VSV-NDV hybrid viruses modulating immune checkpoints for oncolytic viral immunotherapy of cancer

Abstract

The present invention relates to recombinant oncolytic viruses. The present invention further relates to nucleic acids encoding the recombinant oncolytic viruses. The present invention also relates to vectors comprising nucleic acids encoding the recombinant oncolytic viruses. Furthermore, the present invention relates to pharmaceutical compositions comprising the recombinant oncolytic viruses. The present invention further relates to viruses, nucleic acids, vectors, and pharmaceutical compositions for use in medicine (e.g., for use in the prevention and / or treatment of cancer). Furthermore, the present invention relates to the use of viruses, nucleic acids, vectors, and pharmaceutical compositions as gene delivery tools, (non-invasive) imaging of viral biodistribution, and / or for tumor detection.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a recombinant oncolytic virus. The present invention further relates to a nucleic acid encoding the recombinant oncolytic virus. The present invention also relates to a vector comprising a nucleic acid encoding the recombinant oncolytic virus. Furthermore, the present invention relates to a pharmaceutical composition comprising the recombinant oncolytic virus. [Background technology]

[0002] Background of the Invention Despite decades of intensive research, cancer remains a major health concern worldwide, imposing a significant epidemiological, economic, and societal burden. Cancer immunotherapy represents an exciting paradigm shift in cancer treatment, rapidly transforming the landscape of medical oncology by harnessing the patient's immune system as the basis of such treatment. Oncolytic viruses (OVs) have recently earned their place as an exciting subset of cancer immunotherapeutic agents. OVs offer a novel, multi-mechanistic approach through their potential to directly initiate tumor cell oncolysis, modulate the tumor microenvironment, and stimulate adaptive immune responses directed against tumor cells. Therefore, OV approaches are currently under intensive investigation, particularly in the context of rationally designed combination regimens with other immunotherapeutic agents.

[0003] Cancer immunotherapy encompasses a wide range of approaches with the common goal of modulating a patient's immune system to recognize and reject invading tumor cells. Vaccination approaches and adoptive cell therapy (e.g., chimeric antigen receptor (CAR) T cells) rely on the identification of appropriate tumor-associated antigens (TAAs) and neoantigens to target. Due to intratumor heterogeneity and the process of cancer immunoediting, strategies targeting a single antigen can result in the selection of tumor cells that do not express the targeted antigen, which can escape treatment. Furthermore, because each tumor has its own distinct genetic signature, targeted therapy often requires expensive and time-consuming screening of tumor biopsies and the subsequent creation of personalized treatments.

[0004] Immune checkpoints are compensatory controls that function to prevent the constant activation of immune-inflammatory processes against foreign antigens by suppressing T cell activation and creating a state of T cell "exhaustion." Tumor cells exploit these immune checkpoints to create an immunosuppressive microenvironment in which they can evade immune clearance and continue to invade the host. Immune checkpoints are increasingly being considered as promising targets for cancer immunotherapy. Interference using antibodies against key checkpoint molecules has been marketed for limited cancer indications, but appears to be effective only in a small subset of cancer patients, typically in tumors with highly inflammatory and high mutational loads. Furthermore, the high doses of these antibodies required for systemic application can result in severe immune-related toxicity.

[0005] Oncolytic viruses (OVs) offer a simple, multimodal approach to cancer therapy through their ability to induce direct tumor cell lysis while stimulating an immune response directed against the tumor. Nevertheless, the potential of OVs as monotherapy is limited by the relatively rapid onset of antiviral immune responses and insufficient adoptive immune stimulation to provide systemic tumor clearance and protection from recurrence. Over the past decade, significant progress has been made in the development of enhanced OV therapy, and several vectors have entered clinical trials. To date, one OV has received Federal Drug Administration (FDA) and European Medical Association (EMA) approval for use as a clinical drug. This virus, a recombinant herpes simplex virus I (HSV-I) vector, is currently approved for only one indication: unresectable melanoma. Furthermore, US Patent No. 10604574 B2 relates to oncolytic viral delivery of therapeutic polypeptides. However, clinical trial results are often disappointing due to inadequate tumor response to most OV treatments in immunocompetent hosts.

[0006] The inventors have previously engineered a hybrid oncolytic virus technology (WO 2017 / 198779) that combines the beneficial features of oncolytic vesicular stomatitis virus (VSV) with those of Newcastle disease virus (NDV) while eliminating the safety concerns of each.

[0007] In recent years, strategies combining OVs with immune checkpoint therapy have emerged. The rationale is that local viral infection within tumors can convert a "cold" tumor microenvironment into an inflamed "hot" one, indicated by high levels of cytokines and immune cell infiltration, thereby sensitizing the tumor to the effects of immune checkpoint blockade. It has been hypothesized that tumor treatment with oncolytic viruses leads to the upregulation of PD-L1, a ligand for the co-inhibitory T cell receptor, programmed cell death protein 1 (PD-1), within the tumor, and that disruption of the interaction between PD-1 and PD-L1 enhances the efficacy of oncolytic immunotherapy. A strategy utilizing an oncolytic myxoma virus expressing sPD-1 has been described [1].

[0008] However, to fully exploit the potential of combining OVs with immune checkpoint therapy, various obstacles must be addressed. First, traditional immune checkpoint-modulating drugs consist of antibodies, which must be administered systemically and at high concentrations, often resulting in intolerable side effects. Second, the prohibitively high cost of immunotherapeutic agents substantially limits the feasibility of combining two individual drug products in this class. Third, to optimize synergistic effects, it is challenging to select an efficient oncolytic virus that combines potent tumor reduction with immunogenic cell death and inflammation.

[0009] Therefore, there is a need in the art for improved means and methods for oncolytic virus therapy, and improved oncolytic viruses. It is an object of the present invention to provide an excellent cancer therapeutic agent that combines optimal virus-mediated oncolysis with potent immune-mediated effects in the absence of systemic toxicity. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent No. 10,604,574 [Patent Document 2] International Publication No. 2017 / 198779 [Non-patent literature]

[0011] [Non-Patent Document 1] Bartee MY, Dunlap KM, Bartee E. Tumor-Localized Secretion of Soluble PD1 Enhances Oncolytic Virotherapy. Cancer Res. 2017;77(11):2952-63. Summary of the Invention [Means for solving the problem]

[0012] Summary of the Invention Below, elements of the present invention are described. While these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The description should be understood to support and encompass embodiments combining two or more of the explicitly described embodiments, or combining one or more of the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all described elements in this application should be deemed to be disclosed by the description of this application, unless the context indicates otherwise.

[0013] In a first aspect, the present invention provides a recombinant oncolytic virus, the virus comprising: Vesicular stomatitis virus (VSV), Including, Here, the glycoprotein (G protein) of VSV is deleted, and the virus a modified fusion protein (F protein) of Newcastle disease virus (NDV), and NDV hemagglutinin-neuraminidase (HN) protein, Including, Soluble PD-1 (sPD-1) The present invention relates to a virus further comprising:

[0014] In one embodiment, the recombinant virus further comprises an Fc domain or a fragment thereof, preferably a human IgG1, IgG2, IgG3, and / or IgG4 Fc domain or a fragment thereof.

[0015] In one embodiment, the Fc domain or fragment thereof is fused to the sPD-1.

[0016] In one embodiment, the sPD-1 is high-affinity sPD-1 (HA-sPD-1), preferably having an affinity for its ligands PD-L1 and PD-L2 that is at least 2-fold higher (e.g., 45-fold higher and 30-fold higher) than the affinity of wild-type sPD-1 for the ligands, respectively, and / or a K of <3.2 μM for PD-L1 and / or <0.1 μM for PD-L2. d It has affinity with

[0017] In one embodiment, the sPD-1 comprises a mutation at a position selected from 132 and 41 of SEQ ID NO:2, preferably a mutation selected from A132L, L41I, and L41V.

[0018] In one embodiment, the sPD-1 comprises or consists of a sequence having SEQ ID NO:2, optionally further comprising a mutation at a position selected from 132 and 41 of SEQ ID NO:2, preferably a mutation selected from A132L, L41I, and L41V.

[0019] In one embodiment, the sPD-1 is HA-sPD-1-A132L having the sequence of SEQ ID NO:3.

[0020] In one embodiment, the modified fusion protein (F protein) of the NDV is an F3aa modified F protein having the sequence of SEQ ID NO: 25, and / or comprising at least one amino acid substitution in the protease cleavage site, preferably at position L289 of SEQ ID NO: 25, more preferably L289A; and / or The modified fusion protein (F protein) of the NDV is an F3aa modified F protein having the amino acid substitution L289A having SEQ ID NO: 4; and / or The G protein of the VSV is replaced by a modified fusion protein having the sequence of SEQ ID NO:4 and the HN protein of NDV having the sequence of SEQ ID NO:5.

[0021] In a further aspect, the present invention relates to a nucleic acid encoding a recombinant oncolytic virus as defined above.

[0022] In one embodiment, the nucleic acid comprises a nucleic acid encoding an Fc domain or a fragment thereof, preferably having the sequence of SEQ ID NO:7, fused to the nucleic acid encoding sPD-1, preferably having SEQ ID NO:6, wherein the fusion product preferably has the nucleic acid sequence of SEQ ID NO:8.

[0023] In this aspect, the recombinant oncolytic virus, the Fc domain, the fragment, and the sPD-1 are as defined above.

[0024] In a further aspect, the present invention relates to a vector comprising a nucleic acid as defined above, preferably having the sequence SEQ ID NO: 9, as needed, - a reporter gene such as one of the following: HSV1-sr39TK, sodium iodide symporter (NIS), somatostatin receptor 2 (SSTR2), luciferase (firefly or Renilla), green fluorescent protein (GFP), lacZ, and tyrosinase; - a gene to be delivered to tumor cells and / or tumor tissue, such as any of the following: immune stimulatory genes (e.g., IFN-α, IFN-β, or granulocyte-macrophage colony-stimulating factor (GM-CSF)), IL-12, or IL-15; an immune checkpoint inhibitor antibody (e.g., PD-1, PD-L1, CTLA-4, LAG-3, or B7-H3); and / or Tumor-associated antigens (TAA) (specific for the tumor being targeted) for vaccination; or - combinations of these, The present invention relates to a vector further comprising any one of the following:

[0025] In a further aspect, the present invention relates to a nucleic acid as defined above or a vector as defined above, the nucleotide sequence of SEQ ID NO: 9 or 15, or a nucleotide sequence having at least 60%, preferably at least 70% or 80%, more preferably at least 90% or 95% sequence identity with the nucleotide sequence of SEQ ID NO: 9 or 15; or consisting of the nucleotide sequence of SEQ ID NO: 16, or a nucleotide sequence having at least 60%, preferably at least 70% or 80%, more preferably at least 90% or 95% sequence identity with the nucleotide sequence of SEQ ID NO: 16; comprising or consisting of Concerning nucleic acids or vectors.

[0026] In a preferred embodiment, the nucleic acid (as defined above) or the vector (as defined above) comprises or consists of the nucleotide sequence of SEQ ID NO: 9 or 15, or a nucleotide sequence having at least 60%, preferably at least 70% or 80%, more preferably at least 90% or 95% sequence identity to the nucleotide sequence of SEQ ID NO: 9 or 15.

[0027] In a further aspect, the present invention provides a pharmaceutical composition comprising: (i) a recombinant oncolytic virus as defined above, a nucleic acid as defined above, or a vector as defined above; and (ii) optionally, a pharmaceutically acceptable carrier and / or excipient; (iii) Additional medications, as needed, such as: chemotherapy agents, radiotherapeutic agents, tumor vaccines, immune checkpoint inhibitors (e.g., anti-CTLA4), adoptive cell therapy systems (e.g., T cells or dendritic cells), Cell carrier systems, small molecule inhibitors, embolic factors, Shielding polymers, The present invention relates to a pharmaceutical composition comprising:

[0028] In one embodiment, the pharmaceutical composition as defined above comprises for systemic delivery, tumor injection, intravenous administration, and intra-arterial administration; and / or For any of the following injections: intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavity, intracerebral, intraventricular, or intravitreal; It is formulated in

[0029] In a further aspect, the present invention relates to a recombinant oncolytic virus (as defined above), a nucleic acid (as defined above), a vector (as defined above), or a pharmaceutical composition (as defined above) for use in medicine.

[0030] In a further aspect, the present invention relates to a recombinant oncolytic virus (as defined above), a nucleic acid (as defined above), a vector (as defined above), or a pharmaceutical composition (as defined above) for use in the prevention and / or treatment of cancer, for example, the prevention and / or treatment of a cancer selected from hepatocellular carcinoma, pancreatic cancer, and melanoma.

[0031] In one embodiment, the recombinant oncolytic virus for the above use (as defined above), for the above use (as defined above), the nucleic acid for the above use (as defined above), the vector for the above use (as defined above), the pharmaceutical composition for the above use (as defined above) is administered in combination with other therapies such as: Cell carrier systems, e.g., T cells, dendritic cells, NK cells, mesenchymal stem cells, immunotherapy, e.g., tumor vaccines or immune checkpoint inhibitors; adoptive cell therapy (e.g., with T cells or dendritic cells), and / or Standard tumor therapy, e.g., radiofrequency ablation, chemotherapy, embolization, small molecule inhibitors, It is used in combination with.

[0032] In a further aspect, the present invention relates to a recombinant oncolytic virus (as defined above), a nucleic acid (as defined above), a vector (as defined above), or a pharmaceutical composition (as defined above) for use in diagnosing cancer, for example, a cancer selected from hepatocellular carcinoma, pancreatic cancer, and melanoma.

[0033] In a further aspect, the present invention relates to the use of a recombinant oncolytic virus (as defined above), or a nucleic acid (as defined above), or a vector (as defined above), or a pharmaceutical composition (as defined above) as a gene delivery tool, for (non-invasive) imaging of viral biodistribution, and / or for tumor detection. In one embodiment, such use is in vitro use.

[0034] In a further aspect, the present invention relates to a method for the diagnosis, prevention and / or treatment of cancer, said method comprising administering to a subject in need thereof a therapeutic amount of a recombinant oncolytic virus (as defined above), or a nucleic acid (as defined above), or a vector (as defined above), or a pharmaceutical composition (as defined above).

[0035] In one embodiment, the administering step is via systemic, intravenous, intra-arterial, tumor injection, and / or via intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavity, intracerebral, intraventricular, and intravitreal injection.

[0036] In this aspect, the cancer and the administering step are as defined above.

[0037] In a further aspect, the present invention relates to the use of a recombinant oncolytic virus (as defined above), or a nucleic acid (as defined above), or a vector (as defined above), or a pharmaceutical composition (as defined above) for the manufacture of a medicament for diagnosing, preventing and / or treating cancer. In this aspect, the cancer and the diagnosing, preventing and / or treating steps are as defined above. DETAILED DESCRIPTION OF THE INVENTION

[0038] Detailed Description The enhanced virus of the present invention is a modified VSV-NDV that provides several beneficial features over conventional oncolytic viruses generated by the induction of cell-cell fusion reactions, such as rapid and efficient tumor cell oncolysis, while maintaining an excellent safety profile. Not only do infected cells fuse with neighboring tumor cells, thereby killing them, but they also trigger a chain of events that causes the patient's immune system to attack remaining uninfected tumor cells, creating an inflammatory tumor microenvironment. Because VSV-NDV can replicate well in all tumor cells, it has broad therapeutic potential in numerous tumor indications.

[0039] The present invention provides a potent oncolytic virus carrying an effective immune checkpoint blockade molecule within a single therapeutic agent. The inventors used the safe and immune-stimulating rVSV-NDV virus as a vector to express a soluble PD-1 (sPD-1) molecule, which contains the signaling domain and soluble extracellular domain of human PD-1. The recombinant vector infects tumor cells, where it replicates and induces the secretion of sPD-1. sPD-1 then binds to its ligands, PD-L1 and PD-L2, on the surface of surrounding tumor cells and immune cells (i.e., dendritic cells), thereby acting as a decoy that competes for T cell binding to these inhibitory ligands, allowing T cells to remain active without the need for antibodies (Figure 1).

[0040] Compared to traditional antibody-mediated immune checkpoint blockade (ICB) therapy, this invention is innovative in that it combines oncolytic virus therapy and ICB in one therapeutic agent, which acts to simultaneously debulk tumors while alleviating immune suppression within the microenvironment through a direct oncolytic effect. Furthermore, this approach allows for self-amplification of the therapeutic agent through viral replication and local release of PD-1 directly at the tumor site, which is a major safety advantage compared to antibody approaches.

[0041] The present invention has the following advantages: first, the vector contains an enzymatically fused NDV fusion (F) protein (e.g., NDV / F3aa(L289A)), which acts synergistically with potent immunogenic cell death and, optimally, immune checkpoint blockade; second, the sPD-1 used in the construct is preferably a "high-affinity" version with increased affinity compared to wild-type sPD-1. Such high-affinity versions are generated, for example, through the introduction of a single alanine-to-leucine substitution at amino acid 132 (A132L) to produce 45-fold and 30-fold higher affinity binding to its ligands, PD-L1 and PD-L2 [2], respectively, or are high-affinity consensus variants encoding, for example, an isoleucine or valine at position 41, which have 40,000-fold higher affinity for PD-L1 than wild-type [3]. Third, HA-sPD-1 is fused to the Fc domain of human IgG for enhanced stability. In one embodiment, the Fc region directly follows the sPD-1 sequence in frame, where the stop codon of sPD-1 is removed to generate an sPD-1-Fc fusion protein with an Fc domain. In one embodiment, high-affinity sPD-1 (HA-sPD-1) has increased affinity compared to wild-type sPD-1 and / or compared to wild-type PD-1 (e.g., a 2-fold increase in affinity for each ligand). In one embodiment, the "high-affinity" version of sPD-1 has an affinity that is at least twice as high as that of wild-type sPD-1 (e.g., 5-fold higher affinity, 10-fold higher affinity, 30-fold higher affinity, or 45-fold higher affinity). In one embodiment, wild-type human PD-1 has K s of 6.36 μM and 0.19 μM for PD-L1 and PD-L2, respectively. d In one embodiment, HA-sPD-1 (high affinity sPD-1) has a K of <3.2 μM for PD-L1 and <0.1 μM for PD-L2. dand preferably a K of <0.5 μM for PD-L1 and <0.01 μM for PD-L2 (e.g., 0.14 μM and 0.0065 μM for PD-L1 and PD-L2, respectively). d It has an affinity with

[0042] The binding of wild-type sPD-1 to its ligand is likely too weak to support its application as a therapeutically effective approach. An advantage of the present invention is that the high-affinity version of sPD-1 used in the vectors of the present invention is more effective in cancer treatment and therefore has higher efficiency.

[0043] The vectors of the present invention offer several advantages over antibody-based ICB therapy. For example, they provide localized release of PD-1 specifically at the tumor site, potentially avoiding the severe side effects attributed to systemic delivery of these antibodies. The vectors of the present invention also offer a synergistic mechanism of action through tumor debulking, induction of antitumor immune responses, and therapeutic modulation of immunosuppression within the tumor microenvironment. Furthermore, rVSV-NDV-sPD-1 drug products could potentially be produced at a fraction of the price of PD-1 antibodies, making them more cost-effective than administering a combination therapy of OV in addition to a separate PD-1 antibody. Furthermore, the vectors of the present invention are advantageously safe for human use.

[0044] The present invention has additional advantages (e.g., advantages over oncolytic myxoma viruses expressing sPD-1). First, the rVSV-NDV vector is an optimal oncolytic virus platform because it is extremely safe and highly effective in tumor killing through direct oncolytic effects and induction of more immunogenic cell death, resulting in an abscopal effect. These superior therapeutic effects are attributed to VSV glycoprotein substitutions in the NDV envelope proteins (including the fusion (F) and hemagglutinin-neuraminidase (HN) proteins), and more specifically, through the use of a modified hyperfusogenic fusion (F) protein engineered into the viral construct. In one embodiment, the modified NDV fusion protein (F protein) is, for example, an F3aa-modified F protein having the sequence of SEQ ID NO: 25, optionally in a protease cleavage site, preferably further comprising at least one amino acid substitution at position L289 of SEQ ID NO: 25, more preferably L289A. In one embodiment, the use of the L289A modified version of the NDV fusion (F) protein, i.e., NDV / F3aa(L289A) (e.g., a protein having the amino acid sequence of SEQ ID NO: 4), provides increased superfusogenicity. These properties are the basis for rVSV-NDV being an ideal OV vector for use in combination with ICB. Furthermore, the rVSV-NDV-sPD-1 vector utilizes a high-affinity version of sPD-1, preferably human sPD-1. This provides a stronger interaction of sPD-1 with its ligand and therefore a higher potential for therapeutic efficacy. Finally, the high-affinity sPD-1 is fused to the Fc domain of human IgG, which allows for greater stability for an improved pharmacokinetic profile in vivo. In one embodiment, the nucleic acid and / or vector of the present invention comprises a nucleotide sequence having SEQ ID NO: 9 or 15, wherein SEQ ID NOs: 9 and 15 together encode VSV-NDV-HAsPD1-Fc, and wherein SEQ ID NO: 9 further comprises a non-coding region.

[0045] Concentrations, amounts, and other numerical data may be expressed or presented herein in range format. It should be understood that such range format is used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical ranges explicitly recited as range boundaries, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. By way of illustration, a numerical range of "20 to 100 nucleotides" should be interpreted not only to include the explicitly recited value of 20 to 100, but also to include each individual numerical value and subrange within the recited range. Thus, included within this numerical range are individual values ​​such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, ... 97, 98, 100, and subranges such as 25 to 35, 20 to 40, 25 to 50, etc. This same principle applies to ranges reciting a single numerical value (e.g., "at least 25 nucleotides"). Moreover, such an interpretation would apply regardless of the breadth of the range or the characteristics described.

[0046] The term "VSV" as used herein refers to vesicular stomatitis virus (VSV), a negative-strand RNA virus of the Rhabdoviridae family. Due to their inherent tumor specificity and rapid replication cycle, VSV vectors are highly attractive oncolytic agents, resulting in high intratumoral titers and subsequent tumor cell lysis. The VSV genome is a single molecule of negative-sense RNA encoding five major proteins: glycoprotein (G), large polymerase protein (L), phosphoprotein (P), matrix protein (M), and nucleoprotein (N). The entire genome is approximately 11,000 nucleotides. The VSV G protein enables viral entry. It mediates viral binding to the LDL receptor (LDLR) or LDLR family members present on host cells. After binding, the VSV-LDLR complex undergoes rapid endocytosis. It then mediates fusion of the viral envelope with the endosomal membrane. VSV enters cells via partially clathrin-coated vesicles; virus-containing vesicles contain more clathrin and clathrin adaptors than conventional vesicles. Virus-containing vesicles recruit components of the actin machinery for their interaction, thus inducing their own uptake. Replication occurs in the cytoplasm. The VSV L protein is encoded by half of the genome and, in combination with phosphoproteins, catalyzes mRNA replication. The VSV M protein is 831 nucleotides long and encoded by mRNA that translates into a 229-amino acid protein. The predicted M protein sequence does not contain any hydrophobic or nonpolar domains of any length that could promote membrane association. The protein is rich in basic amino acids and contains a highly basic amino-terminal domain. The term "rVSV" refers to recombinant vesicular stomatitis virus (VSV).

[0047] VSV Indiana complete genome SEQ ID NO: 17 NCBI GenBank accession number J02428.1 VSV Indiana G protein SEQ ID NO: 18 For the nucleotide and amino acid sequences, see GenBank Accession No. X03633.1.

[0048] The term "NDV," as used herein, refers to Newcastle disease virus (NDV), an avian virus of the Paramyxoviridae family. Members of this family possess a single-stranded, linear RNA. The entire genome is approximately 16,000 nucleotides. The virus replicates in the cytoplasm of host cells. It is a negative-strand RNA virus and has been developed as an oncolytic virus due to its innate ability to replicate and cause lysis in tumor cells while leaving healthy cells unharmed. Phase I-II clinical trials suggest that there is minimal toxicity associated with the therapy. The primary benefit of NDV as an oncolytic agent is that the viral envelope, which is composed of hemagglutinin-neuraminidase (HN) and fusion (F) proteins, not only mediates viral binding and fusion to target cells but also triggers fusion of infected cells with their neighboring uninfected cells, providing a potent mechanism for viral spread and tumor cell killing. Furthermore, emerging evidence indicates that syncytia formation triggered by cell-cell fusion generates a multimodal cell death response that may act synergistically with the direct oncolytic effect of the virus for a potent tumor-destroying mechanism.

[0049] Two proteins of Newcastle disease virus are inserted into the envelope: the hemagglutinin / neuraminidase protein (HN) and the fusion protein (F). These two proteins are important in determining the virulence of the virus and how it infects host cells. The hemagglutinin / neuraminidase protein has two important parts: (1) the hemagglutinin portion, which is a binding protein that binds to receptors on the outside of the membrane of host cells, including red blood cells; and (2) the neuraminidase portion, which is the active site of an enzyme that helps release the virus from the host cell membrane. The activity of this enzyme affects the time it takes for the virus to eluate from the red blood cell.

[0050] The fusion protein F fuses the viral envelope to the host cell membrane, allowing the viral genome to penetrate the host cell. For fusion to occur, the native fusion protein must change shape. This occurs when a host cell protease cleaves the protein at a specific cleavage site. After this occurs, the fusion protein is activated and can then fuse to the host cell membrane. The amino acid sequence around the cleavage site determines the range of proteases that can activate cleavage of the protein. This sequence therefore determines virulence. The NDV F protein is responsible for cell-to-cell spread of the virus through viral fusion with the cell membrane and syncytium formation. The presence of a multibasic cleavage site within the F protein allows protein cleavage and activation by a wide range of proteases and is a determinant of virulence in velogenic virus strains. We previously showed that a single amino acid substitution of leucine to alanine at amino acid 289 (L289A) in the F3aa-modified fusion protein resulted in substantially greater syncytia formation and tumor necrosis than viruses with only the F3aa mutation, without any additional toxicity [4]. The fusogenic and oncolytic activities of rNDV / F3aa strains can be further enhanced by a point mutation in the F protein from leucine to alanine at residue 289, generating rNDV / F3aa(L289A). In an orthotopic, immunocompetent rat model of liver tumors, administration of the mutant virus via hepatic artery infusion resulted in significant syncytia formation and necrosis, which accounted for a significant 20% prolonged survival over treatment with the original rNDV / F3aa virus [4].

[0051] NDV Hitchner B1 complete genome SEQ ID NO: 20 GenBank accession number AF375823 NDV HN protein SEQ ID NOs: 5 and 19 See GenBank Accession No. AF375823 and NCBI Gene ID 912270 for nucleic acid and amino acid sequences. NDV F protein SEQ ID NOs: 21 and 22 See GenBank Accession No. AF375823 and NCBI Gene ID 912271 for nucleic acid and amino acid sequences. NDV F3aa-modified fusion protein SEQ ID NOs: 24 and 25 NDV F3aa-modified fusion protein with L289A SEQ ID NOs: 23 and 4

[0052] As discussed above, the present invention provides a recombinant oncolytic VSV virus, wherein the VSV glycoprotein is pseudotyped and further comprises soluble PD-1. In one embodiment, when referring to a virus "further comprising soluble PD-1," this is preferably meant to refer to a situation in which the viral nucleic acid, preferably the viral genome, encodes the soluble PD-1; for example, the viral genome of VSV and / or VSV-NDV encodes soluble PD-1. For example, the nucleic acid encoding the soluble PD-1 can be incorporated into the viral genome, for example, the genome of a VSV or VSV-NDV hybrid virus, by genetic engineering. In one embodiment, the soluble PD-1 is expressed from such a viral genome, for example, by cells (e.g., cancer cells). In one embodiment, the recombinant oncolytic virus further comprising soluble PD-1 comprises a nucleic acid sequence encoding the soluble PD-1. In one embodiment, the recombinant oncolytic virus further comprises soluble PD-1 as part of the viral genome of the recombinant oncolytic virus, preferably as part of the VSV viral genome. In one embodiment, soluble PD-1 is expressed as a recombinant protein. In one embodiment, cells, such as cancer cells, infected with the recombinant oncolytic virus express the soluble PD-1. In one embodiment, the recombinant oncolytic virus further comprising soluble PD-1 encodes the soluble PD-1; preferably, the recombinant oncolytic virus encodes the soluble PD-1 in the viral genome. The concept of exchanging viral glycoproteins with glycoproteins of a heterologous virus ("pseudotyping") has previously been shown as an effective means of altering viral tropism. Using this approach, the viral backbone remains intact, and therefore viral replication in susceptible cells should be minimized.

[0053] In one embodiment, the G protein of VSV is replaced by a modified fusion protein, preferably a modified fusion (F) protein having an amino acid substitution at position L289 (e.g., L289A), and the HN protein of NDV. In one embodiment, the recombinant oncolytic virus further comprises the remaining proteins of VSV, i.e., the large polymerase protein (L), phosphoprotein (P), matrix protein (M), and nucleoprotein (N). For example, endogenous glycoproteins of VSV can be deleted from a plasmid encoding a full-length VSV genome. The NDV glycoproteins, including the modified fusion protein (NDV / F(L289A)) and hemagglutinin-neuraminidase (NDV / HN), can be inserted as a discontinuous transcription unit between the VSV matrix (M) gene and the large polymerase (L) gene (see FIG. 2A and WO 2017 / 198779). In one embodiment, the modified fusion protein (F protein) of NDV is an F3aa modified F protein, optionally an F3aa modified F protein comprising at least one amino acid substitution in the protease cleavage site, preferably at position L289 (e.g., L289A).

[0054] In one embodiment of the vector of the present invention, preferably a recombinant VSV (rVSV) vector, the modified fusion protein (F protein) of the NDV has the amino acid sequence of SEQ ID NO: 25 [= aa sequence of F3aa protein] or the amino acid sequence of SEQ ID NO: 4 [= aa sequence of F3aa protein / L289A], or an amino acid sequence having at least 60%, or preferably at least 70%, 80%, 90%, or 95% sequence identity with the amino acid sequence of SEQ ID NO: 25 or 4, and / or wherein the modified fusion protein (F protein) of the NDV has the nucleotide sequence of SEQ ID NO: 24 [= nucleotide sequence of F3aa protein] or the nucleotide sequence of SEQ ID NO: 23 [= nucleotide sequence of F3aa protein / L289A], or a nucleotide sequence having at least 60%, or preferably at least 70%, 80%, 90%, or 95% sequence identity with the nucleotide sequence of SEQ ID NO: 24 or 23; is coded by

[0055] In one embodiment of the vector of the present invention, preferably a rVSV vector, the HN protein of NDV is the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 60%, or preferably at least 70%, 80%, 90%, or 95% sequence identity with the amino acid sequence of SEQ ID NO: 5; and / or The HN protein of the NDV is encoded by the nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence having at least 60%, or preferably at least 70%, 80%, 90%, or 95% sequence identity to the nucleotide sequence of SEQ ID NO: 19.

[0056] The present invention includes nucleic acids encoding the oncolytic viruses of the present invention. The present invention further includes vectors comprising the nucleic acids of the present invention.

[0057] In one embodiment, the vector of the present invention comprises: - a reporter gene such as one of the following: HSV1-sr39TK, sodium iodide symporter (NIS), somatostatin receptor 2 (SSTR2), luciferase (firefly or Renilla), green fluorescent protein (GFP), lacZ, and tyrosinase; - a gene to be delivered to tumor cells and / or tumor tissue, such as any of the following: immune stimulatory genes (e.g., IFN-α, IFN-β, or granulocyte-macrophage colony-stimulating factor (GM-CSF)), IL-12, or IL-15 immune checkpoint inhibitor antibodies (e.g., PD-1, PD-L1, CTLA-4, LAG-3, or B7-H3); and Tumor-associated antigens (TAAs) (specific for the tumor being targeted) for vaccination; and - combinations of these, The present invention further includes any one of the following:

[0058] The present invention provides the recombinant oncolytic virus, the nucleic acid of the present invention, the vector of the present invention, and / or the pharmaceutical composition of the present invention for use in medical treatment. The present invention further provides the recombinant oncolytic virus, the nucleic acid of the present invention, the vector of the present invention, and / or the pharmaceutical composition of the present invention for use in the diagnosis, prevention, and / or treatment of cancer. The present invention also provides the recombinant oncolytic virus, the nucleic acid of the present invention, the vector of the present invention, and / or the pharmaceutical composition of the present invention for use in oncolytic therapy, particularly oncolytic virotherapy. The term "oncolytic virotherapy" as used herein refers to cancer therapy by administering an oncolytic virus, a nucleic acid encoding the same, or the respective vector to induce tumor regression. In one embodiment, the recombinant oncolytic virus of the present invention, the nucleic acid of the present invention, the vector of the present invention, and / or the pharmaceutical composition of the present invention is used in combination with other therapies, such as: Cell carrier systems, e.g., T cells, dendritic cells, NK cells, mesenchymal stem cells, immunotherapy, e.g., tumor vaccines or immune checkpoint inhibitors; adoptive cell therapy (e.g., with T cells or dendritic cells), and / or Standard tumor therapy, e.g., radiofrequency ablation, chemotherapy, embolization, small molecule inhibitors, is provided for use in combination with

[0059] In one embodiment, the recombinant oncolytic virus, nucleic acid, vector, and / or pharmaceutical composition is administered to a patient in need thereof via systemic, intravenous, intraarterial, or tumor injection, and / or via intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavity, intracerebral, intraventricular, and intravitreal injection. In a preferred embodiment, a pharmaceutical composition comprising a recombinant oncolytic virus of the present invention is formulated as a liquid composition for administration, e.g., intravenous or intratumoral administration. Such a composition is administered to a patient in need thereof at appropriate intervals (e.g., once weekly for a period of 1 to 15 weeks).

[0060] The present invention further provides a method for diagnosing, preventing, and / or treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the recombinant oncolytic virus, nucleic acid, or vector of the present invention, or a pharmaceutical composition of the present invention. A therapeutically effective amount of the recombinant oncolytic virus, nucleic acid, or vector of the present invention is an amount that produces a desired therapeutic result, in particular, tumor regression.

[0061] The recombinant virus, nucleic acid, vector, or pharmaceutical composition thereof is preferably administered in multiple cycles over a period of time, such as several days to several weeks. In one embodiment, the pharmaceutical composition of the present invention includes a carrier and / or excipient (e.g., a physiological buffer, and / or a polymer and / or lipid for improved stability and tumor transduction efficiency).

[0062] The present invention also relates to the in vitro and / or ex vivo use of the recombinant oncolytic viruses, nucleic acids, vectors and / or pharmaceutical compositions of the invention as gene delivery tools, (non-invasive) imaging of viral biodistribution and / or for tumor detection.

[0063] The vector of the present invention contains a modified highly fusogenic F protein inserted into a VSV G deletion vector along with the NDV HN binding protein. By creating a hybrid of these two potent oncolytic vectors, the beneficial features of each virus are combined while simultaneously eliminating the safety concerns of each. The resulting vector has a VSV backbone and thus maintains the rapid replication cycle of wild-type VSV. Furthermore, due to the incorporation of the NDV HN and highly fusogenic F proteins, the recombinant virus induces enhanced syncytium formation, allowing for efficient intratumoral spread of the virus and the induction of potent tumor cell killing mechanisms and antitumor immune responses. Using this strategy, the benefits of a fusogenic virus can be achieved without the environmental threats associated with NDV. Furthermore, because endogenous VSV glycoproteins are deleted, there is no neurotoxicity associated with the vector. Finally, because NDV binds to target cells via sialic acid residues, which are upregulated on tumor cells, the present invention achieves further transduction of tumors by targeting with pseudotyped vectors. Specific viral modifications not only make the virus safer, but also provide a highly potent virus due to the replacement of the VSV envelope protein with the NDV envelope protein. A mutated version of the NDV F protein has been introduced to further improve the efficiency of the resulting recombinant virus without negatively impacting safety. In addition, the vector further contains a highly affine soluble PD-1, which allows for highly efficient inhibition of immune checkpoints.

[0064] The benefits of vectors with glycoprotein exchange are three-fold: 1. The neurotropism associated with endogenous VSV glycoproteins can be avoided by deletion of the VSV envelope and introduction of non-neurotropic NDV envelope proteins; 2. Tumor cells can be targeted through the upregulation of sialic acid residues, which are the natural receptors for NDV; and 3. Viral spread and tumor cell killing can be significantly enhanced through the introduction of a highly fusogenic mutant version of the NDV F protein.

[0065] The viruses of the present invention provide improved safety and enhanced efficiency over other vectors. Furthermore, the use of the rVSV-NDV vector backbone is advantageous due to its high fusogenicity characteristics, lack of pre-existing immunity in the general population, and predicted lack of attenuation compared to VSV or NDV. Furthermore, the advantages of fusing sPD-1 with Fc are, first, increased stability of sPD-1, and second, the Fc domain allows the complex to interact with Fc receptors on immune cells, further contributing to immunotherapy. Preferably, the recombinant oncolytic virus of the present invention is a recombinant oncolytic vesicular stomatitis virus (VSV). In one embodiment, the recombinant oncolytic virus of the present invention includes an oncolytic virus as defined in WO 2017 / 198779, further comprising soluble PD-1 (sPD-1), preferably high-affinity sPD-1 (HA-sPD-1) (e.g., a high-affinity sPD-1 variant having SEQ ID NO: 3), and optionally further comprising an Fc domain, wherein the optional Fc domain is preferably fused to the sPD-1. In one embodiment, the recombinant oncolytic virus of the present invention includes an oncolytic virus as defined in WO 2017 / 198779, further comprising sPD-1, preferably high-affinity sPD-1 (HA-sPD-1) (e.g., a high-affinity sPD-1 variant having SEQ ID NO: 3), and further comprising an Fc domain fused to the sPD-1. In one embodiment, the virus of the invention comprises the N, P, M, and L proteins of VSV, modified F and HN proteins of NDV, and an HA-sPD-1-Fc fusion protein and / or a gene encoding such a protein.

[0066] The term "Fc domain," as used herein, refers to a fragment crystallizable region of an antibody, which is the tail region of the antibody that interacts with cell surface receptors (e.g., Fc receptors and some proteins of the complement system). In one embodiment, the Fc domain is any of human IgG1-Fc domain, IgG2-Fc domain, IgG3-Fc domain, and IgG4-Fc domain. In one embodiment, when referring to an "Fc domain" and / or an "Fc domain or fragment thereof," the term also includes a biologically functional fragment of the Fc domain (e.g., a CH3 fragment of the Fc domain). That is, the term includes the entire Fc domain and fragments thereof (e.g., a CH3 fragment). A fragment of an Fc domain is typically a biologically functional fragment of the Fc domain. That is, the fragment retains the ability to interact with a cell surface receptor (e.g., an Fc receptor) and / or a component of the complement system (e.g., a CH3 fragment).

[0067] In one embodiment, a patient's response to infection with a recombinant virus of the invention induces a highly inflamed tumor microenvironment, thereby sensitizing the tumor to immune checkpoint inhibition. In one embodiment, the terms "subject" and "patient" are used interchangeably and preferably refer to a mammalian patient, more preferably a human patient.

[0068] In one embodiment, the term "soluble PD-1" refers to a soluble form of programmed cell death protein 1 (PD-1). PD-1 is a protein that plays a role in immune checkpoints, i.e., regulating immune system responses by downregulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-1 typically has the sequence set forth in SEQ ID NO: 1. PD-1 is a protein found on the cell surface, and soluble PD-1 is its soluble form that can be secreted from cells. Soluble PD-1 typically has the sequence set forth in SEQ ID NO: 2. In one embodiment, the soluble PD-1 is high-affinity sPD-1, which has the sequence set forth in SEQ ID NO: 3. In one embodiment, the virus of the present invention and / or pharmaceutical composition of the present invention comprises an sPD-1 having at least 60%, preferably 80%, and more preferably 95% or greater (e.g., 99%) sequence identity to an amino acid sequence having any of SEQ ID NOs: 1-3, preferably SEQ ID NO: 3. In one embodiment, the virus of the present invention, nucleic acid of the present invention, vector of the present invention, and / or pharmaceutical composition of the present invention comprises an sPD-1 having at least 60%, preferably 80%, and more preferably 95% or greater (e.g., 99%) sequence identity to a nucleic acid encoding an amino acid sequence having any of SEQ ID NOs: 1-3, preferably SEQ ID NO: 3 (e.g., a nucleic acid having the sequence of SEQ ID NO: 6). Due to codon degeneracy in the genetic code, reference to a nucleic acid also encompasses alternative nucleic acid sequences that encode the same amino acid sequence but contain alternative codons.

[0069] The terms "fused" and / or "fusion protein," as used herein in the context of sPD-1 and Fc, refer to a fusion of proteins (e.g., sPD-1 and Fc) created through the joining of two or more genes that normally encode separate proteins. Translation of such a fusion gene (e.g., sPD-1-Fc) results in one or multiple polypeptides possessing functional properties derived from each of the original proteins. Protein fusion typically involves removing the stop codon from the cDNA sequence encoding the first protein and then adding the cDNA sequence of the second protein in frame through methods known to those of skill in the art (e.g., ligation or overlap extension PCR). The resulting DNA sequence is then expressed by cells as a single protein. The protein can be engineered to contain the entire sequence of both original proteins, or only one of the proteins. Optionally, a linker can be included between the components of the fusion protein. In one embodiment, the term "fusion product" refers to a product made by fusing two or more genes (e.g., a fusion gene comprising a gene comprising a nucleic acid encoding an Fc domain, preferably having the sequence of SEQ ID NO: 7, and a gene comprising a nucleic acid encoding sPD-1, preferably having the sequence of SEQ ID NO: 6, and / or a product encoded by such a fused gene). In one embodiment, an exemplary fusion product of HA-sPD-1 and Fc has the nucleic acid sequence of SEQ ID NO: 8 and / or the amino acid sequence of SEQ ID NO: 13. In one embodiment, the Fc domain, or a fragment thereof, is fused to sPD-1 (e.g., HA-sPD-1), meaning that a nucleic acid encoding an Fc domain, or a fragment thereof, and a nucleic acid encoding sPD-1 (e.g., HA-sPD-1) are used.In one embodiment, when used in the context of the fusion (F) protein of NDV, the term "fusion protein" does not relate to two or more proteins joined together; rather, the term relates to a protein that induces fusion of the viral envelope with a cellular receptor.

[0070] The term "high affinity," as used herein, refers to an increased affinity of a modified protein (e.g., HA-sPD-1) for its binding partner (e.g., PD-L1) compared to the affinity of the respective wild-type protein (e.g., sPD-1) for its respective binding partner. In one embodiment, a "high affinity" version of sPD-1 (HA-sPD-1) has increased affinity for its ligand (e.g., a two-fold increased affinity) compared to wild-type sPD-1. Such high affinity versions are generated, for example, through the introduction of a single alanine-to-leucine substitution at amino acid 132 of sPD-1 (A132L), which results in higher affinity (e.g., 45-fold and 30-fold higher affinity) for binding to its ligands, PD-L1 and PD-L2, respectively, or are high-affinity consensus variants encoding an isoleucine or valine at position 41, which results in higher affinity (e.g., 40,000-fold higher affinity for PD-L1) compared to wild-type sPD-1. In one embodiment, the high affinity sPD-1 has a K of <3.2 μM for PD-L1 and <0.1 μM for PD-L2. d In one embodiment, the mutation is preferably a point mutation.

[0071] SEQ ID NO: 1 represents the amino acid sequence of human PD-1. SEQ ID NO: 2 represents the amino acid sequence of human PD-1 (soluble). SEQ ID NO: 3 represents the amino acid sequence of an exemplary high-affinity PD-1 (soluble) that is HA-sPD-1-A132L. SEQ ID NO: 4 represents the amino acid sequence of the modified fusion protein (F protein) of F3aa-NDV with the amino acid substitution L289A. SEQ ID NO: 5 represents the amino acid sequence of the HN protein of NDV. SEQ ID NO: 6 represents the nucleic acid sequence encoding an exemplary high affinity PD-1 (soluble), which is HA-sPD-1-A132L. SEQ ID NO: 7 represents the nucleic acid sequence of human IgG1-Fc; the CH2 and CH3 domains of the human IgG1 heavy chain and hinge region. SEQ ID NO: 8 represents the nucleic acid sequence of an exemplary high-affinity soluble PD-1-IgG1-Fc fusion gene. SEQ ID NO: 9 represents the nucleic acid sequence of an exemplary vector of the invention comprising the construct VSV-NDV-HA-sPD-1-Fc. SEQ ID NO: 10 represents the amino acid sequence of the N protein. SEQ ID NO: 11 represents the amino acid sequence of the P protein. SEQ ID NO: 12 represents the amino acid sequence of the M protein. SEQ ID NO: 13 represents the amino acid sequence of the HA-sPD-1-Fc protein. SEQ ID NO: 14 represents the amino acid sequence of the L protein. SEQ ID NO: 15 represents the nucleic acid sequence of the construct VSV-NDV-HA-sPD-1-Fc. SEQ ID NO: 16 represents the entire nucleic acid sequence of rVSV-NDV-sPD-1, where the sPD-1 is normal sPD-1 without high-affinity modifications, and where the sequence does not include the Fc coding sequence. SEQ ID NO: 17 represents the complete genome of VSV Indiana. SEQ ID NO: 18 represents the amino acid sequence of the VSV Indiana G protein. SEQ ID NO: 19 represents the nucleic acid sequence of the NDV HN protein. SEQ ID NO: 20 represents the complete genome of NDV Hitchner B1. SEQ ID NO: 21 represents the nucleic acid sequence of the NDV F (unmodified) protein. SEQ ID NO: 22 represents the amino acid sequence of the NDV F (unmodified) protein. SEQ ID NO: 23 represents the nucleic acid sequence of the NDV F3aa-modified fusion protein with L289A. SEQ ID NO: 24 represents the nucleic acid sequence of the NDV F3aa-modified fusion protein. SEQ ID NO: 25 represents the amino acid sequence of the NDV F3aa-modified fusion protein. SEQ ID NO: 26 represents the nucleic acid sequence of human soluble PD-1 (sPD-1). [Brief explanation of the drawings]

[0072] The invention will now be further described by reference to the following drawings.

[0073] All methods referred to in the following figure descriptions were performed as detailed in the Examples.

[0074] [Figure 1] Figure 1 illustrates the PD-1 / PD-L1 interaction and the interference of soluble PD-1. When exhausted T cells come into contact with tumor cells expressing PD-L1, PD-1 on the T cells binds and becomes inactivated, allowing the tumor cells to avoid immune clearance. Local expression of soluble PD-1 competes with PD-1 expressed by the T cells for binding to its PD-L1 ligand, interfering with the interaction and allowing the T cells to remain functional and exert their cytotoxic effector function against the tumor cells [5].

[0075] [Figure 2]Figure 2 shows the rVSV-NDV backbone (A) and the rVSV-NDV-sPD-1 construct of the present invention (B). A) A recombinant pseudotyped VSV construct expressing NDV glycoproteins (see also WO 2017 / 198779 A1). Endogenous glycoproteins of VSV were deleted from a plasmid encoding the full-length VSV genome. NDV glycoproteins, including an engineered fusion protein (NDV / F(L289A)) and hemagglutinin-neuraminidase (NDV / HN), were inserted as discontinuous transcription units between the VSV matrix (M) gene and large polymerase (L) gene. Each pseudotyped VSV vector was rescued using an established reverse genetics system. B) The construct of the present invention contains a soluble human PD-1 gene, preferably a high-affinity soluble human PD-1 gene (HA-sPD-1), fused to the Fc domain of human IgG. The high-affinity soluble human PD-1 gene fused to the Fc domain of human IgG (HA-sPD-1) was cloned as an additional transcription unit between the NDV binding protein (HN) and VSV large polymerase (L) genes, and the resulting viral genome is shown.

[0076] [Figure 3] Figure 3 shows that rVSV-NDV-HA-sPD-1-Fc replicates in mouse melanoma cells with slight attenuation. The mouse melanoma cell line, B16-OVA, was infected with rVSV-NDV-GFP or rVSV-NDV-HA-sPD-1-Fc at a multiplicity of infection (MOI) of 0.01. One hour after infection, the cells were washed, and fresh medium was added to the cells. At various time points, post-infection aliquots of the supernatant were collected for measurement of virus titer by TCID50 assay. Experiments were performed in triplicate, and data are presented as the mean ± standard error of the mean.

[0077] [Figure 4]Figure 4 shows that rVSV-NDV-HA-sPD-1-Fc efficiently kills mouse melanoma cells. The mouse melanoma cell line, B16-OVA, was infected with rVSV-NDV-GFP or rVSV-NDV-HA-sPD-1-Fc at a multiplicity of infection (MOI) of 0.01. One hour after infection, the cells were washed, and fresh medium was added to the cells. At various time points, post-infection aliquots of the supernatant were collected for LDH assays for cytotoxicity. Experiments were performed in triplicate, and data are presented as the mean ± standard error of the mean.

[0078] [Figure 5] Figure 5 shows that rVSV-NDV-HA-sPD-1-Fc causes tumor growth delay in immunocompetent mice bearing syngeneic B16-OVA melanoma cells. Male C57B1 / 6 mice were implanted subcutaneously in the flank with 2.4 x 10 B16-OVA cells. On days 7, 10, and 13 after tumor implantation, PBS or 10 TCID of rVSV-NDV-GFP or rVSV-NDV-HA-sPD-1-Fc was injected into the tumor in a volume of 50 μl. Tumor size was monitored daily, and tumor volume was calculated using the formula: 4 / 3 x PI() x ((L + W) / 4)3. Individual tumor growth curves for each treatment are shown. Each curve represents an individual mouse.

[0079] [Figure 6]Figure 6 shows that intratumoral injection of rVSV-NDV-HA-sPD-1-Fc significantly prolongs the survival of mice bearing B16-OVA. Male C57B1 / 6 mice were subcutaneously implanted with 2.4 x 10 B16-OVA cells in the flank. On days 7, 10, and 13 after tumor implantation, PBS or 10 TCID of rVSV-NDV-GFP or rVSV-NDV-HA-sPD-1-Fc was injected into the tumor in a 50 μl volume. Mice were monitored daily and euthanized when the tumor diameter reached 1.5 cm or when the skin broke due to tumor growth. Survival times after treatment were plotted as Kaplan-Meier survival curves, and statistical significance was determined by the log-rank test. The p-value for rVSV-NDV-HA-sPD-1-Fc versus PBS was <0.05.

[0080] [Figure 7] Figure 7 shows an exemplary recombinant VSV-NDV vector encoding soluble PD1 (sPD1). The human sPD1 gene was cloned into the VSV-NDV vector as a unique transcription unit between the hemagglutinin-neuraminidase (HN) gene and the large polymerase (L) gene. Additional modifications, including high-affinity (HA) mutations and fusion with a human Fc fragment, were further engineered. The resulting genome is shown.

[0081] [Figure 8]Figure 8 shows that rVSV-NDV-sPD1 variants have similar growth kinetics and cytotoxic effects in B16 melanoma cells compared to the control rVSV-NDV-GFP. Murine B16 melanoma cells were infected in vitro with rVSV-NDV-GFP, rVSV-NDV-sPD1, rVSV-NDV-HA-sPD1, rVSV-NDV-sPD1-Fc, or rVSV-NDV-HA-sPD1-Fc at an MOI of 0.01. Top panel: Representative photomicrographs were obtained at 200x magnification at 24 and 48 hours post-infection. Uninfected B16 cells served as a control. Bottom left: Cytotoxicity was quantified at various time points post-infection using a lactate dehydrogenase assay (LDH, Promega). The mean + standard error of the mean of duplicate experiments is shown. Bottom right: Aliquots of supernatant taken at multiple time points post-infection were subjected to TCID50 analysis for analysis of virus growth kinetics. Mean + standard error of the mean of triplicate experiments is shown.

[0082] [Figure 9] Figure 9 shows that recombinant sPD1-expressing VSV-NDV vectors produce and secrete human PD1. B16 mouse melanoma cells were infected with VSV-NDV-GFP or variants of VSV-NDV expressing soluble human PD1 (VSV-NDV-sPD1, VSV-NDV-HA-sPD1, VSV-NDV-sPD1-Fc, or VSV-NDV-HA-sPD1-Fc) at an MOI of 0.01, or mock-infected. Left: Aliquots of cell lysates and supernatants were collected at various time points post-infection and subjected to Western blot analysis for human PD1 or GAPDH. Right: Aliquots of supernatants were collected at 24 hours and subjected to ELISA assays for quantification of released human PD1. Means + standard error of the mean are shown.

[0083] [Figures 10A-10B]Figure 10 shows that treatment of immunocompetent tumor-bearing mice with rVSV-NDV-HA-sPD1-Fc results in improved control of tumor growth and enrichment of tumor-specific T cells compared with VSV-NDV controls. A) Female C57B1 / 6 mice were subcutaneously implanted with syngeneic B16 melanoma cells in the contralateral flank. PBS, 107 TCID50 of rVSV-NDV, or rVSV-NDV-HA-sPD1-Fc were injected into the tumor on the right flank, while the tumor on the left flank was left untreated. Tumor volume was monitored daily, and blood was collected on day 15. B) Circulating OVA-specific T cells were quantified by flow cytometry using an OVA-specific tetramer to stain peripheral blood mononuclear cells (PBMCs) obtained from the blood. Individual data points, means, and standard errors of the means are shown. C) Tumor volume was significantly reduced in the rVSV-NDV-HA-sPD1-Fc group compared with the rVSV-NDV and PBS groups. [Figure 10C-1] Figure 10 shows that treatment of immunocompetent tumor-bearing mice with rVSV-NDV-HA-sPD1-Fc results in improved control of tumor growth and enrichment of tumor-specific T cells compared with VSV-NDV controls. A) Female C57B1 / 6 mice were subcutaneously implanted with syngeneic B16 melanoma cells in the contralateral flank. PBS, 107 TCID50 of rVSV-NDV, or rVSV-NDV-HA-sPD1-Fc were injected into the tumor on the right flank, while the tumor on the left flank was left untreated. Tumor volume was monitored daily, and blood was collected on day 15. B) Circulating OVA-specific T cells were quantified by flow cytometry using an OVA-specific tetramer to stain peripheral blood mononuclear cells (PBMCs) obtained from the blood. Individual data points, means, and standard errors of the means are shown. C) Tumor volume was significantly reduced in the rVSV-NDV-HA-sPD1-Fc group compared with the rVSV-NDV and PBS groups. [Figure 10C-2]Figure 10 shows that treatment of immunocompetent tumor-bearing mice with rVSV-NDV-HA-sPD1-Fc results in improved control of tumor growth and enrichment of tumor-specific T cells compared with VSV-NDV controls. A) Female C57B1 / 6 mice were subcutaneously implanted with syngeneic B16 melanoma cells in the contralateral flank. PBS, 107 TCID50 of rVSV-NDV, or rVSV-NDV-HA-sPD1-Fc were injected into the tumor on the right flank, while the tumor on the left flank was left untreated. Tumor volume was monitored daily, and blood was collected on day 15. B) Circulating OVA-specific T cells were quantified by flow cytometry using an OVA-specific tetramer to stain peripheral blood mononuclear cells (PBMCs) obtained from the blood. Individual data points, means, and standard errors of the means are shown. C) Tumor volume was significantly reduced in the rVSV-NDV-HA-sPD1-Fc group compared with the rVSV-NDV and PBS groups. [Figure 10C-3] Figure 10 shows that treatment of immunocompetent tumor-bearing mice with rVSV-NDV-HA-sPD1-Fc results in improved control of tumor growth and enrichment of tumor-specific T cells compared with VSV-NDV controls. A) Female C57B1 / 6 mice were subcutaneously implanted with syngeneic B16 melanoma cells in the contralateral flank. PBS, 107 TCID50 of rVSV-NDV, or rVSV-NDV-HA-sPD1-Fc were injected into the tumor on the right flank, while the tumor on the left flank was left untreated. Tumor volume was monitored daily, and blood was collected on day 15. B) Circulating OVA-specific T cells were quantified by flow cytometry using an OVA-specific tetramer to stain peripheral blood mononuclear cells (PBMCs) obtained from the blood. Individual data points, means, and standard errors of the means are shown. C) Tumor volume was significantly reduced in the rVSV-NDV-HA-sPD1-Fc group compared with the rVSV-NDV and PBS groups.

[0084] In the following, reference is made to examples, which are presented for the purpose of illustrating, but not limiting, the present invention. [Example]

[0085] Example 1: Preparation of rVSV-NDV-sPD-1 virus To engineer the rVSV-NDV-sPD-1 virus, the signaling and extracellular domains of PD-1 were first amplified from human PBMCs by RT-PCR. The resulting RT-PCR product served as a template for a round of overlapping PCR to introduce the A132L mutation for high affinity, generating two overlapping PCR fragments containing the mutated base pair. These fragments were annealed and subjected to a final extension step to generate the full-length HA-sPD-1 gene. The HA-sPD-1 fragment was cloned into pFUSE-hIgG1-Fc1 to create a fusion gene between HA sPD-1 and the human Fc fragment. To insert the HA-sPD-1-Fc construct into rVSV-NDV, appropriate restriction sites were introduced using forward and reverse oligonucleotides and amplified by PCR. Finally, the insert was ligated into the full-length VSV-NDV genome as an additional transcription unit via the multiple cloning site between the NDV-HN and VSV-L genes. The viral construct is shown in Figure 2B. The corresponding infectious recombinant virus was rescued using established methods of reverse genetics.

[0086] Example 2: Characterization of rVSV-NDV-sPD-1 virus The recombinant VSV-NDV-HA-sPD-1-Fc virus was characterized in the mouse melanoma cell line B16-OVA. Comparison of the growth kinetics of the virus with that of the parental rVSV-NDV virus revealed a slight attenuation due to transgene expression, with peak replication reaching approximately 48 hours postinfection at a multiplicity of infection (MOI) of 0.01 (Figure 3). Consistent with these findings, cytotoxicity assays in the same cells revealed a slight delay in tumor cell killing by infection with rVSV-NDV-HA-sPD-1-Fc, but nearly complete killing of the cell monolayer by approximately 72 hours postinfection (Figure 4). Thus, the VSV-NDV-HA-sPD-1-Fc virus provides enhanced efficacy after 72 hours postinfection compared with the virus without HA-sPD-1-Fc.

[0087] Example 3: Anti-cancer effect of rVSV-NDV-sPD-1 virus in vivo To determine in vivo efficacy, experiments were performed in immunocompetent C57B1 / 6 mice bearing subcutaneous B16-OVA tumors in the flank. On days 7, 10, and 13 after tumor implantation, mice were treated with PBS or 10 7 TCID50 of rVSV-NDV-GFP or rVSV-NDV-HA-sPD-1-Fc was injected into the tumor in a volume of 50 μl. Tumors were measured daily and tumor volume was calculated using the following formula: V=4 / 3*PI()*((L+W) / 4) 3 The data revealed a significant delay in tumor growth in those mice treated with rVSV-NDV-GFP, which was even more pronounced in mice receiving rVSV-NDV-HA-sPD-1-Fc compared to PBS (Figure 5).

[0088] Example 4: Enhanced survival of mice treated with rVSV-NDV-HA-sPD-1-Fc In survival studies, mice were euthanized at a humane endpoint when tumors reached 1.5 cm in diameter or when tumor growth breached the skin. Plotting survival time relative to the first treatment dose revealed significantly prolonged survival for mice treated with rVSV-NDV-HA-sPD-1-Fc compared to PBS, with median survival times of 14 days and 26 days, respectively (Figure 6). The light gray line represents survival time after treatment with PBS; the medium gray represents that of rVSV-NDV-GFP treatment; and the black line represents that of rVSV-NDV-HA-sPD-1-Fc treatment. The data show that treatment with rVSV-NDV-HA-sPD-1-Fc resulted in a substantial delay in tumor growth and prolonged survival compared to treatment with buffer or the parental rVSV-NDV virus.

[0089] Example 5: Recombinant VSV-NDV vector encoding soluble PD1 (sPD1) A panel of VSV-NDV recombinant vectors encoding soluble PD1 with (sPD1-Fc) or without (sPD1) a human Fc fragment, and with or without a high-affinity mutation (HA-sPD1), were engineered and rescued by an established reverse genetics system (Figure 7). These rVSV-NDV-sPD1 variants were fully characterized in vitro in comparison to a control rVSV-NDV vector, and the rVSV-NDV-HA-sPD1-Fc vector was selected for further in vitro characterization in an immunocompetent model of murine melanoma.

[0090] Example 6: rVSV-NDV-sPD1 variants replicate well and cause cytotoxicity in B16 mouse melanoma cells To characterize the panel of rVSV-NDV-sPD1 variants in vitro, the B16 mouse melanoma cell line was selected as a representative. B16 cells were infected with various sPD1 variants or the VSV-NDV-GFP control virus at a multiplicity of infection (MOI) of 0.01. Cells were examined microscopically at various time points post-infection to visualize cytotoxic effects. In addition, supernatant samples were collected for analysis of cell viability by lactate dehydrogenase (LDH) assay and quantification of viral replication by tissue culture infectious dose 50 (TCID50) assay. These data did not reveal substantial changes in viral replication or tumor cell killing kinetics among the sPD1 variants or in comparison to rVSV-NDV (Figure 8).

[0091] Example 7: rVSV-NDV-sPD1 variants generate and release soluble human PD1 in vitro To confirm that the recombinant constructs produced and released soluble human PD1, Western blot and ELISA assays were performed. B16 cells were infected with the sPD1-expressing VSV-NDV construct or control VSV-NDV-GFP at an MOI of 0.01 or left uninfected, and lysates and supernatants were collected at various time points postinfection and subjected to Western blot analysis for sPD1. Additionally, GAPDH was analyzed to control for protein loading. As expected, sPD1 virus-infected samples produced bands of the expected size, while the virus expressing the sPD1-Fc fusion protein had a substantial shift in size corresponding to the additional sizes of sPD1 and the Fc fragment (Figure 9, left panel). An additional sample of the supernatant collected at 24 hours was subjected to an ELISA assay to quantify the amount of PD1 secreted into the supernatant. As expected, only samples from cells infected with VSV-NDV expressing sPD1 produced detectable levels of PD1, while those expressing sPD1 fused to an Fc fragment (sPD1-Fc) had even higher levels than those without the Fc fragment, supporting the hypothesis that the presence of the Fc fragment stabilizes extracellular sPD1 (Fig. 9, right panel).

[0092] Example 8: In vivo rVSV-NDV-HA-sPD1-Fc treatment of syngeneic murine melanoma results in enhanced tumor-specific T cell responses and delayed tumor growth For in vivo analysis of VSV-NDV-mediated expression of sPD1, we focused on the HA-sPD1-Fc variant due to the potential benefits of high-affinity binding via HA mutations and stability in the blood provided by the fusion of sPD1 with an Fc fragment. We utilized a subcutaneous model of B16 melanoma, in which tumors were implanted in the contralateral flank and injected with PBS, rVSV-NDV, or rVSV-NDV-HA-sPD1-Fc for 10 min. 7rVSV-NDV-HA-sPD1-Fc-treated mice were injected intratumorally into the right flank lesions on days 7, 10, and 13 after tumor implantation at a dose of TCID50 (Figure 10A). Peripheral blood mononuclear cells (PBMCs) were isolated from blood collected on day 15 and subjected to FACS analysis of CD8+ T cells specific for a model antigen (ovalbumin) expressed by B16 cells. VSV-NDV treatment resulted in a statistically significant increase in OVA-specific T cells compared to PBS, and this effect was even further enhanced in mice treated with rVSV-NDV-HA-sPD1-Fc (Figure 10B). Furthermore, analysis of tumor growth in both injected and contralateral uninjected lesions revealed delayed growth in rVSV-NDV-HA-sPD1-Fc-treated animals compared to rVSV-NDV- or PBS-treated animals (Figure 10C). These results indicate that rVSV-NDV-HA-sPD1-Fc is capable of enhancing T cell activation, resulting in an immune-mediated abscopal effect.

[0093] References [1] Bartee MY, Dunlap KM, Bartee E. Tumor-Localized Secretion of Soluble PD1 Enhances Oncolytic Virotherapy. Cancer Res. 2017;77(11):2952-63. [2] Lazar-Molnar E, Scandiuzzi L, Basu I, Quinn T, Sylvestre E, Palmieri E, et al. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy. EBioMedicine. 2017;17:30-44. [3] Maute RL, Gordon SR, Mayer AT, McCracken MN, Natarajan A, et al. PD-1 variants for immunotherapy and PET imaging. PNAS. Nov 2015, 112 (47) E6506-E6514; DOI: 10.1073 / pnas.1519623112. [4] Altomonte, J., S. Marozin, et al. (2010). "Engineered newcastle disease virus as an improved oncolytic agent against hepatocellular carcinoma." Mol Ther 18(2): 275-284. [5] Malini Guha, The Pharmaceutical Journal (2014).

[0094] The features of the present invention disclosed in this specification, claims and / or the accompanying drawings are: Both separately and in any combination thereof may be necessary to realize the invention in its various forms. In certain embodiments, for example, the following are provided: (Item 1) A recombinant oncolytic virus, said virus comprising: Vesicular stomatitis virus (VSV), wherein the glycoprotein (G protein) of VSV is deleted, and the virus comprises A modified fusion protein (F protein) of Newcastle disease virus (NDV) and NDV hemagglutinin-neuraminidase (HN) protein, Including, Soluble PD-1 (sPD-1) A recombinant oncolytic virus further comprising: (Item 2) 2. The recombinant oncolytic virus according to item 1, wherein the recombinant virus further comprises an Fc domain or a fragment thereof, preferably an Fc domain of human IgG1, IgG2, IgG3, and / or IgG4, or a fragment thereof. (Item 3) 3. The recombinant oncolytic virus of item 1 or 2, wherein the Fc domain or fragment thereof is fused to the sPD-1. (Item 4) The sPD-1 is a high-affinity sPD-1 (HA-sPD-1), which preferably has an affinity for its ligands PD-L1 and PD-L2 that is at least 2-fold higher (e.g., 45-fold higher and 30-fold higher) than the affinity of wild-type sPD-1 for said ligands, and / or a K of <3.2 μM for PD-L1 and / or <0.1 μM for PD-L2.d The recombinant oncolytic virus according to any of the preceding items, having tropism for (Item 5) 2. The recombinant oncolytic virus of any of the preceding items, wherein the sPD-1 comprises a mutation at a position selected from 132 and 41 of SEQ ID NO:2, preferably a mutation selected from A132L, L41I, and L41V. (Item 6) 3. The recombinant oncolytic virus of any of the preceding items, wherein the sPD-1 is HA-sPD-1-A132L having the sequence of SEQ ID NO:3. (Item 7) the modified fusion protein (F protein) of NDV is an F3aa modified F protein having the sequence of SEQ ID NO: 25 and / or comprises at least one amino acid substitution in the protease cleavage site, preferably at position L289 of SEQ ID NO: 25, and more preferably L289A; and / or The modified fusion protein (F protein) of the NDV is an F3aa modified F protein having the amino acid substitution L289A having SEQ ID NO: 4; and / or The G protein of the VSV is replaced by the modified fusion protein having the sequence of SEQ ID NO: 4 and the HN protein of the NDV having the sequence of SEQ ID NO: 5; The recombinant oncolytic virus according to any of the preceding items. (Item 8) A nucleic acid encoding the recombinant oncolytic virus of any of the preceding items. (Item 9) 9. The nucleic acid of Item 8, comprising a nucleic acid encoding an Fc domain or a fragment thereof, preferably having the sequence of SEQ ID NO: 6, fused to a nucleic acid encoding sPD-1, wherein the fusion product preferably has the nucleic acid sequence of SEQ ID NO: 8. (Item 10) 10. A vector comprising a nucleic acid according to item 8 or 9, preferably having the sequence of SEQ ID NO: 9, as needed, - a reporter gene such as one of the following: HSV1-sr39TK, sodium iodide symporter (NIS), somatostatin receptor 2 (SSTR2), luciferase (firefly or Renilla), green fluorescent protein (GFP), lacZ, and tyrosinase; - a gene to be delivered to tumor cells and / or tumor tissue, such as any of the following: immune stimulatory genes (e.g., IFN-α, IFN-β, or granulocyte-macrophage colony-stimulating factor (GM-CSF)), IL-12, or IL-15; an immune checkpoint inhibitor antibody (e.g., PD-1, PD-L1, CTLA-4, LAG-3, or B7-H3); and / or Tumor-associated antigens (TAA) (specific for the tumor being targeted) for vaccination; or - combinations of these, A vector further comprising any one of the following: (Item 11) the nucleotide sequence of SEQ ID NO: 9 or 15, or A nucleotide sequence having at least 60%, preferably at least 70% or 80%, more preferably at least 90% or 95% sequence identity with the nucleotide sequence of SEQ ID NO: 9 or 15. or consisting of the nucleotide sequence of SEQ ID NO: 16, or a nucleotide sequence having at least 60%, preferably at least 70% or 80%, more preferably at least 90% or 95% sequence identity with the nucleotide sequence of SEQ ID NO: 16; comprising or consisting of The nucleic acid according to any one of items 8 to 9 or the vector according to item 10. (Item 12) 1. A pharmaceutical composition comprising: (i) a recombinant oncolytic virus according to any one of Items 1 to 7, a nucleic acid according to any one of Items 8 to 9 and 11, or a vector according to any one of Items 10 to 11; and (ii) optionally, a pharmaceutically acceptable carrier and / or excipient; (iii) Additional medications, as needed, such as: chemotherapy agents, radiotherapeutic agents, tumor vaccines, immune checkpoint inhibitors (e.g., anti-CTLA4), adoptive cell therapy systems (e.g., T cells or dendritic cells); Cell carrier systems, small molecule inhibitors, embolic factors, Shielding polymers, 10. A pharmaceutical composition comprising: (Item 13) for systemic delivery, tumor injection, intravenous administration, and intra-arterial administration; and / or For intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavity, intracerebral, intraventricular, or intravitreal injection 13. The pharmaceutical composition according to item 12, formulated as follows: (Item 14) 14. The recombinant oncolytic virus according to any one of Items 1 to 7, the nucleic acid according to any one of Items 8 to 9 and 11, the vector according to any one of Items 10 to 11, or the pharmaceutical composition according to any one of Items 12 to 13, for use in medicine. (Item 15) The recombinant oncolytic virus according to any one of Items 1 to 7, the nucleic acid according to any one of Items 8 to 9 and 11, the vector according to any one of Items 10 to 11, or the pharmaceutical composition according to any one of Items 12 to 13, for use in the prevention and / or treatment of cancer, for example, the prevention and / or treatment of cancer selected from hepatocellular carcinoma, pancreatic cancer, and melanoma. (Item 16) Use of the recombinant oncolytic virus according to any one of items 1 to 7, the nucleic acid according to any one of items 8 to 9 and 11, the vector according to any one of items 10 to 11, or the pharmaceutical composition according to any one of items 12 to 13 as a gene delivery tool, for (non-invasive) imaging of viral biodistribution and / or for tumor detection.

Claims

1. A recombinant oncolytic virus, said virus comprising: Vesicular stomatitis virus (VSV), wherein the glycoprotein (G protein) of VSV is deleted, and the virus comprises A modified fusion protein (F protein) of Newcastle Disease Virus (NDV), which is an F3aa modified F protein having the sequence of SEQ ID NO: 25 or an F3aa modified F protein having the amino acid substitution L289A having the sequence of SEQ ID NO: 4; and the hemagglutinin-neuraminidase (HN) protein of NDV; Including, Soluble PD-1 (sPD-1) further comprising wherein the sPD-1 is a high-affinity sPD-1 (HA-sPD-1), wherein the HA-sPD-1 (i) has an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, (ii) comprises a mutation at position 132 of SEQ ID NO: 2, wherein the mutation is A132L, and (iii) has affinity for its ligands PD-L1 and PD-L2 that is at least 45-fold and 30-fold greater than the affinity of wild-type sPD-1 for the ligands; the recombinant oncolytic virus further comprises an Fc domain; wherein the Fc domain is fused to the sPD-1.

2. 2. The recombinant oncolytic virus of claim 1, wherein the sPD-1 is HA-sPD-1-A132L having the sequence of SEQ ID NO:

3.

3. the modified fusion protein (F protein) of NDV is an F3aa modified F protein having the sequence of SEQ ID NO: 25 and comprising at least one amino acid substitution at position L289 of SEQ ID NO: 25; and / or The G protein of the VSV is replaced by the modified fusion protein having the sequence of SEQ ID NO: 4 and the HN protein of the NDV having the sequence of SEQ ID NO: 5; The recombinant oncolytic virus according to any one of claims 1 to 2.

4. A nucleic acid encoding the recombinant oncolytic virus according to any one of claims 1 to 3.

5. The nucleic acid of claim 4, comprising a nucleic acid encoding an Fc domain fused to a nucleic acid encoding sPD-1.

6. A vector comprising the nucleic acid of claim 4 or 5.

7. the nucleotide sequence of SEQ ID NO: 9 or 15, or A nucleotide sequence having at least 90% or 95% sequence identity with the nucleotide sequence of SEQ ID NO: 9 or 15 comprising or consisting of encoding the recombinant oncolytic virus and the sPD-1; The nucleic acid according to any one of claims 4 to 5 or the vector according to claim 6.

8. 1. A pharmaceutical composition comprising: (i) a recombinant oncolytic virus according to any one of claims 1 to 3, a nucleic acid according to any one of claims 4 to 5 and 7, or a vector according to any one of claims 6 to 7; and (ii) a pharmaceutically acceptable carrier and / or excipient; 10. A pharmaceutical composition comprising:

9. for systemic delivery, tumor injection, intravenous administration, and intra-arterial administration; and / or 9. The pharmaceutical composition of claim 8, formulated for intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavity, intracerebral, intraventricular, or intravitreal injection.

10. A composition comprising the recombinant oncolytic virus of any one of claims 1 to 3, a composition comprising the nucleic acid of any one of claims 4 to 5 and 7, a composition comprising the vector of any one of claims 6 to 7, or a pharmaceutical composition of any one of claims 8 to 9, for use in medicine.

11. A composition comprising the recombinant oncolytic virus of any one of claims 1 to 3, a composition comprising the nucleic acid of any one of claims 4 to 5 and 7, a composition comprising the vector of any one of claims 6 to 7, or a pharmaceutical composition of any one of claims 8 to 9, for use in the prevention and / or treatment of cancer.

12. A composition comprising a recombinant oncolytic virus according to any one of claims 1 to 3, a composition comprising a nucleic acid according to any one of claims 4 to 5 and 7, a composition comprising a vector according to any one of claims 6 to 7, or a pharmaceutical composition according to any one of claims 8 to 9, for use as a gene delivery tool, for (non-invasive) imaging of viral biodistribution and / or for tumor detection.

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