL21 fragment.More preferably, biological activity is determined using a VSV-GP encoding a CCL21 variant or CCL21 fragment.

[0107] Biological activity may include one or more of the following abilities: chemoattractant activity, antitumor activity, modulation of cytokine expression, such as increasing the expression of interferon-gamma (IFN-gamma) polypeptides or decreasing the expression of transforming growth factor-beta (TGF-beta) polypeptides in a population of syngeneic mammalian cells including CD8-positive T cells, CD4-positive T cells, antigen-presenting cells, and tumor cells. Testing for biological activity may be carried out according to, but is not limited to, protocols such as those shown in the Examples. For purposes of the present invention, a functional variant or fragment of a CCL21 protein is biologically active if it exhibits at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the activity of a CCL21 protein with the sequence shown in SEQ ID NO: 1 or 2 (with or without the signal peptide sequence, respectively) when tested in the same assay and under the same conditions.

[0108] Without wishing to be bound by theory, the present inventors have found that several chemokines and cytokines are upregulated in response to treatment of tumor cells with VSV-GP. CCL21 is one of the chemokines that is not upregulated in tumor cells after VSV-GP treatment. The present data indicate that VSV-GP, which encodes the CCL21 protein in its genome, is particularly effective in cancer treatment and is suitable for further improving immune cell infiltration into tumors infected with oncolytic viruses, thereby further enhancing antitumor immunity.

[0109] Even more surprisingly, recombinant rhabdoviruses encoding CCL21 proteins comprising or containing the sequence of SEQ ID NO: 3 or 4, particularly SEQ ID NO: 5, were even more potent and effective in treating tumors compared to the full-length CCL21 protein, and are active without the need for proteolytic processing.

[0110] Such CCL21 protein is more preferable than full-length CCL21 protein because of its smaller size.Larger or multiple transgenes can negatively affect the survival, stability, oncolytic ability, manufacturability or expression of rhabdovirus and transgene itself.By utilizing smaller transgenes, it also provides the possibility of adding additional transgenes to rhabdoviruses that may have limitations in their capacity to accommodate additional transgenes.

[0111] Rhabdoviruses have negative-strand single-stranded RNA (ssRNA) as their genetic material (genome). Negative-strand ssRNA viruses require RNA polymerase to form positive-strand RNA. Positive-strand RNA acts as viral mRNA, which is translated into protein for the production of new viral material. Using the newly formed virus, more negative-strand RNA molecules are produced.

[0112] A typical rhabdovirus genome encodes at least five structural proteins in the order 3'-NPMGL-5'. The genome may contain short intergenic regions or additional genes between the structural proteins, and therefore may vary in length and organization.

[0113] According to the present invention, the CCL21 gene can be introduced into any position of the rhabdovirus genome. Depending on the insertion site, the transcription efficiency of the CCL21 gene can be affected. Generally, the transcription efficiency of the CCL21 gene decreases from 3' insertion to 5' prime insertion. The CCL21 gene can be inserted into the following genomic positions: 3'-CCL21-NPMGL-5', 3'-N-CCL21-PMGL-5', 3'-NP-CCL21-MGL-5', 3'-NPM-CCL21-GL-5', 3'-NPMG-CCL21-L-5', or 3'-NPMGL-CCL21-5'. In a preferred embodiment, the CCL21 gene is inserted between the G protein and the L protein.

[0114] After infection of tumor cells, the CCL21 gene encoded in the genome of the recombinant rhabdovirus is transcribed into positive-strand RNA, which is then translated into CCL21 protein by tumor cells. The term "encoding" or "coding" refers to the inherent property of a specific nucleotide sequence in a nucleic acid, which serves as a template for the synthesis of a defined nucleotide sequence (e.g., an RNA molecule) or other polymers and macromolecules in biological processes with amino acids and the biological properties resulting therefrom. Thus, when a desired protein is produced in a cell or another biological system by transcription and subsequent translation of mRNA, a gene encodes a protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence) and the non-coding strand can serve as a template for transcription of a gene and can be referred to as encoding the protein or other product of that gene. Protein-encoding nucleic acids and nucleotide sequences can contain introns.

[0115] Transcription of the CCL21 gene is preferably not under the control of its own promoter but is only strictly linked to viral replication, thereby ensuring targeted expression of CCL21 to the site of viral replication and spread (tumor). Thus, transcription of the CCL21 gene is not controlled by additional elements, such as promoters or inducible gene expression elements.

[0116] It will be understood that nucleic acid sequences may be varied with or without altering the primary sequence of the encoded polypeptide. Nucleic acids encoding proteins include any nucleic acids that have different nucleotide sequences but, due to the degeneracy of the genetic code, encode the same amino acid sequence of a protein. It is within the knowledge of one of ordinary skill in the art to select a nucleic acid sequence that results in expression of a CCL21 protein, particularly any specific CCL21 protein disclosed herein. Nucleic acid molecules encoding the amino acid sequence of a CCL21 protein are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from natural sources or preparation of previously prepared CCL21 proteins by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis.

[0117] Pharmaceutical Compositions

[0118] The actual pharmaceutically effective amount or therapeutic dose will, of course, depend on factors known to those skilled in the art, such as the age and weight of the patient, the route of administration, and the severity of the disease, etc. In any case, the recombinant rhabdovirus is administered in a dosage and manner that allows delivery of a pharmaceutically effective amount based on the patient's unique condition.

[0119] Generally, for the treatment and / or alleviation of diseases, disorders and conditions referred to herein, and depending on the specific disease, disorder or condition that is treated, the efficacy of the specific recombinant rhabdovirus of the present invention that is used, specific route of administration and the specific pharmaceutical preparation or composition that is used, the recombinant rhabdovirus of the present invention is generally administered in dosages such as twice a week, every week or every month, but can vary significantly, especially depending on the parameters mentioned above.Thus, in some cases, it may be sufficient to use a dosage that is less than the minimum dosage given above, while in other cases, it will have to exceed the upper limit.When administering large amounts, it may be advisable to divide them into a number of smaller dosages that are distributed throughout a day.

[0120] For use in treatment, recombinant rhabdovirus of the present invention is formulated in suitable pharmaceutical composition for facilitating administration to animals or humans.Typical formulation can be prepared by mixing recombinant virus with physiologically acceptable carrier, excipient or stabilizer in the form of aqueous solution or aqueous or non-aqueous suspension.Carrier, excipient, modifier or stabilizer are non-toxic at the dosage and concentration used. They may contain buffer systems such as phosphate, citrate, acetate, and other inorganic or organic acids and their salts; antioxidants (including ascorbic acid and methionine); preservatives such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone or polyethylene. Examples of excipients include glycols (PEG), amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; mono-, di-, oligo-, or polysaccharides and other sugars (including glucose, mannose, sucrose, trehalose, dextrin, or dextran); chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or ionic or non-ionic surfactants such as TWEEN™ (polysorbate), PLURONICS™, or fatty acid esters, fatty acid ethers, or sugar esters. Excipients may also have release-modifying or absorption-modifying functions.

[0121] In one embodiment, the recombinant rhabdovirus of the present invention is formulated in a pharmaceutical composition comprising Tris, arginine, and optionally citrate.Tris is preferably used at a concentration of about 1mM to about 100mM.Arginine is preferably used at a concentration of about 1mM to about 100mM.Citrate can be present at a concentration of up to 100mM.A preferred formulation comprises about 50mM Tris and 50mM arginine.

[0122] The pharmaceutical composition may be provided as a liquid, frozen, or in lyophilized form. Frozen liquids may be stored at temperatures between about 0°C and about -85°C (including between -70°C and -85°C, and temperatures of about -15°C, -16°C, -17°C, -18°C, -19°C, -20°C, -21°C, -22°C, -23°C, -24°C, or about -25°C).

[0123] Recombinant rhabdovirus or pharmaceutical composition of the present invention is not necessary, but may be formulated with one or more drugs currently used to prevent or treat the disorder in question.The effective amount of such other drugs depends on the amount of recombinant antibody present in the formulation, the type of disorder or treatment, and other factors as described above.They are generally used in the same dosage and administration route as described herein, or at about 1 to 99% of the dosage described herein, or at any dosage and by any route that is empirically / clinically determined to be appropriate.

[0124] For disease prevention or treatment, the appropriate dosage of recombinant rhabdovirus or pharmaceutical compositions of the present invention (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease to be treated, the type of recombinant rhabdovirus, the severity and course of disease, whether recombinant rhabdovirus is administered for prevention or treatment, previous treatment, patient's medical history and response to recombinant rhabdovirus, and the judgment of attending physician.Recombinant rhabdovirus or pharmaceutical compositions of the present invention is appropriately administered to patient at one time or over a series of treatments.

[0125] Depending on the type and severity of the disease, the TCID of the recombinant rhabdovirus 50 measured by about 10 8 From 10 13 The infectious particles of can be the initial candidate dose for administration to a patient, for example, by one or more separate administrations or by continuous infusion. For repeated administration over several days or more, depending on the condition, treatment will generally be continued until the desired suppression of disease symptoms occurs. One exemplary dosage of recombinant rhabdovirus is TCID 50 measured by about 10 8 From 10 13 Thus, the TCID 50 measured by about 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , or 10 13 The patient may be administered one or more doses of infectious particles (or any combination thereof). Such doses may be administered intermittently, for example, every week or every three weeks (e.g., so that the patient receives about 2 to about 20, or, for example, about 6 doses of recombinant rhabdovirus). An initial higher loading dose may be administered, followed by one or more lower doses, or vice versa. However, other dosing regimens may be useful. The progress of this treatment is easily monitored by conventional techniques and assays.

[0126] The effectiveness of the recombinant rhabdovirus of the present invention and the composition comprising it can be tested by any suitable in vitro assay, cell-based assay, in vivo assay and / or known animal model, or any combination thereof, depending on the specific disease involved.Suitable assay and animal model are clear to those skilled in the art, and include, for example, the assay and animal model used in the following examples.

[0127] The actual pharmaceutically effective amount or therapeutic dose will, of course, depend on factors known to those skilled in the art, such as the age and weight of the patient, the route of administration, and the severity of the disease, etc. In any case, the recombinant rhabdovirus of the present invention will be administered in a dosage and manner that allows for the delivery of a pharmaceutically effective amount based on the patient's unique condition.

[0128] Alternatively, the recombinant rhabdovirus or pharmaceutical composition of the present invention may be delivered in a volume of about 50 μl to about 100 ml (including all numbers within the range), depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method.

[0129] For intratumoral administration, the volume is preferably between about 50 μl and about 5 ml (including volumes of about 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, 600 μl, 700 μl, 800 μl, 900 μl, 1000 μl, 1100 μl, 1200 μl, 1300 μl, 1400 μl, 1500 μl, 1600 μl, 1700 μl, 1800 μl, 1900 μl, 2000 μl, 2500 μl, 3000 μl, 3500 μl, 4000 μl, or about 4500 μl). In a preferred embodiment, the volume is about 1000 μl.

[0130] For systemic administration (e.g., by injection of the recombinant rhabdovirus), the volume may naturally be high. Alternatively, a concentrated solution of the recombinant rhabdovirus can be diluted in a larger volume of injection solution immediately prior to injection.

[0131] Particularly for intravenous administration, the volume is preferably between 1 ml and 100 ml (including volumes of about 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml, 19 ml, 20 ml, 25 ml, 30 ml, 35 ml, 40 ml, 45 ml, 50 ml, 55 ml, 60 ml, 70 ml, 75 ml, 80 ml, 85 ml, 90 ml, 95 ml, or about 100 ml). In preferred embodiments, the volume is between about 5 ml and 15 ml, and more preferably, the volume is about 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, or about 14 ml.

[0132] Preferably, the same formulation is used for intratumoral and intravenous administration. The dose and / or volume ratio between intratumoral and intravenous administration can be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or about 1:20. For example, a dose and / or volume ratio of 1:1 means that the same dose and / or volume is administered intratumorally and intravenously, whereas a dose and / or volume ratio of about 1:20 means that the intravenous dose and / or volume is 20 times higher than the intratumoral dose and / or volume. Preferably, the dose and / or volume ratio between intratumoral and intravenous administration is about 1:9.

[0133] The effective concentration of the recombinant rhabdovirus is preferably about 10 per milliliter. 8 and 10 14 The range of vector genome (vg / mL) is between 1.5×10 and 1.5×10. Infectious units may be measured as described in McLaughlin et al., J. Virol.; 62(6):1963-73 (1988). Preferably, the concentration is between about 1.5×10 and 1.5×10. 9 to approximately 1.5 x 10 13 and more preferably about 1.5×10 9 to approximately 1.5 x 10 11In one embodiment, the effective concentration is about 1.5 x 10 9 In another embodiment, the effective concentration is about 1.5×10 10 In another embodiment, the effective concentration is about 1.5×10 11 In yet another embodiment, the effective concentration is about 1.5 x 10 12 In another embodiment, the effective concentration is about 1.5×10 13 In another embodiment, the effective concentration is about 1.5×10 14 In order to reduce the risk of undesired effects, it may be desirable to use the lowest effective concentration. Further dosages within these ranges can be selected by the attending physician, taking into account the physical condition of the subject, preferably a human, being treated, the age of the subject, the particular type of cancer, and the extent to which the cancer (if advanced) has developed.

[0134] The effective target concentration of recombinant rhabdovirus is TCID 50 It can also be expressed as TCID. 50 can be determined, for example, by using the Spearman-Karber method. Preferably, the range is 1×10 8 / ml and 1 × 10 14 / ml TCID 50 Preferably, the effective target concentration is from about 1 x 10 to about 1 x 10 12 / ml, more preferably about 1 x 10 9 to approximately 1 × 10 11 / ml. In one embodiment, the effective target concentration is about 1 x 10 10 / ml. In a preferred embodiment, the target concentration is 5 x 10 10 / ml. In another embodiment, the effective target concentration is about 1.5 x 10 11 / ml. In one embodiment, the effective target concentration is about 1 x 10 12 / ml. In another embodiment, the effective target concentration is about 1.5 x 10 13 / ml.

[0135] The effective target dose of recombinant rhabdovirus is also TCID 50 Preferably, the range is 1×10 8 and 1×10 14 TCID between 50 Preferably, the target dose is about 1 x 10 9 to approximately 1 × 10 13 and more preferably about 1 × 10 9 to approximately 1 × 10 12 In one embodiment, the effective concentration is about 1×10 10 In a preferred embodiment, the effective concentration is about 1×10 11 In one embodiment, the effective concentration is about 1×10 12 In another embodiment, the effective concentration is about 1 x 10 13 is.

[0136] In another aspect, a kit or kit components are provided, which contain materials useful for treating, preventing, and / or diagnosing the disorders described herein.The kit or kit components include a container and a label or package insert on or associated with the container.Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.The container can be made of various materials, for example, glass or plastic.The container can hold the composition by itself or in combination with another composition that is effective for treating, preventing, and / or diagnosing disorders, and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle).At least one active agent in the composition is the recombinant rhabdovirus of the present invention or a pharmaceutical composition.The label or package insert indicates that the composition is used to treat the selected condition.

[0137] Furthermore, the kit or kit component may include (a) a first container with a composition contained therein (wherein the composition comprises a recombinant rhabdovirus or pharmaceutical composition of the present invention); and (b) a second container with a composition contained therein (wherein the composition comprises an additional cytotoxic agent or other therapeutic agent, such as a PD-1 pathway inhibitor or a SMAC mimetic). The kit or kit component in this embodiment of the present invention may further include a package insert indicating that the composition can be used to treat a specific condition, particularly cancer. Alternatively, or in addition, the kit or kit component may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials (including other buffers, diluents, filters, needles, and syringes) that are desirable from a commercial and user standpoint.

[0138] In another embodiment, the recombinant rhabdovirus of the present invention is used in combination with the device that is useful for administering recombinant rhabdovirus, such as syringe, injector pen, micropump or other device.Preferably, the recombinant rhabdovirus of the present invention is included in kit components (for example, also includes the package insert that includes the instructions for using recombinant rhabdovirus).

[0139] medical use

[0140] A further aspect of the present invention provides a recombinant rhabdovirus encoding in its genome at least one CCL21 protein or a functional variant thereof for use in medicine.

[0141] The recombinant rhabdovirus of the present invention can effectively induce tumor cell lysis, which is combined with immunogenic cell death and the stimulation of innate immune cells in tumor microenvironment.Therefore, the recombinant rhabdovirus of the present invention is useful for treating and / or preventing cancer.

[0142] In a further aspect, the recombinant rhabdoviruses of the present invention can be used in methods for treating and / or preventing cancer, including administering a therapeutically effective amount of the recombinant rhabdovirus to an individual suffering from cancer, thereby ameliorating one or more symptoms of the cancer.

[0143] In yet another aspect, the present invention further provides the use of a recombinant rhabdovirus according to the present invention for the manufacture of a medicament for the treatment and / or prevention of cancer.

[0144] In yet another aspect, the recombinant rhabdovirus of the invention can be used in a method for treating and / or preventing gastrointestinal cancer, lung cancer, or head and neck cancer, comprising administering a therapeutically effective amount of the recombinant rhabdovirus to an individual suffering from gastrointestinal cancer, lung cancer, or head and neck cancer, thereby ameliorating one or more symptoms of gastrointestinal cancer, lung cancer, or head and neck cancer.

[0145] For the prevention or treatment of disease, the appropriate dosage of recombinant rhabdovirus, as defined above, can depend on various factors, such as the type of disease to be treated, the severity and course of the disease, whether the recombinant rhabdovirus is administered for prophylactic or therapeutic purposes, previous treatment, the patient's medical history and response to the recombinant rhabdovirus, and the judgment of the attending physician, etc. Recombinant rhabdovirus is suitably administered to the patient at one time or over a series of treatments.

[0146] In one embodiment, the cancer is a solid cancer.The solid cancer may be brain cancer, colorectal cancer, oropharyngeal squamous cell carcinoma, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal cancer, hepatocellular carcinoma, pancreatic adenocarcinoma, bile duct carcinoma, bladder urothelial carcinoma, metastatic melanoma, prostate cancer, breast cancer, head and neck squamous cell carcinoma (HNSCC), glioblastoma, non-small cell lung cancer, brain tumor, or small cell lung cancer.Preferably, gastrointestinal cancer, lung cancer, and head and neck cancer are treated.

[0147] Recombinant rhabdovirus can be administered by any suitable means, including oral, parenteral, subcutaneous, intratumoral, intravenous, intradermal, intraperitoneal, intrapulmonary and intranasal.Parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.Recombinant rhabdovirus can also be suitably administered by pulse infusion.In one aspect, dosage is given by injection, most preferably intravenous or subcutaneous injection, depending in part on whether administration is short-term or chronic.

[0148] Depending on the specific recombinant rhabdovirus of the present invention and its specific pharmacokinetic and other properties, it can be administered every day, every 2 days, every 3 days, every 4 days, every 5 days, or every 6 days, weekly, monthly, etc. The administration regimen can include long-term weekly treatment. By "long-term" is meant a period of at least 2 weeks, preferably several months or years.

[0149] Treatment schedule can include various schemes, but typically requires multiple doses to be administered to patients over a period of 1, 2, 3 or 4 weeks, and optionally one or more additional treatments thereafter.In one embodiment, the recombinant rhabdovirus of the present invention is administered to patients at most 1, 2, 3, 4, 5 or 6 doses within a given period.Preferably, the first treatment is completed within 3 weeks.During the course of 3 weeks of treatment, recombinant rhabdovirus can be administered to patients as described in the following scheme:(i) day 0;(ii) day 0 and 3;(iii) day 0, 3 and 6;(iv) day 0, 3, 6 and 9;(v) day 0 and 5;(vi) day 0, 5 and 10;(vii) day 0, 5, 10 and 15.These schemes can be repeated, and a second or third treatment may be required depending on the outcome of the first treatment. Calculated based on the first treatment, the second treatment preferably includes additional treatments on days 21, 42, and 63. In a preferred embodiment, the recombinant rhabdovirus of the present invention is administered to patients according to the following scheme: day 0, day 3, day 21, day 42, and day 63.

[0150] The term " suppression " is used herein in the same context as " remission " and " alleviation ", and refers to the alleviation or reduction of one or more characteristics of disease.The recombinant rhabdovirus or pharmaceutical composition of the present invention is formulated, dosed and administered in a manner consistent with good medical practice.The factors to be considered in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of individual patient, the cause of disorder, the delivery site of drug, administration method, administration schedule and other factors known to physicians.The " therapeutically effective amount " of the recombinant rhabdovirus administered is influenced by such considerations, and is the minimum amount necessary to prevent, alleviate or treat the clinical symptoms of cancer, particularly the minimum amount that is effective for these disorders.

[0151] In another embodiment, recombinant rhabdovirus of the present invention can be administered multiple times and in several doses.In one embodiment, the first dose of recombinant rhabdovirus is administered intratumorally, and the subsequent doses of recombinant rhabdovirus are administered intravenously.In another embodiment, the first dose of recombinant rhabdovirus and at least one or more subsequent doses are administered intratumorally, and the subsequent doses of recombinant rhabdovirus are administered intravenously.Subsequent administrations can be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or 31 days after the first intratumor administration.

[0152] In another embodiment, the first dose of recombinant rhabdovirus is administered intravenously, and the subsequent doses of recombinant rhabdovirus are administered intratumorally.Subsequent administrations can be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or 31 days after the first intravenous administration.

[0153] In another embodiment, the recombinant rhabdovirus is administered intravenously and subsequent doses of the recombinant rhabdovirus are administered intratumorally.

[0154] In another embodiment, the recombinant rhabdovirus is administered intravenously and intratumorally at each time point.

[0155] As mentioned above, recombinant rhabdovirus of the present invention has many uses for stimulating immune response to cancer cell.It has been observed that strong immune activation ability is limited to tumor microenvironment.Thus, in a preferred embodiment, recombinant rhabdovirus of the present invention can be administered to patient systemically.Systemic applicability is an important attribute because many cancers are highly metastatic, and it allows the treatment of tumor lesions that are difficult to reach and inaccessible.Due to this inherent immune stimulating property, recombinant rhabdovirus of the present invention is particularly useful for the treatment of metastatic tumor.

[0156] It has been observed that some patients develop resistance to checkpoint inhibitor treatment, and these patients appear to accumulate mutations in IFN pathway.Therefore, in one aspect, the recombinant rhabdovirus of the present invention, particularly the recombinant vesicular stomatitis virus of the present invention, is useful for treating the patient who develops resistance to checkpoint inhibitor treatment.Due to the inherent immune-stimulating properties of recombinant rhabdovirus, and particularly the recombinant vesicular stomatitis virus of the present invention, the patient who has been treated in this way can be qualified for the continuation of checkpoint inhibitor treatment.

[0157] In a preferred embodiment, the recombinant rhabdoviruses of the invention, particularly the recombinant vesicular stomatitis viruses of the invention, are useful for the treatment of patients with non-small cell lung cancer who have completed checkpoint inhibitor therapy with either a PD-1 inhibitor or a PD-L1 inhibitor (e.g., an antagonistic antibody to PD-1 or PD-L1).

[0158] It is understood that any of the above pharmaceutical formulations or therapeutic methods may be carried out using any one of the recombinant rhabdoviruses or pharmaceutical compositions of the present invention.

[0159] combination

[0160] The present invention also provides combination treatments / methods that offer certain advantages over treatments / methods currently in use and / or known in the prior art. These advantages may include in vivo efficacy (e.g., improved clinical response, prolonged response, increased rate of response, duration of response, rate of disease stabilization, duration of stabilization, time to disease progression, progression-free survival (PFS) and / or overall survival (OS), subsequent development of resistance, etc.), safe and well-tolerated administration, and reduced frequency and severity of adverse events.

[0161] The recombinant rhabdoviruses of the present invention may be used in combination with other pharmacologically active ingredients, such as state-of-the-art or standard of care compounds (e.g., cytostatic or cytotoxic agents, cytostatic agents, antiangiogenic agents, steroids, immunomodulators / checkpoint inhibitors, etc.).

[0162] Cytostatic and / or cytotoxic active substances that may be administered in combination with the recombinant rhabdovirus of the invention include hormones, hormone analogs and antihormonal agents, aromatase inhibitors, LHRH agonists and antagonists, growth factors (e.g., platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), human epidermal growth factor (HER), and HER2 inhibitors. HER2, HER3, HER4) and hepatocyte growth factor (HGF) inhibitors, and the like, include, but are not limited to, inhibitors of (anti) growth factor antibodies, (anti) growth factor receptor antibodies and tyrosine kinase inhibitors, such as cetuximab, gefitinib, afatinib, nintedanib, imatinib, lapatinib, bosutinib, and trastuzumab; antimetabolites (e.g., antifolates, such as methotrexate, raltitrexate; pyrimidine analogs, such as 5-fluorouracil (5-FU); gemcitabine, irinotecan, doxorubicin, TAS-102, capecitabine, and gemcitabine; purine and adenosine analogs, such as mercaptopurine, thioguanine, cladribine, pentostatin, cytarabine (arabinose); C); antitumor antibiotics (e.g., anthracyclines); platinum derivatives (e.g., cisplatin, oxaliplatin, carboplatin); alkylating agents (e.g., estramustine, mechlorethamine, melphalan, chlorambucil, busulfan, dacarbazine, cyclophosphamide, ifosfamide, temozolomide, nitrosoureas such as carmustine and lomustine, thiotepa); mitotic inhibitors (e.g., vinca alkaloids such as vinblastine, vindesine, vinorelbine, vincristine; and taxanes such as paclitaxel and docetaxel); angiogenesis inhibitors (including bevacizumab, ramucirumab, and aflibercept), tubulin inhibitors;DNA synthesis inhibitors, PARP inhibitors, topoisomerase inhibitors (e.g., epipodophyllotoxins such as etoposide and etopophos, teniposide, amsacrine, topotecan, irinotecan, mitoxantrone), serine / threonine kinase inhibitors (e.g., PDK1 inhibitors, Raf inhibitors, A-Raf inhibitors, B-Raf inhibitors, C-Raf inhibitors, mTOR inhibitors, mTORC1 / 2 inhibitors, PI3K inhibitors, PI3Kα inhibitors, dual mTOR / PI3K inhibitors, STK33 inhibitors) agents, AKT inhibitors, PLK1 inhibitors (e.g., volasertib), CDK inhibitors (including CDK9 inhibitors), Aurora kinase inhibitors, tyrosine kinase inhibitors (e.g., PTK2 / FAK inhibitors), protein-protein interaction inhibitors, MEK inhibitors, ERK inhibitors, FLT3 inhibitors, BRD4 inhibitors, IGF-1R inhibitors, Bcl-xL inhibitors, Bcl-2 inhibitors, Bcl-2 / Bcl-xL inhibitors, ErbB receptor inhibitors, BCR-ABL inhibitors, ABL inhibitors, Src inhibitors, rapamycin isin analogues (e.g., everolimus, temsirolimus, ridaforolimus, sirolimus), androgen synthesis inhibitors, androgen receptor inhibitors, DNMT inhibitors, HDAC inhibitors, ANG1 / 2 inhibitors, CYP17 inhibitors, radiopharmaceuticals, immunotherapeutic agents such as immune checkpoint inhibitors (e.g., CTLA4, PD1, PD-L1, LAG3, and TIM3 binding molecules / immunoglobulins, such as ipilimumab, nivolumab, pembrolizumab, etc.) and various chemotherapeutic agents, such as amifostine, Anagrelide, clodronate, filgrastin, interferon, interferon alpha, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate, and porfimer; proteasome inhibitors (such as bortezomib); Smac and BH3 mimetics; agents that restore p53 functionality (including mdm2-p53 antagonists); inhibitors of the Wnt / beta-catenin signaling pathway; and / or cyclin-dependent kinase 9 inhibitors.

[0163] The recombinant rhabdoviruses of the present invention can be used in combination treatments with either PD-1 pathway inhibitors or SMACm / IAP antagonists. Such combination treatments can be given as a non-fixed (e.g., free) combination of agents or in the form of a fixed combination (e.g., kit of parts).

[0164] In this context, "combination" or "combined" within the meaning of the present invention refers to, but is not limited to, the mixing or combining of more than one active agent, and includes both fixed and non-fixed (e.g., free) combinations (including kits) and uses (e.g., simultaneous, concomitant, sequential, consecutive, alternating, or separate use of components or agents). The term "fixed combination" means that both active agents are administered to a patient at the same time in the form of a single entity or dosage. The term "non-fixed combination" means that both active agents are administered to a patient as separate entities simultaneously, concomitantly, or sequentially, without any specific time limit, such administration providing therapeutically effective levels of the two compounds in the patient's body. The latter also applies to cocktail therapy (e.g., the administration of three or more active agents).

[0165] The present invention provides a recombinant rhabdovirus in combination with a PD-1 pathway inhibitor or a SMACm / IAP antagonist for use in the treatment of cancers described herein, preferably for the treatment of solid tumors.

[0166] The present invention also provides the use of a recombinant rhabdovirus in combination with a PD-1 pathway inhibitor or a SMACm / IAP antagonist for the manufacture of a medicament for the treatment and / or prevention of cancers described herein, preferably for the treatment of solid tumors.

[0167] The present invention further provides a method for treating and / or preventing cancer, comprising administering a therapeutically effective amount of the recombinant rhabdovirus of the present invention and a PD-1 pathway inhibitor or SMACm / IAP antagonist to an individual suffering from cancer, thereby alleviating one or more symptoms of cancer.The recombinant rhabdovirus of the present invention and the PD-1 pathway inhibitor or SMACm / IAP antagonist can be administered simultaneously, sequentially, or alternately.

[0168] The recombinant rhabdovirus of the present invention and PD-1 pathway inhibitor or SMACm / IAP antagonist can be administered by the same administration route or by different administration routes.Preferably, PD-1 pathway inhibitor or SMACm / IAP antagonist is administered intravenously, and the recombinant rhabdovirus of the present invention is administered intratumorally.In another embodiment, the PD-1 pathway inhibitor or SMACm / IAP antagonist is administered intravenously, and the recombinant rhabdovirus of the present invention is administered intratumorally at least once, and the subsequent doses of recombinant rhabdovirus are administered intravenously. Subsequent doses may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 31 days after the initial intratumoral administration. In a preferred embodiment, the PD-1 pathway inhibitor or SMACm / IAP antagonist is administered 21 days after the initial intratumoral administration.

[0169] Particularly preferred is treatment with a recombinant rhabdovirus of the invention in combination with:

[0170] (i) SMAC mimetics (SMACm) / IAP antagonists;

[0171] (ii) Immunotherapeutic agents (including anti-PD-1 agents and anti-PD-L1 agents), and anti-LAG3 agents (such as pembrolizumab and nivolumab), and antibodies disclosed in WO2017 / 198741.

[0172] Combinations provided herein include (i) a recombinant rhabdovirus of the invention, and (iia) a PD-1 pathway inhibitor, preferably an antagonist antibody directed against PD-1 or PD-L1, or (iib) a SMACm / IAP antagonist. Further provided is the use of such combinations including (i) and (iia) or (i) and (iib) for the treatment of the cancers described herein.

[0173] In another aspect, the present invention provides a combination treatment comprising (i) the recombinant rhabdovirus of the present invention and (iia) a PD-1 pathway inhibitor or (iib) a SMACm / IAP antagonist. In such a combination treatment, the recombinant rhabdovirus of the present invention can be administered simultaneously, sequentially, or alternately with the PD-1 pathway inhibitor or SMACm / IAP antagonist.

[0174] For example, "concomitant" administration includes administering active agents within the same general time period, e.g., on the same day, but not necessarily simultaneously. Alternating administration includes administering one agent over a period of time, e.g., over the course of several days or a week, followed by administering the other agent over a subsequent period of time, e.g., over the course of several days or a week, and then repeating the pattern for one or more cycles. Sequential or consecutive administration includes administering one agent during a first period of time (e.g., over the course of several days or a week) using one or more doses, followed by administering the other agent during a second period of time (e.g., over the course of several days or a week) using one or more doses. Overlapping schedules may also be used, which include administering active agents on different days throughout the treatment period, not necessarily in a regular order. Variations on these general guidelines may also be used depending on the agents used and the subject's condition.

[0175] A sequential treatment schedule includes administering a recombinant rhabdovirus of the present invention, followed by administering a PD-1 pathway inhibitor or SMACm / IAP antagonist. A sequential treatment schedule also includes administering a PD-1 pathway inhibitor or SMACm / IAP antagonist, followed by administering a recombinant rhabdovirus of the present invention. A sequential treatment schedule can include administration 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 31 days after each other.

[0176] A PD-1 pathway inhibitor in the sense of the present invention and all of its embodiments is a compound that inhibits the interaction between PD-1 and its receptor. PD-1 pathway inhibitors can impair PD-1 pathway signaling, preferably mediated by the PD-1 receptor. PD-1 inhibitors can be any inhibitor directed against any member of the PD-1 pathway that can antagonize PD-1 pathway signaling. The inhibitor can be an antagonist antibody targeting any member of the PD-1 pathway, but is preferably directed against the PD-1 receptor, PD-L1, or PD-L2. The PD-1 pathway inhibitor can also be a fragment of the PD-1 receptor that blocks the activity of the PD-1 receptor or PD-1 ligand.

[0177] PD-1 antagonists are well known in the art and are reviewed, for example, by Li et al., Int. J. Mol. Sci. 2016, 17, 1151 (incorporated herein by reference). Any PD-1 antagonist, particularly an antibody (such as the antibody disclosed by Li et al.), as well as the additional antibodies disclosed herein below, can be used in accordance with the present invention. Preferably, the PD-1 antagonist of the present invention and all its embodiments is selected from the group consisting of the following antibodies: · Pembrolizumab (anti-PD-1 antibody); · Nivolumab (anti-PD-1 antibody); · Pidilizumab (anti-PD-1 antibody); ·PDR-001 (anti-PD-1 antibody); PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5 (anti-PD-1 antibodies) disclosed herein below Atezolizumab (anti-PD-L1 antibody); Avelumab (anti-PD-L1 antibody); Durvalumab (anti-PD-L1 antibody).

[0178] For example, Hamid, O. et al. (2013) New England Journal of Medicine 369(2):134-44 discloses pembrolizumab (formerly known as lambrolizumab; trade name Keytruda; also known as MK-3475), a humanized IgG4 monoclonal antibody that binds to PD-1; it contains a C228P mutation designed to prevent Fc-mediated cytotoxicity. Pembrolizumab is disclosed, for example, in US 8,354,509 and WO2009 / 114335. It has been approved by the FDA for the treatment of patients with unresectable or metastatic melanoma and patients with metastatic NSCLC.

[0179] Nivolumab (CAS Registry Number: 946414-94-4; BMS-936558 or MDX1106b) is a fully human IgG4 monoclonal antibody that specifically blocks PD-1 and lacks detectable antibody-dependent cellular cytotoxicity (ADCC). Nivolumab is disclosed, for example, in US Pat. No. 8,008,449 and WO2006 / 121168. It has been approved by the FDA for the treatment of patients with unresectable or metastatic melanoma, metastatic NSCLC, and advanced renal cell carcinoma.

[0180] Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pidilizumab is disclosed, for example, in WO2009 / 101611.

[0181] PDR-001 or PDR001 is a high-affinity, ligand-blocking, humanized anti-PD-1 IgG4 antibody that blocks the binding of PD-L1 and PD-L2 to PD-1. PDR-001 is disclosed in WO2015 / 112900 and WO2017 / 019896.

[0182] Antibodies PD1-1 to PD1-5 are antibody molecules defined by the sequences shown in Table 1, where HC denotes the (full-length) heavy chain and LC denotes the (full-length) light chain:

[0183] [Table 1] TIFF0007754891000008.tif132169

[0184] Specifically, the anti-PD-1 antibody molecules described herein above have: (PD1-1:) a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain comprising the amino acid sequence of SEQ ID NO: 15; or (PD1-2:) a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO: 17; or (PD1-3:) a heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain comprising the amino acid sequence of SEQ ID NO: 19; or (PD1-4:) a heavy chain comprising the amino acid sequence of SEQ ID NO: 20 and a light chain comprising the amino acid sequence of SEQ ID NO: 21; or (PD1-5:) A heavy chain comprising the amino acid sequence of SEQ ID NO: 22 and a light chain comprising the amino acid sequence of SEQ ID NO: 23.

[0185] Atezolizumab (also known as Tecentriq, MPDL3280A) is a phage-derived human IgG1k monoclonal antibody that targets PD-L1 and is described, for example, in Deng et al. mAbs 2016;8:593-603. It has been approved by the FDA for the treatment of patients with urothelial carcinoma.

[0186] Avelumab is a fully human anti-PD-L1 IgG1 monoclonal antibody and is described, for example, in Boyerinas et al. Cancer Immunol. Res. 2015;3:1148-1157.

[0187] Durvalumab (MEDI4736) is a human IgG1k monoclonal antibody with high specificity for PD-L1 and is described, for example, in Stewart et al. Cancer Immunol. Res. 2015;3:1052-1062 or in Ibrahim et al. Semin. Oncol. 2015;42:474-483.

[0188] Additional PD-1 antagonists disclosed by Li et al. (supra) or known to be in clinical trials, such as AMP-224, MEDI0680 (AMP-514), REGN2810, BMS-936559, JS001-PD-1, SHR-1210, BMS-936559, TSR-042, JNJ-63723283, MEDI4736, MPDL3280A, and MSB0010718C, may be used as an alternative to, or in addition to, the antagonists mentioned above.

[0189] As used herein, INN is also meant to encompass all biosimilar antibodies that have the same or substantially the same amino acid sequence as the originator antibody, including, but not limited to, those biosimilar antibodies approved under 42 U.S.C. §262 subsection (k) in the United States and equivalent regulations in other jurisdictions.

[0190] The PD-1 antagonists listed above, along with their respective preparations, therapeutic uses, and properties, are known in the art.

[0191] In one embodiment, the PD-1 antagonist is pembrolizumab.

[0192] In another embodiment, the PD-1 antagonist is nivolumab.

[0193] In another embodiment, the PD-1 antagonist is pidilizumab.

[0194] In another embodiment, the PD-1 antagonist is atezolizumab.

[0195] In another embodiment, the PD-1 antagonist is avelumab.

[0196] In another embodiment, the PD-1 antagonist is durvalumab.

[0197] In another embodiment, the PD-1 antagonist is PDR-001.

[0198] In another embodiment, the PD-1 antagonist is PD1-1.

[0199] In another embodiment, the PD-1 antagonist is PD1-2.

[0200] In another embodiment, the PD-1 antagonist is PD1-3.

[0201] In another embodiment, the PD-1 antagonist is PD1-4.

[0202] In another embodiment, the PD-1 antagonist is PD1-5.

[0203] A SMAC mimetic in the sense of this invention and all its embodiments is a compound that binds to IAP proteins and induces their degradation. Preferably, the SMAC mimetic in the sense of this invention and all its embodiments is selected from the group consisting of: · SMAC mimetics (i.e. compounds) disclosed (generaly and / or specifically) in WO2013 / 127729, or pharmaceutically acceptable salts thereof; · SMAC mimetics (i.e. compounds) disclosed (generic and / or specifically) in WO2015 / 025018, or pharmaceutically acceptable salts thereof; · SMAC mimetics (i.e. compounds) disclosed (generic and / or specifically) in WO2015 / 025019, or pharmaceutically acceptable salts thereof; · SMAC mimetics (i.e. compounds) disclosed (generic and / or specifically) in WO2016 / 023858, or pharmaceutically acceptable salts thereof; · SMAC mimetics (i.e. compounds) disclosed (generaly and / or specifically) in WO2008 / 0016893, or pharmaceutically acceptable salts thereof; LCL161, i.e., compound A in Example 1 of WO 2008 / 016893 (pages 28 / 29;

[0122] ), or a pharmaceutically acceptable salt thereof; the SMAC mimetic known as Debio-1143, or a pharmaceutically acceptable salt thereof; the SMAC mimetic known as birinapant, or a pharmaceutically acceptable salt thereof; the SMAC mimetic known as ASTX-660, or a pharmaceutically acceptable salt thereof; the SMAC mimetic known as CUDC-427, or a pharmaceutically acceptable salt thereof; Any one of SMAC mimetics 1 to 26 in Table 2, or a pharmaceutically acceptable salt thereof:

[0204] [Table 2] TIFF0007754891000010.tif224169 TIFF0007754891000011.tif228169 TIFF0007754891000012.tif238169 TIFF0007754891000013.tif50169

[0205] Examples of compounds 1 to 10 in Table 2 are disclosed in WO2013 / 127729. Examples of compounds 11 to 26 in Table 2 are disclosed in WO2016 / 023858.

[0206] As used herein, the term "SMAC mimetic / IAP antagonist" also includes the SMAC mimetics listed above in the form of tautomers, pharmaceutically acceptable salts, hydrates, or solvates (including pharmaceutically acceptable salts). It also includes SMAC mimetics in all their solid, preferably crystalline, forms, and all crystalline forms of their pharmaceutically acceptable salts, hydrates, and solvates (including hydrates and solvates of pharmaceutically acceptable salts).

[0207] All of the SMAC mimetics listed above, along with their respective syntheses and properties, are known in the art. All of the above-referenced patent applications are incorporated by reference in their entirety.

[0208] In one embodiment, the SMAC mimetic is LCL161 or a pharmaceutically acceptable salt thereof (A1).

[0209] In another embodiment, the SMAC mimetic is compound 1 in Table 2 or a pharmaceutically acceptable salt thereof (A2).

[0210] In another embodiment, the SMAC mimetic is compound 2 in Table 2 or a pharmaceutically acceptable salt thereof (A3).

[0211] In another embodiment, the SMAC mimetic is compound 3 in Table 2 or a pharmaceutically acceptable salt thereof (A4).

[0212] In another embodiment, the SMAC mimetic is compound 4 in Table 2 or a pharmaceutically acceptable salt thereof (A5).

[0213] In another embodiment, the SMAC mimetic is compound 5 in Table 2 or a pharmaceutically acceptable salt thereof (A6).

[0214] In another embodiment, the SMAC mimetic is compound 6 in Table 2 or a pharmaceutically acceptable salt thereof (A7).

[0215] In another embodiment, the SMAC mimetic is compound 7 in Table 2 or a pharmaceutically acceptable salt thereof (A8).

[0216] In another embodiment, the SMAC mimetic is compound 8 in Table 2 or a pharmaceutically acceptable salt thereof (A9).

[0217] In another embodiment, the SMAC mimetic is compound 9 in Table 2 or a pharmaceutically acceptable salt thereof (A10).

[0218] In another embodiment, the SMAC mimetic is compound 10 in Table 2 or a pharmaceutically acceptable salt thereof (A11).

[0219] In another embodiment, the SMAC mimetic is compound 11 in Table 2 or a pharmaceutically acceptable salt thereof (A12).

[0220] In another embodiment, the SMAC mimetic is compound 12 in Table 2 or a pharmaceutically acceptable salt thereof (A13).

[0221] In another embodiment, the SMAC mimetic is compound 13 in Table 2 or a pharmaceutically acceptable salt thereof (A14).

[0222] In another embodiment, the SMAC mimetic is compound 14 in Table 2 or a pharmaceutically acceptable salt thereof (A15).

[0223] In another embodiment, the SMAC mimetic is compound 15 in Table 2 or a pharmaceutically acceptable salt thereof (A16).

[0224] In another embodiment, the SMAC mimetic is compound 16 in Table 2 or a pharmaceutically acceptable salt thereof (A17).

[0225] In another embodiment, the SMAC mimetic is compound 17 in Table 2 or a pharmaceutically acceptable salt thereof (A18).

[0226] In another embodiment, the SMAC mimetic is compound 18 in Table 2 or a pharmaceutically acceptable salt thereof (A19).

[0227] In another embodiment, the SMAC mimetic is compound 19 in Table 2 or a pharmaceutically acceptable salt thereof (A20).

[0228] In another embodiment, the SMAC mimetic is compound 20 in Table 2 or a pharmaceutically acceptable salt thereof (A21).

[0229] In another embodiment, the SMAC mimetic is compound 21 in Table 2 or a pharmaceutically acceptable salt thereof (A22).

[0230] In another embodiment, the SMAC mimetic is compound 22 in Table 2 or a pharmaceutically acceptable salt thereof (A23).

[0231] In another embodiment, the SMAC mimetic is compound 23 in Table 2 or a pharmaceutically acceptable salt thereof (A24).

[0232] In another embodiment, the SMAC mimetic is compound 24 in Table 2 or a pharmaceutically acceptable salt thereof (A25).

[0233] In another embodiment, the SMAC mimetic is compound 25 in Table 2 or a pharmaceutically acceptable salt thereof (A26).

[0234] In another embodiment, the SMAC mimetic is compound 26 in Table 2 or a pharmaceutically acceptable salt thereof (A27).

[0235] All embodiments (A1) to (A27) are preferred embodiments of embodiment (A0) in terms of SMAC mimetic properties.

[0236] In a preferred embodiment for combination treatment, the recombinant rhabdovirus is a recombinant vesicular stomatitis virus encoding in its genome at least one CCL21 protein or functional variant thereof, preferably human CCL21, selected from the group comprising: (i) a plasmin-treated CCL21 protein, (ii) a c-terminally truncated CCL21 protein, (iii) a CCL21 protein comprising SEQ ID NO: 2 or a CCL21 protein with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 161 (iv) a protein comprising SEQ ID NO:3 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:3; (v) a protein comprising SEQ ID NO:4 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:4. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1; (vi) a protein according to any of (i) to (v) further comprising a signal peptide sequence; (vii) a protein comprising SEQ ID NO: 1 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1; or (viii) a protein according to SEQ ID NO: SEQ ID NO: 5 or a protein having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5, wherein the gene encoding the recombinant vesicular stomatitis virus glycoprotein G is replaced by the gene encoding the lymphocytic choriomeningitis virus (LCMV) glycoprotein GP and / or glycoprotein G is replaced by the LCMV glycoprotein GP.

[0237] In a further preferred embodiment of the combination treatment, the recombinant rhabdovirus is a recombinant vesicular stomatitis virus encoding in its genome the vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M), glycoprotein (G), and at least one CCL21 protein or functional variant thereof, preferably human CCL21, wherein the CCL21 protein or functional variant thereof is selected from the group comprising: (i) plasmin-treated CCL21 protein, (ii) c a truncated CCL21 protein; (iii) a protein comprising SEQ ID NO:2 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:2; (iv) a protein comprising SEQ ID NO:3 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:3. (v) a protein comprising SEQ ID NO: 4 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4; (vi) a protein according to any of (i) to (v) further comprising a signal peptide sequence; (vii) a protein comprising SEQ ID NO: 1 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1. or (viii) a protein comprising or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5, wherein the gene encoding vesicular stomatitis virus glycoprotein G is replaced by the gene encoding lymphocytic choriomeningitis virus (LCMV) glycoprotein GP;and / or glycoprotein G is replaced by glycoprotein GP of LCMV, wherein the nucleoprotein (N) comprises the amino acids set forth in SEQ ID NO:7 or a functional variant at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7, and the phosphoprotein (P) comprises the amino acids set forth in SEQ ID NO:8 or a functional variant at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8. includes 99% identical functional variants, the large protein (L) includes the amino acids set forth in SEQ ID NO:9 or a functional variant at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9, and the matrix protein (M) includes the amino acids set forth in SEQ ID NO:10 or a functional variant at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:10.

[0238] In a more preferred embodiment of the combination treatment, the recombinant rhabdovirus is a recombinant vesicular stomatitis virus encoding at least one CCL21 protein or functional variant thereof, preferably human CCL21, in its genome, wherein the CCL21 protein or functional variant thereof comprises SEQ ID NO:5 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:5, and wherein the gene encoding glycoprotein G of the recombinant vesicular stomatitis virus is replaced by the gene encoding glycoprotein GP of lymphocytic choriomeningitis virus (LCMV) and / or glycoprotein G is replaced by glycoprotein GP of LCMV.

[0239] The combination of the recombinant rhabdovirus of the present invention, particularly the vesicular stomatitis virus of the present invention, with a PD-1 inhibitor or SMACm / IAP antagonist was exceptionally effective in treating cancer. However, the present inventors have discovered that a combination of a vesicular stomatitis virus that does not encode an additional cargo, i.e., does not encode the CCL21 protein, is also effective when combined with a PD-1 pathway inhibitor or SMACm / IAP antagonist. In particular, combined treatment with VSV-GP (a glycoprotein of vesicular stomatitis virus and LCMV) and a PD-1 pathway inhibitor or SMACm / IAP antagonist, both of which are effective for the treatment of cancer, preferably solid cancer, as described herein. Therefore, also provided herein is a combination comprising a VSV-GP that does not encode the CCL21 protein and a PD-1 pathway inhibitor, preferably an antagonist antibody or SMACm / IAP antagonist directed against PD-1 or PD-L1. Further provided is the use of such a combination for the treatment of cancer as described herein. Further provided is a combination treatment comprising the use of a VSV-GP that does not encode the CCL21 protein and a PD-1 pathway inhibitor or a SMACm / IAP antagonist.

[0240] In connection with combination treatment with VSV-GP that does not encode the CCL21 protein, the recombinant rhabdovirus is preferably a recombinant vesicular stomatitis virus in which the gene encoding glycoprotein G of the recombinant vesicular stomatitis virus has been replaced by the gene encoding glycoprotein GP of lymphocytic choriomeningitis virus (LCMV) and / or in which glycoprotein G has been replaced by glycoprotein GP of LCMV.

[0241] Further in connection with the combination treatment of VSV-GP that does not encode the CCL21 protein, the recombinant rhabdovirus is preferably a recombinant vesicular stomatitis virus encoding in its genome the vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M), and glycoprotein (G), wherein the gene encoding the glycoprotein G of vesicular stomatitis virus is replaced by the gene encoding the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), and / or the glycoprotein G is replaced by the glycoprotein GP of LCMV, and wherein the nucleoprotein (N) comprises the amino acids set forth in SEQ ID NO: 7 or a functional variant at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. The phosphorylated protein (P) comprises the amino acids set forth in SEQ ID NO:8 or a functional variant thereof at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8. The large protein (L) comprises the amino acids set forth in SEQ ID NO:9 or a functional variant thereof at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, or 99% identical to SEQ ID NO:9. , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and matrix protein (M) comprises the amino acids set forth in SEQ ID NO: 10 or a functional variant at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10.

[0242] Virus generation, production, and virus-producing cells

[0243] The present invention also provides a virus-producing cell, characterized in that the cell produces a recombinant rhabdovirus or a recombinant vesicular stomatitis virus according to the invention.

[0244] Cells can be of any origin, and can exist as isolated cells or as cells contained in cell populations.Preferably, the cells that produce recombinant rhabdovirus or recombinant vesicular stomatitis virus are mammalian cells.In a more preferred embodiment, the virus-producing cells of the present invention are characterized in that mammalian cells are multipotent adult progenitor cells (MAPCs), neural stem cells (NSCs), mesenchymal stem cells (MSCs), HeLa cells, HEK cells, any HEK293 cells (e.g., HEK293F or HEK293T), Chinese hamster ovary cells (CHO), baby hamster kidney (BHK) cells, or Vero cells or bone marrow-derived tumor-infiltrating cells (BM-TICs).

[0245] Alternatively, virus-producing cells can be human cells, monkey cells, mouse cells, or hamster cells. Those skilled in the art will be aware of suitable methods for use in testing whether a given cell produces virus and, thus, whether a particular cell falls within the scope of the present invention. In this regard, the amount of virus produced by the cells of the present invention is not particularly limited. A preferred virus titer is ≧1×10 in the crude supernatant of a given cell culture after infection, without further downstream processing. 7 TCID 50 / ml or ≥ 1 × 10 8 Genome copies / ml.

[0246] In a specific embodiment, the virus producer cell of the invention is characterized in that the cell contains one or more expression cassettes for the expression of at least one gene selected from the group consisting of the genes n, l, p, and m encoding the proteins N, L, P, and M of VSV and the gene gp encoding the LCMV-GP, Dandenong-GP, or Mopeia-GP glycoprotein.

[0247] Virus producer cell in the meaning of the present invention includes the classical packaging cell that is used for producing recombinant rhabdovirus from non-replicating vector, and the producer cell that is used for producing recombinant rhabdovirus from replicable vector.Packaging cell usually lacks each of the vectors that are packaged, and / or contains one or more plasmids that are used for expressing the essential genes that are required for producing virus.Such cell is known to those skilled in the art, and can select suitable cell line that is suitable for desired purpose.

[0248] The recombinant rhabdovirus of the present invention can be produced according to methods known to those skilled in the art, including but not limited to: (1) using cDNA transfected into cells, or (2) a combination of cDNA transfected into helper cells, or (3) cDNA transfected into cells (cells are further infected with helper / minivirus to provide the remaining components or activities required in trans for producing either infectious or non-infectious recombinant rhabdovirus).When using any of these methods (e.g., helper / minivirus, helper cell line, or cDNA transfection only), the minimum required components are (1) the DNA molecule that contains the cis-acting signal for the encapsidation of rhabdovirus N protein, P protein, and L protein of genome (or antigenome) RNA, and (2) the replication of genome or antigenome (replicative intermediate) RNA equivalent.

[0249] Replication element or replicon is a strand of RNA that minimally contains the leader sequence and trailer sequence of rhabdovirus at the 5' and 3' ends. In genome sense, the leader is at the 3' end and the trailer is at the 5' end. Any RNA placed between these two replication signals will be replicated in sequence. The leader and trailer regions must also contain the minimum cis-acting elements for the purpose of encapsidation by N protein and polymerase binding, which are necessary for initiating transcription and replication. To prepare recombinant rhabdovirus, miniviruses containing G gene can also contain the G gene with the leader region, trailer region, and appropriate initiation and termination signals for producing G protein mRNA. If miniviruses further contain M gene, appropriate initiation and termination signals for producing M protein mRNA must also be present.

[0250] Any gene contained in recombinant rhabdovirus genome can be flanked by appropriate transcription initiation and termination signals, which allow the expression of these genes and the production of protein products (Schnell et al., Journal of Virology, p.2318-2323, 1996).To produce " non-infectious " recombinant rhabdovirus, recombinant rhabdovirus must have minimum replicon elements and N, P and L proteins, and it must contain M gene.This will produce virus particles that bud from cells, but they are non-infectious particles.To produce " infectious " particles, virus particles must additionally contain proteins that can mediate the binding and fusion of virus particles, for example, through the use of attachment protein or receptor ligand.The natural receptor ligand of rhabdovirus is G protein.

[0251] Any cell that allows recombinant rhabdovirus to be constructed can be used.One method for preparing infectious virus particles comprises that the plasmid that encodes T7 RNA polymerase or other suitable bacteriophage polymerase, such as T3 or SP6 polymerase, is transfected with suitable cell line.Cells can then be transfected with individual cDNAs that comprise the genes that encode G, N, P, L and M rhabdovirus proteins.These cDNAs provide the proteins that are used to construct recombinant rhabdovirus particles.Cells can be transfected by any method known in the art.

[0252] Also, the "polycistronic cDNA" that contains the rhabdovirus genomic RNA equivalent is transfected into cell lines.If infectious recombinant rhabdovirus particles are intended to be lytic in infected cells, then the genes encoding N, P, M and L proteins and heterologous nucleic acid segments must be present.If infectious recombinant rhabdovirus particles are not intended to be lytic, then the gene encoding M protein is not contained in polycistronic DNA.By "polycistronic cDNA", it is meant a cDNA that comprises a transcription unit that at least comprises the genes encoding N, P and L proteins.Recombinant rhabdovirus polycistronic DNA can also contain genes encoding protein variants or polypeptide fragments thereof, or therapeutic nucleic acids or proteins.Alternatively, any protein that is originally associated with the virus particles or fragments that are initially produced can be supplied in trans.

[0253] Also contemplated is a polycistronic cDNA containing a gene encoding CCL21.Contemplated polycistronic cDNA can contain a gene encoding a protein variant, a gene encoding a reporter, a therapeutic nucleic acid, and / or either an NPL gene or an NPLM gene.The first step in producing recombinant rhabdovirus is the expression of RNA, which is genome equivalent or antigenome equivalent, from cDNA.The RNA is then packaged by N protein and replicated by P / L protein.The recombinant virus thus produced can be recovered.If G protein does not exist in recombinant RNA genome, then it is typically supplied in trans.If both G protein and M protein do not exist, then both are supplied in trans.To prepare "non-infectious rhabdovirus" particles, the procedure can be the same as above, except that the polycistronic cDNA transfected into cells only contains the N, P, and L genes of rhabdovirus.The polycistronic cDNA of non-infectious rhabdovirus particles can additionally contain a gene encoding a protein.

[0254] The transfected cells are typically incubated at a desired temperature, typically about 37°C, for at least 24 hours. For non-infectious viral particles, the supernatant is collected and the viral particles are isolated. For infectious viral particles, the virus-containing supernatant is collected and transferred to fresh cells. The fresh cells are incubated for about 48 hours, and the supernatant is collected.

[0255] Other features and advantages of the present invention will become apparent from the following more detailed examples which illustrate, by way of example, the principles of the invention.

[0256] Example

[0257] Example 1

[0258] Induction of immune infiltration / activation and immune checkpoint expression in VSV-GP-infected tumors.

[0259] Expression analysis / NanoString (Figure 1)

[0260] To better understand the impact of therapeutic intervention using the VSV-GP platform, immune cell infiltration (T cells: CD3 epsilon, CD4, and CD8), activation (CD69, granzyme B (GzmB), and perforin (Prf1)), and immune checkpoint (PD-L1 (CD274), PD-1 (Pdcd1), Ctla-4, Tigit, Lag3) expression were analyzed in control or VSV-GP-infected tumors. For this purpose, C57BL / 6 mice bearing established LLC1-IFNARKO (LLC1 tumor cells deleted for interferon alpha receptor) tumors were used as controls or 1 × 10 8 TCID 50 The tumors were treated with a single intravenous injection of VSV-GP. Tumors were excised 7 days after treatment; total RNA was extracted and analyzed using the "Pan Cancer Immune Profiling Panel" from NanoString according to the manufacturer's instructions. As depicted in Figure 1, VSV-GP treatment resulted in a strong upregulation of the expression of the analyzed genes, including the PD-L1 and PD-1 genes.

[0261] Example 2

[0262] Efficacy: VSV-GP combo with anti-PD-1

[0263] Tumor growth (Figure 2A-D)

[0264] Building on the clinical success of PD-1 or PD-L1 blocking antibodies in cancer patients and our own data demonstrating that treatment with the VSV-GP platform resulted in the activation of tumor-infiltrating T cells in close association with the upregulation of immune checkpoints such as PD-1 and PD-L1 (see Figure 1), the therapeutic potential of combining VSV-GP-derived therapeutics with PD-1 blocking antibodies was analyzed using the CT26.CL25-IFNARKO tumor model (CT26.CL25 tumor cells deleted for interferon alpha receptor).

[0265] The engraftment rate of subcutaneously injected CT26Cl25IFNAR- / - tumor cells was 100% (50 / 50 mice). On day 8 (the day of virus application), the median tumor size was 0.05 cm3. Tumor growth was followed over 60-day intervals. Sham (=untreated) controls exhibited spontaneous remission in 10% (1 / 10 mice), with one tumor growing much slower. In mice treated intravenously with VSV-GP alone, complete remission was observed in 30% (3 / 10 mice). PD-1 treatment alone, starting on day 11 post-engraftment, had no effect on tumor growth. The combination of VSV-GP and anti-PD-1 resulted in a high tumor remission rate. When anti-PD-1 was applied after VSV-GP treatment, complete remission was observed in 70% (7 / 10 mice). Mice remained tumor-free for at least 60 days. Overall, a high survival rate was achieved in the combination group (not shown).

[0266] Example 3

[0267] Memory formation: VSV-GP & VSV-GP / anti-PD-1 combo.

[0268] Tumor growth / rechallenge (Figure 3A-C)

[0269] Cured mice from the VSV-GP / anti-PD-1 combination experiment are protected from rechallenge. Mice from the VSV-GP / anti-PD-1 combination group (n = 7) or the VSV-GP alone (low dose, n = 3) group were rechallenged. Briefly, CT26Cl25IFNAR- / - cells were subcutaneously injected into the left flank of mice, and tumor growth was monitored over time. As a positive control, age- and sex-matched naive mice (n = 10) were subcutaneously implanted with CT26Cl25IFNAR- / - tumors. As previously observed, tumors grew consistently, with one spontaneous remission. No tumor growth was observed in the VSV-GP / anti-PD-1 combination group (n = 7) or the VSV-GP alone (low dose, n = 3) group, indicating that the cured mice developed specific immunological memory.

[0270] Example 4

[0271] Selection of CCL21 cargo and VSV-GP-induced chemokine expression in tumors.

[0272] NanoString® Expression Analysis (Figure 4).

[0273] The impact of therapeutic intervention using the VSV-GP platform in preclinical tumor models was analyzed by measuring the expression of multiple chemokines (Figure 4). Expression of CCL5, CXCL9, CXCL10, and other chemokines was significantly elevated in tumors aged 1 × 10 8 TCID 50 was strongly upregulated in LLC1-IFNARKO tumors on days 3 or 7 after a single intravenous treatment with VSV-GP. On the other hand, the expression of the CCR7 ligands CCL19 and CCL21 was not upregulated by VSV-GP infection.

[0274] Example 5

[0275] Generation of VSV-CCL21 recombinants.

[0276] Virus rescue (Figure 5).

[0277] Building on the findings described in Example 4, the genome of the oncolytic virus VSV-GP was engineered to encode the CCL21 gene (see Figure 5), which locally expresses the CCL21 chemokine at tumor sites during viral replication, filling the "immunotherapeutic gap" of VSV-GP (VSV-GP failed to upregulate the CCR7 ligands CCL19 and CCL21), and further improving immune cell infiltration and the therapeutic efficacy of the oncolytic virus VSV-GP.

[0278] Replication-competent VSV-GP-CCL21 virus mutants were generated by reverse genetics (cloning of the gene of interest (GOI), virus rescue, and repeated plaque purification) from bacterial plasmids containing cDNA for the complete viral genome of VSV-GP and versions of mouse or human CCL21. The pVSV-GP-CCL21 plasmid was based on the plasmid pVSV-XN1 (Schnell et al.), which contains the complete cDNA genome of the VSV Indiana serotype under the control of a T7 promoter. To generate the pVSV-GP-CCL21 variant, the entire sequence for the VSV G envelope protein was replaced with the codon-optimized sequence of the GP envelope protein from lymphocytic choriomeningitis virus (LCMV, strain WE-HPI). In addition, a synthetic nucleic acid encoding the CCL21 gene was inserted between the glycoprotein GP and the viral polymerase L by Gibson assembly. Transcription of the CCL21 gene in the context of viral infection is ensured by an extra VSV start signal sequence at the 3′ end of the CCL21 open reading frame and an additional stop signal sequence at the 5′ end (Fig. 5A).

[0279] Infectious virus was recovered (or rescued) from the plasmid cDNA by transfection of HEK293T or any other VSV-permissive cell line using standard transfection methods (e.g., CaPO precipitation, liposomal DNA delivery). Briefly, HEK293T cells were transfected with a pSF-CAG-amp-based expression plasmid encoding VSV proteins N, P, and L and a codon-optimized T7 polymerase. In addition, plasmids encoding the viral genome cDNA of VSV-GP, VSV-GP-CCL21, or their mutants were cotransfected (Figure 5B). In the first step of the rescue process, T7 polymerase transcribes the viral RNA genome from the plasmid-encoded viral cDNA. In the second step, VSV-L and P proteins are exogenously expressed from the cotransfected plasmid, further amplifying the viral RNA genome. The viral RNA genome is cotranscriptionally encapsidated by the VSV-N protein. In addition, the P / L polymerase complex allows transcription of the complete set of viral gene products N, P, M, GP, and L, as well as the inserted CCL21 variant. The viral RNA genome is then packaged into infectious VSV particles containing ribonucleoproteins, matrix protein, and the viral envelope GP. Viral particles are released from the cell by budding.

[0280] Rescued viruses were first passaged in permissive cell lines, such as HEK293T, BHK21C1.13, or VERO. Several rounds of plaque purification were performed before generating viral seed stocks by standard methods. Briefly, HEK293T, BHK21C1.13, or VERO cells were infected with serial 10-fold dilutions of the rescued pre-seed. After approximately 2 hours, the cell monolayers were washed twice and overlaid with medium containing 0.8% low-melting-point agarose. 24 to 48 hours post-infection, plaques were picked, and the virus was used for additional rounds of plaque purification or to generate viral seed stocks.

[0281] Example 5.1

[0282] Validation of viral fitness in vitro.

[0283] TCID 50 (Figures 19A-C)

[0284] One day before infection, Vero, BHK21, and HEK293 cells were seeded in 6-well plates with the following culture media: (a) Vero cells: DMEM (Gibco, #31966-021) + 5% heat-inactivated FBS (Gibco, #10500-064); (b) BHK21 cells: GMEM (Life Technologies, #21710-082 / 025) + 10% heat-inactivated FBS (Gibco, #10500-064) + 5% TPB tryptose phosphate broth (Life Technologies, #18050-039); (c) HEK293 cells: Freestyle™ 293 Expression Medium (ThermoFisher Scientific, #12338018).

[0285] On the day of infection, all cell lines were 60-70% confluent. One well per cell line was counted (Countess™ Cell Counter, Invitrogen) before infecting other wells with one of the viral constructs VSV-GP (GP), VSV-GP-huCCL21 (21), or VSV-GP-huCCL21(1-79) (21k) at an MOI of 0.005.

[0286] Culture supernatants (3 mL total volume) were collected at 0, 24, or 48 hours post-infection and analyzed by TCID 50 Viral replication capacity was determined by measuring the viral load / mL.

[0287] TCID 50The median tissue culture infectious dose (TDC) was determined on 96-well plates of VERO cells seeded 1 day prior to infection. 22 serial half-log dilutions (ranging from 1E-1.0 to 1E-11.5) of all supernatants were prepared and titrated in quadruplicate. On day 6 post-infection, cytopathic effect (CPE) was read by microscopic examination of the plates.

[0288] TCID 50 / mL was calculated according to the Spearman-Karber formula M = x + d[0.5-(1 / n)(r)], where x is the positive index at the highest dilution tested; d is the interval between dilutions; n is the number of wells per dilution; and r is the total number of negative responses.

[0289] Example 5.2

[0290] Cargo expression in vitro.

[0291] ELISA / Western blot (Figures 6A-B, 12A-B, and 18)

[0292] To confirm and quantify the expression of the viral CCL21 cargo (transgene) and to better characterize different CCL21 variants, we performed CCL21-specific ELISA and Western blot analyses. As depicted in Figure 6A and Figure 12A, supernatants from HEK293 cells infected with VSV-GP-muCCL21 (Figure 6A; VSV-GP expressing full-length mouse CCL21) or VSV-GP-huCCL21 (Figure 12A; VSV-GP expressing full-length human CCL21) were analyzed at different time points postviral infection using mouse- or human-specific ELISAs, respectively. Human CCL21 variants, i.e., full-length human CCL21 and a C-terminal truncated version resembling the first 79 amino acids (without the signal sequence) of human CCL21 = CCL21(1-79), were also characterized using human CCL21-specific Western blots. As depicted in Figure 18 (from left), supernatants from HEK293 cells transfected with plasmids encoding CCL21(1-79) or full-length CCL21 protein, as well as supernatants from HEK293 cells infected with the indicated viruses, were analyzed. The chemokine mutants were expressed well in both systems (plasmid and virus). The full-length CCL21 sample contained multiple CCL21 species or degradation / cleavage products, whereas the CCL21(1-79) protein presented as a clean single band.

[0293] Example 5.3

[0294] In vitro cargo activity

[0295] Transwell (T cells / DC) (Figure 6A&12A&16&17)

[0296] To confirm and further characterize the biological functionality of viral CCL21 cargoes (transgenes), their ability to attract T cells or monocyte-derived dendritic cells (moDCs) was analyzed in a Transwell migration assay. To this end, mouse and human CCL21 containing supernatants, migration medium alone (background control), recombinant CCL21 (positive control), or the corresponding supernatants from VSV-GP-infected HEK293 cells (VSV-GP background control), as described in Example 5.2 (Figures 6A and 12A), respectively, were added to the lower wells of a Transwell migration assay setup, and CD3 / 28-stimulated mouse (Figure 6; right panel) or human (Figure 12; right panel) T cells were added to the upper chamber (Transwell insert). After incubation, cells in the lower wells were quantified using the Promega® CellTiter-Glo® cell viability assay. Results are depicted as "fold increase" compared to the migration medium-only control. Further experiments included the shorter cargo versions of CCL21, CCL21(1-79) (aa 1-79 of human CCL21) and the shortest naturally occurring CCL21 fragment resulting from plasmin-mediated processing; CCL21(1-81) (aa 1-81 of human CCL21) (see Figure 15 ), using the assay described above. To this end, supernatants from HEK293 cells transfected with expression plasmids were generated and analyzed (see Figure 16 ). In a final step, supernatants from VSV-GP-, VSV-GP-huCCL21 (full-length), and VSV-GP-huCCL21(1-79)-infected HEK293 cells were compared using the Transwell migration assay described above and moDCs as responder cells. In this assay, both full-length human CCL21 and CCL21(1-79) induced similar levels of moDC migration (see Figure 17 ).

[0297] Example 5.4

[0298] In vitro species cross-reactivity (human vs. mouse & rat)

[0299] Transwell (T cells) (Figure 13)

[0300] To confirm the cross-species reactivity of human CCL21 to the mouse and rat CCL21 receptor (CCR7), the Transwell migration assay described above was used with mouse (left) or rat (right) T cells as responders (see Figure 13). Migration assays were performed using human vs. mouse (left) and human vs. rat (right) recombinant chemokines at the indicated concentrations (lower wells). In conclusion, human CCL21 is active in mouse and rat systems, respectively, like the corresponding mouse and rat chemokines, demonstrating that human molecules can be tested in preclinical rodent (mouse / rat) models.

[0301] Example 6:

[0302] Cargo CCL21 expression in vivo

[0303] RNAseq (Figure 10)

[0304] The expression of virally encoded CCL21 was analyzed / confirmed in rodent tumors in control or VSV-GP or VSV-GP-muCCL21 infected tumors. For this purpose, C57BL / 6 mice with established LLC1-IFNARKO tumors were used as controls or 1 × 10 8 TCID 50 Mice were treated with a single intravenous injection of VSV-GP or VSV-GP-muCCL21. Tumors were excised 7 days after treatment; total RNA was extracted and analyzed using RNAseq. CD3epsilon and CXCL10 were used as controls. Virus-encoded CCL21 was specifically detected using a codon-optimized DNA sequence as readout (see Figure 10).

[0305] Example 6.1:

[0306] Lack of neurovirulence of VSV-GP and VSV-GP-CCL21.

[0307] Survival rate (Figure 11)

[0308] Wild-type VSV infection can cause neurological symptoms if the virus gains access to the brain. These neurological complications include severe encephalitis, which can lead to the death of infected subjects. An advantage of using chimeric VSV-GP is the almost complete absence of neuroinfection, making the VSV backbone a safe oncolytic agent. The reason for the attenuated phenotype is thought to be due to altered viral tropism, facilitated via the viral envelope glycoprotein. Although VSV-GP does not exhibit neuroinfection or spread in the brain, it is unclear whether viral gene expression in other cell types, such as glial cells or astrocytes, is completely absent. Because inadvertent expression of the CCL21 transgene by VSV GP could attract immune cells and cause deleterious effects in the brain, we evaluated the neurotoxicity of VSV-GP-muCCL21.

[0309] Swiss CD-1 mice were injected with 1 x 10 6 TCID 50 In the control group, mice received a single intracranial injection of 3 μl containing the VSV-G DsRed antibody. PBS was administered intracardially in the control group. Animals were monitored daily for signs of neurovirulence and general health. The survival rates of mice in the PBS (dots), VSV-G DsRed (diamonds), VSV-GP (squares), and VSV-GP muCCL21 experimental groups were plotted as Kaplan-Meier curves (Figure 11B). Kaplan-Meier analysis indicates that none of the tested viral mutants exhibited neurovirulence in mice. Only the VSV-G DsRed control group, which contains wild-type VSV glycoprotein on the viral surface, exhibited increased weight loss (Figure 11A) and developed neurological signs leading to euthanasia within the first week after cardiac infection.

[0310] Example 6.2

[0311] In vivo efficacy of VSV-GP and VSV-GP-CCL21.

[0312] tumor growth (Figure 14).

[0313] The therapeutic potential of VSV-GP and VSV-GP-huCCL21 was evaluated / compared using the CT26.CL25-IFNARKO tumor model. For this purpose, established tumors were grown in a volume of 2 × 10 7 TCID 50 Mice were treated with two intravenous injections (days 0 and 3) of VSV-GP or VSV-GP-huCCL21. Survival rates of mice treated as indicated are depicted in FIG.

[0314] Example 6.3

[0315] In vivo efficacy of VSV-GP-huCCL21 and VSV-GP-CCL21(1-79).

[0316] Tumor growth / survival (Fig. 20 / 21).

[0317] The therapeutic potential of VSV-GP-huCCL21 and VSV-GP-huCCL21(1-79) was evaluated and compared using the CT26.CL25-IFNARKO tumor model. For this purpose, established tumors were grown in a volume of 2 × 10 7 TCID 50 Mice were treated with two intravenous injections (days 0 and 3) of VSV-GP-huCCL21 or VSV-GP-huCCL21(1-79). The cumulative tumor growth and 30-day survival rates of mice treated as indicated are depicted in Figures 20 / 21. Treatment with the short CCL21 mutant (CCL21(1-79)), which corresponds to a fully plasmin-processed (lesser aa 80 / 81) and freely diffusible form of human CCL21, resulted in better control of tumor growth and improved survival compared with the full-length CCL21 with the VSV-GP mutant.

[0318] Example 6.4

[0319] MoA: T cell infiltration induced by VSV-GP-huCCL21 and VSV-GP-huCCL21(1-79).

[0320] IHC (FIG.22 / 23)

[0321] Tumors treated under Example 6.3 were analyzed for T cell infiltration. FFPE tumor sections were stained for CD4 and CD8, as well as VSV-N and cleaved caspase 3. Total T cells (CD4+ & CD8+ cells) in viable (non-necrotic) tumor areas were quantified. As depicted in Figure 22, a short CCL21 variant (CCL21(1-79)), corresponding to a fully plasminized (lesser aa 80 / 81) and freely diffusible form of human CCL21, was able to attract more T cells into the tumor, providing an explanation for the increased efficacy observed (Example 6.3).

[0322] In addition, virally expressed CCL21 was able to attract dendritic cells (CD11c positive) into infected CT26.CL25 tumors. Established CT26.CL25 tumors were infected with 2 × 10 7 TCID 50 VSV-GP or VSV-GP-muCCL21 was injected locally (intrathecally). FFPE sections of each tumor were analyzed for dendritic cell infiltration (tumor areas with active viral replication = necrotic rim) (see Figure 24).

[0323] Example 7

[0324] Efficacy: VSV-GP and VSV-GP-CCL21 combo with SMACm.

[0325] Tumor growth / survival (Figures 7-9).

[0326] Based on the encouraging data from the combination of VSV-GP and PD-1-blocking antibodies (Example 2), further combinations of VSV-GP (Figures 7 / 9) and VSV-GP-muCCL21 (Figures 8 / 9) with SMAC mimetics (SMACm), a modulator of the cell death pathway that renders tumor cells more susceptible to cell death-inducing stimuli / drugs, were tested. The therapeutic interactions of the compounds were analyzed using the CT26.CL25-IFNARKO tumor model. To this end, Balb / c mice bearing established CT26.CL25-IFNARKO tumors were cultured in 4x10 6 TCID 50 Patients received a single intravenous treatment with 100 mg / kg of VSV-GP or VSV-GP-muCCL21 and / or 100 mg / kg of SMACm orally (po) once daily for a 2-week period, starting on the same day as the VSV-GP or VSV-GP-muCCL21 treatment. The combination of VSV-GP and SMACm resulted in improved efficacy compared with the corresponding monotherapies. When VSV-GP-muCCL21 was combined with SMACm, the combination effect was even more pronounced, resulting in cure of all treated animals.

Claims

1. A pharmaceutical composition for the treatment of solid cancer, comprising: an effective amount of a recombinant vesicular stomatitis virus; wherein the gene encoding the glycoprotein G of the recombinant vesicular stomatitis virus is replaced by the gene encoding the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), and / or the glycoprotein G is replaced by the glycoprotein GP of LCMV; Following administration of the pharmaceutical composition, a PD-1 pathway inhibitor that is an antagonist antibody to PD-1 is administered. Pharmaceutical compositions.

2. the recombinant vesicular stomatitis virus encodes at least a nucleoprotein (N), a large protein (L), a phosphorylated protein (P), a matrix protein (M), and a glycoprotein (G) of a vesicular stomatitis virus in its genome; the gene encoding the glycoprotein G of the recombinant vesicular stomatitis virus has been replaced by the gene encoding the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), and / or the glycoprotein G has been replaced by the glycoprotein GP of LCMV; The pharmaceutical composition of claim 1.

3. 3. The pharmaceutical composition of claim 2, wherein the nucleoprotein (N) comprises the amino acid sequence set forth in SEQ ID NO: 7, or a functional variant that is at least 98% identical to SEQ ID NO:

7.

4. 4. The pharmaceutical composition according to claim 2, wherein the phosphorylated protein (P) comprises the amino acid sequence set forth in SEQ ID NO: 8 or a functional variant that is at least 98% identical to SEQ ID NO:

8.

5. 5. The pharmaceutical composition according to claim 2, wherein the large protein (L) comprises the amino acid sequence set forth in SEQ ID NO: 9 or a functional variant at least 98% identical to SEQ ID NO:

9.

6. 6. The pharmaceutical composition of claim 2, wherein the matrix protein (M) comprises the amino acid sequence set forth in SEQ ID NO: 10, or a functional variant that is at least 98% identical to SEQ ID NO:

10.

7. The nucleoprotein (N) comprises the amino acid sequence set forth in SEQ ID NO: 7 or a functional variant thereof that is at least 98% identical to SEQ ID NO: 7; The phosphorylated protein (P) comprises the amino acid sequence set forth in SEQ ID NO: 8 or a functional variant thereof that is at least 98% identical to SEQ ID NO: 8; The large protein (L) comprises the amino acid sequence set forth in SEQ ID NO: 9 or a functional variant that is at least 98% identical to SEQ ID NO: 9; and The matrix protein (M) comprises the amino acid sequence set forth in SEQ ID NO: 10, or a functional variant thereof that is at least 98% identical to SEQ ID NO:

10. A pharmaceutical composition according to any one of claims 2 to 6.

8. The pharmaceutical composition of claim 1 , wherein the recombinant vesicular stomatitis virus is replication-competent.

9. 2. The pharmaceutical composition of claim 1, wherein the solid cancer is selected from the list consisting of glioblastoma, non-small cell lung cancer, reproductive system tumor, ovarian tumor, testicular tumor, endocrine tumor, gastrointestinal tumor, pancreatic tumor, liver tumor, kidney tumor, colon tumor, colorectal tumor, bladder tumor, prostate tumor, skin tumor, melanoma, respiratory tumor, lung tumor, breast tumor, head and neck tumor, head and neck squamous cell carcinoma (HNSCC) and bone tumor.

10. 2. The pharmaceutical composition of claim 1, wherein the antagonist antibody is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, PDR-001, PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5.

11. 11. The pharmaceutical composition of any one of claims 1 to 10, wherein the recombinant vesicular stomatitis virus is administered via a different route of administration than the PD-1 pathway inhibitor.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the recombinant vesicular stomatitis virus or pharmaceutical composition is administered intratumorally at least once, and the PD-1 pathway inhibitor is administered intravenously.

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