Recombinant rhabdovirus encoding cd80 ectodomain fc fusion protein

Abstract

The present invention relates to the field of oncolytic viruses, and in particular to a recombinant rhabdovirus, such as a vesicular stomatitis virus encoding a CD80 ectodomain Fc fusion protein in its genome. The invention further relates to the use of the recombinant virus in the treatment of cancer, and also to methods for producing such viruses.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of oncolytic viruses and in particular to a recombinant rhabdovirus encoding a CD80 ectodomain Fc fusion protein in its genome. The invention further relates to the use of the recombinant rhabdovirus in the treatment of cancer and to methods for producing such viruses. BACKGROUND

[0002] Oncolytic viruses are an emerging class of biologicals that selectively replicate in and kill cancer cells and are able to spread within tumors. Attempts to further improve oncolytic viruses to increase their therapeutic potential have led to the generation of so-called armed viruses that encode tumor antigens or immunomodulatory transgenes in their genome to improve their efficacy in tumor therapy.

[0003] In many cases, there are very small amounts of T cells in tumors, and thus a microenvironment that has been termed an "immune desert" - an immune desert is a tumor microenvironment in which T cells of the immune system cannot or do not penetrate the tumor to kill out-of-control cells. It has been hypothesized that, to evade immune surveillance, tumors establish an immunosuppressive microenvironment by recruiting myeloid-derived suppressor cells or secreting factors including TGFp, which exerts a dual role of inducing expression of extracellular matrix genes and suppressing expression of chemokines and cytokines required to promote T cell infiltration into tumors (Pickup M, Novitskiy S, Moses HL. The roles of TGFbeta in the tumour microenvironment. Nat Rev Cancer 2013; 13: 788-99). Furthermore, studies have found that tumors exhibit high expression of genes corresponding to an immunosuppressive microenvironment are associated with poor outcomes in multiple 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 have been linked to clinical outcome. Clin Cancer Res 2008; 14: 5198-208). Hallmarks of an adaptive immune response are specificity and memory.Cellular responses are mediated by T cells that express cell-surface T cell receptors (TCRs) that recognize peptide antigens complexed with major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs). However, the interaction of homologous TCRs with individual MHC-peptide complexes (Signal 1) does not trigger optimal T cell activation. In addition to Signal 1, the binding of positive and negative costimulatory receptors to their homologous ligands regulates T cell activation. This complex signaling network provides optimal T cell activation on the one hand, and prevents aberrant T cell activation under physiological conditions on the other. CD28 (Signal 2) is the major positive costimulatory receptor on T cells. When Signal 2 (CD28 interacting with B7.1 (CD80) or B7.2 (CD86)) is lacking, for example, on TAA-presenting tumor cells, long-term interaction with TAA-specific T cells leads to T cell unresponsiveness (non-responsiveness): even when TCRs interact with TAAs, T cells struggle to transduce signals. This condition has been described in cancer patients, particularly in long-term advanced disease. However, potent T-cell co-stimulation can reactivate TAA-specific T cells in patients with advanced metastatic cancer.

[0004] A recent approach envisions an oncolytic virus that encodes the IFN-β protein as a payload in its genome. In another approach, the expression of the tumor antigen MAGE-A3 is being explored clinically. Besides identifying suitable and effective payloads, expressing other payloads from the viral backbone always carries the risk of enhancing not only antitumor efficacy but also antiviral immunity. It must be noted that the payload does not limit the oncolytic potential of the virus to the extent that the benefits gained from the expression of a therapeutic payload are offset by a loss of oncolytic efficacy. Therefore, there is a need in the art for further modified armed oncolytic viruses that can be used for effective cancer therapy. There is also a need in the art for selectively improving the infiltration of T cells and / or dendritic cells into the immunosuppressive tumor microenvironment. Summary of the Invention

[0005] The present invention addresses the above needs by providing a recombinant rhabdovirus, such as vesicular stomatitis virus, which encodes a CD80 extracellular domain Fc fusion protein or a functional variant thereof in its genome, preferably the human CD80 extracellular domain.

[0006] It should be understood that any implementation scheme relating to a particular aspect may also be combined with another implementation scheme that also involves that particular aspect, or even present multiple levels and combinations of implementation schemes relating to that particular aspect.

[0007] In a first aspect, the present invention relates to a recombinant rhabdovirus that encodes in its genome at least one CD80 extracellular domain Fc fusion protein or a functional variant thereof, wherein the CD80 extracellular domain Fc fusion protein comprises the extracellular domain of CD80 and further comprises the Fc domain of IgG.

[0008] In one embodiment of the first aspect, the CD80 extracellular domain Fc fusion protein or a functional variant thereof is selected from the group comprising: (i) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1; (ii) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the CD80 extracellular domain comprises or is composed of or has at least 80% similarity to SEQ ID NO:1; (iii) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the Fc domain comprises or is composed of or has at least 80% similarity to SEQ ID NO:2; (iv) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the CD80 extracellular domain comprises or is composed of or has at least 80% similarity to SEQ ID NO:1. NO:1 has at least 80% identity and the Fc domain contains or is composed of or has at least 80% identity with SEQ ID NO:2, (v) CD80 extracellular domain Fc fusion protein containing a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the CD80 extracellular domain is composed of amino acids 1-207 of SEQ ID NO:4 or has at least 80% identity with amino acids 1-207 of SEQ ID NO:4 and the Fc domain is composed of amino acids 208-433 of SEQ ID NO:4 or has at least 80% identity with amino acids 208-433 of SEQ ID NO:4, (vi) CD80 extracellular domain Fc fusion protein according to any of (i)-(v) further contains a signal peptide sequence, (vii) CD80 extracellular domain Fc fusion protein containing or having at least 80% identity with SEQ ID NO:3.

[0009] In one implementation of the first aspect, the recombinant rhabdovirus is a vesicular virus.

[0010] In one embodiment of the first aspect, the vesicular virus is selected from the group comprising: vesicular stomatitis alagoas virus (VSAV), carajás virus (CJSV), chandipura virus (CHPV), cocal virus (COCV), vesicular stomatitis Indiana virus (VSIV), isfahan virus (ISFV), maraba virus (MARAV), vesicular stomatitis New Jersey virus (VSNJV), or piry virus (PIRYV), preferably vesicular stomatitis Indiana virus (VSIV) or preferably vesicular stomatitis New Jersey virus (VSNJV).

[0011] In one implementation of the first aspect, the recombinant rhabdovirus is capable of replication.

[0012] In one implementation of the first aspect, the CD80 extracellular domain is the human CD80 extracellular domain.

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

[0014] In a preferred embodiment of the first aspect, the present invention provides a recombinant vesicular stomatitis virus that encodes at least one CD80 extracellular domain Fc fusion protein or a functional variant thereof in its genome, wherein the CD80 extracellular domain Fc fusion protein comprises the extracellular domain of CD80 and further comprises the Fc domain of IgG. In the relevant embodiments, the CD80 extracellular domain Fc fusion protein is selected from the group comprising: (i) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1; (ii) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the CD80 extracellular domain comprises or is composed of SEQ ID NO:1 or has at least 80% similarity to SEQ ID NO:1; (iii) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the Fc domain comprises or is composed of SEQ ID NO:2 or has at least 80% similarity to SEQ ID NO:2; (iv) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the CD80 extracellular domain comprises or is composed of SEQ ID NO:1 or has at least 80% similarity to SEQ ID NO:2. NO:1 has at least 80% identity and the Fc domain comprises or is composed of or has at least 80% identity with SEQ ID NO:2, (v) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the CD80 extracellular domain is composed of amino acids 1-207 of SEQ ID NO:4 or has at least 80% identity with amino acids 1-207 of SEQ ID NO:4 and the Fc domain is composed of amino acids 208-433 of SEQ ID NO:4 or has at least 80% identity with amino acids 208-433 of SEQ ID NO:4, (vi) a CD80 extracellular domain Fc fusion protein according to any of (i)-(v) further comprising a signal peptide sequence, or (vii) a CD80 extracellular domain Fc fusion protein comprising or is composed of SEQ ID NO:3 or has at least 80% identity with SEQ ID NO:2, (vii) a CD80 extracellular domain Fc fusion protein comprising ... NO:3 has at least 80% identity; and the gene encoding the glycoprotein G of the recombinant vesicular stomatitis virus is replaced by the gene encoding the glycoprotein GP of lymphocyte choriomeningovirus (LCMV), and / or the glycoprotein G is replaced by the glycoprotein GP of LCMV.

[0015] In a second aspect, the present invention relates to a recombinant vesicular stomatitis virus that encodes in its genome at least a vesicular stomatitis virus nucleoprotein (N), a large protein (L), a phosphoprotein (P), a matrix protein (M), a glycoprotein (G), and at least one CD80 extracellular domain Fc fusion protein or a functional variant thereof, wherein the CD80 extracellular domain Fc fusion protein comprises the extracellular domain of CD80 and further comprises the Fc domain of IgG.

[0016] In one embodiment of the second aspect, the nucleoprotein (N) comprises an amino acid sequence as shown in SEQ ID NO:7 or a functional variant that is consistent with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:7.

[0017] In one embodiment of the second aspect, the phosphoprotein (P) comprises an amino acid sequence as shown in SEQ ID NO:8 or a functional variant consistent with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:8.

[0018] In one embodiment of the second aspect, the large protein (L) comprises an amino acid sequence as shown in SEQ ID NO:9 or a functional variant consistent with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:9.

[0019] In one embodiment of the second aspect, the matrix protein (M) comprises an amino acid sequence as shown in SEQ ID NO:10 or a functional variant consistent with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:10.

[0020] In a preferred embodiment of the second aspect, the nucleoprotein (N) comprises an amino acid sequence as shown in SEQ ID NO:7 or a functional variant consistent with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:7; the phosphoprotein (P) comprises an amino acid sequence as shown in SEQ ID NO:8 or a functional variant consistent with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:8; the large protein (L) comprises an amino acid sequence as shown in SEQ ID NO:9 or a functional variant consistent with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:8; IDNO:9 at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the functional variants consistent with SEQ ID NO:10; and the matrix protein (M) contains an amino acid sequence as shown in SEQ ID NO:10 or a functional variant consistent with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:10.

[0021] In one implementation of the second aspect, the recombinant vesicular stomatitis virus is capable of replication.

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

[0023] In one embodiment of the second aspect, the CD80 extracellular domain Fc fusion protein is selected from the group comprising: (i) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1; (ii) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the CD80 extracellular domain comprises or is composed of SEQ ID NO:1 or has at least 80% similarity to SEQ ID NO:1; (iii) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the Fc domain comprises or is composed of SEQ ID NO:2 or has at least 80% similarity to SEQ ID NO:2; (iv) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the CD80 extracellular domain comprises or is composed of SEQ ID NO:1 or has at least 80% similarity to SEQ ID NO:2. NO:1 has at least 80% identity and the Fc domain contains or is composed of or has at least 80% identity with SEQ ID NO:2, (v) CD80 extracellular domain Fc fusion protein containing a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the CD80 extracellular domain is composed of amino acids 1-207 of SEQ ID NO:4 or has at least 80% identity with amino acids 1-207 of SEQ ID NO:4 and the Fc domain is composed of amino acids 208-433 of SEQ ID NO:4 or has at least 80% identity with amino acids 208-433 of SEQ ID NO:4, (vi) CD80 extracellular domain Fc fusion protein according to any of (i)-(v) further contains a signal peptide sequence, or (vii) CD80 extracellular domain Fc fusion protein containing or having at least 80% identity with SEQ ID NO:3.

[0024] In a preferred embodiment of the second aspect, the present invention provides a recombinant vesicular stomatitis virus encoding in its genome a vesicular stomatitis virus nucleoprotein (N), a large protein (L), a phosphoprotein (P), a matrix protein (M), a glycoprotein (G), and at least one CD80 extracellular domain Fc fusion protein or a functional variant thereof, wherein the CD80 extracellular domain Fc fusion protein comprises an extracellular domain of CD80 and further comprises an Fc domain of IgG and is selected from the group consisting of: (i) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with an Fc domain of IgG1; (ii) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the CD80 extracellular domain comprises or consists of or has at least 80% identity with SEQ ID NO:1; and (iii) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the Fc domain comprises SEQ ID NO:1. (iv) A CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused to the Fc domain of IgG1, wherein the CD80 extracellular domain comprises or is composed of or has at least 80% identity with SEQ ID NO:1 and the Fc domain comprises or is composed of or has at least 80% identity with SEQ ID NO:2; (v) A CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused to the Fc domain of IgG1, wherein the CD80 extracellular domain is composed of amino acids 1-207 of SEQ ID NO:4 or has at least 80% identity with amino acids 1-207 of SEQ ID NO:4 and the Fc domain is composed of amino acids 208-433 of SEQ ID NO:4 or has at least 80% identity with SEQ ID NO:2; The amino acid sequence 208-433 of NO:4 has at least 80% identity, (vi) the CD80 extracellular domain Fc fusion protein according to any one of (i)-(v) further includes a signal peptide sequence, or (vii) the CD80 extracellular domain Fc fusion protein includes SEQ ID NO:3 or has at least 80% identity with SEQ ID NO:3.Furthermore, the gene encoding the glycoprotein G of the 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 the nucleoprotein (N) contains amino acids as shown in SEQ ID NO:7 or functional variants identical to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:7; the phosphoprotein (P) contains amino acids as shown in SEQ ID NO:8 or functional variants identical to SEQ ID NO:7. NO:8 at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the functional variants are identical; the large protein (L) contains amino acids as shown in SEQ ID NO:9 or functional variants identical to SEQ ID NO:9 at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the functional variants; and the matrix protein (M) contains amino acids as shown in SEQ ID NO:10 or functional variants identical to SEQ ID NO:9 at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the functional variants; and the matrix protein (M) contains amino acids as shown in SEQ ID NO:10 or functional variants identical to SEQ ID NO:9 at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 99% of the functional variants. NO:10 Functional variants with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% consistent accuracy.

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

[0026] In a fourth aspect, the present invention provides a pharmaceutical composition of a recombinant rhabdovirus according to any of the first aspect or embodiments thereof, or a recombinant vesicular stomatitis virus according to any of the second aspect or embodiments thereof, or a pharmaceutical composition according to any of the third aspect or embodiments thereof, which is suitable as a pharmaceutical agent.

[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 treating cancer, preferably solid cancer. In a preferred embodiment, the solid cancer is selected from the list comprising: reproductive tumors, ovarian tumors, pancreatic tumors, testicular tumors, endocrine tumors, gastrointestinal tumors, liver tumors, kidney tumors, colon tumors, colorectal tumors, bladder tumors, prostate tumors, skin tumors, melanoma, respiratory tumors, lung tumors, breast tumors, head and neck tumors, head and neck squamous cell carcinoma (HNSCC), and bone tumors.

[0028] In one embodiment of the fourth aspect, the recombinant rhabdovirus, the recombinant vesicular stomatitis virus, or the pharmaceutical composition is administered intratumorally or intravenously. In another related embodiment, the recombinant rhabdovirus, the recombinant vesicular stomatitis virus, or the pharmaceutical composition is administered intratumorally at least once and subsequently intravenously. In another related embodiment, subsequent intravenous administrations of the recombinant rhabdovirus, the recombinant vesicular stomatitis virus, or the pharmaceutical composition are given on days 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, or 31 after the initial intratumoral administration.

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

[0030] In one embodiment of the fifth aspect, the PD-1 pathway inhibitor is an antagonistic antibody targeting PD-1 or PD-L1. In another related embodiment, the SMAC mimic is selected from the group consisting of any one of compounds 1 to 26 from Table 2 or a pharmaceutically acceptable salt of one of these compounds. In yet another related embodiment, the PD-1 pathway inhibitor is selected from antagonists of 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). In the preferred embodiment, the PD-1 pathway inhibitor is BI-754091.

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

[0032] In a seventh aspect, the present invention provides a combination therapy comprising: a) a recombinant rhabdovirus according to any of the first aspect or embodiments thereof, or a recombinant vesicular stomatitis virus according to any of the second aspect or embodiments thereof, or a pharmaceutical composition according to any of the third aspect or embodiments thereof, and b) a PD-1 pathway inhibitor or SMAC mimic. In one embodiment of the seventh aspect, a) and b) may be administered simultaneously, sequentially, or alternately. In related embodiments, a) and b) are administered via different routes of administration. In another 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 antagonistic antibody targeting PD-1 or PD-L1. In a related embodiment, the PD-1 pathway inhibitor is selected from the group consisting of: pembrolizumab, nivolumab, pirikizumab, atezolizumab, avelumab, duvalumab, PDR-001, PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5 (see Table 1). In another related embodiment, the SMAC mimicry 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 any one of the first aspects or embodiments thereof, or a recombinant vesicular stomatitis virus according to any one of the second aspects or embodiments thereof.

[0035] In one implementation of the eighth aspect, the virus-producing cells are Vero cells, HEK cells, HEK293 cells, Chinese hamster ovary cells (CHO) or young hamster kidney (BHK) cells.

[0036] In a ninth aspect, the present invention provides a method for producing recombinant rhabdovirus in cell cultures:

[0037] (i) Infecting host cells with recombinant rhabdovirus, preferably vesicular stomatitis virus.

[0038] (ii) Culture the host cell under conditions that allow the recombinant rhabdovirus to replicate.

[0039] (iii) Harvest the recombinant rhabdovirus from the cell culture.

[0040] (iv) Optionally, the virus harvested material may be subjected to enzymatic treatment, preferably with a totipotent nuclease (benzonase).

[0041] (v) The rhabdovirus harvest is captured by loading an adsorbent or resin onto a cation exchange monolayer membrane followed by elution.

[0042] (vi) Polishing the rhabdovirus by particle size analysis, multimodal particle size analysis / ion exchange, or tangential flow filtration of the eluent from step (v).

[0043] (vii) Buffer replacement of the conditioned rhabdovirus by ultrafiltration / permeation

[0044] (viii) Perform sterile filtration of rhabdoviruses.

[0045] In one implementation of the ninth aspect, the host cell is a HEK293 cell.

[0046] In one implementation of the ninth aspect, the host cell is cultured in a suspension.

[0047] In one embodiment of the ninth aspect, the recombinant rhabdovirus is formulated into the 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 into the pharmaceutical composition. Attached Figure Description

[0048] Figure 1: Schematic illustration of the components of the immune synapse between antigen-presenting cells (APCs) and T cells. CD80 is a key co-stimulatory molecule during T cell activation. Efficient T cell stimulation requires two polymeric molecular signals within the immune synapse: Signal 1: antigen specificity (TCR:MHC / peptide) and Signal 2: antigen independence (CD28:CD80).

[0049] Figure 2: Draft of the CD80 extracellular domain (ECD) Fc fusion protein (divalent). The fusion protein is expressed in transduced tumor cells after viral infection. Two CD80 extracellular domain Fc fusion monomers are covalently linked together by disulfide bonds formed between cysteine ​​residues in each monomer, thereby forming a CD80 extracellular domain Fc fusion protein dimer.

[0050] Figure 3A-B compares the replication (A) and survival (B) of VSV-GP-CD80-Fc relative to its parental virus VSV-GP. HEK293F cells were infected with either VSV-GP or VSV-GP-CD80-Fc. The Y-axis shows cell survival or genome copies / ml as a percentage. Cell survival and replication were monitored up to 48 h post-infection (x-axis: hours post-infection).

[0051] Figure 4 shows the detection of soluble CD80-Fc by ELISA in the supernatant of tissue culture from HEK293 cells infected with VSV-GP or VSV-GP-CD80-Fc (each with an infection fold of 1 (MOI 1)). CD80-Fc expression was measured at different time points (x-axis) up to 48 hours after infection (y-axis in μg / ml).

[0052] Figure 5A-B compares the in vivo efficacy (A) and weight gain (B) of mice treated with VSV-GP-CD80-Fc in a CT26.CL25-IFNARKO tumor model (intravenous) with that of the parental virus VSV-GP. (A) Mice were treated with a low viral dose of 2x10 on days 0 and 3. 7 TCID 50 Treatment. The X-axis shows time (in days) and the y-axis shows the percentage of surviving mice. (B) Shows the weight development of the same treated mice as in Figure 6A. The X-axis shows time (in days) and the y-axis shows weight (in g).

[0053] Figure 6A-C shows the use of a certain viral dose on days 0 and 3. 8 TCID 50 In B16-F1-OVA tumor models (intramural) treated with VSV-GP (B), the same viral dose of VSV-GP-CD80-Fc (C), or PBS (A), the in vivo efficacy of VSV-GP-CD80-Fc was compared with that of the parental virus VSV-GP in treated mice. The X-axis shows the number of days after treatment (in days), and the Y-axis shows the tumor volume (in cm). 3 (Unit: )

[0054] Figures 7A-B show the use of the EMT-6 tumor model (intracavitary) and low viral doses of 2x10 on days 0 and 3. 7 TCID 50 The in vivo efficacy of VSV-GP-CD80-Fc in treated mice (B) was compared with that of the parental virus VSV-GP (A). The X-axis shows the number of days after treatment (in days), and the Y-axis shows the tumor volume (in cm). 3(Unit: ). Dashed lines represent mice treated with a catalyst, and solid lines represent mice treated with a virus.

[0055] Figure 8. VSV-GP-CD80-Fc replication in infected CT26.CL25-IFNARKO tumors of treated mice, as determined by viral genome replication quantification using qPCR on days 3 and 7 post-infection. Mice were treated with PBS or 10 8 TCID 50 VSV-GP-CD80-Fc treatment and determination of viral genome copy / tumor (y-axis) after 3 or 7 days.

[0056] Figure 9. Nanostring-based measurements of VSV-GP N-protein and CD80-Fc transcripts in LLC-IFNARKO tumors infected with VSV-GP or VSV-GP-CD80-Fc from control mice, or mice obtained from treated mice. Mice were used as controls or 10 8 TCID 50 VSV-GP or VSV-GP-CD80-Fc treatment was followed by determination of the relative expression (y-axis) of viral N-protein or CD80-Fc 3 days later.

[0057] Figure 10 shows IHC-based detection of VSV-GP N-protein and CD80-Fc load (protein) in LLC-IFNARKO tumors infected with VSV-GP or VSV-GP-CD80-Fc from control mice, and mice treated with the modified VSV-GP or VSV-GP-CD80-Fc. Mice were treated as shown in Figure 9, and IHC was performed using a standard protocol on day 3 post-treatment.

[0058] Figures 11A-C depict a sketch of the mechanism of action (MoA) of CD80-Fc in tumors. A hot tumor (A) with mature, activated dendritic cells (DCs) provides highly effective T-cell co-stimulation. A cold tumor (B) lacking DCs and / or controlled by an immature, tolerant DC subgroup. The absence of DCs or the presence of an immature, tolerant DC subgroup leads to poor T-cell immunity, clonal unresponsiveness, T-cell dysfunction, and cell death. CD80-Fc transforms cold tumors into hot tumors by compensating for the lack of potent T-cell co-stimulation (C).

[0059] Figure 12. Human mixed leukocyte cultures (co-culture of T cells and immature dendritic cells from two genetically distinct individuals, resulting in allogeneic T cell stimulation) were used to assess T cell co-stimulation via recombinant CD80-Fc. For this purpose, cultures were stimulated with increased amounts of recombinant CD80-Fc protein, and IFNγ secretion was used as a readout.

[0060] Figures 13A-D show human mixed leukocyte cultures (co-culture of T cells and monocytes from two genetically different individuals) stimulated with recombinant CD80-Fc protein (10 μg / ml) with or without FcγR-blockers (and in the absence of human serum), using IFNγ secretion as a reading. Different subplots (AD) depict different donor pairs.

[0061] Figures 14A-F show human PBMC cultures stimulated with or without low doses of anti-CD3 and increased concentrations of recombinant CD80-Fc protein (F(ab)2(A,D), Fc=IgG4(B,E), or Fc=IgG1(C,F)), with IFNγ(AC) or IL2(DF) secretion measured as readings, which were detected by standard ELISA on the supernatant.

[0062] Figure 15. Nanostring-based measurements of FcγR in control or VSV-GP-infected LLC1-IFNARKO tumors on day 7 post-infection. Mice were either untreated or treated with a specific viral dose of 10 8 TCID 50 VSV-GP infection. The X-axis shows measurements of different FcγR (1, 2b, 3, or 4) and the Y-axis shows relative expression after 7 days.

[0063] Figures 16A-C show the modified induction of tumor-specific T cells via VSV-GP-mCD80-Fc relative to VSV-GP in CT26.CL25-IFNARKO tumor-carrying mice using ELISPOT and tetramer staining. gp70-specific α-tumor T cells were increased in the spleen of VSV-GP-mCD80-Fc-treated mice compared to VSV-GP-treated mice. Hollow symbols indicate intravenous treatment. Solid symbols indicate intravenous / intracavitary treatment. (A) gp70-specific T cells detected from the spleen by ELISPOT or (B) from blood by Dextramer. (C) Experimental overview.

[0064] Figure 17. Luminescence-based readings: co-culture of Jurkat PD-1 reporter cells and CHO-K1-α-CD3 / -PDL1. CD80-Fc was unable to prevent PD1:PDL1-mediated inhibition of the Jurkat reporter cell line compared to the anti-PDL1 antibody avirumab.

[0065] Figure 18A-B Reporter cell analysis: Jurkat-PD1 (luciferase reporter cells) were co-cultured with THP-1-PDL1 cells (expressing FcgR). T cell activation was triggered by CD33XCD3BiTE (10 nM) in this system. (A) Treatment with specified concentrations of specified reagents (anti-PD1 = pembrolizumab; anti-Dig = isotype control and recombinant CD80-Fc). (B) Anti-PD1 (10 nM) and increased concentrations of recombinant CD80-Fc improved Jurkat T cell activation beyond the activity of individual compounds.

[0066] Figures 19A-C show the in vivo efficacy (A), tumor growth curve (B), and body weight changes (C) of mice treated with VSV-GP-muCD80-Fc, parental VSV-GP, and recombinant murine CD80-Fc in a CT26.CL25-IFNARKO tumor model (intravenous). (A) Mice were administered a certain viral dose of 1x10 on day 0 and day 3, respectively. 8 TCID 50 Mice were treated with 1 mg / kg recombinant mouse CD80-Fc on days 0, 3, and 6. The X-axis shows time (in days) and the Y-axis shows the percentage of surviving mice. (B) Shows the mean tumor volume over time in the same treated mice as in Figure 19A. The X-axis shows time (in days after treatment began) and the Y-axis shows tumor volume (in mm). 3 (in units). Data show the group average, with the last observation carried over until 70% group size (70% LOCF). (C) shows the same weight changes of treated mice as in Figure 19A. The x-axis shows time (in days after treatment began) and the y-axis shows the weight change (in percentage) compared to the initial weight at the start of treatment. Detailed Implementation

[0067] In the following detailed description, numerous specific details are set forth to provide a full understanding of the invention. However, it will be apparent to those skilled in the art that the invention can 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 invention. Titles are included for convenience only and should not be construed as limiting the invention to certain aspects or embodiments.

[0068] Rhabdomyovirus

[0069] The rhabdovirus family comprises 18 genera and 134 species, with an antisense single-stranded RNA genome of approximately 10–16 kb (Walke et al., ICTV Virus Taxonomy Profile: Rhabdoviridae, Journal of General Virology, 99:447–448 (2018)).

[0070] Characteristic features of members of the rhabdovirus family include one or more of the following: bullet-shaped or rod-shaped particles, 100-430 nm in length and 45-100 nm in diameter, comprising a helical nucleocapsid surrounded by a matrix layer and a lipid envelope; some rhabdoviruses are non-enveloped filamentous viruses. A predominantly unsegmented 10.8-16.1 kb antisense single-stranded RNA. A genome encoding at least five genes encoding structural proteins: nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M), and glycoprotein (G).

[0071] As used in this article, rhabdoviruses may belong to the following genera: almendravirus, curiovirus, cytorhabdovirus, dichorhavirus, ephemerovirus, Hapavirus, ledantevirus, lyssavirus, novirhabdovirus, nucleorhabdovirus, perhabdovirus, sigmavirus, sprivivirus, sripuvirus, tibrovirus, tupavirus, varicosavirus, or vesicular virus.

[0072] Within the genera mentioned in this article, rhabdoviruses can belong to any of the listed species. The genus *Arboretum almendravirus* includes: *Balsa almendravirus*, *Coot Bay almendravirus*, *Puerto Almendras almendravirus*, and *Rio Chicoalmendravirus*. The genus *Curiovirus* includes: *Curiopolis curiovirus*, *Iriri curiovirus*, *Itacaiunas curiovirus*, and *Rochambeau curiovirus*. The genus *Cytoplasmic rhabdovirus* includes: *Alfalfa dwarf cytorhabdovirus* and *Barley yellow striate mosaic*. Cytorhabdovirus, Broccoli necrotic yellows cytorhabdovirus, Colocasia bobone disease-associated cytorhabdovirus, Festuca leaf streak cytorhabdovirus, Lettuce necrotic yellows cytorhabdovirus, Lettuce yellow mottle cytorhabdovirus, Northern cerealmosaic cytorhabdovirus, Sonchus cytorhabdovirus 1, Strawberry crinkle cytorhabdovirus, Wheat American striate mosaic cytorhabdovirus cytorhabdovirus);The genus *Dichorhavirus* includes: Coffee ringspot dichorhavirus and Orchid fleckdichorhavirus; the genus *Ephemerovirus* includes: Adelaide River ephemerovirus, Berrimah ephemerovirus, Bovinefever ephemerovirus, Kimberley ephemerovirus, Koolpinyah ephemerovirus, Kotonkan ephemerovirus, Obodhiang ephemerovirus, and Yataephemerovirus; the genus *Happavirus* includes: Flanders hapavirus, Gray Lodge hapavirus, and Hart Park hapavirus. The following viruses are listed: hapavirus, Joinjakaka hapavirus, Kamese hapavirus, La Joya hapavirus, Landjia hapavirus, Manitoba hapavirus, Marco hapavirus, Mosqueirohapavirus, Mossuril hapavirus, Ngainganhapavirus, Ord River hapavirus, Parry Creekhapavirus, and Wongabel hapavirus.The genus *Leadantevirus* includes: Barur ledantevirus, Fikirini ledantevirus, Fukuoka ledantevirus, Kanyawara ledantevirus, Kern Canyon ledantevirus, Keuralibaledantevirus, Kolente ledantevirus, Kumasiledantevirus, Le Dantec ledantevirus, Mount Elgon bat ledantevirus, Nishimuro ledantevirus, Nkolbisson ledantevirus, Oitaledantevirus, Wuhan ledantevirus, and Yongjia ledantevirus.The genus *Lyssavirus* includes: Aravan lyssavirus, Australian bat lyssavirus, Bokeloh bat lyssavirus, Duvenhage lyssavirus, European bat 1 lyssavirus, European bat 2 lyssavirus, Gannoruwa bat lyssavirus, Ikoma lyssavirus, Irkut lyssavirus, Khujand lyssavirus, Lagos bat lyssavirus, Lleida bat lyssavirus, Mokola lyssavirus, and Rabies lyssavirus. Lyssavirus), Shimoni bat lyssavirus, West Caucasian bat lyssavirus; Noravirus genus includes: Hirame novirhabdovirus, Piscine novirhabdovirus, Salmonid novirhabdovirus, Snakehead novirhabdovirus;The genus *Nucleorhabdovirus* includes: *Datura yellow vein nucleorhabdovirus*, *Eggplant mottled dwarf nucleorhabdovirus*, *Maize fine streak nucleorhabdovirus*, *Maize Iranian mosaic nucleorhabdovirus*, *Maize mosaic nucleorhabdovirus*, *Potato yellow dwarf nucleorhabdovirus*, *Rice yellow stunt nucleorhabdovirus*, *Sonchus yellownet nucleorhabdovirus*, *Sowthistle yellow vein nucleorhabdovirus*, and *Taro vein nucleorhabdovirus*. The genus *Chloris nucleorhabdovirus* includes: *Anguillidperhabdovirus*, *Perch perhabdovirus*, and *Sea troutperhabdovirus*. The genus *Sigmavirus* includes: *Drosophila affinissigmavirus*, *Drosophila ananassae sigmavirus*, *Drosophila immigrans sigmavirus*, *Drosophila melanogaster sigmavirus*, *Drosophila obscura sigmavirus*, *Drosophila tristis sigmavirus*, and *Muscinastabulans*. sigmavirus); the genus Sprivivirus includes: Carp sprivivirus, Pike fry sprivivirus;The genus *Sripuvirus* includes: Almpiwar sripuvirus, Chaco sripuvirus, Niakha sripuvirus, Sena Madureirasripuvirus, and Sripur sripuvirus. The genus *Tibrovirus* includes: Bas-Congo tibrovirus, Beatrice Hilltibrovirus, Coastal Plains tibrovirus, Ekpoma 1 tibrovirus, Ekpoma 2 tibrovirus, and Sweetwater Branch tibrovirus. Tibrovirus), Tibrogargantibrovirus; Tupavirus genus includes: Durham tupavirus, Klamath tupavirus, Tupaia tupavirus; Megavein virus genus includes: Lettuce big-vein associated varicosavirus;The genus *Vecilobovirus* includes: Alagoas vesiculovirus, American bat vesiculovirus, Carajas vesiculovirus, Chandipura vesiculovirus, Cocal vesiculovirus, Indiana vesiculovirus, Isfahan vesiculovirus, Jurona vesiculovirus, Malpais Spring vesiculovirus, Maraba vesiculovirus, Morreton vesiculovirus, New Jersey vesiculovirus, Perinet vesiculovirus, and Piry vesiculovirus. vesiculovirus, radivesiculovirus, Yug Bogdanovac vesiculovirus, or Moussa virus.

[0073] Preferably, the recombinant rhabdovirus of the present invention is an oncolytic rhabdovirus. In this context, oncolysis has its conventional meaning as known in the art and refers to a rhabdovirus capable of infecting and lysing (degrading) cancer cells but not normal cells (to any significant extent). Preferably, the oncolytic rhabdovirus is capable of replicating within cancer cells. Oncolytic activity can be tested in various analytical systems known to those skilled in the art (exemplary in vitro analyses are described by Muik et al., Cancer Res., 74(13), 3567-78, 2014). It should be understood that the oncolytic rhabdovirus may only infect and lyse specific types of cancer cells. Furthermore, the oncolytic effect can vary depending on the type of cancer cell.

[0074] In a preferred embodiment, the rhabdovirus belongs to the genus *Vesculovirus*. *Vesculovirus* species have been defined primarily through serological methods combined with genomic phylogenetic analysis. Biological characteristics such as host range and transmission mechanisms are also used to distinguish viral species within the genus. Therefore, the genus *Vesculovirus* forms a unique monophyletic group fully supported by a maximum likelihood tree inferred from complete L sequences.

[0075] Viruses belonging to different species within the genus *Vesicella* may possess 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 by serological tests; and E) occupy different ecological niches, as confirmed by differences in hosts and / or arthropod vectors.

[0076] The preferred formulation is vesicular stomatitis virus (VSV), and more particularly VSV-GP (a recombinant of GP with LCMV). The advantageous properties of VSV-GP include one or more of the following: extremely potent and rapid killer (<8h); oncolytic virus; systemic administration capability; reduced neurotropism / neurotoxicity; lytic replication and induction of immunogenic cell death; attributed to interferon (IFN) response without replication in healthy human cells; strong activation of innate immunity; approximately 3kb of space for immunomodulatory payload and antigens; recombination with isopyrviral glycoproteins from lymphocytic choriomeningovirus (LCMV); advantageous safety profile of reduced neurotoxicity and lower sensitivity to neutralizing antibody response and complement destruction than wild-type VSV (VSV-G); specific replication in tumor cells that can no longer establish and respond to antiviral innate immune responses (e.g., type I IFN signaling); inability to replicate in “healthy cells” for rapid elimination from normal tissues; viral replication in tumor cells that can induce immunogenic cell death, release tumor-associated antigens, cause local inflammation, and induce antitumor immunity.

[0077] The present invention is further embodied by recombinant vesicular stomatitis virus, which encodes in its genome at least vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M), glycoprotein (G) and at least one CD80 extracellular domain Fc fusion protein or a functional variant thereof, preferably including the human CD80 extracellular domain.

[0078] In a preferred embodiment, the recombinant vesicular stomatitis virus encodes at least the following vesicular stomatitis virus proteins in its genome: a nucleoprotein (N) comprising the amino acid sequence shown in SEQ ID NO:7 or a functional variant consistent with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:7; and a phosphoprotein (P) comprising the amino acid sequence shown in SEQ ID NO:8 or a functional variant consistent with SEQ ID NO:7. NO:8 At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the functional variants are identical to SEQ ID NO:9; a large protein (L) comprising the amino acid sequence shown in SEQ ID NO:9 or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the functional variants identical to SEQ ID NO:9; and a matrix protein (M) comprising the amino acid sequence shown in SEQ ID NO:10 or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the functional variants identical to SEQ ID NO:9; NO:10 is a functional variant with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% consistent percentages.

[0079] Those skilled in the art will understand that the sequences of the vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M), or glycoprotein (G) can be modified without losing the essential functions of those proteins. Such functional variants, as used herein, retain all or part of their essential functions or activities. For example, protein L is a polymerase and has essential functions during viral transcription and replication. Its functional variants must retain at least some of this ability. A good indication of retention of essential functions or activities is the successful generation of viruses (including these functional variants) that are still able to replicate and infect tumor cells. Virus generation and infection and replication assays in tumor cells can be tested in various analytical systems known to those skilled in the art (illustrative in vitro analyses are described by Muik et al., Cancer Res., 74(13), 3567-78, 2014).

[0080] 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 CD80 extracellular domain Fc fusion protein or a functional variant thereof, wherein the large protein (L) comprises an amino acid sequence having ≥80% sequence identity with SEQ ID NO:9.

[0081] In a preferred embodiment, the recombinant vesicular stomatitis virus encodes in its genome at least a vesicular stomatitis virus nucleoprotein (N), a large protein (L), a phosphoprotein (P), a matrix protein (M), a glycoprotein (G), and at least one CD80 extracellular domain Fc fusion protein or a functional variant thereof, wherein the nucleoprotein (N) comprises an amino acid sequence having ≥90% sequence identity with SEQ ID NO:7.

[0082] In another preferred embodiment, the recombinant vesicular stomatitis virus encodes in its genome at least a vesicular stomatitis virus nucleoprotein (N), a large protein (L), a phosphoprotein (P), a matrix protein (M), a glycoprotein (G), and at least one CD80 extracellular domain Fc fusion protein or a functional variant thereof, wherein the large protein (L) comprises an amino acid sequence having 80% or more sequence identity as SEQ ID NO:9 and the nucleoprotein (N) comprises an amino acid sequence having ≥90% sequence identity as SEQ ID NO:7.

[0083] In a preferred embodiment of the invention, the RNA genome of the recombinant rhabdovirus of the present invention comprises or is composed of the sequence shown in SEQ ID NO:24. Alternatively, the RNA genome of the recombinant rhabdovirus of the present invention may also consist of or comprise those sequences in which the nucleic acids of the RNA genome undergo exchange according to genetic code degeneracy (this does not alter the individual amino acid sequences). In another preferred embodiment, the RNA genome of the recombinant rhabdovirus of the present invention comprises or is composed of a coding sequence that is 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 or composed of the coding sequence shown in SEQ ID NO:24.

[0084] It should be understood that the recombinant rhabdovirus of the present invention may encode other payloads in its genome, such as tumor antigens, other chemokines, cytokines or other immune regulatory components.

[0085] In another embodiment, the recombinant rhabdovirus of the present invention further encodes a sodium iodide syn-transporter protein (NIS) in its genome. NIS is expressed and, for example, [the protein is then used in conjunction with other proteins].125 I. Together, we can cultivate a method that allows the use of NIS as an imaging reporter (Carlson et al., Current Gene Therapy, 12, 33-47, 2012).

[0086] Recombinant rhabdovirus

[0087] Certain wild-type rhabdovirus strains, such as wild-type VSV strains, are known to be neurotoxic. It has also been reported that infected individuals can rapidly establish a strong humoral response with high antibody titers primarily targeting glycoproteins. Neutralizing antibodies targeting glycoprotein G of rhabdoviruses in general and specifically VSV can limit viral spread and thereby mediate protection against reinfection. However, viral neutralization limits the repeated administration of rhabdoviruses to cancer patients.

[0088] To eliminate these drawbacks, wild-type glycoprotein G of rhabdovirus can be replaced by a glycoprotein from another virus. In this regard, glycoprotein replacement means (i) replacing the gene encoding wild-type glycoprotein G with the gene encoding glycoprotein GP of another virus, and / or (ii) replacing wild-type glycoprotein G with glycoprotein GP of another virus.

[0089] In a preferred embodiment, the rhabdovirus glycoprotein G is replaced by the glycoprotein GP of lymphocytic choriomeningovirus (LCMV), preferably by the WE-HPI strain. In an even more preferred embodiment, the rhabdovirus is a vesicular stomatitis virus having the glycoprotein GP of lymphocytic choriomeningovirus (LCMV), preferably having the WE-HPI strain. Such VSVs are described, for example, in WO2010 / 040526 and named VSV-GP. The advantages provided are (i) removal of VSV-G-mediated neurotoxicity and (ii) no antibody-neutralizing vector (as shown in mice).

[0090] The glycoprotein GP of lymphocytic choriomeningitis virus (LCMV) can be GP1 or GP2. This invention includes glycoproteins from different LCMV strains. In particular, LCMV-GP can be derived from wild-type LCMV or LCMV strains LCMV-WE, LCMV-WE-HPI, and LCMV-WE-HP1 opt. In a preferred embodiment, the gene encoding the LCMV glycoprotein GP encodes a protein having the amino acid sequence shown in SEQ ID NO:11 or having an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to the amino acid sequence of SEQ ID NO:11, while the functional characteristics of the recombinant rhabdovirus containing the glycoprotein GP encoding the amino acid sequence shown in SEQ ID NO:11 are maintained.

[0091] In another embodiment, the recombinant rhabdovirus glycoprotein G is replaced with the glycoprotein GP of Dandenong virus (DANDV) or Mopeya virus (MOPV). In a more preferred embodiment, the recombinant rhabdovirus is a vesicular stomatitis virus, wherein the glycoprotein G is replaced with the glycoprotein GP of Dandenong virus (DANDV) or Mopeya virus (MOPV). The advantages provided are (i) removal of VSV-G-mediated neurotoxicity and (ii) no vector antibody neutralization (as shown in mice).

[0092] DANDV is an old-world arenavirus. To date, only a single strain known to those skilled in the art exists that contains the glycoprotein GP and can be used as a donor for the glycoprotein GP contained in the recombinant rhabdovirus of this invention. The DANDV glycoprotein GP contained in the recombinant rhabdovirus of this invention has more than six glycosylation sites, particularly seven. The glycoprotein GP is exemplarily preferred to be that contained in DANDV, as is available under Genbank number EU136038. In one embodiment, the gene encoding the DNADV glycoprotein GP encodes an amino acid sequence as shown in SEQ ID NO:12 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO:12, thereby maintaining the functional characteristics of the recombinant rhabdovirus containing the glycoprotein GP encoding the amino acid sequence as shown in SEQ ID NO:12.

[0093] Mopea virus (MOPV) is an Old World arenavirus. Several strains known to those skilled in the art exist that contain the glycoprotein GP and can be used as donors of the glycoprotein GP contained in the recombinant rhabdovirus of this invention. The MOPV glycoprotein GP contained in the recombinant rhabdovirus of this invention has more than 6 glycosylation sites, particularly 7 glycosylation sites. The glycoprotein GP exemplarily preferred is that contained in Mopea virus, as obtained under Genbank number AY772170. In one embodiment, the gene encoding the glycoprotein GP of MOPV encodes an amino acid sequence as shown in SEQ ID NO:13 or a sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO:13, and the functional characteristics of the recombinant rhabdovirus containing the glycoprotein GP encoding the amino acid sequence shown in SEQ ID NO:13 are maintained.

[0094] CD80 extracellular domain Fc fusion protein

[0095] CD80, also known as B7-1, is a 60kD single-chain type I glycoprotein belonging to the immunoglobulin superfamily. CD80 is expressed on activated / mature antigen-presenting cells, such as dendritic cells. CD80 binds to CD28 and CD152 (CTLA-4). Together with CD86 (B7-2), CD80 plays a crucial role in regulating T cell activation. The interaction between CD80 and CD28 provides a co-stimulatory signal for T cell activation in the TCR conjugation complex. Its interaction with CTLA-4 (e.g., expressed on regulatory T cells), which has a higher affinity for CD80 than CD28 and acts as a decoy receptor for CD80 rather than an inhibitory signaling receptor, deprives T cells of the crucial co-stimulatory CD28 signal.

[0096] In the past, it has been proposed that oncolytic viruses, particularly VSV-GP, can induce tumor cell lysis and immunogenic cell death and stimulate innate immune cells in the tumor microenvironment. Further proposals have suggested that immunomodulatory proteins be encoded into the genome of the oncolytic virus, and that the expression of these immunomodulatory proteins can support the oncolytic effect of the virus through local immune stimulation.

[0097] One challenge in expressing immunostimulatory molecules (such as chemokines and / or cytokines) from the viral backbone is that not only must enhanced antitumor immunity be achieved, but antiviral immune responses induced by these immunostimulatory molecules must also be avoided. It is important to note that additional immunostimulatory molecules should not limit the oncolytic potential of the virus to the extent that the potential benefits gained from the expression of the therapeutic payload are outweighed by a loss of oncolytic efficacy.

[0098] The inventors hypothesize that the CD80 extracellular domain Fc fusion protein can provide effective T cell costimulation upon T cell receptor binding, while simultaneously preventing the activation of natural killer cells (activated by, for example, IL2, IL15, and CD137), thus limiting viral replication and / or persistence in the early stages. The CD80 extracellular domain Fc fusion protein is a potent costimulatory molecule active in the initiation and reactivation of antigen-specific T cells. This stimulation is crucial because T cell costimulatory signals are typically underexpressed in tumors, leading to clonal T cell unresponsiveness, loss of effector function, and T cell death.

[0099] By providing the recombinant rhabdovirus according to the invention, tumor-restricted replication of the CD80 extracellular domain Fc fusion protein can induce local expression of a T cell co-stimulatory fusion protein, which further enhances anti-tumor T cell immunity by providing activation signals to T cells in an FcγR-dependent manner in the case of T cell receptor binding (e.g., tumor cell recognition).

[0100] The inventors unexpectedly discovered that the recombinant rhabdovirus according to the invention, encoding a CD80 extracellular domain Fc fusion protein, can induce tumor cell lysis and immunogenic cell death and stimulate innate immune cells in the tumor microenvironment. Furthermore, prolonged survival was observed in established mouse tumor models treated with such recombinant rhabdoviruses armed with CD80 extracellular domain Fc fusion proteins.

[0101] Unexpectedly, infection with the recombinant rhabdovirus according to the invention encoding the CD80 extracellular domain Fc fusion protein resulted in a strong increase in FcγR expression within the infected tumor. The inventors demonstrated that the optimal biological activity of the CD80 extracellular domain Fc fusion protein is largely dependent on FcγR.

[0102] Without being bound by theory, it is believed that the potent antitumor effect obtained by the recombinant rhabdovirus according to the invention, which encodes the CD80 extracellular domain Fc fusion protein, is at least in part based on the FcγR-dependent activity of the CD80 extracellular domain Fc fusion protein, which is enhanced by the increased expression of FcγR in infected tumors after infection with the recombinant rhabdovirus according to the invention.

[0103] Alternatively, in this case, it has also been considered to provide a CD86(B7-2) fusion protein, namely a recombinant rhabdovirus encoding a CD86 extracellular domain Fc fusion protein, and in particular a VSV-GP encoding a CD86 extracellular domain Fc fusion protein, wherein the gene encoding glycoprotein G of recombinant vesicular stomatitis virus is replaced by the gene encoding glycoprotein GP of lymphocyte choriomeningovirus (LCMV), and / or glycoprotein G is replaced by glycoprotein GP of LCMV.

[0104] As used in this article, "CD80 extracellular domain Fc fusion protein" refers to a fusion protein or a functional variant thereof that contains or is composed of a CD80 extracellular domain fused to the Fc domain of IgG.

[0105] The “CD80 extracellular domain” includes naturally occurring polypeptides such as different isoforms and their functional variants or components thereof, preferably the human CD80 extracellular domain.

[0106] In one aspect, recombinant rhabdoviruses encoding at least one CD80 extracellular domain Fc fusion protein or a functional variant thereof in their genome can enhance the recruitment of T cells and dendritic cells to the tumor environment.

[0107] On the other hand, the genomic expression of at least one CD80 extracellular domain Fc fusion protein or a functional variant thereof by a recombinant rhabdovirus provides a therapeutic option for patients with cold tumors and low mutational burden to enhance T cell-mediated anti-tumor T cell responses against poorly immunogenic tumors.

[0108] In another respect, the genome of a self-recombinant rhabdovirus expressing at least one CD80 extracellular domain Fc fusion protein or a functional variant thereof does not induce additional natural killer cells (exceeding the effect of the parental VSV-GP virus) and selectively activates antigen-specific T cells.

[0109] In another instance, the expression of at least one CD80 extracellular domain Fc fusion protein or a functional variant thereof by the genome of a self-recombinant rhabdovirus did not induce super-efficacy.

[0110] On the other hand, the expression of at least one CD80 extracellular domain Fc fusion protein or a functional variant thereof in the genome of a recombinant rhabdovirus does not increase early antiviral immunity against the recombinant rhabdovirus.

[0111] On the other hand, in addition to being locally expressed in tumors due to its solubility, the CD80 extracellular domain Fc fusion protein can also reach tumor-draining lymphatic vessels (e.g., lymph nodes).

[0112] Human CD80 protein (UniProtKB-P33681|CD80_human T lymphocyte activation antigen CD80) contains a total of 288 amino acids or is composed of a total of 288 amino acids and contains a signal peptide, an extracellular domain, and a transmembrane / topological domain:

[0113]

[0114] In one embodiment, the CD80 extracellular domain of the CD80 extracellular domain Fc fusion protein comprises or is composed of amino acids 1-242 of SEQ ID NO:6, 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%, or 99% identity with amino acids 1-242 of SEQ ID NO:6.

[0115] In another embodiment, the CD80 extracellular domain of the CD80 extracellular domain Fc fusion protein comprises or is composed of the following sequences:

[0116]

[0117] Or a sequence that is 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%, or 99% identical to SEQ ID NO:1.

[0118] In one embodiment, the CD80 extracellular domain Fc fusion protein includes a signal peptide sequence. In another embodiment, the CD80 extracellular domain Fc fusion does not include a signal peptide sequence.

[0119] The term "signal peptide" or "signal peptide sequence" describes a peptide sequence typically 10 to 30 amino acids in length located at the N-terminus of a newly synthesized secretory or membrane polypeptide, which guides the polypeptide across or into the cell membrane (plasma membrane in prokaryotes and endoplasmic reticulum membrane in eukaryotes). It is usually subsequently removed. In particular, signal peptides can be able to guide polypeptides into the cell's secretory pathway.

[0120] It should be understood that, for the purposes of this invention, other (i.e., other than wild-type) signal peptide sequences may be used in conjunction with CD80 extracellular domain Fc fusion proteins. Such other signal peptide sequences may replace the original wild-type signal peptide sequence. Signal peptides include peptides that guide newly synthesized proteins in the ribosome to the ER and further to the Golgi complex for delivery to the plasma membrane or out of the cell. They generally comprise a hydrophobic amino acid string and include immunoglobulin leader sequences and other sequences known to those skilled in the art. Signal peptides particularly include peptides capable of being acted upon by signal peptidases, which are specific proteases located on the cisternae of the endoplasmic reticulum. Signal peptides are well known to those skilled in the art and may include any known signal peptide. The signal peptide is incorporated at the N-terminus of the protein and processed by a signal peptidase into the CD80 extracellular domain Fc fusion protein to produce an active biological form.

[0121] In one embodiment, the CD80 extracellular domain Fc fusion protein comprises a wild-type CD80 signal peptide sequence. In a preferred embodiment, the CD80 extracellular domain Fc fusion protein comprises a wild-type human CD80 signal peptide sequence, which is amino acids 1-34 of SEQ ID NO:6 or a sequence 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%, or 99% identity with amino acids 1-34 of SEQ ID NO:6.

[0122] In another embodiment, the CD80 extracellular domain Fc fusion protein comprises a signal peptide sequence having the following sequence:

[0123] MGWSCIILFLVATATGVHS(SEQ ID NO:5)

[0124] Or a signal peptide sequence that is 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%, or 99% identical to SEQ ID NO:5.

[0125] In relevant embodiments, the CD80 extracellular domain of the CD80 extracellular domain Fc fusion protein comprises or is composed of a sequence identical to 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%, or 99% of SEQ ID NO:1, and further comprises a signal peptide sequence according to or identical to SEQ ID NO:1. NO:5 has a signal peptide sequence with 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%, or 99% homology.

[0126] CD80 extracellular domain Fc fusion proteins may also include fusion proteins having a truncated signal peptide sequence. In this context, truncation refers to a signal peptide sequence that is shorter than the original signal peptide sequence but retains at least a portion of its functionality to act as a signal peptide. For example, the human CD80 signal peptide sequence comprises or consists of amino acids 1-34 of SEQ ID NO:6. CD80 extracellular domain Fc fusion proteins having a truncated signal peptide sequence may have amino acids 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of SEQ ID NO:6. In another embodiment, the signal peptide may comprise or consist of the sequence shown in SEQ ID NO:5. CD80 extracellular domain Fc fusion proteins with truncated signal peptide sequences may have amino acids 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 from amino acids 1-18 of SEQ ID NO:5.

[0127] The CD80 extracellular domain Fc fusion protein having a truncated signal peptide sequence may also be a protein containing SEQ ID No:1 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%, or 99% similarity to SEQ ID NO:1 and additionally containing a signal peptide sequence shorter than the original signal peptide sequence. Furthermore, for example, the signal peptide sequence may have amino acids 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 from amino acids 1-18 of SEQ ID NO:6, or in another embodiment, the signal peptide may comprise or consist of the sequence shown in SEQ ID NO:5. The CD80 extracellular domain Fc fusion protein having a truncated signal peptide sequence may have amino acids 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 from amino acids 1-18 of SEQ ID NO:5.

[0128] The CD80 extracellular domain can have any source, including mice and rats. Preferably, the CD80 extracellular domain protein is derived from a human source.

[0129] The Fc domain of IgG may be covalently fused to the N-terminal or C-terminal portion of the CD80 extracellular domain or fused at an internal location. In some embodiments, the Fc domain of the IgG molecule may be fused to the CD80 extracellular domain via a linker peptide (such as a GS linker). Preferably, the Fc domain is fused to the C-terminal portion of the CD80 extracellular domain.

[0130] In some embodiments, the Fc domain has a wild-type sequence. In other embodiments, the Fc domain is a natural or engineered variant. In some embodiments, the Fc domain contains one or more mutations, substitutions, and / or deletions compared to its wild-type sequence. In some embodiments, an Fc domain that has altered interactions with one or more Fcγ receptors (FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB) is selected. Preferably, the Fc domain is derived from human IgG, such as IgG1, IgG2, IgG3, or IgG4. More preferably, the Fc domain is derived from human IgG1.

[0131] In a preferred embodiment, the Fc domain of the CD80 extracellular domain Fc fusion protein comprises or is composed of the following sequences:

[0132]

[0133] Or a sequence that is 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%, or 99% identical to SEQ ID NO:2.

[0134] In a preferred embodiment, the CD80 extracellular domain Fc fusion protein comprises or is composed of the following sequences:

[0135]

[0136]

[0137] Or a sequence that is 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%, or 99% identical to SEQ ID NO:4.

[0138] In another preferred embodiment, the CD80 extracellular domain Fc fusion protein comprises or is composed of a CD80 extracellular domain fused to or formed with the Fc domain of IgG1, wherein the CD80 extracellular domain comprises or is composed of amino acids 1-207 of SEQ ID NO:4 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%, or 99% homology with amino acids 1-207 of SEQ ID NO:4, and the Fc domain comprises or is composed of amino acids 208-433 of SEQ ID NO:4 or has at least 70%, 72%, 74%, 76%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 98%, or 99% homology with amino acids 1-207 of SEQ ID NO:4, and the Fc domain comprises or is composed of amino acids 208-433 of SEQ ID NO:4, or has at least 70%, 72%, 74%, 76%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with amino acids 1-207 of SEQ ID NO:4, and the Fc domain ...2%, 93%, 94%, The amino acid 208-433 of NO:4 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%, or 99% consistency.

[0139] In another preferred embodiment, the CD80 extracellular domain Fc fusion protein comprises or consists of the following sequences:

[0140]

[0141] Or a sequence that is 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%, or 99% identical to SEQ ID NO:3.

[0142] As used herein, in the case of two or more nucleic acid or polypeptide sequences, the term "consistent" or "consistency percentage" refers to two or more sequences or subsequences being identical or having a specified percentage of identical nucleotide or amino acid residues when compared and aligned for maximum correspondence. To determine the consistency percentage, sequences are aligned for optimal comparison purposes (e.g., a gap may be introduced in the first amino acid sequence or nucleic acid sequence to achieve optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid 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, the molecule is consistent at that position. The consistency percentage between two sequences is a function of the number of consistent positions common to the sequences (i.e., consistency % = number of consistent positions / total number of positions (i.e., overlapping positions) × 100). In some embodiments, where appropriate, after introducing a gap within the sequences, the two sequences being compared are of the same length (e.g., excluding any additional sequences extending beyond the compared sequences).

[0143] Mathematical algorithms can be used to determine the percentage of identity or similarity between two sequences. A preferred, non-limiting embodiment of the mathematical algorithm used to compare two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as described in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. A BLAST nucleotide search can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid encoding the relevant protein. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the relevant protein. To obtain gap alignments for comparative purposes, gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used for iterative searches to detect distant relationships between (identical) molecules. When using BLAST, gapped BLAST, and PSI-Blast programs, preset parameters of individual programs (e.g., XBLAST and NBLAST) can be used. Another preferred, non-limiting embodiment of the mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, CABIOS (1989). Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weighted residue table, vacancy length penalty 12, and vacancy penalty 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Roboti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA as 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. If ktup = 2, similar regions in the two compared sequences are obtained by observing aligned residue pairs; if ktup = 1, single aligned amino acids are examined. For protein sequences, ktup can be set to 2 or 1, or for DNA sequences, it can be set from 1 to 6. If ktup is not specified, the default value is 2 for proteins and 6 for DNA.Alternatively, the CLUSTAL W algorithm can be used for protein sequence alignment, as described by Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0144] Functional variants of CD80 extracellular domain Fc fusion proteins include the aforementioned biologically active variants and biologically active fragments of CD80 extracellular domain Fc fusion proteins. Functional variants may have variations in the CD80 extracellular domain, Fc domain, and / or both domains. For example, some CD80 extracellular domain functional variants have been described in WO2017181152. In another embodiment, functional variants of the Fc domain have been described in WO17079117 and include, for example, a human IgG1 Fc domain with amino acid substitutions of L234F, L235E, and / or P331S.

[0145] For example, variants may have one or more different amino acids at the location specifically describing a CD80 extracellular domain or Fc domain protein. Variants may share at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher amino acid identity with such CD80 extracellular domains or Fc domains. Fragments may have the same amino acids as a given specific CD80 extracellular domain or Fc domain protein, but may lack specific portions or regions of the CD80 extracellular domain or Fc domain protein.

[0146] Functional variants of the CD80 extracellular domain Fc fusion protein include only variants and fragments of the biologically active CD80 extracellular domain Fc fusion protein. For the purposes of this invention, the biological activity of CD80 extracellular domain Fc fusion protein variants or fragments encoded in the genome of a recombinant rhabdovirus is determined after its expression in individual cells or tumor cells. This means that biological activity is determined in the case of recombinant rhabdoviruses encoding CD80 extracellular domain Fc fusion protein variants or fragments (e.g., in Transwell assays or in vitro tumor models). Preferably, biological activity is determined using vesicular virus encoding CD80 extracellular domain Fc fusion protein variants or fragments. More preferably, biological activity is determined using VSV-GP encoding CD80 extracellular domain Fc fusion protein variants or fragments.

[0147] Bioactivity may include one or more of the following capabilities: chemical inducer activity, antitumor activity, and regulation of cytokine expression, such as increased expression of interferon-γ (IFN-γ) peptide or decreased expression of transforming growth factor-β (TGF-β) peptide in syngeneic mammalian cell populations (including CD8-positive T cells, CD4-positive T cells, antigen-presenting cells, and tumor cells). Bioactivity may be tested, for example, but not limited to, according to the protocols shown in the examples. For the purposes of this invention, if a functional variant or fragment of a CD80 extracellular domain Fc fusion protein exhibits at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the CD80 extracellular domain Fc fusion protein activity as shown in SEQ ID NO:3 or 4 (with or without a signal peptide sequence, respectively), then it is biologically active.

[0148] Rhabdoviruses possess antisense single-stranded RNA (ssRNA) as their genetic material (genome). Antisense ssRNA viruses require RNA polymerase to form sense RNA. The sense RNA acts as viral mRNA, which is translated into proteins used to produce new viral material. Using the newly formed virus, more antisense RNA molecules are produced.

[0149] A typical rhabdovirus genome encodes at least five structural proteins in the order 3′-NPMGL-5′. The genome may contain other short intergenic regions or additional genes between structural proteins, and therefore its length and organization may vary.

[0150] According to the present invention, the CD80 extracellular domain Fc fusion protein gene can be introduced into any location in the rhabdovirus genome. Depending on the insertion site, the transcription efficiency of the CD80 extracellular domain Fc fusion protein gene can be affected. Generally, the transcription efficiency of the CD80 extracellular domain Fc fusion protein gene decreases from 3′ insertion to 5′ insertion. The CD80 extracellular domain Fc fusion protein gene can be inserted into the following genomic locations: 3′-CD80 extracellular domain Fc fusion protein-NPMGL-5', 3′-N-CD80 extracellular domain Fc fusion protein-PMGL-5', 3′-NP-CD80 extracellular domain Fc fusion protein-MGL-5', 3′-NPM-CD80 extracellular domain Fc fusion protein-GL-5', 3′-NPMG-CD80 extracellular domain Fc fusion protein-L-5', or 3′-NPMGL-CD80 extracellular domain Fc fusion protein-5'. In a preferred embodiment, the CD80 extracellular domain Fc fusion protein gene is inserted between the G protein and the L protein.

[0151] Following infection by tumor cells, the CD80 extracellular domain Fc fusion protein gene, encoded in the genome of a recombinant rhabdovirus, is transcribed into positive-sense RNA and subsequently translated by the tumor cells into the CD80 extracellular domain Fc fusion protein. The term "encoding" refers to the inherent property of a specific nucleotide sequence in a nucleic acid to serve as a template for the synthesis of other polymers and macromolecules in biological processes, possessing a defined sequence of nucleotides (e.g., RNA molecules) or amino acids and the biological characteristics derived from them. Therefore, a gene encodes a protein if the desired protein is produced in a cell or another biological system through transcription and subsequent translation of mRNA. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence) and the non-coding strand can serve as templates for gene transcription and can be referred to as the encoded protein or other products of the gene. The nucleic acid and nucleotide sequence encoding a protein may include introns.

[0152] The transcription of the CD80 extracellular domain Fc fusion protein gene is preferably not under the control of its own promoter and is strictly related only to viral replication, ensuring that the CD80 extracellular domain Fc fusion protein is targeted to the site of viral replication and spread (tumor). Therefore, the transcription of the CD80 extracellular domain Fc fusion protein gene is not controlled by other components such as promoters or inducible gene expression components.

[0153] Translated CD80 extracellular domain Fc fusion proteins typically exist in solution as dimeric fusion proteins comprising two identical monomeric mature CD80 extracellular domain Fc fusion proteins. In this embodiment, each monomeric CD80 extracellular domain Fc fusion protein comprises a CD80 extracellular domain fused to the C-terminus and N-terminus of the IgG Fc domain. The two CD80 extracellular domain Fc fusion monomers are covalently linked together by disulfide bonds formed between cysteine ​​residues in each monomer, thereby forming a CD80 extracellular domain Fc fusion protein dimer.

[0154] It should be understood that nucleic acid sequences can vary with or without alteration of the major sequence of the encoded polypeptide. Nucleic acids encoding proteins include any nucleic acid with different nucleotide sequences but encoding the same amino acid sequence of protein due to degeneracy of the genetic code. The selection of nucleic acid sequences that will result in the expression of CD80 extracellular domain Fc fusion proteins, and especially any specific CD80 extracellular domain Fc fusion proteins disclosed herein, is within the knowledge of those skilled in the art. Nucleic acid molecules encoding the amino acid sequence of CD80 extracellular domain Fc fusion proteins 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 via oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cartridge mutagenesis of previously prepared CD80 extracellular domain Fc fusion proteins.

[0155] Pharmaceutical Composition

[0156] Of course, the actual pharmaceutically effective or therapeutic dose will depend on factors known to those skilled in the art, such as the patient's age and weight, route of administration, and severity of disease. In any case, recombinant rhabdovirus will be administered at a dosage and in a manner that allows for the delivery of a pharmaceutically effective dose, based on the patient's unique condition.

[0157] Generally, for the treatment and / or relief of the diseases, conditions, and symptoms mentioned herein, and depending on the specific disease, condition, or symptom to be treated, the efficacy of the specific recombinant rhabdovirus of the present invention to be used, the specific route of administration, and the specific pharmaceutical formulation or composition used, the recombinant rhabdovirus of the present invention will typically be administered at a dose, for example, twice a week, once a week, or once a month, but this can be significantly varied, particularly depending on the parameters mentioned below. Therefore, in some cases, using a dose less than the minimum dose given above may be sufficient, while in others it may exceed the upper limit. When administering a larger dose, it may be preferable to divide it into multiple smaller doses within a day.

[0158] For use in therapy, the recombinant rhabdovirus of the present invention is formulated into a pharmaceutical composition suitable for facilitating administration to animals or humans. Typical formulations are prepared by mixing the recombinant virus with a physiologically acceptable carrier, excipient, or stabilizer in an aqueous or aqueous or non-aqueous suspension. The carrier, excipient, modifier, or stabilizer is non-toxic at the dose and concentration used. It includes buffer systems such as phosphates, citrates, acetates, and other inorganic or organic acids and their salts; antioxidants, including ascorbic acid and methionine; preservatives such as octadecyl dimethylbenzyl ammonium chloride; hexahydroxyquaternary ammonium chloride; benzyl ammonium chloride, benzoyl ammonium chloride; phenol, butanol, or benzyl alcohol; alkyl p-hydroxybenzoate, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; proteins such as serum albumin, gelatin, or immunoglobulins; and acetones. Aqueous polymers, such as polyvinylpyrrolidone or polyethylene glycol (PEG); amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, oligosaccharides, or polysaccharides and other carbohydrates, including glucose, mannose, sucrose, trehalose, dextrin, or dextran; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming ions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or ionic or nonionic surfactants, such as TWEEN. TM (polysorbate), PLURONICS TM These may be fatty acid esters, fatty acid ethers, or sugar esters. Excipients may also have the function of regulating release or absorption.

[0159] In one embodiment, the recombinant rhabdovirus of the present invention is formulated into a pharmaceutical composition comprising Tris, arginine, and optionally a citrate. Tris is preferably used at a concentration of about 1 mM to about 100 mM. Arginine is preferably used at a concentration of about 1 mM to about 100 mM. The citrate may be present at a concentration of up to 100 mM. Preferably, the formulation comprises about 50 mM Tris and 50 mM arginine.

[0160] The pharmaceutical composition may be provided as a liquid, a frozen liquid, or in lyophilized form. The frozen liquid may be stored at temperatures between about 0°C and about -85°C, including temperatures between -70°C and -85°C and 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.

[0161] The recombinant rhabdovirus or pharmaceutical composition of the present invention does not need to be formulated, but optionally may be, with one or more agents currently used for the prevention or treatment of said conditions. The effective amount of such other agents depends on the amount of recombinant antibody present in the formulation, the type of condition or treatment, and other factors as described above. It is generally used at the same dose and via the route of administration as described herein, or at about 1% to 99% of the dose described herein, or at any dose and via any route determined empirically / clinically.

[0162] For the prevention or treatment of disease, the appropriate dosage of the recombinant rhabdovirus or pharmaceutical composition of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of recombinant rhabdovirus, the severity and course of the disease, whether the recombinant rhabdovirus is administered for prophylactic or therapeutic purposes, prior therapy, the patient's clinical history and response to the recombinant rhabdovirus, and the judgment of the attending physician. The recombinant rhabdovirus or pharmaceutical composition of the present invention is suitable for administration to a patient in a single dose or in a series of treatments.

[0163] Depending on the type and severity of the disease, TCID using recombinant rhabdoviruses can be used. 50 Approximately 10 measured 8 Up to 10 13 One infectious particle can be the initial candidate dose administered to the patient, whether, for example, by one or more single administrations or by continuous infusion. Treatment generally continues until the required suppression of disease symptoms occurs, depending on the condition, when repeated administrations are given over several days or longer. An exemplary dose of one type of recombinant rhabdovirus will be approximately 10 8 Up to 10 13 One by TCID 50 Within the range of infectious particles measured. Therefore, it is possible to administer TCID to patients. 50 Approximately 10 measured 8 109 10 10 10 11 10 12 Or 10 13 One or more doses (or any combination thereof) of an infectious particle. Such doses may be administered intermittently, for example weekly or every three weeks (e.g., to give the patient about two to about twenty, or for example about six, doses of recombinant rhabdovirus). A higher initial starting dose may be administered, followed by one or more lower doses, or vice versa. However, other dosing regimens may be applicable. The progress of this therapy can be easily monitored using routine techniques and analysis.

[0164] The efficacy of the recombinant rhabdovirus of the present invention and compositions comprising it may be tested using any suitable in vitro assay, cell-based assay, in vivo assay, and / or animal models known per se, or any combination thereof, depending on the specific disease involved. Suitable assays and animal models will be apparent to those skilled in the art, and include, for example, the assays and animal models used in the following examples.

[0165] Of course, the actual pharmaceutically effective dose or therapeutic dose will depend on factors known to those skilled in the art, such as the patient's age and weight, route of administration, and severity of disease. In any case, the recombinant rhabdovirus of the present invention will be administered at a dosage and in a manner that allows for the delivery of a pharmaceutically effective dose, based on the patient's unique condition.

[0166] Alternatively, the recombinant rhabdovirus or pharmaceutical composition of the present invention may be delivered in volumes of about 50 μl to about 100 ml (inclusive of all numbers within this 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.

[0167] For intratumoral administration, the volume is preferably between about 50 μl and about 5 ml, including the following volumes: 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.

[0168] For systemic administration, such as via infusion of recombinant rhabdovirus, the volume can naturally be higher. Alternatively, a concentrated solution of recombinant rhabdovirus can be diluted directly in a larger volume of infusion solution prior to infusion.

[0169] In particular, for intravenous administration, the volume is preferably between 1 ml and 100 ml, including the following volumes: 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 a preferred embodiment, the volume is between about 5 ml and 15 ml, more preferably about 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, or about 14 ml.

[0170] Preferably, the same formulation is used for both 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 1:1 dose and / or volume ratio means that the same dose and / or volume is administered intratumorally and intravenously, while a dose and / or volume ratio of about 1:20 means that the intravenous dose and / or volume is twenty 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.

[0171] The ideal effective concentration of recombinant rhabdovirus is between approximately 10. 8 With 10 14 The concentration ranges between vector genomes per milliliter (vg / mL). Infectivity units can be measured as described in McLaughlin et al., J Virol.; 62(6):1963-73 (1988). Preferably, the concentration is about 1.5 × 10⁻⁶. 9 To approximately 1.5 × 10 13 More preferably, about 1.5 × 10 9 To approximately 1.5 × 10 11 In one implementation scheme, the effective concentration is approximately 1.5 × 10⁻⁶. 9 In another embodiment, the effective concentration is approximately 1.5 × 10⁻⁶. 10 In another embodiment, the effective concentration is approximately 1.5 × 10⁻⁶. 11 In yet another embodiment, the effective concentration is approximately 1.5 × 10⁻⁶. 12 In another embodiment, the effective concentration is approximately 1.5 × 10⁻⁶. 13 In another embodiment, the effective concentration is approximately 1.5 × 10⁻⁶.14 The lowest effective concentration may need to be used to reduce the risk of unwanted effects. Other doses within these ranges may be selected by the attending physician considering the individual's (preferably human) physical condition, age, specific type of cancer, and the extent of cancer progression (if any).

[0172] The effective target concentration of recombinant rhabdovirus can be obtained using TCID. 50 Expression. TCID 50 For example, by using the Spyman-Caber The method is used to determine this. Ideally, the range includes 1×10⁻⁶. 8 / ml and 1×10 14 / ml of TCID 50 The effective target concentration is between [a certain value]. Preferably, the effective target concentration is approximately 1 × 10⁻⁶. 9 To approximately 1×10 12 / ml, and more preferably about 1×10 9 To approximately 1×10 11 / ml. In one implementation, the effective target concentration is approximately 1×10⁻⁶. 10 / ml. In a preferred embodiment, the target concentration is 5 × 10⁹ / ml. 10 / ml. In another embodiment, the effective target concentration is approximately 1.5 × 10⁹ / ml. 11 / ml. In one implementation, the effective target concentration is approximately 1×10⁻⁶. 12 / ml. In another embodiment, the effective target concentration is approximately 1.5 × 10⁹ / ml. 13 / ml.

[0173] The effective target dose of recombinant rhabdovirus can also be TCID. 50 Expression. Ideally, the range includes 1×10. 8 With 1×10 14 TCID 50 The target dose is between [amount missing]. Preferably, the target dose is approximately 1 × 10 [units missing]. 9 To approximately 1×10 13 And more preferably about 1×10 9 To approximately 1×10 12 In one implementation scheme, the effective concentration is approximately 1 × 10⁻⁶. 10 In the preferred embodiment, the effective concentration is approximately 1 × 10⁻⁶. 11 In one implementation scheme, the effective concentration is approximately 1 × 10⁻⁶. 12 In another embodiment, the effective concentration is approximately 1 × 10⁻⁶. 13 .

[0174] In another aspect, a kit or kit containing materials that can be used to treat, prevent, and / or diagnose the conditions described above is provided. The kit or kit includes a container and a label or instruction manual on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from various materials, such as glass or plastic. The container contains a composition, alone or in combination with another composition effective in treating, preventing, and / or diagnosing the condition, and may have a sterile dispensing port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is the recombinant rhabdovirus or pharmaceutical composition of the present invention. The label or instruction manual indicates that the composition is used to treat the selected condition.

[0175] Furthermore, the kit or kit may comprise (a) a first container containing a composition comprising the recombinant rhabdovirus or pharmaceutical composition of the present invention; and (b) a second container containing a composition comprising another cytotoxic agent or other therapeutic agent, such as a PD-1 pathway inhibitor or SMAC mimic. The kit or kit in this embodiment of the invention may further comprise a pharmaceutical label indicating that the composition may be used to treat a specific condition, particularly cancer. Alternatively or additionally, the kit or kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextran solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0176] In another aspect, the recombinant rhabdovirus of the present invention is used in combination with a suitable device for administering the recombinant rhabdovirus, such as a syringe, injection pen, micropump, or other device. Preferably, the recombinant rhabdovirus of the present invention is included in a kit, for example, a pharmaceutical instruction manual containing instructions for use of the recombinant rhabdovirus.

[0177] Medical use

[0178] Another aspect of the invention provides a recombinant rhabdovirus encoding at least one CD80 extracellular domain Fc fusion protein or a functional variant thereof in its genome for use in medicine.

[0179] The recombinant rhabdovirus of this invention effectively induces tumor cell lysis and immunogenic cell death, and stimulates innate immune cells in the tumor microenvironment. Therefore, the recombinant rhabdovirus of this invention can be used for the treatment and / or prevention of cancer.

[0180] In another aspect, the recombinant rhabdovirus of the present invention can be used in a method for treating and / or preventing cancer, the method comprising administering a therapeutically effective amount of the recombinant rhabdovirus to an individual suffering from cancer, thereby improving symptoms of one or more cancers.

[0181] In another aspect, the present invention further provides the use of the recombinant rhabdovirus according to the invention for the manufacture of agents for the treatment and / or prevention of cancer.

[0182] In another aspect, in a method of treating and / or preventing gastrointestinal cancer, lung cancer, or head and neck cancer using the recombinant rhabdovirus of the present invention, the method comprises administering a therapeutically effective amount of the recombinant rhabdovirus to an individual suffering from gastrointestinal cancer, lung cancer, or head and neck cancer, thereby improving one or more symptoms of gastrointestinal cancer, lung cancer, or head and neck cancer.

[0183] The appropriate dose of recombinant rhabdovirus for the prevention or treatment of disease will depend on various factors, such as the type of disease to be treated, the severity and course of the disease as defined above, whether the recombinant rhabdovirus is being administered for preventative or therapeutic purposes, prior therapy, the patient's clinical history and response to the recombinant rhabdovirus, and the judgment of the attending physician. Recombinant rhabdovirus is suitable for administration to the patient either as a single dose or as part of a series of treatments.

[0184] In one aspect, the cancer is a solid tumor. Solid tumors can be brain cancer, endometrial cancer, vaginal cancer, anal cancer, colorectal cancer, oropharyngeal squamous cell carcinoma, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal cancer, hepatocellular carcinoma, pancreatic adenocarcinoma, bile duct cancer, bladder urothelial carcinoma, metastatic melanoma, prostate cancer, breast cancer, ovarian cancer, head and neck squamous cell carcinoma (HNSCC), glioblastoma, non-small cell lung cancer, brain tumor, or small cell lung cancer. Treatment of gastrointestinal cancer, lung cancer, and head and neck cancer is preferred.

[0185] Recombinant rhabdovirus may be administered by any suitable means, including oral, non-enteral, subcutaneous, intratumoral, intravenous, intradermal, intraperitoneal, intrapulmonary, and intranasal administration. Non-enteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Additionally, recombinant rhabdovirus is suitable for administration by pulsatile infusion. In one aspect, depending partly on whether the administration is short-term or long-term, administration may be provided by injection, preferably intravenous or subcutaneous.

[0186] Depending on the specific recombinant rhabdovirus of the present invention and its specific pharmacokinetic and other properties, the recombinant rhabdovirus may be administered once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once weekly, once monthly, or in a similar manner. Treatment courses may include long-term treatment once weekly. "Long-term" means a duration of at least two weeks, preferably several months or years.

[0187] Treatment durations may include various courses and typically require administering multiple doses to the patient over periods of one, two, three, or four weeks, optionally followed by one or more further rounds of treatment. In one aspect, the recombinant rhabdovirus of the present invention is administered to the patient in up to 1, 2, 3, 4, 5, or 6 doses over a predetermined period. Preferably, the first round of treatment is completed within three weeks. During the three-week treatment period, the recombinant rhabdovirus may be administered to the patient as described in the following protocols: (i) once on day 0; (ii) on day 0 and day 3; (iii) on day 0, day 3, and day 6; (iv) on day 0, day 3, day 6, and day 9; (v) on day 0 and day 5; (vi) on day 0, day 5, and day 10; (vii) on day 0, day 5, day 10, and day 15. These courses of treatment may be repeated, and a second or third round of treatment may be required depending on the outcome of the first round of treatment. Based on the first round of treatment, the second round of treatment preferably includes further treatment on days 21, 42, and 63. In a preferred embodiment, the recombinant rhabdovirus of the present invention is administered to the patient according to the following schedule: days 0, 3, 21, 42, and 63.

[0188] The term "inhibition" is used herein in the same context as "improvement" and "relief," meaning the reduction or decrease of one or more characteristics of a disease. The recombinant rhabdovirus or pharmaceutical composition of the present invention will be formulated, administered, and applied in accordance with good medical practice. In this context, considerations include the specific disease being treated, the specific mammal being treated, the individual patient's clinical symptoms, the cause of the disease, the site of drug delivery, the method of administration, the timing of administration, and other factors known to a medical practitioner. The "therapeuticly effective amount" of the recombinant rhabdovirus to be administered will be adjusted for these considerations and will be the minimum amount required to prevent, improve, or treat clinical symptoms of cancer, particularly the minimum amount effective for these conditions.

[0189] In another aspect, the recombinant rhabdovirus of the present invention can be administered multiple times and in several doses. In one aspect, a first dose of the recombinant rhabdovirus is administered intratumorally, followed by an intravenous administration of subsequent doses of the recombinant rhabdovirus. In another aspect, a first dose and at least one or more subsequent doses of the recombinant rhabdovirus are administered intratumorally, followed by an intravenous administration of subsequent doses of the recombinant rhabdovirus. Subsequent doses may be administered on days 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, or 31 after the initial intratumoral administration.

[0190] In another embodiment, the first dose of recombinant rhabdovirus is administered intravenously, followed by subsequent doses intratumorally. Subsequent doses may be administered on days 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, or 31 after the initial intravenous administration.

[0191] In another case, recombinant rhabdovirus was administered intravenously and a subsequent dose of recombinant rhabdovirus was administered intratumorally.

[0192] In another instance, recombinant rhabdovirus was administered intravenously and intratumorally at various time points.

[0193] As stated above, the recombinant rhabdovirus of the present invention has numerous uses for stimulating an immune response against cancer cells. Strong immune activation potential has been observed, but this is limited by the tumor microenvironment. Therefore, in a preferred aspect, the recombinant rhabdovirus of the present invention can be administered systemically to a patient. Systemic applicability is a key property because many cancers are highly metastatic and allow for treatment of tumor lesions that are difficult or impossible to reach. Due to this unique immunostimulatory property, the recombinant rhabdovirus according to the present invention is particularly useful for treating metastatic tumors.

[0194] Some patients develop resistance to checkpoint inhibitor therapy, and these patients appear to have accumulated mutations in the IFN pathway. Therefore, in one aspect, the recombinant rhabdovirus of the present invention, and especially the recombinant vesicular stomatitis virus of the present invention, can be used to treat patients resistant to checkpoint inhibitor therapy. Due to the unique immunomodulatory properties of the recombinant rhabdovirus of the present invention, and especially the recombinant vesicular stomatitis virus, such treated patients may become suitable for continued checkpoint inhibitor therapy.

[0195] In a preferred embodiment, the recombinant rhabdovirus of the present invention, and especially the recombinant vesicular stomatitis virus of the present invention, can be used to treat patients with non-small cell lung cancer who have completed checkpoint inhibitor therapy using PD-1 or PD-L1 inhibitors (e.g., PD-1 or PD-L1 antagonist antibodies).

[0196] It should be understood that any of the recombinant rhabdoviruses or pharmaceutical compositions of the present invention can be used to implement any of the above-described pharmaceutical formulations or treatment methods.

[0197] combination

[0198] This invention also provides combination therapies / methods to offer certain advantages compared to those currently in use and / or known in the background art. These advantages may include in vivo efficacy (e.g., improved clinical response, prolonged response, increased response rate, duration of response, rate of disease stabilization, duration of stabilization, time to disease progression, progression-free survival (PFS) and / or overall survival (OS), later-onset resistance and the like), safety and well-tolerated administration, and reduced frequency and severity of adverse events.

[0199] The recombinant rhabdovirus of this invention can be used in combination with other pharmacologically active ingredients, such as currently advanced or standard care compounds, such as cell growth inhibitors or cytotoxic substances, cell proliferation inhibitors, anti-angiogenic substances, steroids, immunomodulators / checkpoint inhibitors, and the like. The recombinant rhabdovirus of this invention can also be used in combination with radiotherapy.

[0200] Cell growth inhibitory and / or cytotoxic active substances that can be combined with the recombinant rhabdovirus of the present invention include (but are not limited to) hormones, hormone analogs and anti-hormones, aromatase inhibitors, LHRH agonists and antagonists, growth factor inhibitors (such as 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, such as HER2, HER3, HER4) and hepatocyte growth factor (HGF)), such as (anti) growth factor antibodies, (anti) growth factor receptor antibodies, and tyrosine kinase inhibitors (such as cetuximab, gefitinib, afatinib, nintedanib, imatini). b) Lapatinib, bosutinib, and trastuzumab; antimetabolites (e.g., antifolates such as methotrexate and raltitrexed); pyrimidine analogues such as 5-fluorouracil (5-FU), gemcitabine, irinotecan, doxorubicin, TAS-102, capecitabine, and gemcitabine); purine and adenosine analogues such as mecaptopurine, thioguanine, cladribine, pentostatin, and cytarabine. C) Fludarabine; antitumor antibiotics (e.g., anthracyclines); platinum derivatives (e.g., cisplatin, oxaliplatin, carboplatin); alkylating agents (e.g., estradiol mustard, meclorethamine, melphalan, chlorambucil, busulphan, dakarpa). (dacarbazin), cyclophosphamide, ifosfamide, temozolomide, nitrosoureas (such as carmustin and lomustin, thiotepa); antimitotic agents (such as vinblastine, vindesin, vincristine, and vinorelbine; and taxanes, such as paclitaxel and docetaxel); angiogenesis inhibitors,This includes bevacizumab, ramucirumab, and aflibercept; microtubule inhibitors; DNA synthesis inhibitors, PARP inhibitors, topoisomerase inhibitors (e.g., epipodophyllotoxin) (such as etoposide and etoposide phosphate), teniposide, amsacrin, topotecan, irinotecan, and mitoxa. ntrone), 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, AKT inhibitors, PLK1 inhibitors (such as vorasetib), CDK inhibitors (including CDK9 inhibitors), Aurora kinase inhibitors), tyrosine kinase inhibitors (e.g., PTK2 / FAK inhibitors), protein-protein interaction 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 analogs (e.g., everolimus, tesiromolimus, sirolimus), androgen synthesis inhibitors, androgen receptor inhibitors, DNMT inhibitors, HDAC inhibitors, ANG1 / 2 inhibitors, CYP17 inhibitors, radiopharmaceuticals, and immunotherapeutic agents (such as immune checkpoint inhibitors (e.g., CTLA4, PD1, PD-L1, LAG3, and TIM3 binding molecules / immunoglobulins)).Drugs such as ipilimumab, nivolumab, and pembrolizumab, as well as various chemotherapy agents (such as amifostin, anagrelid, clodronat, filgrastin, interferon, interferon-alpha, leucovorin, rituximab, procarbazine, and levamisole) Drugs containing: mesna, mitotane, pamidronate, and porfimer; proteasome inhibitors (such as bortezomib); Smac and BH3 mimics; agents that restore p53 function, including MDM2-p53 antagonists; inhibitors of the Wnt / β-catenin signaling pathway; Flt3L and Flt3-stimulating antibodies or ligand mimics; SIRPα and CD47 blocking therapies; and / or cyclin-dependent kinase 9 inhibitors.

[0201] Furthermore, the potential transformation in immune “cold” to “hot” tumors, bone marrow / dendritic cell activation, and CD80-Fc-mediated T cell activation further interact advantageously with therapeutic modalities such as T cell conjugates. Therefore, in one embodiment, the recombinant rhabdovirus of the present invention can be used in combination therapy with T cell conjugates (such as bispecific DLL3 / CD3 binders) that provide T cell receptor stimulation but without co-stimulation. Additionally, potential clinical combination therapies may also include tumor-angiogenic modulators. Therefore, in another embodiment, the recombinant rhabdovirus of the present invention can be used in combination therapy with tumor angiogenic modulators (such as bispecific VEGF / ANG2 binders).

[0202] The recombinant rhabdovirus of the present invention can be used in combination with treatments using PD-1 pathway inhibitors or SMACm / IAP antagonists. Such combination therapies can be given in non-fixed (e.g., free) combinations of substances or in fixed combinations (including kits).

[0203] In this context, the term "combination" or "in combination" as used in the context of this invention includes, but is not limited to, products resulting from mixing or combining more than one active agent, and includes fixed and non-fixed (e.g., free) combinations (including kits) and uses, such as, for example, the simultaneous, parallel, sequential, continuous, alternating, or individual use of components or agents. The term "fixed combination" means the simultaneous administration of the active agent to a patient in the form of a single entity or dose. The term "non-fixed combination" means the simultaneous, parallel, or sequential administration of the active agent to a patient in the form of a single entity without a specific time limit, wherein such administration provides a therapeutically effective amount of two compounds in the patient's body. The latter also applies to mixture therapies, such as the administration of three or more active agents.

[0204] This invention provides a combination of recombinant rhabdovirus with a PD-1 pathway inhibitor or an SMACm / IAP antagonist for the treatment of cancers as described herein, preferably for the treatment of solid tumors.

[0205] The present invention also provides the use of a combination of recombinant rhabdovirus and PD-1 pathway inhibitor or SMACm / IAP antagonist for the manufacture of agents for the treatment and / or prevention of cancers as described herein, preferably for the treatment of solid cancers.

[0206] The present invention further provides a method for treating and / or preventing cancer, comprising administering to an individual suffering from cancer a therapeutically effective amount of the recombinant rhabdovirus and PD-1 pathway inhibitor or SMACm / IAP antagonist of the present invention, thereby improving one or more symptoms of cancer. The recombinant rhabdovirus and PD-1 pathway inhibitor or SMACm / IAP antagonist of the present invention may be administered simultaneously, sequentially, or alternately.

[0207] The recombinant rhabdovirus and PD-1 pathway inhibitor or SMACm / IAP antagonist of the present invention can be administered via the same route of administration or via different routes of administration. Preferably, the 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 subsequent doses of the recombinant rhabdovirus are administered intravenously. Subsequent doses may be administered on days 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, or 31 after the initial intratumoral administration. In a preferred embodiment, a PD-1 pathway inhibitor or an SMACm / IAP antagonist is administered 21 days after the initial intratumoral administration.

[0208] Treatment is particularly preferred using the recombinant rhabdovirus of the present invention in combination with the following:

[0209] (i) SMAC mimic (SMACm) / IAP antagonist,

[0210] (ii) Immunotherapy agents, including anti-PD-1 and anti-PD-L1 agents and anti-LAG3 agents, such as pembrolizumab and nivolumab and antibodies as disclosed in WO2017 / 198741.

[0211] The combinations provided herein comprise (i) the recombinant rhabdovirus of the present invention and (iia) a PD-1 pathway inhibitor, preferably an antagonistic antibody against PD-1 or PD-L1, or (iib) an SMACm / IAP antagonist. Further, use is provided for such combinations comprising (i) and (iia) or (i) and (iib) for the treatment of cancers as described herein.

[0212] In another aspect, a combination therapy is provided comprising using (i) the recombinant rhabdovirus of the present invention and (iia) a PD-1 pathway inhibitor or (iib) an SMACm / IAP antagonist. In such a combination therapy, the recombinant rhabdovirus of the present invention may be administered simultaneously, sequentially, or alternately with the PD-1 pathway inhibitor or SMACm / IAP antagonist.

[0213] For example, “simultaneous” administration includes administering the active agent within the same general time period (e.g., on the same day, but not necessarily simultaneously). Alternating administration includes administering one agent during a time period (e.g., over a course of several days or a week), followed by administering another agent during a subsequent time period (e.g., over a course of several days or a week), and then repeating the pattern for one or more cycles. Sequential or continuous administration includes administering one agent with one or more doses during a first time period (e.g., over a course of several days or a week), followed by administering another agent with one or more doses during a second time period (e.g., over a course of several days or a week). Overlapping schedules may also be used, which include administering the active agent on different days within the treatment period, not necessarily in a conventional order. Variations of these general guidelines may also be used, for example, based on the agent used and the individual’s condition.

[0214] The sequential treatment schedule includes administering the recombinant rhabdovirus of the present invention followed by the administration of a PD-1 pathway inhibitor or an SMACm / IAP antagonist. The sequential treatment schedule also includes administering the recombinant rhabdovirus of the present invention followed by the administration of a PD-1 pathway inhibitor or an SMACm / IAP antagonist. The sequential treatment schedule may include administration on days 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, or 31 after each other.

[0215] In the context of this invention and all embodiments, a PD-1 pathway inhibitor is a compound that inhibits the interaction between PD-1 and its receptor. PD-1 pathway inhibitors can attenuate PD-1 pathway signaling, preferably mediated by the PD-1 receptor. A PD-1 inhibitor can be any inhibitor targeting any member of the PD-1 pathway capable of antagonizing PD-1 pathway signaling. The inhibitor can be an antagonistic antibody targeting any member of the PD-1 pathway, preferably targeting the PD-1 receptor, PD-L1, or PD-L2. Alternatively, a PD-1 pathway inhibitor can be a fragment of the PD-1 receptor or the PD-1 receptor itself that blocks the activity of the PD-1 ligand.

[0216] PD-1 antagonists are well known in the art, for example, as reviewed by Li et al., Int. J. Mol. Sci. 2016, 17, 1151 (incorporated herein by reference). According to the present invention, any PD-1 antagonist, especially antibodies, such as those disclosed by Li et al., and other antibodies disclosed below, can be used. Preferably, the PD-1 antagonists of the present invention and all embodiments thereof are selected from the group consisting of the following antibodies:

[0217] ● Pembrolizumab (anti-PD-1 antibody);

[0218] ● Nivolumab (anti-PD-1 antibody);

[0219] ● Pilizumab (anti-PD-1 antibody);

[0220] ●PDR-001 (anti-PD-1 antibody);

[0221] ●The following are PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5 (anti-PD-1 antibodies) disclosed below.

[0222] ● Atezolizumab (anti-PD-L1 antibody);

[0223] ●Avilumab (anti-PD-L1 antibody);

[0224] ● Duvalumab (anti-PD-L1 antibody).

[0225] For example, pembrolizumab (formerly known as lambrolizumab; trademark name Keytruda; also known as MK-3475), disclosed in Hamid, O. et al. (2013) New England Journal of Medicine 369(2):134-44, is a humanized IgG4 monoclonal antibody that binds to PD-1 and contains a mutation at C228P designed to prevent Fc-mediated cytotoxicity. Pembrolizumab is disclosed (for example) in US 8,354,509 and WO2009 / 114335. It is approved by the FDA for the treatment of patients with unresectable or metastatic melanoma and patients with metastatic NSCLC.

[0226] Nivolumab (CAS Registry No.: 946414-94-4; BMS-936558 or MDX1106b) is a fully human IgG4 monoclonal antibody that specifically blocks PD-1 and lacks detectable antibody-dependent cytotoxicity (ADCC). Nivolumab is disclosed (for example) in US 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.

[0227] Pilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pilizumab is disclosed, for example, in WO2009 / 101611.

[0228] 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.

[0229] Antibodies PD1-1 to PD1-5 are antibody molecules defined by sequences as shown in Table 1, where HC represents the (full-length) heavy chain and LC represents the (full-length) light chain:

[0230] Table 1:

[0231]

[0232]

[0233]

[0234]

[0235] In particular, the anti-PD-1 antibody molecule described above has the following characteristics:

[0236] (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

[0237] (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

[0238] (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

[0239] (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

[0240] (PD1-5:) heavy chain containing the amino acid sequence of SEQ ID NO:22, and light chain containing the amino acid sequence of SEQ ID NO:23.

[0241] Atezolizumab (Tecentriq, also known as MPDL3280A) is a phage-derived human IgG1k monoclonal antibody targeting 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.

[0242] Avirumab 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.

[0243] Duvalumab (MEDI4736) is a human IgG1k monoclonal antibody with high specificity against PD-L1 and is described, for example, in Stewart et al. Cancer Immunol. Res. 2015; 3:1052-1062 or Ibrahim et al. Semin. Oncol. 2015; 42:474-483.

[0244] Other PD-1 antagonists disclosed by Li et al. (see above) or known 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 to replace or supplement the antagonists mentioned above.

[0245] As used herein, INN means that it also covers all biosimilar antibodies having the same or substantially the same amino acid sequence as the source antibody, including but not limited to those biosimilar antibodies authorized under subsection (k) of USC §262 and equivalent provisions in other jurisdictions.

[0246] The above-listed PD-1 antagonists, their individual manufacturing processes, therapeutic uses, and characteristics are known in the art.

[0247] In one implementation, the PD-1 antagonist is pembrolizumab.

[0248] In another implementation, the PD-1 antagonist is nivolumab.

[0249] In another implementation, the PD-1 antagonist is pilizumab.

[0250] In another embodiment, the PD-1 antagonist is atezolizumab.

[0251] In another embodiment, the PD-1 antagonist is avirutumab.

[0252] In another implementation, the PD-1 antagonist is durvalumab.

[0253] In another embodiment, the PD-1 antagonist is PDR-001.

[0254] In another embodiment, the PD-1 antagonist is PD1-1.

[0255] In another embodiment, the PD-1 antagonist is PD1-2.

[0256] In another embodiment, the PD-1 antagonist is PD1-3.

[0257] In another embodiment, the PD-1 antagonist is PD1-4.

[0258] In another embodiment, the PD-1 antagonist is PD1-5.

[0259] The SMAC mimicry within the meaning of this invention and all its embodiments is a compound that binds to and induces the degradation of IAP proteins. Preferably, the SMAC mimicry within the meaning of this invention and all its embodiments is selected from the group consisting of (A0):

[0260] ● SMAC analogues (i.e. compounds) or pharmaceutically acceptable salts thereof disclosed (generally and / or specifically) as in WO 2013 / 127729;

[0261] ● SMAC analogues (i.e. compounds) or pharmaceutically acceptable salts thereof disclosed (generally and / or specifically) as in WO 2015 / 025018;

[0262] ● SMAC analogues (i.e. compounds) or pharmaceutically acceptable salts thereof disclosed (generally and / or specifically) as in WO 2015 / 025019;

[0263] ● SMAC analogues (i.e. compounds) or pharmaceutically acceptable salts thereof disclosed (generally and / or specifically) as in WO 2016 / 023858;

[0264] ● SMAC analogues (i.e. compounds) or pharmaceutically acceptable salts thereof disclosed (generally and / or specifically) as in WO 2008 / 0016893;

[0265] ● LCL161 (i.e., compound A) or a pharmaceutically acceptable salt thereof in Example 1 of WO 2008 / 016893 (pages 28 / 29;

[122] );

[0266] ● An SMAC analogue known as Debio-1143 or a pharmaceutically acceptable salt thereof;

[0267] ● SMAC analogues known as birinapant or their pharmaceutically acceptable salts;

[0268] ● An SMAC analogue known as ASTX-660 or a pharmaceutically acceptable salt thereof;

[0269] ● An SMAC analogue known as CUDC-427 or a pharmaceutically acceptable salt thereof

[0270] ● Any of the SMAC analogs 1 to 26 in Table 2 or their pharmaceutically acceptable salts:

[0271] Table 2:

[0272]

[0273]

[0274]

[0275]

[0276]

[0277] Examples of compounds 1 to 10 in Table 2 are disclosed in WO 2013 / 127729. Examples of compounds 11 to 26 in Table 2 are disclosed in WO 2016 / 023858.

[0278] As used herein, the term "SMAC mimic / IAP antagonist" also includes the SMAC mimics listed above in the form of tautomers, pharmaceutically acceptable salts, hydrates, or solvates (including pharmaceutically acceptable hydrates or solvates of salts). It also includes SMAC mimics in all their solid, preferably crystalline, and pharmaceutically acceptable crystalline, hydrate, and solvate forms (including pharmaceutically acceptable hydrates and solvates of salts).

[0279] All SMAC analogs listed above, along with their individual synthesis and properties, are known in the art. All patent applications mentioned above are incorporated herein by reference in their entirety.

[0280] In one implementation, the SMAC mimic is LCL161 or a pharmaceutically acceptable salt thereof (A1).

[0281] In another embodiment, the SMAC mimic is compound 1 in Table 2 or a pharmaceutically acceptable salt thereof (A2).

[0282] In another embodiment, the SMAC mimic is compound 2 in Table 2 or a pharmaceutically acceptable salt thereof (A3).

[0283] In another embodiment, the SMAC mimic is compound 3 in Table 2 or a pharmaceutically acceptable salt thereof (A4).

[0284] In another embodiment, the SMAC mimic is compound 4 in Table 2 or a pharmaceutically acceptable salt thereof (A5).

[0285] In another embodiment, the SMAC mimic is compound 5 in Table 2 or a pharmaceutically acceptable salt thereof (A6).

[0286] In another embodiment, the SMAC mimic is compound 6 in Table 2 or a pharmaceutically acceptable salt thereof (A7).

[0287] In another embodiment, the SMAC mimic is compound 7 in Table 2 or a pharmaceutically acceptable salt thereof (A8).

[0288] In another embodiment, the SMAC mimic is compound 8 in Table 2 or a pharmaceutically acceptable salt thereof (A9).

[0289] In another embodiment, the SMAC mimic is compound 9 in Table 2 or a pharmaceutically acceptable salt thereof (A10).

[0290] In another embodiment, the SMAC mimic is compound 10 in Table 2 or a pharmaceutically acceptable salt thereof (A11).

[0291] In another embodiment, the SMAC mimic is compound 11 in Table 2 or a pharmaceutically acceptable salt thereof (A12).

[0292] In another embodiment, the SMAC mimic is compound 12 in Table 2 or a pharmaceutically acceptable salt thereof (A13).

[0293] In another embodiment, the SMAC mimic is compound 13 in Table 2 or a pharmaceutically acceptable salt thereof (A14).

[0294] In another embodiment, the SMAC mimic is compound 14 in Table 2 or a pharmaceutically acceptable salt thereof (A15).

[0295] In another embodiment, the SMAC mimic is compound 15 in Table 2 or a pharmaceutically acceptable salt thereof (A16).

[0296] In another embodiment, the SMAC mimic is compound 16 in Table 2 or a pharmaceutically acceptable salt thereof (A17).

[0297] In another embodiment, the SMAC mimic is compound 17 in Table 2 or a pharmaceutically acceptable salt thereof (A18).

[0298] In another embodiment, the SMAC mimic is compound 18 in Table 2 or a pharmaceutically acceptable salt thereof (A19).

[0299] In another embodiment, the SMAC mimic is compound 19 in Table 2 or a pharmaceutically acceptable salt thereof (A20).

[0300] In another embodiment, the SMAC mimic is compound 20 in Table 2 or a pharmaceutically acceptable salt thereof (A21).

[0301] In another embodiment, the SMAC mimic is compound 21 in Table 2 or a pharmaceutically acceptable salt thereof (A22).

[0302] In another embodiment, the SMAC mimic is compound 22 in Table 2 or a pharmaceutically acceptable salt thereof (A23).

[0303] In another embodiment, the SMAC mimic is compound 23 in Table 2 or a pharmaceutically acceptable salt thereof (A24).

[0304] In another embodiment, the SMAC mimic is compound 24 in Table 2 or a pharmaceutically acceptable salt thereof (A25).

[0305] In another embodiment, the SMAC mimic is compound 25 in Table 2 or a pharmaceutically acceptable salt thereof (A26).

[0306] In another embodiment, the SMAC mimic is compound 26 in Table 2 or a pharmaceutically acceptable salt thereof (A27).

[0307] In terms of the nature of the SMAC analogue, all embodiments (A1) to (A27) are preferred embodiments of embodiment (A0).

[0308] In a preferred embodiment of the combination therapy, the recombinant rhabdovirus is a recombinant vesicular stomatitis virus encoding in its genome at least one CD80 extracellular domain Fc fusion protein selected from the group consisting of: (i) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1; (ii) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the CD80 extracellular domain comprises or is composed of SEQ ID NO:1 or has at least 80% identity with SEQ ID NO:1; (iii) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the Fc domain comprises or is composed of SEQ ID NO:2 or has at least 80% identity with SEQ ID NO:1. NO:2 has at least 80% identity; (iv) CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the CD80 extracellular domain comprises or is composed of or has at least 80% identity with SEQ ID NO:1 and the Fc domain comprises or is composed of or has at least 80% identity with SEQ ID NO:2; (v) CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the CD80 extracellular domain is composed of amino acids 1-207 of SEQ ID NO:4 or has at least 80% identity with amino acids 1-207 of SEQ ID NO:4 and the Fc domain is composed of amino acids 208-433 of SEQ ID NO:4 or has at least 80% identity with SEQ ID NO:2; The amino acid sequence 208-433 of NO:4 has at least 80% identity, (vi) the CD80 extracellular domain Fc fusion protein according to any one of (i)-(v) further comprises a signal peptide sequence, or (vii) the CD80 extracellular domain Fc fusion protein comprising SEQ ID NO:3 or having at least 80% identity with SEQ ID NO:3, wherein the gene encoding glycoprotein G of recombinant vesicular stomatitis virus is replaced by the gene encoding glycoprotein GP of lymphocyte choriomeningovirus (LCMV), and / or the glycoprotein G is replaced by glycoprotein GP of LCMV.

[0309] In another preferred embodiment of the combination therapy, the recombinant rhabdovirus is a recombinant vesicular stomatitis virus that encodes in its genome a vesicular stomatitis virus nucleoprotein (N), large protein (L), phosphoprotein (P), matrix protein (M), glycoprotein (G), and at least one CD80 extracellular domain Fc fusion protein or a functional variant thereof, preferably a human CD80 extracellular domain, wherein the CD80 extracellular domain Fc fusion protein or a functional variant thereof is selected from the group comprising: (i) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1; (ii) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the CD80 extracellular domain comprises or consists of or has at least 80% identity with SEQ ID NO:1; and (iii) a CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused with the Fc domain of IgG1, wherein the Fc domain comprises SEQ ID NO:1. (iv) A CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused to the Fc domain of IgG1, wherein the CD80 extracellular domain comprises or is composed of or has at least 80% identity with SEQ ID NO:1 and the Fc domain comprises or is composed of or has at least 80% identity with SEQ ID NO:2; (v) A CD80 extracellular domain Fc fusion protein comprising a CD80 extracellular domain fused to the Fc domain of IgG1, wherein the CD80 extracellular domain is composed of amino acids 1-207 of SEQ ID NO:4 or has at least 80% identity with amino acids 1-207 of SEQ ID NO:4 and the Fc domain is composed of amino acids 208-433 of SEQ ID NO:4 or has at least 80% identity with SEQ ID NO:2; The amino acid sequence 208-433 of NO:4 has at least 80% identity, and (vi) the CD80 extracellular domain Fc fusion protein according to any one of (i)-(v) further comprises a signal peptide sequence, or (vii) the CD80 extracellular domain Fc fusion protein comprising SEQ ID NO:3 or having at least 80% identity with SEQ ID NO:3, wherein the gene encoding the glycoprotein G of the 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 shown in SEQ ID NO:7 or identical with SEQ ID NO:3. NO:7 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% consistent functional variants;The phosphoprotein (P) comprises amino acids as shown in SEQ ID NO:8 or functional variants identical to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:8; the large protein (L) comprises amino acids as shown in SEQ ID NO:9 or functional variants identical to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:9; and the matrix protein (M) comprises amino acids as shown in SEQ ID NO:10 or functional variants identical to SEQ ID NO:8. NO:10 Functional variants with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% consistent accuracy.

[0310] In a more preferred embodiment of the combination therapy, the recombinant rhabdovirus is a recombinant vesicular stomatitis virus encoding at least one CD80 extracellular domain Fc fusion protein or a functional variant thereof, preferably the human CD80 extracellular domain, in its genome, wherein the CD80 extracellular domain Fc fusion protein or a functional variant thereof comprises or is composed of SEQ ID NO:3 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 with SEQ ID NO:3, wherein the gene encoding the recombinant vesicular stomatitis virus glycoprotein G 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.

[0311] Virus generation, production, and virus-producing cells

[0312] The present invention also provides a virus-producing cell, characterized in that the cell produces recombinant rhabdovirus or recombinant vesicular stomatitis virus according to the present invention.

[0313] The cells can be of any origin and can exist in isolated cell form or as cells contained in a cell population. 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 the mammalian cells are pluripotent 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 (CHOs), young hamster kidney (BHK) cells, or Vero cells or bone marrow-derived tumor-infiltrating cells (BM-TICs).

[0314] Alternatively, the virus-producing cells can be human cells, monkey cells, mouse cells, or hamster cells. Those skilled in the art are familiar with methods suitable for testing whether given cells produce virus and whether such specific cells fall within the scope of this invention. In this regard, there is no specific limitation on the amount of virus produced by the cells of this invention. A preferred viral titer in the crude supernatant of the given cell culture after infection and without further downstream processing is ≥1 × 10⁻⁶. 7 TCID50 / ml or ≥1×10 8 One genome copy / ml.

[0315] In a particular embodiment, the virus-producing cell of the present invention is characterized in that the cell comprises one or more expression cartridges for expressing at least one gene selected from the group consisting of: genes n, l, p and m encoding proteins N, L, P and M of VSV, and gene gp encoding LCMV-GP, Dandenong-GP or Mopeya-GP glycoprotein.

[0316] The virus-generating cells in this invention include classic packaging cells for generating recombinant rhabdoviruses from non-replicating vectors and generating cells for generating recombinant rhabdoviruses from replicating vectors. Packaging cells typically contain one or more plastids expressing genes that are lacking in the individual vectors to be packaged and / or essential for virus generation. Such cells are known to those skilled in the art, who can select appropriate cell lines suitable for the desired purpose.

[0317] The recombinant rhabdovirus of the present invention can be generated according to methods known to those skilled in the art, and includes (but is not limited to) using a combination of (1) cDNA transfected into cells, or (2) cDNA transfected into helper cells, or (3) cDNA transfected into cells further infected with helper virus / microvirus to trans-provide the remaining components or activity required to generate infectious or non-infectious recombinant rhabdovirus. When using any of these methods (e.g., helper virus / microvirus, helper cell lines, or cDNA transfection only), the minimum required component is a DNA molecule containing cis-acting signals for (1) capsidating genomic (or antigenomic) RNA via rhabdovirus N, P, and L proteins and (2) replicating genomic or antigenomic (replication intermediate) RNA equivalents.

[0318] The replication assembly, or replicon, is an RNA strand containing at least the leader and tail sequences of a rhabdovirus at both the 5' and 3' ends. Genomically, the leader is located at the 3' end and the tail at the 5' end. Any RNA located between these two replication signals will then be replicated. The leader and tail regions must also contain at least the minimum cis-acting components necessary for initiating transcription and replication, for N protein capsidation and polymerase binding. For the preparation of recombinant rhabdovirus, microviruses containing the G gene also contain a leader region, a tail region, and a G gene with appropriate initiation and termination signals for the production of G protein mRNA. If the microvirus further contains an M gene, appropriate initiation and termination signals for the production of M protein mRNA must also be present.

[0319] For any gene contained within the genome of a recombinant rhabdovirus, the gene is flanked by appropriate transcription initiation and termination signals that will allow expression of those genes and the production of protein products (Schnell et al., Journal of Virology, pp. 2318-2323, 1996). To produce a “non-infectious” recombinant rhabdovirus, the recombinant rhabdovirus must possess a minimum number of replicon components and N, P, and L proteins, and it must contain the M gene. This produces a cell-derived, but non-infectious, viral particle. To produce an “infectious” particle, the viral particle must additionally contain proteins that can mediate viral particle binding and fusion, such as through the use of attachment proteins or receptor ligands. The natural receptor ligand for rhabdoviruses is the G protein.

[0320] Any cell that will allow the assembly of recombinant rhabdoviruses can be used. One method for preparing infectious viral particles involves infecting a suitable cell line with plasmids encoding T7 RNA polymerase or other suitable phage polymerases, such as T3 or SP6 polymerases. The cells are then transfected with individual cDNAs containing genes encoding G, N, P, L, and M rhabdovirus proteins. These cDNAs will provide the proteins used to construct recombinant rhabdovirus particles. Transfection of cells can be performed using any method known in the art.

[0321] The cell line is also transfected with "polycistronic cDNA" containing the equivalent of the rhabdovirus genomic RNA. If the infectious recombinant rhabdovirus particle intends to lyse in the infected cell, it must contain genes encoding the N, P, M, and L proteins, as well as any heterologous nucleic acid segments. If the infectious recombinant rhabdovirus particle does not intend to lyse, the gene encoding the M protein is not included in the polycistronic cDNA. "Polycistronic cDNA" means cDNA containing at least a transcription unit encoding the genes encoding the N, P, and L proteins. The recombinant rhabdovirus polycistronic cDNA may also contain genes encoding protein variants or polypeptide fragments thereof, or therapeutic nucleic acids or proteins. Alternatively, any protein or fragment thereof intended to initially associate with the first generated viral particle may be trans-supplied.

[0322] Polycistronic cDNA containing genes encoding CD80 extracellular domain Fc fusion proteins is also considered. The considered polycistronic cDNA may contain genes encoding protein variants, genes encoding reporter proteins, therapeutic nucleic acids, and / or NPL or NPLM genes. The first step in generating recombinant rhabdovirus is to express RNA as a genomic or antigenomic equivalent of cDNA. This RNA is then packaged by the N protein and subsequently replicated by the P / L protein. The resulting recombinant virus can be recovered. If the G protein is not present in the recombinant RNA genome, it is usually supplied trans-. If neither the G nor the M protein is present, both are supplied trans-. For the preparation of “non-infectious rhabdovirus” particles, the procedure can be the same as above, except that the polycistronic cDNA transfected into cells will contain only the rhabdovirus N, P, and L genes. The polycistronic cDNA of non-infectious rhabdovirus particles may additionally contain genes encoding proteins.

[0323] Transfected cells are typically incubated at the desired temperature, usually around 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 harvested and transferred to fresh cells. The fresh cells are incubated for approximately 48 hours, and the supernatant is collected.

[0324] Other features and advantages of the invention will become apparent from the following more detailed embodiments, which illustrate the principles of the invention by way of example.

[0325] Example

[0326] Example 1

[0327] Generate VSV-GP-huCD80-Fc(IgG1) virus rescue

[0328] The genome of the oncolytic virus VSV-GP was engineered to encode the CD80-Fc gene to locally express the CD80-Fc fusion protein at the tumor site during viral replication. A replicative VSV-GP-CD80-Fc viral variant was generated from bacterial plasmids containing cDNA for the complete VSV-GP viral genome and human CD80-Fc using reverse genetics (cloning of the gene of interest (GOI), viral rescue, and purification of duplicate plaques). The pVSV-GP-CD80-Fc plastid is based on plastid pVSV-XN1 (Schnell et al.) which contains the complete cDNA genome of the VSV Indiana serotype under T7 promoter control. To generate the pVSV-GP-CD80-Fc variant, the entire sequence of the VSV G envelope protein was replaced with a codon-optimized sequence of the GP envelope protein from lymphocytic choriomeningovirus (LCMV, WE-HPI strain). Additionally, the synthetic nucleic acid encoding the CD80-Fc gene was inserted between the glycoprotein GP and the viral polymerase L via Gibson assembly. Transcription of the CD80-Fc gene in a viral infection context was ensured by an additional VSV start signal sequence at the 3' end and an additional stop signal sequence at the 5' end of the CD80-Fc open reading frame.

[0329] HEK293T cells or any other VSV are allowed to recover (or rescue) infectious virus from plasmonic cDNA by transfection using standard transfection methods (e.g., CaPO4 precipitation, liposomal DNA delivery). In short, HEK293T cells are transfected with pSF-CAG-amp-based expression plasmids encoding VSV proteins N, P, and L, and codon-optimized T7-polymerase. Additionally, plasmids encoding viral genomic cDNA of VSV-GP, VSV-GP-CD80-Fc, or variants thereof are co-transfected. In the first step of the rescue process, the T7 polymerase transcribes the viral RNA genome from the plasmid-encoded viral cDNA. In the second step, the viral RNA genome is further amplified by exogenously expressed VSV-L and -P proteins from the co-transfected plasmids. The viral RNA genome is co-transcribed and lichenified by the VSV-N protein. Furthermore, the P / L polymerase complex allows transcription of the entire viral gene product set N, P, M, GP, and L, as well as the inserted CD80-Fc variant. The viral RNA genome was then packaged into infectious VSV particles containing ribonucleoproteins, matrix proteins, and the viral envelope GP. The viral particles were released from the cell via budding.

[0330] First, the rescued virus is passaged in a permissible cell line (such as HEK293T). Several rounds of plaque purification are performed, followed by the preparation of a virus stock using standard methods. In short, HEK293T cells are infected with a series of tenfold dilutions of the rescued pre-seed. After approximately two hours, the cell monolayer is washed twice and overlaid with medium containing 0.8% low-melting-point agarose. Between 24 and 48 hours post-infection, plaques are selected and subjected to another round of plaque purification or virus stock preparation.

[0331] Example 2

[0332] Validation of Viral Adaptability - TCID 50 / Cell killing

[0333] Figure 3A-B

[0334] Infect suspension cultures (freestyle culture medium) with VSV-GP or VSV-GP-CD80-Fc at a low MOI (0.0005). TMHEK293F cells were grown in 293 expression medium (ThermoFisher Scientific). On the day of infection, cell confluence was 60%–70%. One well was counted (Countess™ cell counter, Invitrogen), and the remaining wells were then infected with either the viral construct VSV-GP (GP) or VSV-GP-CD80-Fc at 0.005 MOI. Culture supernatant (3 mL total volume) was harvested, and samples were analyzed at 8 h, 16 h, 24 h, 32 h, 40 h, and 48 h post-infection for viral replication and cell killing. Viral replication was assessed by detecting the viral genome in the culture supernatant at specified time points using qPCR (Figure 3A). Virus-induced cell killing was assessed by counting viable cells in the culture samples at specified time points (Figure 3B). Both viruses expressed the same pattern, indicating that the addition of the human CD80-Fc transgene did not affect viral fitness.

[0335] Example 3

[0336] Load expression - ELISA

[0337] Figure 4

[0338] The supernatant from HEK293 cells infected with VSV-GP-CD80-Fc was analyzed using ELISA at different time points following viral infection. Expression of the virus-encoded CD80-Fc fusion protein in infected mammalian cells was validated by infecting HEK293 cells with either the parental virus VSV-GP or the novel virus VSV-GP-CD80-Fc encoding CD80-Fc using MOI1. As measured by ELISA, expression of the CD80-Fc transgene in the tissue culture supernatant was readily detectable at 24, 31, and 48 hours post-infection.

[0339] Example 4

[0340] VSV-GP-huCD80-Fc (in vivo)-CT26.CL25-IFNARKO tumor model (high load expression)

[0341] Figure 5A-B

[0342] Using the CT26.CL25-IFARKO tumor model, engineered to lack the interferon alpha receptor (IFNAR) to better reflect human patient outcomes and improve viral replication and load expression in the murine system, a back-to-back comparison was performed between the parental virus VSV-GP and a novel virus encoding CD80-Fc, VSV-GP-CD80-Fc. For this purpose, on days 0 and 3 (survival plot), mice with established tumors were compared at 2 × 10⁻⁶ mmol / L.7 TCID 50 Two viruses were administered intravenously (IV). As depicted in Figure (A), the parental virus VSV-GP did not show a significant improvement in survival in tumor-carrying animals at this low viral dose, while treatment with the novel payload-armed virus VSV-GP-CD80-Fc resulted in a significantly better survival benefit than the control and VSV-GP-treated animals. Furthermore, treatment with the CD80-Fc-encoded virus did not cause increased weight loss compared to the control and VSV-GP-treated animals (B), demonstrating the safety of this novel virus.

[0343] Example 5

[0344] VSV-GP-huCD80-Fc (in vivo)-B16-F1-OVA and EMT-6 tumor models (low loading expression)

[0345] Figures 6A-C and 7A-B

[0346] On days 0 and 3, the low-tolerance tumor models B16-F1-OVA (Fig. 6A-C) and EMT-6 (Fig. 7A-B) were treated twice with intratumoral injections of the parental virus VSV-GP or the novel virus encoding CD80-Fc, VSV-GP-CD80-Fc, which allowed only minimal viral replication and thus permitted payload (CD80-Fc) expression. Only mice with well-established tumors were used for injection. Treatment of the B16-F1-OVA tumor model with VSV-GP-CD80-Fc resulted in improved tumor growth delay compared to control and VSV-GP. In EMT-6, tumor model treatment with VSV-GP-CD80-Fc resulted in increased tumor clearance (33% in treated animals) compared to control (0% of treated animals) and VSV-GP (8% of treated animals). Even in tumors with low levels of viral replication and load expression, these results demonstrate the uplift potential of the novel load-armed virus VSV-GP-CD80-Fc relative to its parent virus VSV-GP, which is closely related to the ability of the virus to replicate.

[0347] Example 6

[0348] In vivo viral persistence, replication, and payload expression

[0349] Figure 8-10

[0350] Balb / c mice with established CT26.CL25-IFNARKO (interferon α receptor-deficient CT26.CL25 tumor cells) tumors were used as controls or treated with 1×10 8 TCID 50A single intravenous injection of VSV-GP-CD80-Fc was administered. Tumors were excised three and seven days after treatment; intact RNA was extracted and analyzed using qPCR primers specific to the VSV n gene (Figure 8). C57BL / 6 mice with established LLC1-IFNARKO (interferon α receptor-deficient LLC1 tumor cells) tumors were used as controls, or 1×10⁻⁶ mice were used. 8 TCID 50 A single intravenous injection of VSV-GP or VSV-GP-CD80-Fc was administered. Three days after treatment, tumors were excised, and RNA was analyzed according to the manufacturer's instructions using a NanoString pan-cancer immunoprofiling graph and virus and payload-specific probes (peaks) (Figure 9). In summary, the results showed active replication of VSV-GP-CD80-Fc and expression of the payload mRNA in the tumors of the treated animals. Peak replication was observed around day three. The virus also persisted until at most day seven. C57BL / 6 mice with established LLC1-IFNARKO tumors were used as controls, or 1×10⁻⁶ mice were used. 8 TCID 50 The tumor was treated with a single intravenous injection of either VSV-GP or VSV-GP-CD80-Fc. Three days after treatment, the tumor was excised, fixed in formalin, and embedded in paraffin. Thin fractions were stained with antibodies specific to either VSV-N protein or human CD80 protein (Figure 10). N protein staining was similar for both VSV-GP and VSV-GP-CD80-Fc, while for the latter only human CD80 was specifically detected.

[0351] Example 7

[0352] CD80-Fc provides co-stimulation of T cells in human mixed leukocyte cultures. Blockade of FcγR reduces T cell activation, which is the main function of Fc.

[0353] Figures 12 and 13A-D

[0354] Human mixed leukocyte cultures (co-cultured leukocyte populations selected from two genetically distinct individuals to generate allogeneic T-cell stimulation) were used to evaluate the T-cell co-stimulatory potential of recombinant CD80-Fc (Figs. 12 and 13) and the contribution of wild-type human IgG1 Fc to T-cell co-stimulation (Fig. 13). For this purpose, IFNγ secretion was used as a readout, and the cultures in Fig. 12 were stimulated with increased amounts of recombinant CD80-Fc. IFNγ secretion was significantly improved in a dose-dependent manner by the addition of CD80-Fc to the cultures, thus validating its T-cell co-stimulatory potential. Based on these data and aiming to elucidate the contribution of Fc in the CD80-Fc fusion protein, human mixed leukocyte cultures were again stimulated with recombinant CD80-Fc protein (10 μg / ml) with or without an FcγR inhibitor (in the absence of human serum), and IFNγ was used as a readout (Fig. 13). Different subplots (ADs) depict different donor pairs. Adding an FcγR blocker significantly reduced CD80-Fc-mediated T cell stimulation, indicating that FcγR interaction and FcγR-mediated clustering are crucial for CD80-Fc activity.

[0355] Example 8

[0356] CD3-activated T cell activation in PBMCs via CD80-Fc Fc-dependent FcγR (F(ab)2 and IgG4)

[0357] Figures 14A-F

[0358] Human PBMC cultures were stimulated with low doses of anti-CD3 (clone OKT3; 10 ng / ml) and increased concentrations of recombinant CD80-Fc protein, with or without these doses. IFNγ (Fig. 14A-C) or IL2 (Fig. 14D-F) were used as readings, detected by standard ELISA. To confirm the T-cell co-stimulatory potential of recombinant CD80-Fc and the contribution of wild-type human IgG1 Fc to T-cell co-stimulation, and to validate Fc selection, the following recombinant CD80-Fc variants were compared back-to-back: CD80-Fc (a recombinant with viral load and wild-type human IgG1 Fc, Fig. 14C and F); CD80 FAB (the F(ab)2 variant of CD80-Fc, lacking Fc but retaining bivalent, Fig. 14A and D); and CD80 IgG4 (similar to CD80-Fc but with human IgG4 Fc, Fig. 14B and E). Although CD80-Fc itself did not significantly stimulate PBMCs, the combination of CD80-Fc with stimulating anti-CD3 antibodies resulted in a dose-dependent increase in T cell stimulation, as demonstrated by IFNγ and IL2 secretion (Fig. 14C and F). Conversely, CD80-Fc of the F(ab)2 variant lacking Fc was inactive and did not improve T cell stimulation, either alone or in combination with anti-CD3 stimulation (Fig. 14A and D). IgG4-based CD80 fusions (Fig. 14B and E) showed potential for co-stimulatory T cells, but to a much lower degree than IgG1-based CD80 fusion constructs (Fig. 14B and F), similar to viral loads engineered into the novel virus VSV-GP-CD80-Fc. These results further confirm the FcγR dependence of CD80-Fc and the selectivity of human IgG1 Fc. Furthermore, the absence of CD80-Fc activity without accompanying TCR stimulation demonstrates its favorable safety profile.

[0359] Example 9

[0360] VSV-GP induces a local increase in FcγR within infected tumors loaded with CD80-FcMoA.

[0361] Figure 15

[0362] Given the dependence of viral CD80-Fc load on FcγR in response to T cell co-stimulation, this study aimed to investigate the effect of VSV-GP infection on FcγR expression in tumors. To this end, FcγR expression was measured using NanoString on day 7 post-infection in control or VSV-GP-infected LLC1-IFNARKO tumors. Mice were either untreated or treated with a specific viral dose of 10... 8 TCID 50VSV-GP infection. The X-axis shows measurements of different FcγRs (1, 2b, 3, or 4), and the Y-axis shows relative expression. As the data clearly show, VSV-GP-infected tumors unexpectedly exhibited strong upregulation of expression of all four analyzed FcγRs. These data provide a mechanistic basis for the favorable therapeutic interaction between oncolytic virus VSV-GP and FcγR-dependent, virus-encoded payload CD80-Fc, which facilitates virus-mediated FcγR upregulation within infected tumors.

[0363] Example 10

[0364] VSV-GP-CD80-Fc induces tumor-specific T-cell immunity superior to that induced by the parental virus VSV-GP.

[0365] Figure 16A-C

[0366] The effect of virus-encoded CD80-Fc payload on tumor antigen-specific T cell immunity was elucidated using the CT26.CL25-IFNARKO tumor model, which was treated with either the parental virus VSV-GP or the novel virus VSV-GP-CD80-Fc. Gp70 / tumor-specific T cells were detected in the spleen and blood of the treated mice depicted in Figure 16C by ELISPOT (Figure 16A) and FACS-based Dextramer staining (Figure 16B), respectively. The significant increase in the frequency of tumor antigen-specific T cells in the VSV-GP-CD80-Fc group compared to the VSV-GP group demonstrates the uplift potential of the novel, payload-armed virus relative to the parental virus, and further provides an immunological / mechanistic basis for the improved antitumor activity of the novel oncolytic virus VSV-GP-CD80-Fc.

[0367] Example 11

[0368] CD80-Fc does not interact with PD-L1.

[0369] Figure 17

[0370] It has been argued that CD80 can directly interact with programmed cell death 1 ligand 1 (PD-L1), thereby blocking the inhibitory interaction between PD-L1 and its receptor, programmed cell death 1 (PD-1), on activated T cells. To determine whether the CD80-Fc fusion protein encoded in VSV-GP-CD80-Fc directly interacts with PD-L1, we performed a binding study on CHO-K1 cells stably transfected with human PD-L1. The PD-L1-specific antibody avelumumab was used as a positive control. Although avelumumab readily binds to PD-L1 on the surface of CHO-K1-PD-L1 cells, the recombinant CD80-Fc protein failed to bind to PD-L1 at all tested dose levels. Therefore, we conclude that CD80-Fc does not directly bind to PD-L1.

[0371] Example 12

[0372] α-PD-1 and CD80-Fc improve T cell stimulation in an additive manner

[0373] Figure 18A-B

[0374] To address the question of whether the combination of CD80-Fc-mediated T-cell co-stimulation and antibody-mediated PD-1 inhibition could provide additional benefit relative to monotherapy, we employed a T-cell reporter system in which Jurkat T cells expressing stable PD-1 respond to T-cell receptor (TCR) stimulation by upregulating biochemically detectable luciferase activity. Jurkat-PD-1 reporter cells were co-cultured with FcγR-positive THP1-PD-L1 cells stably expressing PD-L1 (as opposed to FcγR-negative CHO-K1 cells). The interaction between PD-L1 and PD-1 resulted in inhibition of Jurkat T-cell activation, comparable to T-cell suppression observed in cancer patients. TCR stimulation was achieved by adding a bispecific BiTE molecule that links CD33 on THP1 cells to CD3 on Jurkat T cells. As can be seen from Figure 18A, by blocking the inhibitory PD-1:PD-L1 interaction, the PD-1 blocking antibody pembrolizumab was able to restore CD3xCD33 BiTE-mediated T cell stimulation in a dose-dependent manner. Simultaneously and quite unexpectedly, despite the inhibitory PD-1:PD-L1 interaction, CD80-Fc itself provided co-stimulation of T cells and improved Jurkat T cell activation in a dose-dependent manner. A digitoxin (Dig)-specific antibody was used as an isotype control. In Figure 18B, a fixed anti-PD-1 concentration (10 nM, saturated) was combined with increasing concentrations of recombinant CD80-Fc. Adding CD80-Fc to the anti-PD-1 antibody produced superior T cell activation, providing clear evidence for the beneficial interactions of these different treatment modalities, driven by their complementary modes of action.

[0375] Example 13

[0376] VSV-GP-muCD80-Fc (in vivo)-CT26.CL25-IFNARKO tumor model (high load expression)

[0377] Figures 19A-C

[0378] Using the CT26.CL25-IFNARKO tumor model again, this study compared the in vivo efficacy of recombinant murine CD80Fc, VSV-GP, or VSV-GP-muCD80Fc. For this purpose, a viral dose of 1×10⁻⁶ was administered on day 0 and day 3, respectively. 8 TCID 50 Furthermore, mice with established tumors were treated intravenously with 1 mg / kg of recombinant rodent CD80-Fc on days 0, 3, and 6.

[0379] As depicted in Figure 19(A), the survival curve shows improved treatment outcomes in the VSV-GP-muCD80Fc treatment group. The significant survival benefit compared to recombinant CD80Fc protein or VSV-GP can be measured by 1×10⁻⁶. 8 TCID 50 VSV-GP-muCD80Fc displays (Log-rank (Mantel-Cox) test; P-value 0.0394).

[0380] Figure 19(B) depicts an overview of the average tumor size on a single tumor growth curve and reflects the results of using VSV-GP-muCD80Fc (dose 1×10⁻⁶). 8 TCID 50 The treatment showed strong tumor growth inhibition, with expression levels far exceeding those of VSV-GP and 1 mg / kg recombinant CD80Fc protein.

[0381] Weight loss was observed after the first injection of both viruses (Fig. 19(C)), however, recovery occurred immediately, unaffected by subsequent injections, and there was no significant difference between the parent virus (VSV-GP) and the virus encoding muCD80-Fc (VSV-GP-muCD80Fc).

[0382] In summary, this study specifically compared the in vivo effects of recombinant murine CD80Fc or VSV-GP with those of VSV-GP-muCD80Fc. It demonstrated that implementing muCD80Fc in the viral backbone achieved a synergistic effect on tumor growth and overall survival. sequence list <110> Boehringer Ingelheim International Ltd. <120> Recombinant rhabdovirus encoding CD80 extracellular domain Fc fusion protein <130> 01-3411 <160> twenty four <170> BiSSAP 1.3.6 <210> 1 <211> 208 <212> PRT <213> Homo sapiens <400> 1 Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys 1 5 10 15 Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp 20 25 30 Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn 35 40 45 Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn 50 55 60 Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr 65 70 75 80 Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His 85 90 95 Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser 100 105 110 Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys 115 120 125 Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn 130 135 140 Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu 145 150 155 160 Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr 165 170 175 Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn 180 185 190 Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn 195 200 205 <210> 2 <211> 232 <212> PRT <213> Homo sapiens <400> 2 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr ​​​​​​​​​​​​​​​​​​​​​​​​​​​​Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys 20 25 30 Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu 35 40 45 Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile 50 55 60 Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp 65 70 75 80 Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr 85 90 95 Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly 100 105 110 Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg 115 120 125 Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr 130 135 140 Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile 145 150 155 160 Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu 165 170 175 Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp 180 185 190 Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met 195 200 205 Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg 210 215 220 Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro 225 230 235 240 Asp Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly 465 <210> 4 <{211}> 433 <212> PRT <213> Homo sapiens <400> 4 Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys 1 5 10 15 Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp 20 25 30 Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn 35 40 45 Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn 50 55 60 Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr 65 70 75 80 Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His 85 90 95 Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser 100 105 110 Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys 115 120 125 Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn 130 135 140 Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu 145 150 155 160 Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr 165 170 175 Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn 180 185 190 Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asp 195 200 205 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 210 215 220 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 225 230 235 240 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 245 250 255 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 260 265 270 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 275 280 285 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 290 295 300 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 305 310 315 320 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 325 330 335 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 340 345 350 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 355 360 365 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 370 375 380 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 385 390 395 400 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 405 410 415 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 420 425 430 Gly <210> 5 <211> 19 <212> PRT <213> Mouse <400> 5 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser <210> 6 <211> 288 <212> PRT <213> Homo sapiens <400> 6 Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr 1 5 10 15 Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys 20 25 30 Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu 35 40 45 Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile 50 55 60 Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp 65 70 75 80 Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr 85 90 95 Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly 100 105 110 Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg 115 120 125 [[ID=三十八]]Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr 13 ​Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile 145 150 155 160 Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu 165 170 175 Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp 180 185 190 Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met 195 200 205 Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg 210 215 220 Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro 225 230 235 240 Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly 245 250 255 Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg 260 265 270 Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val 275 280 285 <210> 7 <211> 422 <212> PRT <213> Pimple virus <400> 7 Met Ser Val Thr Val Lys Arg Ile Ile Asp Asn Thr Val Val Val Pro 1 5 10 15 Lys Leu Pro Ala Asn Glu Asp Pro Val Glu Tyr Pro Ala Asp Tyr Phe 20 25 30 Arg Lys Ser Lys Glu Ile Pro Leu Tyr Ile Asn Thr Thr Lys Ser Leu 35 40 45 Ser Asp Leu Arg Gly Tyr Val Tyr Gln Gly Leu Lys Ser Gly Asn Val 50 55 60 Ser Ile Ile His Val Asn Ser Tyr Leu Tyr Gly Ala Leu Lys Asp Ile 65 70 75 80 Arg Gly Lys Leu Asp Lys Asp Trp Ser Ser Phe Gly Ile Asn Ile Gly 85 90 95 Lys Ala Gly Asp Thr Ile Gly Ile Phe Asp Leu Val Ser Leu Lys Ala 100 105 110 Leu Asp Gly Val Leu Pro Asp Gly Val Ser Asp Ala Ser Arg Thr Ser 115 120 125 Ala Asp Asp Lys Trp Leu Pro Leu Tyr Leu Leu Gly Leu Tyr Arg Val 130 135 140 Gly Arg Thr Gln Met Pro Glu Tyr Arg Lys Lys Leu Met Asp Gly Leu 145 150 155 160 Thr Asn Gln Cys Lys Met Ile Asn Glu Gln Phe Glu Pro Leu Val Pro 165 170 175 Glu Gly Arg Asp Ile Phe Asp Val Trp Gly Asn Asp Ser Asn Tyr Thr 180 185 190 Lys Ile Val Ala Ala Val Asp Met Phe Phe His Met Phe Lys Lys His 195 200 205 Glu Cys Ala Ser Phe Arg Tyr Gly Thr Ile Val Ser Arg Phe Lys Asp 210 215 220 Cys Ala Ala Leu Ala Thr Phe Gly His Leu Cys Lys Ile Thr Gly Met 225 230 235 240 Ser Thr Glu Asp Val Thr Thr Trp Ile Leu Asn Arg Glu Val Ala Asp 245 250 255 Glu Met Val Gln Met Met Leu Pro Gly Gln Glu Ile Asp Lys Ala Asp 260 265 270 Ser Tyr Met Pro Tyr Leu Ile Asp Phe Gly Leu Ser Ser Lys Ser Pro 275 280 285 Tyr Ser Ser Val Lys Asn Pro Ala Phe His Phe Trp Gly Gln Leu Thr 290 295 300 Ala Leu Leu Leu Arg Ser Thr Arg Ala Arg Asn Ala Arg Gln Pro Asp 305 310 315 320 Asp Ile Glu Tyr Thr Ser Leu Thr Thr Ala Gly Leu Leu Tyr Ala Tyr 325 330 335 Ala Val Gly Ser Ser Ala Asp Leu Ala Gln Gln Phe Cys Val Gly Asp 340 345 350 Asn Lys Tyr Thr Pro Asp Asp Ser Thr Gly Gly Leu Thr Thr Asn Ala 355 360 365 Pro Pro Gln Gly Arg Asp Val Val Glu Trp Leu Gly Trp Phe Glu Asp 370 375 380 Gln Asn Arg Lys Pro Thr Pro Asp Met Met Gln Tyr Ala Lys Arg Ala 385 390 395 400 Val Met Ser Leu Gln Gly Leu Arg Glu Lys Thr Ile Gly Lys Tyr Ala 405 410 415 Lys Ser Glu Phe Asp Lys 420 <210> 8 <211> 265 <212> PRT <213> Herpes virus <400> 8 Met Asp Asn Leu Thr Lys Val Arg Glu Tyr Leu Lys Ser Tyr Ser Arg 1 5 10 15 Leu Asp Gln Ala Val Gly Glu Ile Asp Glu Ile Glu Ala Gln Arg Ala 20 25 30 Glu Lys Ser Asn Tyr Glu Leu Phe Gln Glu Asp Gly Val Glu Glu His 35 40 45 Thr Lys Pro Ser Tyr Phe Gln Ala Ala Asp Asp Ser Asp Thr Glu Ser 50 55 60 Glu Pro Glu Ile Glu Asp Asn Gln Gly Leu Tyr Ala Pro Asp Pro Glu 65 70 75 80 Ala Glu Gln Val Glu Gly Phe Ile Gln Gly Pro Leu Asp Asp Tyr Ala 85 90 95 Asp Glu Glu Val Asp Val Val Phe Thr Ser Asp Trp Lys Gln Pro Glu 100 105 110 Leu Glu Ser Asp Glu His Gly Lys Thr Leu Arg Leu Thr Ser Pro Glu 115 120 125 Gly Leu Ser Gly Glu Gln Lys Ser Gln Trp Leu Ser Thr Ile Lys Ala 130 135 140 Val Val Gln Ser Ala Lys Tyr Trp Asn Leu Ala Glu Cys Thr Phe Glu 145 150 155 160 Ala Ser Gly Glu Gly Val Ile Met Lys Glu Arg Gln Ile Thr Pro Asp 165 170 175 Val Tyr Lys Val Thr Pro Val Met Asn Thr His Pro Ser Gln Ser Glu 180 185 190 Ala Val Ser Asp Val Trp Ser Leu Ser Lys Thr Ser Met Thr Phe Gln 195 200 205 Pro Lys Lys Ala Ser Leu Gln Pro Leu Thr Ile Ser Leu Asp Glu Leu 210 215 220 Phe Ser Ser Arg Gly Glu Phe Ile Ser Val Gly Gly Asp Gly Arg Met 225 230 235 240 Ser His Lys Glu Ala Ile Leu Leu Gly Leu Arg Tyr Lys Lys Leu Tyr 245 250 255 Asn Gln Ala Arg Val Lys Tyr Ser Leu 260 265 <210> 9 <211> 2109 <212> PRT <213> Pimple virus <400> 9 Met Glu Val His Asp Phe Glu Thr Asp Glu Phe Asn Asp Phe Asn Glu 1 5 10 15 Asp Asp Tyr Ala Thr Arg Glu Phe Leu Asn Pro Asp Glu Arg Met Thr 20 25 30 Tyr Leu Asn His Ala Asp Tyr Asn Leu Asn Ser Pro Leu Ile Ser Asp 35 40 45 Asp Ile Asp Asn Leu Ile Arg Lys Phe Asn Ser Leu Pro Ile Pro Ser 50 55 60 Met Trp Asp Ser Lys Asn Trp Asp Gly Val Leu Glu Met Leu Thr Ser 65 70 75 80 Cys Gln Ala Asn Pro Ile Pro Thr Ser Gln Met His Lys Trp Met Gly 85 90 95 Ser Trp Leu Met Ser Asp Asn His Asp Ala Ser Gln Gly Tyr Ser Phe 100 105 110 Leu His Glu Val Asp Lys Glu Ala Glu Ile Thr Phe Asp Val Val Glu 115 120 125 Thr Phe Ile Arg Gly Trp Gly Asn Lys Pro Ile Glu Tyr Ile Lys Lys 130 135 140 Glu Arg Trp Thr Asp Ser Phe Lys Ile Leu Ala Tyr Leu Cys Gln Lys 145 150 155 160 Phe Leu Asp Leu His Lys Leu Thr Leu Ile Leu Asn Ala Val Ser Glu 165 170 175 Val Glu Leu Leu Asn Leu Ala Arg Thr Phe Lys Gly Lys Val Arg Arg 180 185 190 Ser Ser His Gly Thr Asn Ile Cys Arg Ile Arg Val Pro Ser Leu Gly 195 200 205 Pro Thr Phe Ile Ser Glu Gly Trp Ala Tyr Phe Lys Lys Leu Asp Ile 210 215 220 Leu Met Asp Arg Asn Phe Leu Leu Met Val Lys Asp Val Ile Ile Gly 225 230 235 240 Arg Met Gln Thr Val Leu Ser Met Val Cys Arg Ile Asp Asn Leu Phe 245 250 255 Ser Glu Gln Asp Ile Phe Ser Leu Leu Asn Ile Tyr Arg Ile Gly Asp 260 265 270 Lys Ile Val Glu Arg Gln Gly Asn Phe Ser Tyr Asp Leu Ile Lys Met 275 280 285 Val Glu Pro Ile Cys Asn Leu Lys Leu Met Lys Leu Ala Arg Glu Ser 290 295 300 Arg Pro Leu Val Pro Gln Phe Pro His Phe Glu Asn His Ile Lys Thr 305 310 315 320 Ser Val Asp Glu Gly Ala Lys Ile Asp Arg Gly Ile Arg Phe Leu His 325 330 335 Asp Gln Ile Met Ser Val Lys Thr Val Asp Leu Thr Leu Val Ile Tyr 340 345 350 Gly Ser Phe Arg His Trp Gly His Pro Phe Ile Asp Tyr Tyr Thr Gly 355 360 365 Leu Glu Lys Leu His Ser Gln Val Thr Met Lys Lys Asp Ile Asp Val 370 375 380 Ser Tyr Ala Lys Ala Leu Ala Ser Asp Leu Ala Arg Ile Val Leu Phe 385 390 395 400 Gln Gln Phe Asn Asp His Lys Lys Trp Phe Val Asn Gly Asp Leu Leu 405 410 415 Pro His Asp His Pro Phe Lys Ser His Val Lys Glu Asn Thr Trp Pro 420 425 430 Thr Ala Ala Gln Val Gln Asp Phe Gly Asp Lys Trp His Glu Leu Pro 435 440 445 Leu Ile Lys Cys Phe Glu Ile Pro Asp Leu Leu Asp Pro Ser Ile Ile 450 455 460 Tyr Ser Asp Lys Ser His Ser Met Asn Arg Ser Glu Val Leu Lys His 465 470 475 480 Val Arg Met Asn Pro Asn Thr Pro Ile Pro Ser Lys Lys Val Leu Gln 485 490 495 Thr Met Leu Asp Thr Lys Ala Thr Asn Trp Lys Glu Phe Leu Lys Glu 500 505 510 Ile Asp Glu Lys Gly Leu Asp Asp Asp Asp Leu Ile Ile Gly Leu Lys 515 520 525 Gly Lys Glu Arg Glu Leu Lys Leu Ala Gly Arg Phe Phe Ser Leu Met 530 535 540 Ser Trp Lys Leu Arg Glu Tyr Phe Val Ile Thr Glu Tyr Leu Ile Lys 545 550 555 560 Thr His Phe Val Pro Met Phe Lys Gly Leu Thr Met Ala Asp Asp Leu 565 570 575 Thr Ala Val Ile Lys Lys Met Leu Asp Ser Ser Ser Gly Gln Gly Leu 580 585 590 Lys Ser Tyr Glu Ala Ile Cys Ile Ala Asn His Ile Asp Tyr Glu Lys 595 600 605 Trp Asn Asn His Gln Arg Lys Leu Ser Asn Gly Pro Val Phe Arg Val 610 615 620 Met Gly Gln Phe Leu Gly Tyr Pro Ser Leu Ile Glu Arg Thr His Glu 625 630 635 640 Phe Phe Glu Lys Ser Leu Ile Tyr Tyr Asn Gly Arg Pro Asp Leu Met 645 650 655 Arg Val His Asn Asn Thr Leu Ile Asn Ser Thr Ser Gln Arg Val Cys 660 665 670 Trp Gln Gly Gln Glu Gly Gly Leu Glu Gly Leu Arg Gln Lys Gly Trp 675 680 685 Ser Ile Leu Asn Leu Leu Val Ile Gln Arg Glu Ala Lys Ile Arg Asn 690 695 700 Thr Ala Val Lys Val Leu Ala Gln Gly Asp Asn Gln Val Ile Cys Thr 705 710 715 720 Gln Tyr Lys Thr Lys Lys Ser Arg Asn Val Val Glu Leu Gln Gly Ala 725 730 735 Leu Asn Gln Met Val Ser Asn Asn Glu Lys Ile Met Thr Ala Ile Lys 740 745 750 Ile Gly Thr Gly Lys Leu Gly Leu Leu Ile Asn Asp Asp Glu Thr Met 755 760 765 Gln Ser Ala Asp Tyr Leu Asn Tyr Gly Lys Ile Pro Ile Phe Arg Gly 770 775 780 Val Ile Arg Gly Leu Glu Thr Lys Arg Trp Ser Arg Val Thr Cys Val 785 790 795 800 Thr Asn Asp Gln Ile Pro Thr Cys Ala Asn Ile Met Ser Ser Val Ser 805 810 815 Thr Asn Ala Leu Thr Val Ala His Phe Ala Glu Asn Pro Ile Asn Ala 820 825 830 Met Ile Gln Tyr Asn Tyr Phe Gly Thr Phe Ala Arg Leu Leu Leu Met 835 840 845 Met His Asp Pro Ala Leu Arg Gln Ser Leu Tyr Glu Val Gln Asp Lys 850 855 860 Ile Pro Gly Leu His Ser Ser Thr Phe Lys Tyr Ala Met Leu Tyr Leu 865 870 875 880 Asp Pro Ser Ile Gly Gly Val Ser Gly Met Ser Leu Ser Arg Phe Leu 885 890 895 Ile Arg Ala Phe Pro Asp Pro Val Thr Glu Ser Leu Ser Phe Trp Arg 900 905 910 Phe Ile His Val His Ala Arg Ser Glu His Leu Lys Glu Met Ser Ala 915 920 925 Val Phe Gly Asn Pro Glu Ile Ala Lys Phe Arg Ile Thr His Ile Asp 930 935 940 Lys Leu Val Glu Asp Pro Thr Ser Leu Asn Ile Ala Met Gly Met Ser 945 950 955 960 Pro Ala Asn Leu Leu Lys Thr Glu Val Lys Lys Cys Leu Ile Glu Ser 965 970 975 Arg Gln Thr Ile Arg Asn Gln Val Ile Lys Asp Ala Thr Ile Tyr Leu 980 985 990 Tyr His Glu Glu Asp Arg Leu Arg Ser Phe Leu Trp Ser Ile Asn Pro 995 1000 1005 Leu Phe Pro Arg Phe Leu Ser Glu Phe Lys Ser Gly Thr Phe Leu Gly 1010 1015 1020 Val Ala Asp Gly Leu Ile Ser Leu Phe Gln Asn Ser Arg Thr Ile Arg 1025 1030 1035 1040 Asn Ser Phe Lys Lys Lys Tyr His Arg Glu Leu Asp Asp Leu Ile Val 1045 1050 1055 Arg Ser Glu Val Ser Ser Leu Thr His Leu Gly Lys Leu His Leu Arg 1060 1065 1070 Arg Gly Ser Cys Lys Met Trp Thr Cys Ser Ala Thr His Ala Asp Thr 1075 1080 1085 Leu Arg Tyr Lys Ser Trp Gly Arg Thr Val Ile Gly Thr Thr Val Pro 1090 1095 1100 His Pro Leu Glu Met Leu Gly Pro Gln His Arg Lys Glu Thr Pro Cys 1105 1110 1115 1120 Ala Pro Cys Asn Thr Ser Gly Phe Asn Tyr Val Ser Val His Cys Pro 1125 1130 1135 Asp Gly Ile His Asp Val Phe Ser Ser Arg Gly Pro Leu Pro Ala Tyr 1140 1145 1150 Leu Gly Ser Lys Thr Ser Glu Ser Thr Ser Ile Leu Gln Pro Trp Glu 1155 1160 1165 Arg Glu Ser Lys Val Pro Leu Ile Lys Arg Ala Thr Arg Leu Arg Asp 1170 1175 1180 Ala Ile Ser Trp Phe Val Glu Pro Asp Ser Lys Leu Ala Met Thr Ile 1185 1190 1195 1200 Leu Ser Asn Ile His Ser Leu Thr Gly Glu Glu Trp Thr Lys Arg Gln 1205 1210 1215 His Gly Phe Lys Arg Thr Gly Ser Ala Leu His Arg Phe Ser Thr Ser 1220 1225 1230 Arg Met Ser His Gly Gly Phe Ala Ser Gln Ser Thr Ala Ala Leu Thr 1235 1240 1245 Arg Leu Met Ala Thr Thr Asp Thr Met Arg Asp Leu Gly Asp Gln Asn 1250 1255 1260 Phe Asp Phe Leu Phe Gln Ala Thr Leu Leu Tyr Ala Gln Ile Thr Thr 1265 1270 1275 1280 Thr Val Ala Arg Asp Gly Trp Ile Thr Ser Cys Thr Asp His Tyr His 1285 1290 1295 Ile Ala Cys Lys Ser Cys Leu Arg Pro Ile Glu Glu Ile Thr Leu Asp 1300 1305 1310 Ser Ser Met Asp Tyr Thr Pro Pro Asp Val Ser His Val Leu Lys Thr 1315 1320 1325 Trp Arg Asn Gly Glu Gly Ser Trp Gly Gln Glu Ile Lys Gln Ile Tyr 1330 1335 1340 Pro Leu Glu Gly Asn Trp Lys Asn Leu Ala Pro Ala Glu Gln Ser Tyr 1345 1350 1355 1360 Gln Val Gly Arg Cys Ile Gly Phe Leu Tyr Gly Asp Leu Ala Tyr Arg 1365 1370 1375 Lys Ser Thr His Ala Glu Asp Ser Ser Leu Phe Pro Leu Ser Ile Gln 1380 1385 1390 Gly Arg Ile Arg Gly Arg Gly Phe Leu Lys Gly Leu Leu Asp Gly Leu 1395 1400 1405 Met Arg Ala Ser Cys Cys Gln Val Ile His Arg Arg Ser Leu Ala His 1410 1415 1420 Leu Lys Arg Pro Ala Asn Ala Val Tyr Gly Gly Leu Ile Tyr Leu Ile 1425 1430 1435 1440 Asp Lys Leu Ser Val Ser Pro Pro Phe Leu Ser Leu Thr Arg Ser Gly 1445 1450 1455 Pro Ile Arg Asp Glu Leu Glu Thr Ile Pro His Lys Ile Pro Thr Ser 1460 1465 1470 Tyr Pro Thr Ser Asn Arg Asp Met Gly Val Ile Val Arg Asn Tyr Phe 1475 1480 1485 Lys Tyr Gln Cys Arg Leu Ile Glu Lys Gly Lys Tyr Arg Ser His Tyr 1490 1495 1500 Ser Gln Leu Trp Leu Phe Ser Asp Val Leu Ser Ile Asp Phe Ile Gly 1505 1510 1515 1520 Pro Phe Ser Ile Ser Thr Thr Leu Leu Gln Ile Leu Tyr Lys Pro Phe 1525 1530 1535 Leu Ser Gly Lys Asp Lys Asn Glu Leu Arg Glu Leu Ala Asn Leu Ser 1540 1545 1550 Ser Leu Leu Arg Ser Gly Glu Gly Trp Glu Asp Ile His Val Lys Phe 1555 1560 1565 Phe Thr Lys Asp Ile Leu Leu Cys Pro Glu Glu Ile Arg His Ala Cys 1570 1575 1580 Lys Phe Gly Ile Ala Lys Asp Asn Asn Lys Asp Met Ser Tyr Pro Pro 1585 1590 1595 1600 Trp Gly Arg Glu Ser Arg Gly Thr Ile Thr Thr Ile Pro Val Tyr Tyr 1605 1610 1615 Thr Thr Thr Pro Tyr Pro Lys Met Leu Glu Met Pro Pro Arg Ile Gln 1620 1625 1630 Asn Pro Leu Leu Ser Gly Ile Arg Leu Gly Gln Leu Pro Thr Gly Ala 1635 1640 1645 His Tyr Lys Ile Arg Ser Ile Leu His Gly Met Gly Ile His Tyr Arg 1650 1655 1660 Asp Phe Leu Ser Cys Gly Asp Gly Ser Gly Gly Met Thr Ala Ala Leu 1665 1670 1675 1680 Leu Arg Glu Asn Val His Ser Arg Gly Ile Phe Asn Ser Leu Leu Glu 1685 1690 1695 Leu Ser Gly Ser Val Met Arg Gly Ala Ser Pro Glu Pro Pro Ser Ala 1700 1705 1710 Leu Glu Thr Leu Gly Gly Asp Lys Ser Arg Cys Val Asn Gly Glu Thr 1715 1720 1725 Cys Trp Glu Tyr Pro Ser Asp Leu Cys Asp Pro Arg Thr Trp Asp Tyr 1730 1735 1740 Phe Leu Arg Leu Lys Ala Gly Leu Gly Leu Gln Ile Asp Leu Ile Val 1745 1750 1755 1760 Met Asp Met Glu Val Arg Asp Ser Ser Thr Ser Leu Lys Ile Glu Thr 1765 1770 1775 Asn Val Arg Asn Tyr Val His Arg Ile Leu Asp Glu Gln Gly Val Leu 1780 1785 1790 Ile Tyr Lys Thr Tyr Gly Thr Tyr Ile Cys Glu Ser Glu Lys Asn Ala 1795 1800 1805 Val Thr Ile Leu Gly Pro Met Phe Lys Thr Val Asp Leu Val Gln Thr 1810 1815 1820 Glu Phe Ser Ser Ser Gln Thr Ser Glu Val Tyr Met Val Cys Lys Gly 1825 1830 1835 1840 Leu Lys Lys Leu Ile Asp Glu Pro Asn Pro Asp Trp Ser Ser Ile Asn 1845 1850 1855 Glu Ser Trp Lys Asn Leu Tyr Ala Phe Gln Ser Ser Glu Gln Glu Phe 1860 1865 1870 Ala Arg Ala Lys Lys Val Ser Thr Tyr Phe Thr Leu Thr Gly Ile Pro 1875 1880 1885 Ser Gln Phe Ile Pro Asp Pro Phe Val Asn Ile Glu Thr Met Leu Gln 1890 1895 1900 Ile Phe Gly Val Pro Thr Gly Val Ser His Ala Ala Ala Leu Lys Ser 1905 1910 1915 1920 Ser Asp Arg Pro Ala Asp Leu Leu Thr Ile Ser Leu Phe Tyr Met Ala 1925 1930 1935 Ile Ile Ser Tyr Tyr Asn Ile Asn His Ile Arg Val Gly Pro Ile Pro 1940 1945 1950 Pro Asn Pro Pro Ser Asp Gly Ile Ala Gln Asn Val Gly Ile Ala Ile 1955 1960 1965 Thr Gly Ile Ser Phe Trp Leu Ser Leu Met Glu Lys Asp Ile Pro Leu 1970 1975 1980 Tyr Gln Gln Cys Leu Ala Val Ile Gln Gln Ser Phe Pro Ile Arg Trp 1985 1990 1995 2000 Glu Ala Val Ser Val Lys Gly Gly Tyr Lys Gln Lys Trp Ser Thr Arg 2005 2010 2015 Gly Asp Gly Leu Pro Lys Asp Thr Arg Ile Ser Asp Ser Leu Ala Pro 2020 2025 2030 Ile Gly Asn Trp Ile Arg Ser Leu Glu Leu Val Arg Asn Gln Val Arg 2035 2040 2045 Leu Asn Pro Phe Asn Glu Ile Leu Phe Asn Gln Leu Cys Arg Thr Val 2050 2055 2060 Asp Asn His Leu Lys Trp Ser Asn Leu Arg Arg Asn Thr Gly Met Ile 2065 2070 2075 2080 Glu Trp Ile Asn Arg Arg Ile Ser Lys Glu Asp Arg Ser Ile Leu Met 2085 2090 2095 Leu Lys Ser Asp Leu His Glu Glu Asn Ser Trp Arg Asp 2100 2105 <210> 10 <211> 229 <212> PRT <213> Vesicular stomatitis virus <400> 10 Met Ser Ser Leu Lys Lys Ile Leu Gly Leu Lys Gly Lys Gly Lys Lys 1 5 10 15 Ser Lys Lys Leu Gly Ile Ala Pro Pro Pro Tyr Glu Glu Asp Thr Ser 20 25 30 Met Glu Tyr Ala Pro Ser Ala Pro Ile Asp Lys Ser Tyr Phe Gly Val 35 40 45 Asp Glu Met Asp Thr Tyr Asp Pro Asn Gln Leu Arg Tyr Glu Lys Phe 50 55 60 Phe Phe Thr Val Lys Met Thr Val Arg Ser Asn Arg Pro Phe Arg Thr 65 70 75 80 Tyr Ser Asp Val Ala Ala Ala Val Ser His Trp Asp His Met Tyr Ile 85 90 95 Gly Met Ala Gly Lys Arg Pro Phe Tyr Lys Ile Leu Ala Phe Leu Gly 100 105 110 Ser Ser Asn Leu Lys Ala Thr Pro Ala Val Leu Ala Asp Gln Gly Gln 115 120 125 Pro Glu Tyr His Ala His Cys Glu Gly Arg Ala Tyr Leu Pro His Arg 130 135 140 Met Gly Lys Thr Pro Pro Met Leu Asn Val Pro Glu His Phe Arg Arg 145 150 155 160 Pro Phe Asn Ile Gly Leu Tyr Lys Gly Thr Ile Glu Leu Thr Met Thr 165 170 175 Ile Tyr Asp Asp Glu Ser Leu Glu Ala Ala Pro Met Ile Trp Asp His 180 185 190 Phe Asn Ser Ser Lys Phe Ser Asp Phe Arg Glu Lys Ala Leu Met Phe 195 200 205 Gly Leu Ile Val Glu Lys Lys Ala Ser Gly Ala Trp Val Leu Asp Ser 210 215 220 Ile Gly His Phe Lys 225 <210> 11 <211> 498 <212> PRT <213> Lymphocytic choroid plexus meningitis virus <400> 11 Met Gly Gln Ile Val Thr Met Phe Glu Ala Leu Pro His Ile Ile Asp 1 5 10 15 Glu Val Ile Asn Ile Val Ile Ile Val Leu Ile Ile Ile Thr Ser Ile 20 25 30 Lys Ala Val Tyr Asn Phe Ala Thr Cys Gly Ile Leu Ala Leu Val Ser 35 40 45 Phe Leu Phe Leu Ala Gly Arg Ser Cys Gly Met Tyr Gly Leu Asn Gly 50 55 60 Pro Asp Ile Tyr Lys Gly Val Tyr Gln Phe Lys Ser Val Glu Phe Asp 65 70 75 80 Met Ser His Leu Asn Leu Thr Met Pro Asn Ala Cys Ser Ala Asn Asn 85 90 95 Ser His His Tyr Ile Ser Met Gly Ser Ser Gly Leu Glu Leu Thr Phe 100 105 110 Thr Asn Asp Ser Ile Leu Asn His Asn Phe Cys Asn Leu Thr Ser Ala 115 120 125 Phe Asn Lys Lys Thr Phe Asp His Thr Leu Met Ser Ile Val Ser Ser 130 135 140 Leu His Leu Ser Ile Arg Gly Asn Ser Asn His Lys Ala Val Ser Cys 145 150 155 160 Asp Phe Asn Asn Gly Ile Thr Ile Gln Tyr Asn Leu Ser Phe Ser Asp 165 170 175 Pro Gln Ser Ala Ile Ser Gln Cys Arg Thr Phe Arg Gly Arg Val Leu 180 185 190 Asp Met Phe Arg Thr Ala Phe Gly Gly Lys Tyr Met Arg Ser Gly Trp 195 200 205 Gly Trp Ala Gly Ser Asp Gly Lys Thr Thr Trp Cys Ser Gln Thr Ser 210 215 220 Tyr Gln Tyr Leu Ile Ile Gln Asn Arg Thr Trp Glu Asn His Cys Arg 225 230 235 240 Tyr Ala Gly Pro Phe Gly Met Ser Arg Ile Leu Phe Ala Gln Glu Lys 245 250 255 Thr Lys Phe Leu Thr Arg Arg Leu Ala Gly Thr Phe Thr Trp Thr Leu 260 265 270 Ser Asp Ser Ser Gly Val Glu Asn Pro Gly Gly Tyr Cys Leu Thr Lys 275 280 285 Trp Met Ile Leu Ala Ala Glu Leu Lys Cys Phe Gly Asn Thr Ala Val 290 295 300 Ala Lys Cys Asn Val Asn His Asp Glu Glu Phe Cys Asp Met Leu Arg 305 310 315 320 Leu Ile Asp Tyr Asn Lys Ala Ala Leu Ser Lys Phe Lys Gln Asp Val 325 330 335 Glu Ser Ala Leu His Val Phe Lys Thr Thr Val Asn Ser Leu Ile Ser 340 345 350 Asp Gln Leu Leu Met Arg Asn His Leu Arg Asp Leu Met Gly Val Pro 355 360 365 Tyr Cys Asn Tyr Ser Lys Phe Trp Tyr Leu Glu His Ala Lys Thr Gly 370 375 380 Glu Thr Ser Val Pro Lys Cys Trp Leu Val Thr Asn Gly Ser Tyr Leu 385 390 395 400 Asn Glu Thr His Phe Ser Asp Gln Ile Glu Gln Glu Ala Asp Asn Met 405 410 415 Ile Thr Glu Met Leu Arg Lys Asp Tyr Ile Lys Arg Gln Gly Ser Thr 420 425 430 Pro Leu Ala Leu Met Asp Leu Leu Met Phe Ser Thr Ser Ala Tyr Leu 435 440 445 Ile Ser Ile Phe Leu His Leu Val Lys Ile Pro Thr His Arg His Ile 450 455 460 Lys Gly Gly Ser Cys Pro Lys Pro His Arg Leu Thr Asn Lys Gly Ile 465 470 475 480 Cys Ser Cys Gly Ala Phe Lys Val Pro Gly Val Lys Thr Ile Trp Lys 485 490 495 Arg Arg <210> 12 <211> 498 <212> PRT <213> Dandenong virus <400> 12 Met Gly Gln Leu Ile Thr Met Phe Glu Ala Leu Pro His Ile Ile Asp 1 5 10 15 Glu Val Ile Asn Ile Val Ile Ile Val Leu Val Ile Ile Thr Ser Ile 20 25 30 Lys Ala Val Tyr Asn Phe Ala Thr Cys Gly Ile Ile Ala Leu Ile Ser 35 40 45 Phe Cys Leu Leu Ala Gly Arg Ser Cys Gly Leu Tyr Gly Val Thr Gly 50 55 60 Pro Asp Ile Tyr Lys Gly Leu Tyr Gln Phe Lys Ser Val Glu Phe Asn 65 70 75 80 Met Ser Gln Leu Asn Leu Thr Met Pro Asn Ala Cys Ser Ala Asn Asn 85 90 95 Ser His His Tyr Ile Ser Met Gly Lys Ser Gly Leu Glu Leu Thr Phe 100 105 110 Thr Asn Asp Ser Ile Ile Ser His Asn Phe Cys Asn Leu Thr Asp Gly 115 120 125 Phe Lys Lys Lys Thr Phe Asp His Thr Leu Met Ser Ile Val Ala Ser 130 135 140 Leu His Leu Ser Ile Arg Gly Asn Thr Asn Tyr Lys Ala Val Ser Cys 145 150 155 160 Asp Phe Asn Asn Gly Ile Thr Ile Gln Tyr Asn Leu Ser Phe Ser Asp 165 170 175 Ala Gln Ser Ala Ile Asn Gln Cys Arg Thr Phe Arg Gly Arg Val Leu 180 185 190 Asp Met Phe Arg Thr Ala Phe Gly Gly Lys Tyr Met Arg Ser Gly Tyr 195 200 205 Gly Trp Lys Gly Ser Asp Gly Lys Thr Thr Trp Cys Ser Gln Thr Ser 210 215 220 Tyr Gln Tyr Leu Ile Ile Gln Asn Arg Thr Trp Glu Asn His Cys Glu 225 230 235 240 Tyr Ala Gly Pro Phe Gly Leu Ser Arg Val Leu Phe Ala Gln Glu Lys 245 250 255 Thr Lys Phe Leu Thr Arg Arg Leu Ala Gly Thr Phe Thr Trp Thr Leu 260 265 270 Ser Asp Ser Ser Gly Thr Glu Asn Pro Gly Gly Tyr Cys Leu Thr Lys 275 280 285 Trp Met Leu Ile Ala Ala Glu Leu Lys Cys Phe Gly Asn Thr Ala Val 290 295 300 Ala Lys Cys Asn Ile Asn His Asp Glu Glu Phe Cys Asp Met Leu Arg 305 310 315 320 Leu Ile Asp Tyr Asn Lys Ala Ala Leu Lys Lys Phe Lys Glu Asp Val 325 330 335 Glu Ser Ala Leu His Leu Phe Lys Thr Thr Val Asn Ser Leu Ile Ser 340 345 350 Asp Gln Leu Leu Met Arg Asn His Leu Arg Asp Leu Met Gly Val Pro 355 360 365 Tyr Cys Asn Tyr Ser Lys Phe Trp Tyr Leu Glu His Val Lys Thr Gly 370 375 380 Asp Thr Ser Val Pro Lys Cys Trp Leu Val Ser Asn Gly Ser Tyr Leu 385 390 395 400 Asn Glu Thr His Phe Ser Asp Gln Ile Glu Gln Glu Ala Asp Asn Met 405 410 415 Ile Thr Glu Met Leu Arg Lys Asp Tyr Ile Lys Arg Gln Gly Ser Thr 420 425 430 Pro Leu Ala Leu Met Asp Leu Leu Met Phe Ser Thr Ser Ala Tyr Leu 435 440 445 Ile Ser Val Phe Leu His Leu Met Lys Ile Pro Thr His Arg His Ile 450 455 460 Lys Gly Gly Thr Cys Pro Lys Pro His Arg Leu Thr Ser Lys Gly Ile 465 470 475 480 Cys Ser Cys Gly Ala Phe Lys Val Pro Gly Val Lys Thr Val Trp Lys 485 490 495 Arg Arg <210> 13 <211> 489 <212> PRT <213> Mopeia virus <400> 13 Met Gly Gln Ile Val Thr Phe Phe Gln Glu Val Pro His Ile Leu Glu 1 5 10 15 Glu Val Met Asn Ile Val Leu Met Thr Leu Ser Ile Leu Ala Ile Leu 20 25 30 Lys Gly Ile Tyr Asn Val Met Thr Cys Gly Ile Ile Gly Leu Ile Thr 35 40 45 Phe Leu Phe Leu Cys Gly Arg Ser Cys Ser Ser Ile Tyr Lys Asp Asn 50 55 60 Tyr Glu Phe Phe Ser Leu Asp Leu Asp Met Ser Ser Leu Asn Ala Thr 65 70 75 80 Met Pro Leu Ser Cys Ser Lys Asn Asn Ser His His Tyr Ile Gln Val 85 90 95 Gly Asn Glu Thr Gly Leu Glu Leu Thr Leu Thr Asn Thr Ser Ile Ile 100 105 110 Asp His Lys Phe Cys Asn Leu Ser Asp Ala His Arg Arg Asn Leu Tyr 115 120 125 Asp Lys Ala Leu Met Ser Ile Leu Thr Thr Phe His Leu Ser Ile Pro 130 135 140 Asp Phe Asn Gln Tyr Glu Ala Met Ser Cys Asp Phe Asn Gly Gly Lys 145 150 155 160 Ile Ser Ile Gln Tyr Asn Leu Ser His Ser Asn Tyr Val Asp Ala Gly 165 170 175 Asn His Cys Gly Thr Ile Ala Asn Gly Ile Met Asp Val Phe Arg Arg 180 185 190 Met Tyr Trp Ser Thr Ser Leu Ser Val Ala Ser Asp Ile Ser Gly Thr 195 200 205 Gln Cys Ile Gln Thr Asp Tyr Lys Tyr Leu Ile Ile Gln Asn Thr Ser 210 215 220 Trp Glu Asp His Cys Met Phe Ser Arg Pro Ser Pro Met Gly Phe Leu 225 230 235 240 Ser Leu Leu Ser Gln Arg Thr Arg Asn Phe Tyr Ile Ser Arg Arg Leu 245 250 255 Leu Gly Leu Phe Thr Trp Thr Leu Ser Asp Ser Glu Gly Asn Asp Met 260 265 270 Pro Gly Gly Tyr Cys Leu Thr Arg Ser Met Leu Ile Gly Leu Asp Leu 275 280 285 Lys Cys Phe Gly Asn Thr Ala Ile Ala Lys Cys Asn Gln Ala His Asp 290 295 300 Glu Glu Phe Cys Asp Met Leu Arg Leu Phe Asp Phe Asn Lys Gln Ala 305 310 315 320 Ile Ser Lys Leu Arg Ser Glu Val Gln Gln Ser Ile Asn Leu Ile Asn 325 330 335 Lys Ala Val Asn Ala Leu Ile Asn Asp Gln Leu Val Met Arg Asn His 340 345 350 Leu Arg Asp Leu Met Gly Ile Pro Tyr Cys Asn Tyr Ser Lys Phe Trp 355 360 365 Tyr Leu Asn Asp Thr Arg Thr Gly Arg Thr Ser Leu Pro Lys Cys Trp 370 375 380 Leu Val Thr Asn Gly Ser Tyr Leu Asn Glu Thr Gln Phe Ser Thr Glu 385 390 395 400 Ile Glu Gln Glu Ala Asn Asn Met Phe Thr Asp Met Leu Arg Lys Glu 405 410 415 Tyr Glu Lys Arg Gln Ser Thr Thr Pro Leu Gly Leu Val Asp Leu Phe 420 425 430 Val Phe Ser Thr Ser Phe Tyr Leu Ile Ser Val Phe Leu His Leu Ile 435 440 445 Lys Ile Pro Thr His Arg His Ile Lys Gly Lys Pro Cys Pro Lys Pro 450 455 460 His Arg Leu Asn His Met Ala Ile Cys Ser Cys Gly Phe Tyr Lys Gln 465 470 475 480 Pro Gly Leu Pro Thr Gln Trp Lys Arg 485 <210> 14 <211> 446 <212> PRT <213> Homo sapiens <400> 14 Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Ala Ser 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Gly Gly Gly Gly Asp Thr Tyr Tyr Ser Ser Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Ser Asn Val Asn Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 <210> 15 <211> 218 <212> PRT <213> Homo sapiens <400> 15 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Met Ser Cys Arg Ala Ser Glu Asn Ile Asp Thr Ser 20 25 30 Gly Ile Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Val Ala Ser Asn Gln Gly Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 16 <211> 446 <212> PRT <213> Homo sapiens <400> 16 Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Ala Ser 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Gly Gly Gly Gly Asp Thr Tyr Tyr Ser Ser Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Ser Asn Pro Asn Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 <210> 17 <211> 218 <212> PRT <213> Homo sapiens <400> 17 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Met Ser Cys Arg Ala Ser Glu Asn Ile Asp Thr Ser 20 25 30 Gly Ile Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Val Ala Ser Asn Gln Gly Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser $65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg<00\01901>100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 脯氨酸-精氨酸-谷氨酸-丙氨酸-赖氨酸-缬氨酸-谷氨酰胺-色氨酸-赖氨酸-缬氨酸-天冬酰胺-丙氨酸-亮氨酸-谷氨酰胺-丝氨酸 145 150 155 160 甘氨酸-天冬酰胺-丝氨酸-谷氨酰胺-谷氨酸-丝氨酸-缬氨酸-苏氨酸-谷氨酸-谷氨酰胺-天冬氨酸-丝氨酸-赖氨酸-天冬氨酸-丝氨酸-苏氨酸 165 170 175 酪氨酸-丝氨酸-亮氨酸-丝氨酸-丝氨酸-苏氨酸-亮氨酸-苏氨酸-亮氨酸-丝氨酸-赖氨酸-丙氨酸-天冬氨酸-酪氨酸-谷氨酸-赖氨酸 180 185 190 组氨酸-赖氨酸-缬氨酸-酪氨酸-丙氨酸-半胱氨酸-谷氨酸-缬氨酸-苏氨酸-组氨酸-谷氨酰胺-甘氨酸-亮氨酸-丝氨酸-丝氨酸-脯氨酸 195 200 205 缬氨酸-苏氨酸-赖氨酸-丝氨酸-苯丙氨酸-天冬酰胺-精氨酸-甘氨酸-谷氨酸-半胱氨酸 210 215 <210> 18 <211> 446 <212> PRT <213> Homo sapiens <400> 18 谷氨酸-缬氨酸-甲硫氨酸-亮氨酸-缬氨酸-谷氨酸-丝氨酸-甘氨酸-甘氨酸-甘氨酸-亮氨酸-缬氨酸-谷氨酰胺-脯氨酸-甘氨酸-甘氨酸 1 5 10 15 丝氨酸-亮氨酸-精氨酸-亮氨酸-丝氨酸-半胱氨酸-苏氨酸-丙氨酸-丝氨酸-甘氨酸-苯丙氨酸-苏氨酸-苯丙氨酸-丝氨酸-赖氨酸-丝氨酸 20 25 30 丙氨酸-甲硫氨酸-丝氨酸-色氨酸-缬氨酸-精氨酸-谷氨酰胺-丙氨酸-脯氨酸-甘氨酸-赖氨酸-甘氨酸-亮氨酸-谷氨酸-色氨酸-缬氨酸 35 40 45 丙氨酸-酪氨酸-异亮氨酸-丝氨酸-甘氨酸-甘氨酸-甘氨酸-甘氨酸-天冬氨酸-苏氨酸-酪氨酸-酪氨酸-丝氨酸-丝氨酸-丝氨酸-缬氨酸 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Ser Asn Val Asn Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 <210> 19 <211> 218 <212> PRT <213> Homo sapiens <400> 19 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Met Ser Cys Arg Ala Ser Glu Asn Ile Asp Val Ser 20 25 30 Gly Ile Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Val Ala Ser Asn Gln Gly Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 20 <211> 446 <212> PRT <213> Homo sapiens <400> 20 Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Lys Ser 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Gly Gly Gly Gly Asp Thr Tyr Tyr Ser Ser Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Ser Asn Val Asn Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 <210> 21 <211> 218 <212> PRT <213> Homo sapiens <400> 21 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Met Ser Cys Arg Ala Ser Glu Asn Ile Asp Val Ser 20 25 30 Gly Ile Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Val Ala Ser Asn Gln Gly Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 22 <211> 446 <212> PRT <213> Homo sapiens <400> 22 Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 1'5 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Lys Ser 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Gly Gly Gly Gly Asp Thr Tyr Tyr Ser Ser Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Ser Asn Val Asn Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 <210> 23 <211> 218 <212> PRT <213> Homo sapiens <400> 23 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Met Ser Cys Arg Ala Ser Glu Asn Ile Asp Val Ser 20 25 30 Gly Ile Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Val Ala Ser Asn Gln Gly Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Serving Lys Asp Serving Thr 165 170 175 Tyr Ser Leu Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Valid Thr Lys Ser PhE As An Arg Gly Glu Cys 210 215 <210> 24 <211> 12603 <212> RNA <213> smell the smell <400> 24 ugcuucguu uguuggua uaauaguau uuccgaguc cucuuugaa ugucauuag 60 uuuuacagac auugucaguu cucuuagua cuguguguc agcaucaagg uuuugaagga 120 cguuacucc uaggucaccu uaugggccgu cuaugaagu cuuuuaguuu ccuuaagga 180 gaaauguagu uaugauguuu uucaaacagu guaauucuc cuauacagau gguuccggag 240 uuuaggccuu uacauaguua guauguacag uugucgauga acauaccucg uaauuuccug 300 uaggccccau ucaaccuauu ucuaaccagu ucaaagccuu auuuguagcc cuuucguccc 360 cuauguuagc cuuauaaacu ggaacauagg aacuuucggg accugccgca ugaaggucua 420 ccucauagcc uacgaagguc uuggucgcgu cuacuguuua ccaacggaaa cauagaugaa 480 ccgaauugu cucaccccguc uuguguuuac ggacuuaugu cuuuuuucga guaccuaccc 540 600 cuguaaaaaac uacacacccc uuuacuguca uuaauguguu uuuaacagcg acgucaccug 660 uacaagaagg ugaacaaguu uuuuguacuu acacggagca agucuaugcc uugauaacaa 720 aggucuaagu uucuaacacg acguaaccgu uguaaaccug uggagacguu uuauuggccu 780 uacagauguc uucuacauug cuggaccuag aacuuggcuc uucaacgucu acuuuaccag 840 guuuacuacg aagguccggu ucuuuaacug uuccggcuaa guauguacgg aauaaacuag 900 cugaaaccua acagaagauu cagagguaua agaaggcagu uuuugggacg gaaggugaag 960 acccccguua acugucgaga agacgagucu agguggucuc guuccuuacg ggcugucgga 1020 cuacuguaac ucauauguag agaaugaugu cguccaaaca acaugcgaau acgucauccu 1080 aggagacggc ugaaccgugu ugucaaaaca caaccucuau uguuuaugug aggucuacua 1140 ucauggccuc cuaacugcug auuacguggc gguguuccgu cucuacacca gcuuaccgag 1200 ccuaccaaac uucuaguuuu gucuuuuggc ugaggacuau acuacgucau acgcuuuucu 1260 cgucaguaca gugacguucc ggauucucuc uucuguuaac cguucauacg auucagucuu 1320 aaacuguuua cugggauauu aagagucuag uggauaauau auaauacgau guauacuuuu 1380 uuugauuguc uauaguaccu auuagagugu uuucaagcac ucauagaguu caggauaaga 1440 gcagaccuag uccgccaucc ucucuaucua cucuagcuuc guguugcucg acuuuucagg 1500 uuaauacuca acaagguucu ccuaccucac cuucucguau gauucgggag aauaaaaguc 1560 cgucgucuac uagacugug ucuuagacuu ggucuuuaac ucuguuagu uccgaacaua 1620 cguggucuag gucuucgacu cguucacuu ccgaauauug uccccggaa ucuacugaaua 1680 1740 cugcucguac cuucugga uggcucgu agcgucucc caauucacc ucucgucuu 1800 agggucaccg aaagcugcua auuucgucag cacguucac gguuuauugac cuagaccgu 1860 cucacgugua aacuucguag cccucuuccc caguaacu uccucgcggu cuauugaggc 1920 Cuacauauau Uccagugagg Ucacuacuug Uguguaggca Ggguuagucu Ucgucauagu 1980 weather gagaguu cuguagguuc cuguagguuc cuguagguuc 2040 ggagaguggu auaggaaccu acuuaacaag aguagaucuc cucucaagua gagacagccu 2100 ccacugccug cuacagagu auuucuccgg uaggacgagc cggacucuau guuuuucaac 2160 auguuagucc gcucucaguu uuaagagac aucuaguacu uuuuuucauu gucuuaguug 2220 quagauucac auuagggua gwaaguagua acuquaggaa oucuucuaa gagccagacu 2280 cuckooooooooooooooooooooooooooooooooooooo gooooooooooooooooooooooooooooooooooooooooooooooooooooo lay la La la La pa last 2340 gaucguaccu caacgaggc ucgcgagguu aacuguuuag gauaaaccu caacugcucu 2400 accuguggau acuagcuua guuaauuuua uacucuuuaa gagaaugu cacuuuuacu 2460 gccaaucuag auuagcaggc aagucuugua ugagucuaca ccgucggcga cauaggguaa 2520 cccuagugua cauguagccu uaccgucccu ugcaggga gauguuuuag aaccgaaaaa 2580 acccaagaag auuagauuuc cggugagguc gccauaaccg ucuaguucca guuggucuca 2640 uagugcgagu gacgcuccg ucccgaauaa acgguguauc cuckccuuc ugggagggu 2700 acgaguaca uggcucgug aagucuucgu guaguaua uccagaaug ucccuugcu 2760 aacucgagug uacuggug augcuac acgugaccu uacgucgagga uacuagaccc 2820 waguaagou aaaaaaaaaaaaaaccgacuu ccggaauuac aaaccgacu 2880 aacagcucuu uuuccguaga ccucgcaccc aggaccugag auagccggug aaguuacuc 2940 gaucagauug aagaucgaag acuuguuagg ggccaauuga gaugagggg auuaaggucg 3000 3060. snowflake snowflake 3060. snowflake snowflake agacaaaugc gcagugccua gggggcccga cguccuuaag cggugguacc cggucuagca cugguacaag cuccgggacg ggguguagua gcugcuccac uaguguagc acuaguagca cgaguaguag uaguggucgu aguuccggca cauguugaag cgguggacgc cguaggaccg 3300. ggaccacucg aaggacaagg accggccguc uucgacgccg uacaugccgg acuuaccggg gcuauagaug uucccgcaca to close cucgcaccuc aagcuguacu cgguggacu 3420. gggugguac ggguugcgga cgucgcgguu guuaucggug gugauguagu cguacccguc gucgccggac cucaacugga gucguaggac uugguguuga agacguugga guggucgcgg aaguuguucu uuuggaagcu ggugugggag uacucguagc acucgucgga cguggacucg uagucuccgu ugucguuggu guuccggcac ucgacgcuga aguuguugcc guagugguag gucauguugg acucgaaguc gcuaggaguc ucgcgguagu cggucacguc 3720. ccgucucacg accuguacaa aagccgccgu ucauguacuc uucgccgacc ccgacccggc cgucgcugcc guucuggugg accacgucgg ucuggucgau 3780 ggucauggag uaguaggucu ugucuuggac ccucuuggug acgucuuugc ggccuggaaa 3840 gccguacucg cucuaggaca agcggccu cuuugguc aaggaguggu cucugaccg 3900 gccguggaag uggaccugggg acucgcuguc gucgccgcac cucuugggac cgccgaugac 3960 ggagugguuc accuacuagg accggcggcu cgacuucacg aagccguugu ggcggcaccg 4020 frequency frequency frequency frequency frequency 4080 guuccggcgg gacucguca aguucguccu gcaccucucg cgggacgugc acaaguucug 4140 guggcacuug ucggaguagu cgcuggucga cgaguacucu uugguggacu cucuggagua 4200 cccgcacggg augcguuggcquagcc cucgugcgg ucuggccgcu 4260 cuggucgcac ggguuccacga ccgaccacug guuaccgucg auggacuugc ucugggugaa 4320 gucgcuguc uagcucgucc uucggcuguu guacuagugg cucuacgacu cucuccugau 4380 guaguucucu gucccgucgu ggggggaccg ggaguaccua gacgaguaca agucgugguc 4440 gcggauggag uagucguaga aggacgugga ccacuucuag gggugggugu cuguguaguu cccgccgucg acgggguucg gggugucuga gugguuguuc ccguagacgu cgacgccgcg 4560 gaaguccac gggccgcacu uuugguagac cucuccucu auucgccggc gaugcuggag 4680. cugauacuuu uuuugauugu cuauaggagc ugcgguggua cccggugugu ucuuccgucc 4740. snow-covered snow-covered snow-covered snow-covered snow-covered snow-covered snow-covered snow-covered acucggugaa aacaagaccg cacuaggugc acugguuucu ucacuuucuc cagcggugug acucgacgcc gguguugcaa agucaccuuc uugaccgggu cuggucuuag augaccgucu 4920. uucuuuucuu uuaccacgac ugguacuacu cgccgcugua cuuguagacc gggcucaugu ucuuggccug guagaagcug uagugguugu uggacucgua gcacuaagac cgggacuccg gaagacuacu cccguggaua cucacgcacc acgacuucau gcucuuccug cggaaguucg 5100. cgcucguaga ccggcuucac ugugacucgc acuuccggcu gaaagggugu ggaucguagu 5160. cgcugaagcu cuaggggugg ucguuguagu cugccuagua gacaucgugg ucgccgccga aaggucucgg aguagacaga accgaccuuu ugccgcuccu ugacuugcgg uaguuguggu 5220 ggcacagagu ccuggggcuc ugucucgaca uacggcacag gucguucgac cugaaguugu 5280 acuggugguu ggugucgaag uacacggacu aauucaugcc gguggacucu cacuuggucu 5340 ggaaguugac cuuguggugg uucguucugg ugaaggggcu cguguucugg guggagacag 5400 gagguacagg acgaggucuu gacgagccgc cugggaggca caaagacaag ggagguuucg 5460 5520 gagugcuccu gggucuucac uucaaguuaa ccaugcaaccu gccgcaccuu cacguguugc 5580 gguucugguu cggaucucuc cuugucaugu ugucguggau gucucaccac aggcacgacu 5640 ggcacgacgu aguccugacc gacuugccgu uucucauguu cacguuccac agguuguucc 5700 gggacggacg aggauagcuc uuuugguagu cguuccgguu cccugucggg gcccuuggag 5760 uucacauaug ggacggaga ucggcccuuc ucuacugguu cuuaguccac agggacugga 5820 cggcacuu cccgaaug ggaaggcuau agcggcaccu uacccucucg uuaccggucg 5880 gagucugu gaguucugu agggaggc gcugccgagu ahagg 5940 ugucguuuga cuggcaccug ucucgucua ccgucguccc guuacacaag ucgacgucgc 6000 acuacgugcu ccgggacgug uuggugaugu gggucuuuuc ggacucggac agaggaccga 6060 cuaguugauc gguquaagaa guacaaccu gguuagouug aacacuaugg uacgaguuuc 6120 uccggagoua auauaaaacuc aaaaaaaaaaaacuuuu uuuuogucg uaaguaccuu 6180 caggugcua aacucuggcu gcucaagua cuaaguac uucuacugau acgguucu 6240 cuaaggacu uagggcuacu cgcguacugc augacuuag uacgacuau guuggacuua 6300 agaggau auacacuacu auaacugua auauaguccu uaaaguuag agagguaaa 6360 6420 cgguuagggu aggguaguag agucuacgua uuaccuacc cuocaccaa uuacagacua 6480 uuaguacuac ggucaguucc cauaucaaa auuguacuac accuguuuc ccgucuuuau 6540 uguaaacugc accaccucug gaaguaggcg ccgaccccgu uguuugguua acuuaguag 6600 uuuuuccuuu cuaccugacu gaguaaguuu uaagagcgaa uaaacacagu uuucaaaaac 6660 cugaaugugu ucaacuguaa uuagaauuuua cgacagagacac uccaccuuaa cgaguugaac 6720 cgcuccugaa aguuuccguu ucagucuucu ucaagaguac cuugcuugua uacguccuaa 6780 ucccaagggu cgaacccagg augaaaauaa agucuuccua cccgaaugaa guucuuugaa 6840 cuauaagauu accuggcuuu gaaagacaau uaccaguuuc uacacuaaua ucccuccuac 6900 guuugccacg auagguacca uacaucuuuau cuguuggaca agagucucgu ucuguagaag 6960 agggaaauu uauagauguc uuaaccucua uuuuaacacc ucuccguccc uuuaaaaaga 7020 aaucugaacu aauuuuacca ccuuggcuau aauguacu ucgacuacu uaaucuacu 7080 cuuaguuccg gaaucaggg uguuaaggga guaaaacuuu uaguauaguu cugaagacaa 7140 cuacuucccc guuuuuaacu ggcuccauau ucuaaggg uacuagucua uuacucacac 7200 uuuugucacc uagaguguga ccacuaaua ccuagcaagu cuguaacccc aguaggaaaa 7260 uaucuaauaa ugugaccuga ucuuuuuaau guaaggguuc auuggguacuu cuuucuauaa 7320 cuacacagua uacguuuucg ugaacguuca cuaaaucgag ccuaacaaga uaaaguuguc 7380 aaguuacuag uauuuuucac caagcacuua ccucugaacg agggaguacu aguagggaaa 7440 uuuucaguac aauuucuuuu auguaccggg ugucgacgag uucaaguucu aaaaccucua 7500 uuuaccguac uugaaggcga cuaauuuaca aaacuuuaug ggcugaauga ucuggguagc 7560 uauuauauga gacuguuuuc aguaaguuac uuauccaguc uccacaacuu uguacaggcu 7620 uacuuaggcu ugugagguaa gggaucauuu uuccacaacg ucugauacaa ccuguguuuc 7680 cgaugguuaa ccuuucuuaa agaauuucuc uaacuacucu ucccgaaucu acuacuacua 7740 gauuaauaac cagaauuucc uuuccucucc cuugacuuca accguccauc uaaaaagagg 7800 gauuacagaa ccuuuaacgc ucuuaugaaa cauuaauggc uuauaaacua uuucugagua 7860 aagcagggau acaaauuucc ggacuguuac cgccugcuag auugacguca guaauuuuuc 7920 uacaaucuaa ggaguaggcc gguuccuaac uucaguauac uccguuaaac guaucgguua 7980 guguaacuaa ugcuuuuuac cuuauuggug guuuccuuca auaguuugcc gggucacaag 8040 gcuacauacc cggucaagaa uccaauaggu aggaauuagc ucucuugagu acuuaaaaaa 8100 cucuuuucag aauauaugau guuaccuucu ggucugaacu acgcacaagu guuguugugu 8160 gacuaguuaa guuggaggu ugcucaaaca accguuccug uucucccacc ugaccuucca 8220 gaugccguuu uuccuaccuc auaggaguua gaugaccaau aaguuucucu ccgauuuuag 8280 ucuuugugac gacaguuuca gaaccguguu ccacuauuag uucaauaaac gugugucaua 8340 uuuugcuucu uuagcucuuu gcaacaucuu aaugucccac gagaguuagu uuaccaaaga 8400 uuauuacucu uuuaauacug acguuaguuu uaucccuguc ccuucaaucc ugaaaacuau 8460 uuacugcuac ucugauacgu uagacgucua augaacuuaa uaccuuuuuua uggcuaaaag 8520 gcaccucacu aaucucccaa ucucugguuc ucuaccagug cucacugaac acaguguua 8580 cugguuuaug ggugaacacg auuauauuac ucgagucaaa gguguuuacg agaguggcau 8640 cgaguaaaac gaccuuggg uuaguuacgg uacuauguca uguuaauaaa acccuguaaa 8700 cgaucugaga acaacuacua cguacuagga cgagaagcag uuaguaacau acuucaaguu 8760 cuauucuaug gcccgaacgu gucaagauga aguuuaugc gguacaacau aaaccuggga 8820 agguaccuc cucacagccc guacagaaac agguccaaaa acuaucucg gaagggucua 8880 gggcauuguc uucagagag uaagaccucu aaguagguac auguacgagc uucacucgua 8940 gacuuccucu acucacguca uaaccuuug gggcucuauc gguucaaagc uuauugagug 9000 uaucuguucg aucaucuucu agguuggaga gacuuguagc gauacccuua cucaggucgc 9060 uugaacaauu ucugacucca auuuuuuacg aauuagcuua guucuguuug guaguccuug 9120 guccacuaau uccuacguug guauauaaac auaguacuuc uccuagccga gucuucaaag 9180 aauaccaguu auuuaggaga caagggaucu aaaaaucac uuaaguuuag uccgugaaaa 9240 aacccucagc gucugcccga guagucagau aaaguuuuaa gagcaugaua agccuugagg 9300 aaauucuuuu ucauaguauc ccuuaaccua quaaacuac acuccucacu ccauaggaga 9360 aacuguguaa aucccuuuga aguaaacucu uccccuagua cauuuuacac cuguacaagu 9420 cgaugaguac gacuguguaa uucuauguuu aggaccccgg caugucaaua acccuguuga 9480 cauggguag guaaucuuua caacccaggu guuguagcuu uucucugagg aacacguggu 9540 acauugugua gucccaaguu aauacaaaga cacguaacag gucugcccua gguacugcag 9600 aaaucaagug ccccugguaa cggacgaaua gaucccagau uuuguagacu uagauguaga 9660 uaaaacgucg gaacccuuuc ccuuucguuu cagggugacu aauuuucucg auggcagaa 9720 ucucuacgau agagaaccaa acaacuuggg cugagauuug aucguuacug auugaaaga 9780 uuguagguga gaauugucc gcuucuuacc ugguuuuccg ucguacccaa guuuucuugu 9840 cccagacggg aaguauccaa aagcuguaga gccuacucgg uaccaccccaa gcguagaguc 9900 9960 gucuuaaagc ugaaaaauaa gguucguugc aacgagauac gaguuuaaug guggugacaa 10020 cguucucugc cuaccuagug gucaacaugu cuaguaauag uauaacggac auucaggaca 10080 aacucugggu aucucucua gugggaccug aguucauacc ugaugugcgg gggucuacau 10140 aggguacacg acuucuguac cuccuuaccc cuuccaagca ccccuguucu cuauuuuguc 10200 uagauagga aucuucccuu aucguggac gacucguuag gauaguucag 10320. ccgucuacau auccaaaaga uauaccucug aaccgcauau cuuuagaug aguacggcuc 10380. cugucaagag auaaaggaga uagauauguu ccagcauaau cuccagcucc aaagaauuuu cccaacgauc ugccuaauua cucumber acgacgguuc auuauguggc cucumber cgaguaaacu ucuccggccg guugcgucac augccucca acuaaaugaa cuaacuauuu aacucacaua snow snow snow snow snow snow snow 10620. 10620. 10620. 10620. 10620. 10620. 10620. 10620. 10620. uaacagucuu uaaugguuacg gcagauuaac uuuucccuuu uaugucuagu 10740. 10740. 10740. 10740. 10740. 10740. 10740. 10740. 10740 10800. 10800. 10800. 10800. 10800. 10800. 10800. 10800 uuacucaacu cucucgaccg uuuagaaga aguaacgauu cucucucucu ccccacccuu 10920. 10920. 10920. 10920. 10920. 10920. 10920. 10920. 10920. 10920 cgaacguuca agcccuaacg auuccuauua uuauuucugu acucgauagg gggaaccccu 10980 ucccuuaggu cucccuguua auguuguuag ggacaaauaa uaugcuggug gggaaugggu 11040 uucuacgauc uuacggagg uuuuaggu uuaggggacg acaggccuua guccaacccg 11100 11160 augcccuga agaacucaac accucugccg aggccuccccu aguacgacg uaaugaugcu 11220 cuuuuacacg uaucgucucc uuauaaguua ucagacaauc uuaauagucc cagucaguac 11280 11340 acacauuuac cacuuuguac aacccuuaua gguagacuga auacacuggg uuccugaacc 11400 11460 11520 guggccuaaa accuacucgu uccucaaaaau uagauguucu gaauaccuug uauauaaaca 11580 cucucgcuuu ucuuacguca uuguuaggaa ccaggguaca aguucugcca gcugaaucaa 11640 guuugucuua aaucaucaag aguuugcaga cuucauauau accauacauu uccaaacuuc 11700 uuuaauuagc uacuuggguu agggcuaacc agaagguagu uacuuaggac cuuuuuggac 11760 augcguaagg ucaguagucu uguccuuaaa cggucucguu ucuuccaauc auguaugaaa 11820 uggaacuguc cauaagggag gguuaaguaa ggacuaggaa aacauuugua acucugauac 11880 gauguuuaua agccucaugg gugcccacac agaguacgcc gacggaauuu uaguagacua 11940 ucuggacguc uaaauaacug guaucggaa aaaauauacc gcuaauauag cauaauauug 12000 uaguuaguau agucucaucc uggcuaugga ggcuuggggg guagucuacc uuaacguguu 12060 uuacaccccu agcgauauug accauauucg aaaccgacu caaacuccu cuuucuguaa 12120 ggugauauag uugucacaaa ucgucaauag gucguuagua agggcuaauc cacccuccga 12180 caaagucauu uuccuccuau guucgucuuc accucaugau cuccacuacc cgaggguuuu 12240 cuaugggcuu aaagucugag gaaccggggu uagcccuuga ccuagucuag agaccuuaac 12300 caggcuuugg uucaagcaga uuuagguaag uuacucuaga acaaguuagu cgauacagca 12360 ugucaccuau uaguaaacuu uaccaguuua aacgcuucuu uguguccuua cuaacuuacc 12420 uaguuaucug cuuaaaguuu ucuucuggcc agauaugacu acaacuucuc acuggaugug 12480 cuccuuuuga gaaccucucu aauuuuuuag uacuccucug agguuugaaa uucauacuuu 12540 uuuugaaacu aggaauucug ggagaacacc aaaaauaaaa aauagaccaa aacaccagaa 12600 gca 12603

Claims

1. A recombinant vesicular stomatitis virus encoding at least one CD80 extracellular domain Fc fusion protein in its genome, wherein the CD80 extracellular domain Fc fusion protein comprises the extracellular domain of CD80 and the Fc domain of IgG, and the amino acid sequence of the CD80 extracellular domain Fc fusion protein is shown in SEQ ID NO:

4. The gene encoding the glycoprotein G of the recombinant vesicular stomatitis virus is replaced by the gene encoding the glycoprotein GP of lymphocyte choriomeningovirus (LCMV), and / or the glycoprotein G is replaced by the glycoprotein GP of LCMV.

2. The recombinant vesicular stomatitis virus of claim 1, wherein the CD80 extracellular domain Fc fusion protein further comprises a signal peptide sequence.

3. The recombinant vesicular stomatitis virus according to claim 2, wherein the amino acid sequence of the CD80 extracellular domain Fc fusion protein is shown in SEQ ID NO:

3.

4. A recombinant vesicular stomatitis virus encoding in its genome a vesicular stomatitis virus nucleoprotein N, a large protein L, a phosphoprotein P, a matrix protein M, a glycoprotein G, and at least one CD80 extracellular domain Fc fusion protein, wherein the CD80 extracellular domain Fc fusion protein comprises the extracellular domain of CD80 and the Fc domain of IgG, and the amino acid sequence of the CD80 extracellular domain Fc fusion protein is shown in SEQ ID NO:

4. The gene encoding the glycoprotein G of the recombinant vesicular stomatitis virus is replaced by the gene encoding the glycoprotein GP of lymphocyte choriomeningovirus (LCMV), and / or the glycoprotein G is replaced by the glycoprotein GP of LCMV.

5. The recombinant vesicular stomatitis virus of claim 4, wherein the CD80 extracellular domain Fc fusion protein further comprises a signal peptide sequence.

6. The recombinant vesicular stomatitis virus according to claim 5, wherein the amino acid sequence of the CD80 extracellular domain Fc fusion protein is shown in SEQ ID NO:

3.

7. The recombinant vesicular stomatitis virus according to any one of claims 4 to 6, wherein the nucleoprotein N comprises the amino acid sequence shown in SEQ ID NO:

7.

8. The recombinant vesicular stomatitis virus according to any one of claims 4 to 7, wherein the phosphoprotein P comprises the amino acid sequence shown in SEQ ID NO:

8.

9. The recombinant vesicular stomatitis virus according to any one of claims 4 to 8, wherein the large protein L comprises the amino acid sequence shown in SEQ ID NO:

9.

10. The recombinant vesicular stomatitis virus according to any one of claims 4 to 9, wherein the matrix protein M comprises the amino acid sequence shown in SEQ ID NO:

10.

11. The recombinant vesicular stomatitis virus according to any one of claims 4 to 10, wherein: The nucleoprotein N contains the amino acid sequence shown in SEQ ID NO:

7. The phosphoprotein P contains the amino acid sequence shown in SEQ ID NO:

8. The large protein L contains the amino acid sequence shown in SEQ ID NO:

9. The matrix protein M contains the amino acid sequence shown in SEQ ID NO:

10.

12. The recombinant vesicular stomatitis virus according to any one of claims 4 to 11, wherein it is capable of replication.

13. A recombinant vesicular stomatitis virus encoding in its genome a vesicular stomatitis virus nucleoprotein N, a large protein L, a phosphoprotein P, a matrix protein M, a glycoprotein G, and at least one CD80 extracellular domain Fc fusion protein, wherein the CD80 extracellular domain Fc fusion protein comprises the extracellular domain of CD80 and the Fc domain of IgG, and the amino acid sequence of the CD80 extracellular domain Fc fusion protein is shown in SEQ ID NO:

4. in, The gene encoding the glycoprotein G of the vesicular stomatitis virus was replaced by the gene encoding the glycoprotein GP of lymphocytic choriomeningovirus (LCMV), and / or the glycoprotein G was replaced by the glycoprotein GP of LCMV, and wherein... The nucleoprotein N contains the amino acid sequence shown in SEQ ID NO:

7. The phosphoprotein P contains the amino acid sequence shown in SEQ ID NO:

8. The large protein L contains the amino acid sequence shown in SEQ ID NO:9, and The matrix protein M contains the amino acid sequence shown in SEQ ID NO:

10.

14. The recombinant vesicular stomatitis virus of claim 13, wherein the CD80 extracellular domain Fc fusion protein further comprises a signal peptide sequence.

15. The recombinant vesicular stomatitis virus of claim 14, wherein the amino acid sequence of the CD80 extracellular domain Fc fusion protein is shown in SEQ ID NO:

3.

16. A pharmaceutical composition, characterized in that... The composition comprises recombinant vesicular stomatitis virus according to any one of claims 1 to 15.

17. Use of the recombinant vesicular stomatitis virus according to any one of claims 1 to 15 or the pharmaceutical composition according to claim 16 to prepare a medicament for treating colon tumors.

18. Use of the recombinant vesicular stomatitis virus according to any one of claims 1 to 15 or the pharmaceutical composition according to claim 16 to prepare a medicament for treating colorectal tumors.

19. Use of the recombinant vesicular stomatitis virus according to any one of claims 1 to 15 or the pharmaceutical composition according to claim 16 to prepare a medicament for treating melanoma.

20. Use of the recombinant vesicular stomatitis virus according to any one of claims 1 to 15 or the pharmaceutical composition according to claim 16 to prepare a medicament for treating breast tumors.

21. The use according to any one of claims 17 to 20, wherein the recombinant vesicular stomatitis virus or the pharmaceutical composition is to be administered intratumorally or intravenously.

22. The use according to any one of claims 17 to 21, wherein the recombinant vesicular stomatitis virus or the pharmaceutical composition is administered intratumorally at least once and subsequently intravenously.

23. The use according to claim 22, wherein subsequent intravenous administration is given on days 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 or 31 after the initial intratumoral administration.

24. The use according to any one of claims 17 to 20, wherein the recombinant vesicular stomatitis virus or the pharmaceutical composition is used in combination with a PD-1 pathway inhibitor.

25. The use according to claim 24, wherein the recombinant vesicular stomatitis virus or the pharmaceutical composition is administered simultaneously, sequentially or alternately with the PD-1 pathway inhibitor.

26. The use according to claim 24 or 25, wherein the PD-1 pathway inhibitor is selected from the group consisting of: pembrolizumab, nivolumab, pilithrumab, atezolizumab, avelumab, duvalumab, PDR-001, PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5. in, PD1-1 comprises a heavy chain and a light chain, wherein the heavy chain contains the amino acid sequence of SEQ ID NO:14 and the light chain contains the amino acid sequence of SEQ ID NO:15; PD1-2 comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:16, and the light chain comprising the amino acid sequence of SEQ ID NO:17; PD1-3 comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:18, and the light chain comprising the amino acid sequence of SEQ ID NO:19; PD1-4 comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:20, and the light chain comprising the amino acid sequence of SEQ ID NO:21; PD1-5 comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:22, and the light chain comprising the amino acid sequence of SEQ ID NO:

23.

27. The use according to claim 25 or 26, wherein the recombinant vesicular stomatitis virus or the pharmaceutical composition is administered via a route of administration different from that of the PD-1 pathway inhibitor.

28. The use according to claim 25 or 26, wherein the recombinant vesicular stomatitis virus or the pharmaceutical composition is administered intratumorally at least once and the PD-1 pathway inhibitor is administered intravenously.

29. A composition comprising recombinant vesicular stomatitis virus according to any one of claims 1 to 15, and further comprising a PD-1 pathway inhibitor.

30. The composition of claim 29, wherein the PD-1 pathway inhibitor is an antagonistic antibody that targets PD-1 or PD-L1.

31. The composition of claim 29, wherein 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. in, PD1-1 comprises a heavy chain and a light chain, wherein the heavy chain contains the amino acid sequence of SEQ ID NO:14 and the light chain contains the amino acid sequence of SEQ ID NO:15; PD1-2 comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:16, and the light chain comprising the amino acid sequence of SEQ ID NO:17; PD1-3 comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:18, and the light chain comprising the amino acid sequence of SEQ ID NO:19; PD1-4 comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:20, and the light chain comprising the amino acid sequence of SEQ ID NO:21; PD1-5 comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:22, and the light chain comprising the amino acid sequence of SEQ ID NO:

23.

32. A complete reagent kit, comprising: a) the recombinant vesicular stomatitis virus according to any one of claims 1 to 15 or the pharmaceutical composition according to claim 16, and b) PD-1 pathway inhibitors.

33. The kit according to claim 32, wherein the PD-1 pathway inhibitor is an antagonistic antibody that targets PD-1 or PD-L1.

34. The kit according to claim 32, wherein 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. in, PD1-1 comprises a heavy chain and a light chain, wherein the heavy chain contains the amino acid sequence of SEQ ID NO:14 and the light chain contains the amino acid sequence of SEQ ID NO:15; PD1-2 comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:16, and the light chain comprising the amino acid sequence of SEQ ID NO:17; PD1-3 comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:18, and the light chain comprising the amino acid sequence of SEQ ID NO:19; PD1-4 comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:20, and the light chain comprising the amino acid sequence of SEQ ID NO:21; PD1-5 comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:22, and the light chain comprising the amino acid sequence of SEQ ID NO:

23.

35. A virus-producing cell, characterized in that... The cell produces recombinant vesicular stomatitis virus according to any one of claims 1 to 15.

36. The virus-producing cell of claim 35, wherein the cell is a Vero cell.

37. The virus-producing cell of claim 35, wherein the cell is an HEK cell.

38. The virus-producing cell of claim 35, wherein the cell is a HEK293 cell.

39. The virus-producing cell according to claim 35, wherein the cell is a Chinese hamster ovary (CHO) cell.

40. The virus-producing cell of claim 35, wherein the cell is a young hamster kidney (BHK) cell.

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