VSV chimeric vectors

Abstract

The present invention relates to a VSV chimeric vector, characterized in that said vector comprises a gene encoding the glycoprotein GP of the Dandenong virus (DANDV) or the Mopeia virus (MOPV) and lacks a functional gene encoding the envelope protein G of VSV. The present invention also provides a VSV chimeric vector system. Furthermore, the present invention relates to the use of the VSV chimeric vector and system of the present invention, including in medicine, such as in the treatment of solid tumors.

Description

[0001] The present invention relates to a VSV chimeric vector, characterized in that the vector comprises a gene encoding the glycoprotein GP of the Dandenong virus (DANDV) or the Mopeia virus (MOPV) and lacks a functional gene encoding the envelope protein G of VSV. The present invention also provides a VSV chimeric vector system. Furthermore, the present invention relates to the use of the VSV chimeric vector and system of the present invention, including in medicine, such as in the treatment of solid tumors.

[0002] Description of the prior art

[0003] Over the past decades, the use of viruses in cancer therapy has been intensively investigated. Oncolytic viruses (OVs) are considered as important agents in cancer therapy. OVs offer an attractive therapeutic combination of tumor-specific cell lysis and immune stimulation. Furthermore, OVs can be genetically modified to optimize tumor selectivity and enhance immune stimulation and can be easily used in combination with other agents such as checkpoint inhibitory antibody molecules and other immunotherapeutics. The effectiveness of OVs has been demonstrated in numerous preclinical studies and recently in humans, with the U.S. Food and Drug Administration approval of the oncolytic herpes virus talimogene laherparepvec [1, 2].

[0004] To date, various viruses with different properties are under preclinical and clinical investigation. Oncolytic viruses vary in size and complexity from large double-stranded DNA viruses such as vaccinia virus (190 kb) [3] and herpes simplex virus type 1 (152 kb) [4] to small single-stranded RNA viruses such as vesicular stomatitis virus (VSV) [5, 6], measles virus (MV) [7] or Newcastle disease virus (NDV) [8] with genome sizes between 11 and 16 kb. However, each oncolytic virus platform has its advantages and disadvantages. Apparently, a promising oncolytic approach is to match different viruses to the tumor type in which they are naturally allowed to replicate. On the other hand, certain platforms have a broad natural receptor tropism, for example vaccinia and vesicular stomatitis virus-based vectors, which can be used for many different types of cancer.

[0005] The VSV-based vector platform is considered a very promising viral therapy, not only due to its broad receptor tropism, but also because of its great replicative capacity in permissive cells and the ability to induce strong CPE, leading to local inflammation and generating an immune response within the infected tumor. However, there are several disadvantages in using wild-type VSV in oncolytic virus therapy. First, wild-type VSV staining is considered to be neurotoxic. Wild-type VSV can cause severe encephalitis leading to death if the virus accidentally crosses the blood-brain barrier and spreads within the brain of the treated individual. Second, VSV-infected individuals are able to rapidly elicit a strong humoral response with high antibody titers mainly directed against the nucleoprotein and the glycoprotein. In addition to strong CTL responses generated against epitopes located within the nucleoprotein, neutralizing antibodies binding to the envelope glycoprotein G of VSV are considered to be important for controlling VSV infection. Neutralizing antibodies targeting the glycoprotein G of VSV are able to limit virus spread and they are able to mediate protection of individuals from reinfection with VSV. However, vector neutralization limits the repeated application of oncolytic agents to cancer patients.

[0006] To eliminate these disadvantages of VSV wild-type, a recombinant VSV vector named VSV-GP is described in WO 2010 / 040526. In VSV-GP, the coding region of the endogenous glycoprotein VSV-G, considered to be the main determinant of neurotoxicity and vector neutralization, is replaced by the envelope glycoprotein GP of the lymphocytic choriomeningitis virus (LCMV) strain WE-HPI. The advantages provided by the replacement of the VSV viral envelope protein G with LCMV-GP (WE HPI) are (i) the loss of VSV-G-mediated neurotoxicity [9] and (ii) the lack of antibody-mediated vector neutralization only in mouse models [10, 11]. However, there is still a need for a vector with reduced neutralizing antibody induction and thus improved suitability for therapy.

[0007] Thus, it is a technical problem underlying the present invention to provide improved VSV chimeric vectors and corresponding uses and methods. This technical problem is solved by the embodiments provided and claimed herein. SUMMARY

[0009] The present invention provides a VSV chimeric vector characterized in that the vector comprises a gene encoding the glycoprotein GP of Dandenong virus (DANDV) or Mopeia virus (MOPV) or a functional fragment or variant thereof and lacks a functional gene encoding the envelope protein G of VSV. It is preferred within the scope of the present invention that the envelope protein G of VSV is replaced by the GP of DANDV or MOPV or a functional fragment or variant thereof. In an alternative embodiment of the present invention, a VSV chimeric vector is provided, characterized in that the vector comprises a gene encoding the glycoprotein of Ippy virus (IPPYV), Latino virus (LATV) or Olivero virus (OLIVV) or a functional fragment or variant thereof and lacks a functional gene encoding the envelope protein G of VSV.

[0010] In an attempt to improve VSV-based OV in the art, the inventors surprisingly found that the surface protein GP of DANDV or MOPV in a VSV vector leads to a chimeric vector that does not show neutralizing antibody titers or only shows low neutralizing antibody titers and maintains the replication capacity of the VSV vector-based in tumor cells. Moreover, the lack of tropism and neurotoxicity of the VSV-GP vector is retained.

[0011] The inventors thus surprisingly found that the envelope protein GP of an arenavirus, in particular DANDV and MOPV, when used in the framework of VSV generates a recombinant vector that does not show neurotoxicity after intracranial application in mice. At the same time, the chimeric VSV vector surprisingly maintains the cell tropism of the prior art VSV vector. In contrast to VSV with the GP of LCMV, an improved killing of selected human tumor cell lines can be shown due to virus-induced cytopathic effect (CPE), which was very surprising to the inventors. This effect has been demonstrated in vivo, in particular the efficacy of VSV-G(x)-DANDV and VSV-G(x)-MOPV in a human lung cancer xenograft model was surprisingly improved compared to VSV-LCMV-GP. Moreover, VSV-G(x)-DANDV and VSV-G(x)-MOPV show a significantly delayed induction of neutralizing antibodies to the vector in a rabbit model after intravenous immunization.

[0012] Accordingly, in one embodiment, the present application is directed to a VSV chimeric vector characterized in that the vector comprises a gene encoding the glycoprotein GP of a Dandenong virus (DANDV) or a Mopeia virus (MOPV) and lacks a functional gene encoding the envelope protein G of VSV, wherein the vector exhibits reduced neutralizing antibody induction compared to a VSV vector pseudotyped with the GP of LCMV under the same conditions. In yet another embodiment of the present application, the vector comprises a gene encoding the glycoprotein GP of an Ippy, Olivero or Latino virus. A "neutralizing antibody" is an antibody that binds to an antigen and thereby prevents the biological effect imparted by the antigen. Many antigens foreign to a host induce a response by the host's immune system, including the production of neutralizing antibodies. However, the VSV chimeric vectors of the present application are improved over known chimeric VSV vectors in that they result in reduced induction of neutralizing antibodies only, and thus can exhibit an increased biological effect on targeted host cells. Those skilled in the art are well aware of ways and means of determining whether neutralizing antibodies are induced and the degree of induction. Accordingly, those skilled in the art can readily determine whether a VSV chimeric antibody of the present application induces neutralizing antibodies and whether the induction is reduced compared to a VSV chimeric vector pseudotyped with the GP of LCMV. Such assays can provide quantitative or qualitative results. For example, the assay can provide an absolute measure of neutralizing antibody induction capacity, which can then be compared to a predetermined amount for a VSV chimeric vector pseudotyped with the GP of LCMV. Additionally or alternatively, the assay can quantitatively provide a relative capacity to induce neutralizing antibodies. An exemplary assay that can be used within the present application is provided in Example 5. As shown in Figure 9 Table 5, the results surprisingly and unexpectedly show that the vectors of the present application exhibit significantly reduced induction of neutralizing antibodies. Accordingly, an exemplary assay can include the use of p-nitro-phenyl phosphate (pNPP) in the determination of the presence of neutralizing antibodies in serum samples from different time points after immunization. In the absence of nAbs, GP-pseudotyped viruses carrying a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a different envelope protein (LCMV-GP, DANDV-GP or MOPV-GP, respectively) are able to infect BHK21 Cl.13 cells, resulting in the expression of SEAP. In contrast, if the virus is neutralized by antibodies in the serum, the virus is unable to infect the cells and, thus, will not express SEAP. Accordingly, nAb activity can be determined as a function of SEAP activity. By non-linear curve fitting, EC50values can be calculated.

[0013] In one embodiment of the invention, the enhanced biological effect can be enhanced tumor cell killing compared to a VSV vector pseudotyped with LCMV using GP. Therefore, in one embodiment, the invention relates to a VSV chimeric vector characterized by containing a gene encoding the glycoprotein GP of Dandenong virus (DANDV) or Mopeia virus (MOPV) and lacking a functional gene encoding the envelope protein G of VSV, wherein the vector exhibits enhanced tumor cell killing compared to a VSV vector pseudotyped with LCMV using GP under the same conditions. Those skilled in the art are familiar with ways and methods for determining whether tumor cells are killed and the extent of killing. Therefore, those skilled in the art can readily determine whether the VSV chimeric vector of the present invention kills tumor cells and whether the killing is enhanced compared to a VSV chimeric vector expressing LCMV using GP. Such assays can provide qualitative or quantitative results. For example, an assay can provide an absolute measurement of tumor cell killing ability, which can then be compared to a predetermined number of VSV chimeric vectors expressing LCMV using GP. Alternatively or additionally, an assay can quantitatively provide a relative ability to kill tumor cells. Exemplary assays are provided in Example 4. Such assays may include pre-incubating cells to be infected with IFN and subsequently infecting them with VSV-GP using the VSV chimeric vector and / or LCMV of the present invention. Following incubation, for example, three days, cell viability can be analyzed using an in vitro cytotoxicity assay based on MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), as recommended by the manufacturer. Samples can then be measured at 550 nm in a conventional microplate reader. Values ​​can be normalized to simulated infected cells not pretreated with interferon (IFN) and expressed as a percentage of surviving cells.

[0014] Dandenong virus (DANDV) is an Old World sand virus

[12] . To date, only one strain known to those skilled in the art contains the glycoprotein GP and can be used as a donor of the GP contained in the VSV chimeric vector of the present invention. The DANDV GP contained in the VSV chimeric vector of the present invention has more than 6 glycosylation sites, particularly 7 glycosylation sites. An exemplary preferred GP is one contained in DANDV that can be accessed with Genbank number EU136038. Thus, in one embodiment, the gene encoding the GP of DANDV has a nucleic acid sequence as shown in SEQ ID NO:1 or a sequence having at least 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity with the nucleic acid sequence of SEQ ID NO:1, while maintaining the functional characteristics of the chimeric VSV vector containing the GP encoded by the nucleic acid sequence shown in SEQ ID NO:1.

[0015] Mopeia virus (MOPV) is an Old World arenavirus

[13] . There are several strains known to those skilled in the art that comprise a glycoprotein GP and can be used in the present application as a donor for the GP comprised in the VSV chimeric vectors of the present application. The MOPV GP comprised in the VSV chimeric vectors of the present application has more than 6 glycosylation sites, in particular 7 glycosylation sites. An exemplary, preferred GP is comprised in Mopeia virus accessible under Genbank No. AY772170. Thus, in one embodiment, the gene encoding the GP of MOPV has a nucleic acid sequence as set forth in SEQ ID NO: 3 or a sequence having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 3 while maintaining the functional properties of the chimeric VSV vector comprising the GP encoded by the nucleic acid sequence set forth in SEQ ID NO: 3.

[0016] Ippy virus (IPPYV) is an Old World arenavirus first isolated from wild-caught rodents

[14] . There are several strains known to those skilled in the art that comprise a glycoprotein GP and can be used in the present application as a donor for the GP comprised in the VSV chimeric vectors of the present application. The IPPYV GP comprised in the VSV chimeric vectors of the present application has more than 6 glycosylation sites, preferably more than 8 glycosylation sites, in particular 10 glycosylation sites. An exemplary, preferred GP is comprised in IPPYV accessible under Genbank No. DQ32887. Thus, in one embodiment, the gene encoding the GP of Ippy virus has a nucleic acid sequence as set forth in SEQ ID NO: 5 or a sequence having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 5 while maintaining the functional properties of the chimeric VSV vector comprising the GP encoded by the nucleic acid sequence set forth in SEQ ID NO: 5.

[0017] The Latino virus (LATV) is a New World arenavirus first isolated from adult pregnant calomys callosus

[15] . There are several strains known to the person skilled in the art which comprise a glycoprotein GP and which can be used in the present application as donors for the GP comprised in the VSV chimeric vectors of the present application. The LATV GP comprised in the VSV chimeric vectors of the present application has more than 6 glycosylation sites, preferably more than 8 glycosylation sites, in particular 10 glycosylation sites. An exemplary preferred GP is comprised in LATV which can be accessed under Genbank No. AF485259. Thus, in one embodiment, the gene encoding the GP of LATV has a nucleic acid sequence as set forth in SEQ ID NO: 7 or a sequence having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 7 while maintaining the functional properties of the chimeric VSV vector comprising the GP encoded by the nucleic acid sequence set forth in SEQ ID NO: 7.

[0018] The Olivero virus (OLIVV) is a New World arenavirus first isolated from the rodent Bolomys obscures

[16] . There are several strains known to the person skilled in the art which comprise a glycoprotein GP and which can be used in the present application as donors for the GP comprised in the VSV chimeric vectors of the present application. The OLIVV GP comprised in the VSV chimeric vectors of the present application has more than 6 glycosylation sites, preferably more than 8 glycosylation sites, in particular 9 glycosylation sites. An exemplary preferred GP is comprised in OLIVV which can be accessed under Genbank No. U34248. Thus, in one embodiment, the gene encoding the GP of OLIVV has a nucleic acid sequence as set forth in SEQ ID NO: 9 or a sequence having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 9 while maintaining the functional properties of the chimeric VSV vector comprising the GP encoded by the nucleic acid sequence set forth in SEQ ID NO: 9.

[0019] In view of the surprising and unexpected technical advantages of the VSV chimeric vectors of the application described above and shown in the examples attached, it is within the scope of the application to provide the vectors of the application for medical use and thus to provide vectors specifically designed to be suitable for such use. Thus, in one embodiment of the application, there is provided a vector of the application comprising at least one transgene. A "transgene" is a segment of DNA comprising a gene sequence that has been isolated from one organism and introduced into a different organism. Within the present application, the transgene(s) comprised in the vectors of the application can be of any origin and have any biological effect on the targeted cells.

[0020] In yet another embodiment, the present application relates to a VSV chimeric vector system characterized in that said system comprises at least two complementary replication VSV vectors, wherein said system comprises the genes n, 1, p and m encoding the proteins N, L, P and M of VSV, the gene gp encoding Dandenong-GP or Mopeia-GP and lacks a functional gene encoding the G protein of VSV, wherein each vector of the system lacks one of the genes n, 1, p, m and gp, and wherein the lacking gene is present on any other vector of the system. Each GP gene comprised in the VSV chimeric vectors of the application is one of the genes encoding the GP protein comprised in the VSV chimeric vectors of the application as described above.

[0021] In yet another embodiment, the present application relates to a chimeric VSV virion, characterized in that the virion comprises a GP protein of a Dandenong virus or a Mopeia virus as an envelope protein. In yet another embodiment, the virion can comprise a GP of an Ippy virus, a Latino virus or an Olivero virus as an envelope protein. Accordingly, the present application also provides a virion comprising as an envelope protein any of the GP gene products described above, a viral particle. In this regard, the envelope protein of the virion is known to those skilled in the art to be used to identify and bind to receptor sites on host cell membranes. Accordingly, the virion of the present application comprises as an envelope protein on its surface a chimeric GP protein, i.e. a GP protein that is different from the GP protein of VSV. However, the composition of the nucleic acid sequence content of the virion is not particularly limited. That is, for example, the chimeric VSV virion can comprise a non-functional gene encoding the GP of VSV. The chimeric VSV virion can also lack a gene encoding the GP of VSV. The GP expressed on the surface of the chimeric VSV virion of the present application is the gene product of a gene encoding a GP protein of a Dandenong virus or a Mopeia virus as further described above. In yet another embodiment, the GP expressed on the surface of the chimeric VSV virion of the present application can be the gene product of a gene encoding a GP of an Ippy virus, a Latino virus or an Olivero virus as further described above. Accordingly, the GP protein can comprise an amino acid sequence as set forth in any one of SEQ ID NO: 2 or 4. Alternatively, the GP protein can comprise an amino acid sequence as set forth in any one of SEQ ID NO: 6, 8 or 10. The chimeric VSV virion of the present application can also comprise as an envelope protein a protein comprising an amino acid sequence having 60, 65, 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO: 2, or 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO: 4, or 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO: 6, or 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 8 or 60, 65, 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO: 10, wherein the envelope protein retains the cell tropism and functionality of the chimeric VSV virion comprising as an envelope protein a GP comprising an amino acid sequence as set forth in any one of SEQ ID NO: 2, 4, 6, 8 or 10.

[0022] The present application also provides a virus production cell, characterized in that the cell produces a chimeric VSV virion of the present application. The cell can be of any origin and can exist as an isolated cell or as a cell comprised in a cell population. Preferably, the cell producing a pseudotyped VSV virion of the present application is a mammalian cell. In a more preferred embodiment, the virus production cell of the present application is characterized in that the mammalian cell is a multipotent adult progenitor cell (MAPC), a neural stem cell (NSC), a mesenchymal stem cell (MSC), a HeLa cell, any HEK293 cell, a Vero cell or a bone marrow-derived tumor infiltrating cell (BM-TIC). Alternatively, the virus production cell can be a human cell, a monkey cell, a mouse cell or a hamster cell. Methods suitable for testing whether a given cell produces a virus are known to the skilled person and thus the person skilled in the art is aware whether a particular cell falls within the scope of the present application. The amount of virus produced by the cell of the present application is not particularly limited in this respect. Preferably, the virus titer is > 1 x 10 7 TCID50 / ml or > 1 x 10 8 genome copies / ml in the crude supernatant of a given cell culture without further downstream processing after infection.

[0023] In a particular embodiment, the virus production cell of the present application is characterized in that the cell comprises one or more expression cassettes for expressing at least one gene selected from the group consisting of the genes n, 1, p and m encoding the proteins N, L, P and M of VSV and the gene gp encoding the Dandenong-GP or Mopeia-GP glycoprotein.

[0024] The virus production cell of the present application can be used, for example, in gene therapy. For such purposes, a virus production cell is provided, characterized in that the cell comprises a gene transfer vector for packaging into a VSV virion pseudotyped with the GP of a Dandenong or Mopeia virus, wherein the gene transfer vector comprises a transgene. In this respect, the transgene can be any gene addressing a particular host demand.

[0025] The transgene within the meaning of the present application can be transferred into a cell using the means provided herein. Thus, in one embodiment, the present application relates to a method for transferring a transgene into a cell in vitro or in vivo, characterized in that the cell is transduced with a chimeric virion of the present application, wherein the virion comprises the transgene. The transgene can also be transferred using the virus production cell of the present application. Thus, in a further embodiment, a method for transferring a transgene into a cell in vitro is provided, characterized in that the cell is contacted with a virus production cell of the present application. Preferably, the cell into which the transgene is transferred is a tumor cell.

[0026] In yet another embodiment, the present application relates to a pharmaceutical composition comprising any of the tools provided herein. The pharmaceutical composition is thus characterized in that the composition comprises the VSV chimeric vector of the application, the VSV chimeric vector system of the application, the chimeric VSV virion of the application or the virus production cell of the application.

[0027] In yet another embodiment, the tools provided herein, in particular the VSV chimeric vector of the application, the VSV chimeric virus system of the application, the chimeric VSV virion of the application, the virus production cell of the application or the pharmaceutical composition of the application are provided for use as a medicament.

[0028] The present application further relates to the VSV chimeric vector of the application, the VSV chimeric virus system of the application, the chimeric VSV virion of the application, the virus production cell of the application or the pharmaceutical composition of the application for use in the treatment of cancer. In a preferred embodiment, the cancer is a solid cancer. In a more preferred embodiment, the solid cancer can be brain cancer, colorectal cancer, oropharyngeal squamous cell carcinoma, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal cancer, hepatocellular carcinoma, pancreatic adenocarcinoma, cholangiocarcinoma, bladder urothelial carcinoma, metastatic melanoma, prostate cancer, breast cancer, glioblastoma, non-small cell lung cancer, brain tumor or small cell lung cancer.

[0029] In yet another embodiment of the present application, the VSV chimeric vector, the VSV chimeric vector system, the chimeric VSV virion, the virus production cell or the pharmaceutical composition of the application are provided in combination with a PD-1 or PD-L1 antagonist. Such a combination can be made prior to administration, i.e. the VSV chimeric vector, the VSV chimeric vector system, the chimeric VSV virion, the virus production cell or the pharmaceutical composition of the application can be combined with the PD-1 or PD-L1 antagonist to form a combined preparation prior to administration to a patient in need thereof. Alternatively, the VSV chimeric vector, the VSV chimeric vector system, the chimeric VSV virion, the virus production cell or the pharmaceutical composition of the application can be administered separately to form a combination therapy. Thus, the present application also relates to a pharmaceutical composition comprising the VSV chimeric vector, the VSV chimeric vector system, the chimeric VSV virion, the virus production cell or the pharmaceutical composition of the application in combination with a PD-1 or PD-L1 antagonist for use in a use provided herein. Furthermore, the present application relates to a method of treatment comprising administering the VSV chimeric vector, the VSV chimeric vector system, the chimeric VSV virion, the virus production cell or the pharmaceutical composition of the application and administering a PD-1 or PD-L1 antagonist.

[0030] Within the present application, the skilled person, in particular the clinician, can select a suitable route of administration for the VSV chimeric vector, the VSV chimeric vector system, the chimeric VSV virion, the virus production cell or the pharmaceutical composition of the present application. In a particular embodiment, there is provided a VSV chimeric vector, a VSV chimeric vector system, a chimeric VSV virion, a virus production cell or a pharmaceutical composition of the present application, wherein the VSV chimeric vector, the VSV chimeric vector system, the chimeric VSV virion, the virus production cell or the pharmaceutical composition can be administered intratumorally or intravenously. In another embodiment, there is provided a VSV chimeric vector, a VSV chimeric vector system, a chimeric VSV virion, a virus production cell or a pharmaceutical composition for use according to the present application, wherein the VSV chimeric vector, the VSV chimeric vector system, the chimeric VSV virion, the virus production cell or the pharmaceutical composition is administered intratumorally and subsequently intravenously. That is, the VSV chimeric vector, the VSV chimeric vector system, the chimeric VSV virion, the virus production cell or the pharmaceutical composition of the present application can be suitably formulated for intravenous or intratumoral administration.

[0031] The present application also provides a method for treating a subject, in particular a human subject, wherein the subject is suffering from a cancer, in particular a solid tumor, comprising administering a therapeutically effective amount of a VSV chimeric vector of the present application, a VSV chimeric vector system of the present application, a chimeric VSV virion of the present application, a virus production cell of the present application or a pharmaceutical composition of the present application. In a particular embodiment, the solid cancer is brain cancer, colorectal cancer, oropharyngeal squamous cell carcinoma, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal cancer, hepatocellular carcinoma, pancreatic adenocarcinoma, cholangiocarcinoma, bladder urothelial carcinoma, metastatic melanoma, prostate cancer, breast cancer, glioblastoma, non-small cell lung cancer, brain tumor or small cell lung cancer.

[0032] The present application furthermore relates to a kit comprising the tools provided herein. SUMMARY

[0034] Figure 1 Table (Table 1): Comparison of Old World arenavirus (A) and New World arenavirus clade C (B) GPC sequences.

[0035] Sequences were obtained from NCBI. The number of N-linked glycosylation signals NxT or NxS was deduced from the translated nucleotide sequence and ranges from 6 (LCMV) to 10 (IPPV, OLIVV) signals. The given amino acid positions correspond to the sequence positions of the respective sequences starting with the methionine from the translation start. GPC sequences were compared to LCMV-GP WEHPI by pairwise MUSCLE alignment

[17] . Relative sequence identity (% Seq Id.), absolute identity (n) and sequence similarity are shown. aa: amino acid, nt: nucleotide.

[0036] Figure 2 Multiple sequence alignment of arenavirus glycoproteins.

[0037] Amino acid sequences of LCMV-GP WE HPI, DANDV GP, IPPYV GP, MOPV GP, OLIVV GP and LATV GP were aligned using MUSCLE

[17] . Black arrows indicate cleavage sites for signal peptidase and the cellular subtilisin kexin isozyme 1 (SKI-1) / site 1 protease (S1P) which cleave the precursor glycoprotein GPC into SSP, GP1 and GP2. The predicted N-linked glycosylation signal sequence NxT or NxS within the GP1 peptide of arenavirus GPs is indicated by a dashed box.

[0038] Figure 3 Table (Table 2): Nucleotide and amino acid sequence identity in the GPC region between selected Old World and New World clade C arenaviruses.

[0039] Figure 4 Infectious trans-complementing VSV*M Q GFP expression by ΔG viruses in BHK21C 1.13 cells.

[0040] Non-cytopathogenic VSV*M Q ΔG viruses were trans-complemented in BHK21C 1.13 cells, which carry mutations M33A, M51R, V221F and S226R within the VSV matrix protein and have the complete G protein coding sequence replaced by an eGFP sequence

[18] . VSV*M Q BHK21C 1.13 cells were transiently transfected with expression plasmid pCAG-DANDV-GP, pCAG-IPPYV-GP, pCAG-LATV-GP, pCAG-MOPV-GP or pCAG-OLIVV-GP 24h prior to infection to express the respective arenavirus GP in the cells. For controls, cells were mock transfected. Supernatants containing the transcomplementing viruses were harvested 24h post infection and passaged on BHK21C 1.13 cells (A) or BHK-556 cells (B) at undiluted (neat) or serial ten-fold dilutions ranging from 1:10 to 1:1000. VSCΔG GFP viruses transcomplemented with DANDV-GP, LATV-GP, MOPV-GP or OLIVV-GP were able to spread within the cell culture, even at high dilution, leading to ubiquitous GFP expression 48h post infection in BHK-566 cells stably expressing LCMV-GP. IPPY-GP did not transcomplement VSV*M QΔG virus, thus BHK-566 cells did not express GFP at levels higher than mock control. GFP fluorescence was imaged at 48hpi on a Leica DM2500 fluorescent microscope using the same exposure time.

[0041] Figure 5 Table (Table 3): VSV*M cp CPE of cells infected with ΔG trans-complemented viruses.

[0042] VSV*M cp ΔG virus

[18] was trans-complemented in BHK21 Cl.13 cells. BHK21 Cl.13 were transiently transfected with expression plasmid pCAG-DANDV-GP, pCAG-IPPYV-GP, pCAG-LATV-GP, pCAG-MOPV-GP or pCAG-OLIVV-GP 24h prior to infection with VSV*M cp ΔG at MOI=3. Supernatants containing trans-complemented viruses were collected 24hpi and passaged on BHK21 Cl.13 cells (A) or BHK-556 cells (B) at undiluted (neat) or serial ten-fold dilutions ranging from 1:10 to 1:1000. In BHK-566 cells stably expressing LCMV-GP, VSV*M cp ΔG viruses were able to spread within cell culture even at high dilutions. Cytopathic effect (CPE) was monitored at 48hpi under bright field microscopy. CPE was classified ranging from strong CPE (++++) to no visible CPE (-).

[0043] Figure 6 : Replication kinetics of VSV-G(x) DANDV and MOPV.

[0044] Vero cells were seeded at a density of 4x10 4 cells / cm 2 in T75 cell culture flasks for infection and 24h later infected with VSV-GP as control and two variants VSV-G(x) DANDV and VSV-G(x) MOPV at the indicated MOI (multiplicity of infection, 0.05 or 0.0005). Samples were taken at 24, 30 and 42h and infection titers were determined by TCID50 (A) and genome titers by qPCR (B).

[0045] Figure 7 : Type I interferon (IFN) limits VSV-G(x) replication in human tumor cell lines.

[0046] Human Calu6 (A) and 22Rvl (B) cells were pre-incubated with the indicated amounts of IFN for 16 hours. Subsequently, cells were infected with 0.1, 1 or 10 MOI of VSV-GP, VSV-G(x)DANDV, VSV-G(x)MOPV or VSV-G(x)OLIVV in quadruplicate, or not infected as negative control. Three days post-infection, cell viability was analyzed using the MTT assay. The graph shows the mean ± SEM, and the viability of uninfected cells not pre-treated with IFN was normalized to 100%.

[0047] Figure 8 Type I interferons (IFNs) limit VSV-G(x) replication in murine tumor cell lines.

[0048] Murine SCCVII (A), Ct26 Cl.25 (B) or LLC1 (C) cells were pre-incubated with the indicated amounts of IFN for 16 hours. Subsequently, cells were infected with 0.1, 1 or 10 MOI of VSV-GP, VSV-G(x)DANDV, VSV-G(x)MOPV or VSV-G(x)OLIVV in quadruplicate, or not infected as negative control. Three days post-infection, cell viability was analyzed using the MTT assay. The graph shows the mean ± SEM, and the viability of uninfected cells not pre-treated with IFN was normalized to 100%.

[0049] Figure 9 Schedule of rabbit treatment and monitoring.

[0050] The schedule shows the design of one of the three treatments. All treatment cycles were identical, starting on DO, D14 and D28, with i.v. injection of VSV-GP or VSV-G(x) viruses. The time points at which the rabbits were manipulated are indicated by arrows. nAb, neutralizing antibodies; BW, body weight; BT, body temperature; WBC, white blood cell count.

[0051] Figure 10 Neutralizing antibody induction in rabbits immunized with LCMV-GP, DANDV-GP and MOPV-GP recombinant VSV vectors.

[0052] Neutralizing antibodies against the autologous arenavirus glycoprotein were analyzed in sera collected at day -4, 10 days after each virus treatment (prime, first boost, second boost) and at the end of the experiment. The neutralizing capacity of the sera was tested using VSVAG SEAP pseudotyped with LCMV-GP WE HPI, DANDV-GP or MOPV-GP as described in detail in the methods section. (A) The infection rate [in %] normalized to the no serum control was plotted against serum dilutions of 1 : 10 to 1 : 31.250. None of the rabbits showed nAbs against the arenavirus GPs of LCMV, DANDV or MOPV after the prime immunization (open squares). First boost sera of rabbits immunized with VSV-G(x)DANDV and MOPV showed only partial neutralization of the autologous VSVAG SEAP virus compared to VSV-G immunized rabbit sera and therefore no calculation of respective EC50 values was applicable (n.a.). EC50 values were calculated after non-linear curve fitting (B). Neutralization of VSV-AG SEAP GP by the LCMV-GP neutralizing antibody KL25 was used as inter-assay control (C).

[0053] Figure 11 Efficacy of intratumoral treatment with different arenavirus glycoprotein GP recombinant VSV vectors in a lung cancer xenograft mouse model.

[0054] 5x105Calu6 lung cancer cells were injected subcutaneously into the right flank of 8 weeks old NMRI nude mice. Treatment was started when the mean size of the tumors reached 0.05 to 0.07 cm3. Mice were treated intratumorally with PBS (n=8) (A) or 1x105TCID50VSV-GP (B), VSV-G(x)DANDV or VSV-G(x)MOPV (D) three times with 4 days interval. Tumor growth of the animals was monitored every 2-3 days after start of treatment and animals were sacrificed when the tumor volume reached 0.8 cm3or the tumor ulcerated. Kaplan-Meier survival curves (E). Dotted lines indicate the time points of virus injection. 6 3 7 3

[0055] Figure 12 Table (Table 4): Score for evaluation of virus-induced cytotoxicity.

[0056] Neurotoxicity score of infected mice was based on different categories like general appearance, clinical observations, behavior movement caused by body condition and respiration. Score ranges for different categories were 0-3. Figure 12 Cumulative poison score shown was calculated by adding the score of each category.

[0057] Figure 13 ​​​​Neurotoxicity in Swiss CD1 mice.

[0058] Swiss CD-1 mice received a single i.c. injection of 3 μl containing 1 x 10 6 TCID50. In the control group, PBS was administered i.c. Animals were monitored daily for signs of neurotoxicity and general health for 42 days. (A) Survival rates of mice in the PBS (diamonds), VSV-G DsRed (squares), VSV-GP (dots), VSV-GP (x) Dandenong (triangles) and VSV-GP (x) Mopeia (inverted triangles) experimental groups were plotted as Kaplan-Meier curves. Kaplan-Meier analysis indicated that none of the virus variants tested displayed neurotoxicity in mice. (B) Cumulative toxic scores calculated according to Figure 12 Figure 12 Cumulative toxic scores calculated according to

[0059] Detailed description

[0060] As mentioned above, the present application generally relates to the provision of chimeric VSV vectors, wherein the VSV vector lacks a functional gene encoding the envelope protein GP of VSV, and instead comprises a gene encoding the glycoprotein GP of an arenavirus, in particular a Dandenong virus or a Mopeia (MOPV) virus. Alternatively, the vector can comprise the GP of an Ippy virus, a Latino virus or an Olivero virus. As known to the skilled person, the glycoprotein GP is an envelope protein present on the surface of the virion, which is responsible for the binding between the virion and the cell of the host organism. Thus, the envelope protein determines the tropism of the virion. By changing the tropism of VSV, a chimeric VSV vector can be prepared which is suitable for use in medicine. Such chimeric vectors / virions have already been provided in the prior art, for example in WO 2010 / 040526. However, there is a need for further improved chimeric vectors which provide even more effective treatment options and even better suitability for medical use. The present application meets this need.

[0061] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs and refer to terms as commonly used in the art, which are incorporated herein by reference. The following definitions are provided solely to clarify the meanings of the terms as used herein and, unless otherwise defined, shall be consistent with the ordinary meanings of such terms as used in the art.

[0062] ​The term “a” or “an” refers to one or more, for example, “a gene” is understood to mean one or more such genes. Therefore, the terms “a” (or “an”), “one or more”, and “at least one” are used interchangeably herein. As used herein, the term “about” indicates a difference of 10% from a given reference, unless otherwise indicated.

[0063] Regarding the following description, it is intended that, in another embodiment, each composition described herein is useful in the method of the invention. Additionally, in another embodiment, it is intended that each composition described herein that is useful in the method is itself an embodiment of the invention. While various embodiments in the specification are presented using the language of "comprising," in other instances, it is also intended that the language of "consisting of" or "substantially consisting of" be used to explain and describe the associated embodiments.

[0064] In this respect, the vector of the present invention is based on a virus causing vesicular stomatitis and has at least the following general advantages compared to retroviral vectors:

[0065] (i) The VSV vector is oncolytic and has particularly high oncolytic activity compared to other oncolytic virus vectors.

[0066] (ii) The VSV vector preferentially replicates in tumor cells and has a particularly high replication capacity compared to other oncolytic virus vectors.

[0067] (iii) VSV vector infection of actively dividing cells and resting cells.

[0068] (iv) The VSV vector induces a strong innate humoral and cellular immune response.

[0069] (v) VSV vectors replicate purely in the cytoplasm, meaning that as RNA viruses, they cannot integrate into the host cell genome or recombine to form a virus capable of replication.

[0070] (vi) VSV carriers are easy to package.

[0071] (vii) VSV glycoproteins can be interchanged with foreign envelope proteins. Examples of glycoproteins that have previously bound to the VSV envelope include: HIV gp160

[19] , HCVE1 / E2

[20] , SARS S

[21] , Lassa GP

[22] or LCMV GP.

[0072] However, the carrier of the present invention has other advantages over the prior art, and in particular over the GP pseudomorphic VSV using LCMV.

[0073] Specifically, the vector of the present invention surprisingly exhibits reduced neutralizing antibody induction compared to the VSV vector pseudotyped with LCMV under the same conditions. This can be used by those skilled in the art to determine the induction of neutralizing antibodies.

[0074] By using the assays described in Example 5, the present invention surprisingly found a reduction in the induction of neutralizing antibodies. Such an effect is beyond the reasonable expectation of those skilled in the art. Furthermore, the significance of this effect demonstrates the clear advantages offered by the vectors provided herein and the further tools available. In this respect, the vectors / virions provided herein preferably induce neutralizing antibodies at a lower rate compared to VSV vectors pseudotyped with LCMV.

[0075] Under the same conditions, compared with GP-pseudotyped VSV vectors using LCMV, the vector / virion of the present invention not only demonstrated reduced neutralizing antibodies but also showed enhanced tumor cell killing. Those skilled in the art know that it can be used to measure / quantify tumor cell killing. Preferably, the vector / virion of the present invention exhibits improved tumor cell killing compared with GP-pseudotyped VSV vectors using LCMV.

[0076] To achieve the above surprising effects, the GP of the VSV is non-functional, while the GP of the Dandenong virus or Mopeia (MOPV) virus is incorporated or the GP of the VSV is replaced by the GP of the Dandenong virus or Mopeia (MOPV) virus. Alternatively, the GP of the VSV is non-functional, while the GP of the Ippy virus, Latino virus or Olivero virus is incorporated or the GP of the VSV is replaced by the GP of the Ippy virus, Latino virus or Olivero virus. There are multiple possibilities how to pseudotype a VSV vector with any of the above GPs. Thus, the exact nucleic acid sequence of the inventive vector can vary as long as the GP of the VSV is essentially absent from the virion surface, while the GP of any of the above viruses is expressed on the virion surface. Within the present invention, exemplary preferred vectors include a nucleic acid sequence comprising SEQ ID NO: 1 or 3, 5, 7 or 9 or a nucleic acid sequence comprising at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity to any of SEQ ID NO: 1, 3, 5, 7 or 9. In this regard, SEQ ID NO: 1 corresponds to a preferred nucleic acid sequence encoding the GP of the Dandenong virus, SEQ ID NO: 3 corresponds to a preferred nucleic acid sequence encoding the GP of the Mopeia virus, SEQ ID NO: 5 corresponds to a preferred nucleic acid sequence encoding the GP of the Ippy virus, SEQ ID NO: 7 corresponds to a preferred nucleic acid sequence encoding the GP of the Latino virus, and SEQ ID NO: 9 corresponds to a preferred nucleic acid sequence encoding the GP of the Olivero virus.

[0077] The "percent (%) nucleic acid sequence identity" with respect to a reference nucleic acid sequence is defined as the percentage of nucleotides in a candidate sequence that are identical with nucleotides in the reference sequence. Methods to determine the percentage of identical nucleotides are known to the skilled person. Within the present invention, it is preferred to use computer software, like BLAST. Parameters can be easily determined by the skilled person.

[0078] While the nucleic acid sequence encoding any of the GP genes used for pseudotyping the VSV can vary, it is important to maintain the functionality given by the GPs encoded by the preferred gene sequences provided above. The parameters of the functionality to be maintained are preferably the induction of neutralizing antibodies, the killing of tumor cells and / or the tropism. In this regard, the assays provided above can be used. Furthermore, assays known to the skilled person can be used to determine the virion / virion tropism.

[0079] As the skilled artisan will know, nucleic acid sequences can be altered, with or without altering the primary sequence of the encoded polypeptide. Within the present application, it is preferred that the polypeptide sequence encoded by the gene used for pseudotyping remains unaltered relative to the GP protein encoded by SEQ ID NO: 1, 3, 5, 7, or 9, respectively. That is, it is preferred that the GP protein comprised in the virion of the present application corresponds to the GP protein of the Dandenong or Mopeia virus. More particularly, it is preferred that the virion of the present application comprises a GP protein comprising any one of the amino acid sequences set forth in SEQ ID NO: 2, 4, 6, 8, or 10. However, the skilled artisan knows that the amino acid sequence of a polypeptide can be altered without affecting its functionality. Thus, the present application also encompasses polypeptides comprising alternative sequences, as long as the functionality of any one of the above amino acid sequences is substantially maintained.

[0080] Thus, in certain embodiments, virions comprising amino acid sequence variants of the GP proteins provided herein are contemplated. Amino acid sequence variants can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the respective GP, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the GP. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, as long as the final construct possesses the desired characteristics, e.g., induction of neutralizing antibodies, killing of tumor cells, and / or tropism.

[0081] In certain embodiments, variants having one or more amino acid substitutions are provided. Conservative substitutions are shown under the heading of "preferred substitutions" in Table 5. More substantial modifications are provided in Table 5 under the heading of "exemplary substitutions," and with reference to amino acid side chain classes below. Amino acid substitutions can be introduced into an GP of interest and the products / virions screened for a desired activity.

[0082] Amino acids can be grouped according to common side-chain properties:

[0083] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He;

[0084] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0085] (3) acidic: Asp, Glu;

[0086] (4) basic: His, Lys, Arg;

[0087] (5) residues that influence chain orientation: Gly, Pro;

[0088] (6) aromatic: Trp, Tyr, Phe.

[0089] Table 5

[0090] Amino acid substitutions

[0091]

[0092] Non-conservative substitutions will require exchanging a member of one of these classes for another class. Within the scope of the present application, a variant GP protein contained in a virosome of the present application maintains the functionality conferred to the virosome by the unaltered GP protein of Dandenong virus, Mopeia virus, Ippy virus, Latino virus or Olivero virus, respectively. Such a variant GP protein can have 60, 65, 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO: 2, or 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO: 4, or 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO: 6, or 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 8 or 60, 65, 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO: 10, wherein the envelope protein maintains the cell tropism and functionality of a chimeric VSV virosome comprising as envelope protein a GP comprising the amino acid sequence shown in any one of SEQ ID NO: 2, 4, 6, 8 or 10.

[0093] "Percent (%) amino acid sequence identity" with respect to a reference amino acid sequence is defined as the percentage of amino acids in a candidate sequence that have the same identity to the amino acids in the reference polypeptide sequence after aligning the sequences, if necessary introducing gaps, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0094] In yet another embodiment, the present application relates to a variant GP sequence having the functionality of a Dandenong or Mopeia GP protein, or any one of Ippy, Latino or Olivero GP, wherein the variant GP protein comprises 20, 15, 10 or 5, or preferably 4, 3, 2 or 1 alterations.

[0095] In addition to their inherent oncolytic properties, VSV chimeric vectors or vector systems based thereon and as provided herein can be further improved by the introduction of at least one transgene. Techniques known to the skilled person can be used to introduce such transgenes into the vectors of the present application and where they are introduced. An exemplary method comprises the steps of restriction and ligation or PCR and Gibson assembly. The transgenes that can be used within the present application are not particularly limited as long as the specific need is addressed by the delivery of the transgene. Non-limiting and preferred examples include transgenes encoding suicide proteins, cytokines / chemokines, antibodies or antibody fragments that bind to immune-related receptors, proteins for vaccination purposes, viral fusion proteins, marker proteins or fusions thereof.

[0096] To improve safety during the use of replicable viruses in therapeutic use, a vector system is provided that ensures replication, oncolysis and production of VSV viruses only in cells infected by at least two replication-deficient complementing each other vectors.

[0097] The present application thus in one embodiment relates to a VSV chimeric vector system characterized in that the system comprises at least two complementing replication VSVs, wherein the system comprises the genes n, 1, p and m encoding the proteins N, L, P and M of VSV, the gene gp encoding Dandenong-GP or Mopeia-GP, or alternatively Ippy-GP, Latino-GP or Olivero-GP, and lacks a functional gene encoding the G protein of VSV, wherein each vector of the system lacks one of the genes n, 1, p, m and gp, and wherein the lacking gene is present on any other vector of the system. Such complementing replication (cr) VSV vectors can spread within the targeted cells (e.g. tumor cells) to a limited extent, which improves the efficiency of gene transfer and oncolysis. Thus, the vector system according to the present application allows to make oncolytic VSV chimeric vectors with a limited replication capacity for gene transfer in targeted cells, in particular tumor cells. The gene cp encoding LCMV-GP and possibly other genes (such as therapeutic genes and / or marker genes) can be present on any vector of the system.

[0098] Different variants of the vector system according to the application are possible. For example, the vector system can consist of two vectors or more than two vectors. When the vector system consists of two vectors, the first vector can comprise the GP of a Dandenong virus or a Mopeia virus, or alternatively the GP of an Ippy virus, a Latino virus or an Olivero virus, replacing the GP of VSV, and the gene p encoding the P protein is deleted. In addition, the second vector can not comprise VSV-G, but expresses the P protein of VSV. Each vector can express the nucleoprotein (N) and the polymerase (L) of VSV and a less cell pathogenic variant of the M protein (Mncp). The first vector can additionally carry a marker gene rfp, while the second vector can carry a transgene.

[0099] The application also provides a vector system comprising two vectors, wherein one vector comprises the GP of a Dandenong virus or a Mopeia virus, or alternatively the GP of an Ippy virus, a Latino virus or an Olivero virus, as defined herein, and the second vector comprises the GP of LCMV. The second vector comprising the GP of LCMV can be the vector described in WO 2010 / 040526.

[0100] The application also relates to cells producing the chimeric virions of the application. Viral producer cells in the meaning of the application include classical packaging cells for the production of virions from non-replicable vectors, as well as producer cells for the production of virions from vectors capable of replication. Packaging cells usually comprise one or more plasmids for the expression of essential genes that are not present in the respective vector to be packaged and / or are essential for the production of virions. Such cells can be mammalian cells, in particular human cells, monkey cells, mouse cells or hamster cells, more particularly HEK293 cells, Hela cells or Vero cells. Such cells are known to the skilled person, who can select a suitable cell for the desired purpose.

[0101] In previous studies, packaging cells were used for transfer of viral vectors; however, this mainly involved fibroblasts, which do not move within the tumor (Short et al., 1990, Culver et al., 1992). In contrast, adult stem cells, in particular neuronal (NSC), multipotent adult progenitor cells (MAPC) and mesenchymal stem cells (MSC), have a high migratory potential. They remain confined in the tumor tissue, thus enabling a very efficient but also specific gene transfer into the tumor tissue. However, these stem cells have a limited passaging capacity in vitro.

[0102] A subpopulation of adult mesenchymal stem cells, termed BM-TIC (bone marrow-derived tumor infiltrating cells), infiltrate the entire tumor upon injection into experimentally induced gliomas and, in addition, can track individual tumor cells away from the tumor mass

[23] . BM-TIC are isolated from adult bone marrow, have a high expansion potential and can be used as migratory producer cells for MLV

[24] and VSV vectors.

[0103] Therefore, the subject matter of the present application is a virus producer cell, which produces the VSV chimeric vector of the present application. In particular, these are tumor infiltrating producer cells, which release the vector during migration within the tumor. Preferred cells are adult stem cells, in particular neuronal (NSC) and mesenchymal stem cells (MSC). Particularly preferred cells are BM-TIC cells derived from MSC.

[0104] The virus producer cell of the present application and thus the VSV chimeric vector produced by the cell can comprise a gene encoding a mutated M protein. This vector variant is selectively oncolytic for tumor cells, but not toxic for healthy cells. Preferred are M variants with amino acid exchanges in the 37PSAP40 region of the M protein or with single (M51R) or multiple (V221F and S226R; M33A and M51A) mutations outside the PSAP region of the M protein. M proteins with the mutations M33A, M51R, V22F and S226R are particularly preferred. To ensure efficient virus production in the packaging cell, the M variant can be stably transfected with a viral interferon antagonist.

[0105] In one embodiment, the virus production is characterized in that the cell comprises one or more expression cassettes expressing at least one gene selected from the genes n, 1, p and m encoding the proteins N, L, P and M of VSV, and the gene gp encoding the Dandenong-GP or Mopeia-GP glycoprotein, or, alternatively, the Ippy-GP, Latino-GP or Olivero-GP. The cell can further comprise a gene transfer vector for packaging into a VSV virion pseudotyped with the GP of a Dandenong or Mopeia virus, or, alternatively, an Ippy, Latino or Olivero virus, wherein the gene transfer vector comprises a transgene.

[0106] Furthermore, the subject matter of the present application is an in vitro method for gene transfer, wherein the VSV chimeric vector comprising a transgene according to the present application or the VSV chimeric vector system according to the present application is introduced into a cell directly or via a virus production cell (packaging cell) according to the present application. If a cr vector system with at least two vectors is used, at least two packaging cells are used, wherein each cell produces one (replication incompetent) cr vector. The production of VSV viruses only takes place in cells that are infected with all vectors of the cr vector system and thus contain all essential viral genes.

[0107] Furthermore, the present application relates to the use of the vectors and virus production cells according to the present application as medicaments in a method of treatment. In particular, the vectors and virus production cells according to the present application are used for the treatment of solid cancers. Without being bound by theory, the therapeutic effect is caused by the oncolytic properties of the recombinant vectors and viruses and by the use of therapeutic genes.

[0108] The solid cancer can be brain cancer, colorectal cancer, oropharyngeal squamous cell carcinoma, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal cancer, hepatocellular carcinoma, pancreatic adenocarcinoma, cholangiocarcinoma, urothelial carcinoma of the bladder, metastatic melanoma, prostate cancer, breast cancer, glioblastoma, non-small cell lung cancer, brain tumor or small cell lung cancer.

[0109] The subject matter of the present application is further a pharmaceutical composition comprising the vectors, virosomes or virus production cells of the present application, and optionally additives, such as pharmaceutically acceptable carriers and auxiliary substances.

[0110] To improve the efficiency of viral oncolysis and transfer of therapeutic genes, the tumor infiltrating virus production cells releasing the vectors continuously can be formulated for direct implantation into a tumor. The tools provided herein can thus be formulated for intratumoral or intravenous administration.

[0111] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0112] A "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than the active ingredient, which is nontoxic to the subject to which the formulation would be administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0113] As used herein, "treatment" (and grammatical alterations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the application are used to delay development of a disease or to slow the progression of a disease.

[0114] Pharmaceutical formulations of the vectors, virions, or cells described herein are prepared by mixing the vector, virion, or cell having the desired degree of purity with one or more optional pharmaceutically-acceptable carriers

[25] , in either freeze-dried or aqueous solution form. Pharmaceutically-acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically-acceptable carriers herein further include interstitial drug dispersing agents such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 Hyaluronidase Glycoproteins, such as rHuPH20 (HYLENEX®, Ferring Pharmaceuticals Inc.) Certain exemplary sHASEGPs and methods of use are described in U.S. Pat. Pub. Nos. 2005 / 0260186 and 2006 / 0104968, including rHuPH20. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases (such as a chondroitinase). Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use are described in U.S. Pat. Pub. Nos. 2005 / 0260186 and 2006 / 0104968, including rHuPH20. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases (such as a chondroitinase).

[0115] Formulations herein can also contain more than one active ingredient as desired. Those of the latter type will generally be those with complementary activities that do not adversely affect each other.

[0116] The active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles, and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition (1990), Osol, Ed. (Alibert's Pharmaceutical Mfg. Encyc. 15th edition 1980), and in more recent publications. Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate, which matrices are in the form of shaped articles, e.g., films, or microcapsules. The preparations to be used for in vivo administration are generally sterile. Sterility is readily achieved, for example, by filtration through sterile filtration membranes.

[0117] In one aspect, there is provided a vector, virion, cell or pharmaceutical composition according to the application for use as a medicament. In further aspects, there is provided a vector, virion, cell or pharmaceutical composition according to the application for use in a method of treatment. In certain embodiments, there is provided a vector, virion, cell or pharmaceutical composition according to the application for use in the treatment of cancer. In certain embodiments, the application provides a vector, virion, cell or pharmaceutical composition of the application for use in a method of treating an individual having cancer, the method comprising administering to the individual an effective amount of the vector, virion, cell or pharmaceutical composition. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one other therapeutic agent (e.g., as described below).

[0118] In a further aspect, the application provides the use of a vector, virion, cell or pharmaceutical composition of the application in the manufacture or preparation of a medicament. In one embodiment, the medicament is for use in the treatment of cancer, in particular a solid tumour. In a further embodiment, the medicament is for use in a method of treating cancer, in particular a solid cancer, the method comprising administering to an individual having cancer, in particular a solid cancer, an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one other therapeutic agent (e.g., a therapeutic agent that is an antagonist of PD-1 or PD-L1).

[0119] In a further aspect, the application provides a method of treating cancer, in particular a solid cancer. In one embodiment, the method comprises administering to an individual having cancer an effective amount of a vector, virion, cell or pharmaceutical composition of the application. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one other therapeutic agent as described below.

[0120] The "individual" according to any one of the above embodiments can be a human. However, any mammal in need of such a therapeutic or prophylactic method is included, particularly including humans. Other mammals in need of such treatment or prophylaxis include dogs, cats, or other domesticated animals, horses, livestock, laboratory animals, including non-human primates, and the like. The subject can be male or female. In one embodiment, the subject has cancer, more particularly a solid tumor, or is at risk of developing cancer, more particularly a solid tumor.

[0121] As described above, the present application provides a pharmaceutical composition comprising any of the vectors, virions, or cells provided herein, e.g., for use in any of the methods above. In one embodiment, the pharmaceutical preparation comprises any of the vectors, virions, or cells provided herein and a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical preparation comprises any of the vectors, virions, or cells provided herein and at least one other therapeutic agent, e.g., as described below.

[0122] The vectors, virions, cells, or pharmaceutical compositions of the present application can be used alone in therapy or in combination with other agents. For example, the vectors, virions, cells, or pharmaceutical compositions of the present application can be co-administered with at least one other therapeutic agent.

[0123] A "therapeutic agent" within the meaning of the present application is a molecule, including, but not limited to, polypeptides, peptides, glycoproteins, nucleic acids, synthetic and natural drugs, peptides, polyenes, macrocycles, glycosides, terpenes, terpenoids, aliphatic and aromatic compounds, and derivatives thereof. In a preferred embodiment, the therapeutic agent is an antagonist of PD-1 or PD-L1. In another preferred embodiment, the therapeutic agent is a checkpoint inhibitory antibody or any other immunotherapeutic agent that stimulates an immune response in an individual. In yet another embodiment, the therapeutic agent is a chemotherapeutic agent. The chemotherapeutic agent can be essentially any active agent that exhibits an oncolytic effect against tumor cells in an individual and does not inhibit or diminish the oncolytic effect of the oncolytic virus of the present application. The active agent can be any known or subsequently discovered chemotherapeutic agent. For example, known types of chemotherapeutic agents include, e.g., anthracyclines, alkylating agents, alkyl sulfonates, aziridines, ethyleneimines, methylmelamines, nitrogen mustards, nitrosoureas, antibiotics, antimetabolites, folic acid analogs, purine analogs, pyrimidine analogs, enzymes, podophyllotoxins, platinum-containing active agents, interferons, and interleukins.

[0124] Suitable therapeutic agents include, but are not limited to, those set forth in Goodman and Oilman's The Pharmacological Basis of Therapeutics (e.g., 9th edition) or The Merck Index (e.g., 12th edition). Classes of therapeutic agents include, but are not limited to, drugs affecting the inflammatory response, drugs affecting the composition of body fluids, drugs affecting electrolyte metabolism, chemotherapeutic agents (e.g., for hyperproliferative diseases, particularly cancer, parasitic infections, and microbial diseases), antineoplastic agents, drugs affecting the blood and blood-forming organs, hormones and hormone antagonists, vitamins and nutrients, vaccines, oligonucleotides, and gene therapy agents. It will be understood that the present application also includes compositions comprising combinations, e.g., mixtures or blends of two or more active agents (such as two drugs).

[0125] Such combination therapies described above include combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case the administration of the vector, virion, cell, or pharmaceutical composition of the present application can precede, follow, and / or overlap with the administration of the other therapeutic agent(s) and / or adjuvant(s). The vector, virion, cell, or pharmaceutical composition of the present application can also be used in conjunction with radiation therapy.

[0126] The vector, virion, cell, or pharmaceutical composition of the present application (and any other therapeutic agents) can be administered by any suitable means, including parenterally, intrapulmonary, and intranasally, and, if local treatment is desired, intralesionally, intrauterinely, or intravesically. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Direct intratumoral administration is also contemplated. Intravenous or intratumoral administration is preferred. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether short- or long-term administration is desired. Various dosing schedules are contemplated herein, including but not limited to single or multiple doses at various time points, bolus administration, and pulsed infusion.

[0127] For discrete, solid, accessible tumors, intratumoral injection or direct injection into the tumor vasculature is specifically contemplated. Local, regional, or systemic administration can also be appropriate. For example, for tumors >4 cm, the volume to be administered is approximately 4-10 mL (suitably 10 mL), while for tumors <4 cm, a volume of approximately 1-3 mL can be used (suitably 3 ml). In some embodiments, the volume of active agent administered can be up to 25% of the tumor volume or up to 33%. Multiple injections delivered as a single dose include volumes of about 0.1 to about 0.5 mL. Multiple injections of the tumor at spatial intervals of approximately 1 cm can advantageously contact the viral particles. In the case of surgical intervention, the compositions of the application can be used preoperatively to make inoperable tumors amenable to resection. In appropriate cases, continuous administration can also be applied, for example, by implanting a catheter into the tumor or tumor vasculature. Such continuous perfusion can be carried out for a period of about 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 weeks or more after initiation of treatment. Typically, the dose of the therapeutic composition by continuous perfusion will be equivalent to the dose administered by single or multiple injections adjusted for the period of time over which perfusion occurs. It is further contemplated that limb perfusion can be used for administration of the therapeutic compositions, particularly in the treatment of melanomas and sarcomas.

[0128] The vectors, virions, cells, or pharmaceutical compositions of the application will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The vectors, virions, cells, or pharmaceutical compositions need not, but can optionally be, formulated with one or more agents currently used in preventive or therapeutic medicine. The effective amount of such other agents depends on the amount of the vectors, virions, cells, or pharmaceutical compositions present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages or with about from 1 to 99% of the frequencies of the dosages, or both, as used herewith.

[0129] For the prevention or treatment of disease, the appropriate dosage of a vector, virion, cell, or pharmaceutical composition of the application (when used alone or in combination with one or more additional therapeutic agents) will depend on the type of disease to be treated, the type of vector, virion, cell, or pharmaceutical composition, the severity and course of the disease, whether the vector, virion, cell, or pharmaceutical composition is being used for preventive or therapeutic purposes, whether the vector, virion, cell, or pharmaceutical composition is administered to the patient for a chronic or acute treatment, the patient's clinical history, and the judgment of the treating physician. The vector, virion, cell, or pharmaceutical composition can be suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg - 10 mg / kg) of the vector, virion, cell, or pharmaceutical composition can be an initial candidate dosage, whether, for example, the dosage is to be administered once or multiple times (e.g., daily) or as a continuous infusion. One typical daily dosage might range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations of the vector, virion, cell, or pharmaceutical composition, depending on the condition, the treatment can be repeated until the desired symptoms have been addressed. An exemplary dosage of the vector, virion, cell, or pharmaceutical composition would be in the range from about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses (or any combination thereof) of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg can be administered to the patient. Such doses can be administered intermittently, e.g. every week or every three weeks (e.g., the patient can receive from about two to about twenty, or, e.g., about six doses). Initial higher loading doses, followed by one or more lower doses, can be administered. However, other dosage regimens can be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0130] Alternatively, the vectors, virions, cells, or pharmaceutical compositions of the present application can be delivered in a volume of about 50 μL to about 10 mL, including all numbers subsumed therein, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired method effect. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 70 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 250 μL. In another embodiment, the volume is about 300 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 750 μL. In another embodiment, the volume is about 850 μL. In another embodiment, the volume is about 1000 μL. In another embodiment, the volume is about 2000 μL. In another embodiment, the volume is about 3000 μL. In another embodiment, the volume is about 4000 μL. In another embodiment, the volume is about 5000 μL. In another embodiment, the volume is about 6000 μL. In another embodiment, the volume is about 7000 μL. In another embodiment, the volume is about 8000 μL. In another embodiment, the volume is about 9000 μL. In another embodiment, the volume is about 10000 μL. The effective concentration of virions carrying a nucleic acid sequence encoding a desired transgene under the control of a cell-specific promoter sequence desirably ranges from about 1 x 10 8 to 10 13 vector genomes per milliliter (vg / mL). Infectious units can be measured as described by S. K. McLaughlin et al.

[26] . Preferably, the concentration is about 1.5 x 10 9 vg / mL to about 1.5 x 10 12 vg / mL, and more preferably about 1.5 x 10 9 vg / mL to about 1.5 x 10 11 vg / mL. In one embodiment, the effective concentration is about 1.5 x 10 10 vg / mL. In another embodiment, the effective concentration is about 1.5 x 10 11 vg / mL. In another embodiment, the effective concentration is about 2.8 x 10 11 vg / mL. In yet another embodiment, the effective concentration is about 1.5 x 10 12vg / mL. In another embodiment, the effective concentration is about 1.5 x 10 13 vg / mL. It is desirable to use the lowest effective concentration to reduce the risk of adverse effects. Other dosages within these ranges can also be selected by the attending physician, in accordance with a subject's (preferably a human's) condition, the subject's age, the particular type of cancer, and the extent of the cancer's development (if any).

[0131] It should be understood that any of the above formulations or methods of treatment can be carried out using any of the vectors, virions, cells, or pharmaceutical compositions of the application.

[0132] In another aspect of the application, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or in combination with another composition effective for treating, preventing and / or diagnosing the condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a vector, virion, cell, or pharmaceutical composition of the application. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture can comprise (a) a first container with a composition contained therein, wherein the composition comprises a vector, virion, cell, or pharmaceutical composition of the application; and (b) a second container with a composition contained therein, wherein the composition comprises another cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the application can further comprise a package insert indicating that the compositions can be used to treat the particular condition, particularly cancer. Alternatively, or additionally, the article of manufacture can further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. Examples

[0133] Example 1: Alignment of arenavirus GP sequences

[0134] The arenavirus RNA sequences of the S segment were retrieved from the NCBI nucleotide library in GenBank flat file format as follows: LCMV GP WE HPI (accession number: AJ297484), MOPV GP (accession number: JN561684), DANDV GP (accession number: EU136038), IPPYV GP (accession number: DQ328877), OLIVV GP (accession number: U34248) and LATV (accession number: AF485259). The GPC glycoprotein sequences were obtained by translation of the GPC open reading frame encoded by the S segment. The nucleotide and protein level of the GPC sequences of the Old World and New World clade C arenaviruses were compared using the Geneious software package version 11.0.5 (Biomatters Ltd.).

[0135] The N-linked glycosylation signal in the GP1 peptide of the glycoprotein GP was identified by the glycosylation signal sequence NxS or NxT. The numbering of the glycosylation signal site corresponds to the parental sequence of the GP1 peptide of the GP of the arenavirus LCMV WE HPI (accession number: AJ297484) starting with the ATG methionine translation start codon. Figure 1 ).

[0136] After multiple alignments of the nucleotide sequences or the corresponding GPC amino acid sequences, the absolute and relative sequence identity (% Seq Id) was calculated Figure 2 and 3 ). The sequences were aligned using MUSCLE

[17] with a maximum of eight iterations using a distance matrix clustered by k-mer and pctid. Subsequently, the sequences were clustered using the UPMGA method. The sequence similarity of each arenavirus GPC protein compared to LCMV WE HPI was calculated by pairwise alignment using a blosum62 matrix with a threshold of > 1.

[0137] Example 2: Arenavirus GPs can complement VSV-G

[0138] The GP glycoproteins of DANDV, MOPV as well as LATV and OLIVV were able to transcomplement VSV*MQAG viruses. The resulting VSV pseudotypes were able to infect BHK21 Cl.13 cells in subsequent passages and showed an increased GFP signal at different dilutions when compared to mock controls Figure 4A). The GFP signal observed in BHK21 Cl.13 cells infected with the VSV*MQAG supernatant of IPPYV GP-complemented VSV*MQAG viruses was comparable to mock controls and resulted from the input VSV*MQAG virus. To confirm these results, BHK-566 cells stably expressing LCMV-GP were infected with pseudotyped VSV*MQAG viruses. Viruses complemented in trans with DANDV-GP, LATV-GP, MOPV-GP or OLIVV-GP were able to spread within the cell culture, leading to ubiquitous GFP expression 48h post-infection even at high dilutions Figure 4 B). Cytopathic effect (CPE) of VSV*M cp AG viruses complemented in trans with the GPs of DANDV, MOPVV, IPPYV, OLIVV and LATV confirmed the results obtained with the corresponding VSV*MQAG pseudotypes and are summarized in Figure 5

[0139] For trans-complementation, 0.8-1 x 10 6 BHK21 Cl.13 cells were seeded in six-well plates. The next day, BHK21 Cl.13 cells were transfected with 2,5 μg of pCAG-DANDV-GP, pCAG-IPPYV-GP, pCAG-LATV-GP, pCAG-MOPV-GP or pCAG-OLIVV-GP using TransIT-LT1 transfection reagent (Mirus Bio LCC, Madison, Wisconsin USA) according to the manufacturer's recommendations. Twenty-four hours post-transfection, transiently transfected BHK21 Cl.13 cells were infected with VSV*M cp AG or VSV*M Q AG [9] carrying the mutations M33A, M51R, V221F and S226R within the VSV matrix protein. One hour post-infection, cells were washed twice with cGMEM (containing 10% FCS, 2% glutamine and 1% Tryptose phosphate broth) and incubated for another 24 hours. Supernatants were collected and cell debris removed by centrifugation at 8000 rpm for 5 min in a table centrifuge. Supernatants were then transferred in serial ten-fold dilutions to fresh BHK21 Cl.13 or BHK-566 cells stably expressing LCMV-GP WE HPI in 24-well plates. GFP expression or cytopathic effect from vectors was monitored after 24h and 48h on a Leica DM2500 fluorescence microscope. Pictures were taken using the same exposure time.

[0140] Example 3 - Replication kinetics

[0141] ​The VSV chimeric vectors comprising the VSV vector backbone described in WO 2010 / 040526 and the GP of DANDV, MOPV, OLIVV and LATV. The resulting chimeric VSV-G(x)-DANDV, -MOPV, -OLIVV and LATV vectors replaced the GP of LCMV, without any change in the remaining VSV vector backbone. Although there were significant differences in the replication kinetics of VSV-G(x)-DANDV and -MOPV (VSV-G(x)-LATV and -OLIVV were not tested), both viruses replicated to titers higher than 1 x 10 7 TCID50 / ml Figure 6 A) or 1 x 10 9 Genomic copies / ml Figure 6 B) at 30 hpi. Regarding their replication potential, VSV-G(x)-DANDV and -MOPV can be particularly suitable for OV cancer therapy.

[0142] For comparison of the replication kinetics, Vero cell monolayers in T75 cell culture flasks were infected with VSV-GP, VSV-G(x)-DANDV or VSV-G(x)-MOPV at 0.05 or 0.0005 MOI. 500 μΐ of supernatant of infected cells were collected at the indicated time points. Supernatants were centrifuged at approximately 2000 rpm for 5 min in a table centrifuge to remove cell debris and stored at -80°C until further processing. Viral titration by TCID50 assay

[27] and viral genome analysis by qPCR

[28] were performed as described elsewhere.

[0143] Example 4 - Tumor cell killing by VSV-G(x) variants Figure 7 )

[0144] Cell lines. Human Calu6 lung carcinoma cells were obtained from Dr. Edith Lorenz, OncoTyrol (Department of Internal Medicine, Hematology and Oncology, AG Zwierzina, Innsbruck). Cells were grown in DMEM medium containing 10% fetal calf serum, 2 mM L-glutamine and 1% penicillin / streptomycin (Pen / Strep.). They were subcultured every 2-3 days using EDTA-trypsin 0.05% when they reached 80% confluency. 22Rv1 cells derived from human prostate carcinoma were kindly provided by Prof. Z. Culig (Department of Urology, Medical University of Innsbruck). Cells were subcultured twice a week in RPMI 1640 containing 10% FCS, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate and 1% Pen / Strep. Adherent murine squamous cell carcinoma cells (SCCVII) were obtained from Dr. Lukas Mach (Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna). Cells were grown in DMEM containing 10% FCS, 2 mM glutamine, 0.1 mM non-essential amino acids (NEAA), 1 mM sodium pyruvate and 1% Pen / Strep. SCCVII cells were subcultured three times a week at a ratio of 1 : 10 using EDTA-trypsin 0.05%. CT26 Cl.25 murine colon carcinoma cells derived from Balb / c stably transduced with a LacZ cassette disrupting the IFN-I antiviral response were obtained from ATCC (#CRL-2639). Cells were subcultured twice a week at a ratio of 1 : 10 in RPMI 1640 containing 10% FCS, 2% glutamine, 10 mM HEPES, 0.1 mM NEAA, 1 mM sodium pyruvate, 1% P / S and 400 pg / ml G418. LLC1 cells were established from C57BL / 6 mice bearing lung tumors after implantation of the primary Lewis lung carcinoma. Cells were obtained from ATCC (#CRL-1642) and cultured in DMEM containing 10% FCS, 4 mM glutamine and 1% P / S. Confluent cell cultures were subcultured every 3-4 days at a ratio of 1 :6 to 1 : 10 by resuspending loosely adherent cells.

[0145] VSV killing assay. Cells were seeded in 96-well plates and pre-incubated overnight with 10, 100, 500 and 1000 units of consensus-1 IFN (PBL, Piscataway, NJ, USA) in a volume of 100 μl / well. The next morning, cells were infected with VSV-GP, VSV-G(x)DANDV, VSV-G(x)MOPV or VSV-G(x)OLIVV at 0.1, 1 or 10 MOI in a final volume of 120 μl / well. For each condition, samples were performed in quadruplicate. As a positive killing control, cells were incubated with H202 at a final concentration of 6.67 mM. Three days post-infection, cell viability was analyzed using an in vitro cytotoxicity assay based on MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) according to the manufacturer's recommendations (Sigma-Aldrich, Saint Louis, MI, USA). Plates were measured in a conventional microplate reader at 550 nm and blank values of wells containing no cells were subtracted. Values were normalized to mock-infected cells without pre-treatment with interferon (IFN) and presented as percentage of viable cells.

[0146] Example 5: Neutralizing antibodies after i.v. treatment of NZW rabbits

[0147] This study aimed to evaluate whether VSV G(x) variants induce no or lower levels of neutralizing antibodies (nAbs) compared to VSV-GP after 3 intravenous (i.v.) administrations in healthy non-tumor bearing New Zealand White (NZW) rabbits. NZW rabbits were i.v. treated with 1 x 107TCID50of VSV-GP or different VSV-G(x) candidates at 14 days intervals for three times. As shown in the 9 TCID 50 Example 5: Neutralizing antibodies after i.v. treatment of NZW rabbits Figure 9 After treatment, nAb induction as well as body weight and temperature, viremia, whole blood cells and blood chemistry were monitored at regular intervals. The main objective of this study was to monitor nAb induction by measuring nAb levels 10 days after each administration. The nAb assay was based on the work previously published by Kaku et al.

[29] J. Virol Methods (2012) 179(1):226-32. The VSVAG SEAP virus used in the nAb assay expresses a secreted embryonic alkaline phosphatase (SEAP) instead of the envelope glycoprotein G. This virus is complemented by LCMVCP or each other arenavirus glycoprotein ectopically expressed on the cell surface of the producer cell. Neutralization of these complemented viruses was subsequently tested on collected rabbit sera Figure 10 ).

[0148] Safety parameters, such as the health status of the animals, including body weight (BW) and body temperature (BT), white blood cell (WBC) count, blood chemistry (optional) and viremia (TCID50 and N-specific qPCR in plasma / blood) are secondary objectives.

[0149] Rabbit handling and housing: Crl:KBL (NZW) rabbits were purchased from Charles River Laboratories (France). Upon arrival at the animal facility, rabbits were approximately 7 weeks old. Animals were housed in pairs in rabbit cages (R-Suite Enriched Rabbit Housing, Techniplast). Individuals were identified by patterns on the ears. Rabbit housing was controlled daily and overall health status was monitored. During the experiment, food was supplied ad libitum and drinking water was changed daily. Rabbits were acclimatized for at least 4 days before they were suitable for handling. After rabbits were acclimatized and handled for at least one week, virus treatment was initiated.

[0150] Virus and treatment of NZW rabbits: Virus was diluted in PBS. The final concentration of VSV-GP or VSV-G(x) solution was 1 x 10 9 TCID50 / ml. Virus dilutions were kept on ice until shortly before injection. Rabbits were immobilized by wrapping them with a cotton towel. The injection site of the patterned left ear should be shaved appropriately and then cleaned with a sterile alcohol swab. 1 ml of 1 x 10 9 TCID 50 Virus was slowly infused into the left ear vein.

[0151] Rabbit monitoring: Body weight and body temperature were measured regularly according to the schedule presented. Figure 9 Body weight and body temperature were measured regularly according to the schedule presented. Temperature was measured rectally with a digital thermometer. Body weight was recorded using an Ultra MBS C balance.

[0152] Blood collection and processing: Blood was collected from the ear artery using a 23G micro-needle (Sarstedt, Germany). The ear was shaved appropriately and disinfected with an alcohol pad before puncturing the artery. At least 2 ml of blood was collected in a 15 ml test tube containing 50 μl of heparin (5000 IE / ml). 100 μl of heparin blood was stored at -80°C for analysis of viral titers by qPCR, the remaining blood was processed into heparin plasma (200 μl) for TCID50determination or clinical chemistry (300 μl). An additional 2-3 ml of blood was collected in a 15 ml Falcon for serum preparation on day -4 before the first treatment and on day 10 after each treatment cycle. Additionally, two pre-coated EDTA microhematocrit tubes (Sarstedt, Germany) were collected for blood smears on day -4 before the first treatment and on day 10 after each treatment cycle. Approximately 500 μΐ blood was collected in EDTA tubes (Sarstedt, Germany). EDTA blood was used for complete blood count and the minimum archive amount was 100 μΐ and for qPCR. EDTA plasma was prepared from the second microtainer and stored at -80°C until use.

[0153] Virusemia was determined by TCID50 assay in plasma 50 Virusemia was determined by TCID50 assay in plasma

[0154] Leukocyte count: Leukocyte counts using EDTA blood were measured using a ScilVet ABC blood counter. The assay requires approximately 10-20 μΐ of fresh EDTA blood. Measurements were performed according to the manufacturer.

[0155] Sacrifice: Rabbits were anesthetized with ketamine / xylazine according to FELASA and GVSOLAS recommendations. After anesthesia, a catheter was inserted in the right ear vein and rabbits were terminally bled by cardiac puncture. Blood without anticoagulant and heparin blood were collected. Rabbits were released from anesthesia by intravenous catheter with a lethal dose of pentobarbital according to FELASA and GVSOLAS recommendations. After sacrifice of the rabbits, the spleen was removed and splenocytes were isolated by standard procedures for optional downstream analysis (e.g. VSV-N specific T cell responses). Blood was processed into serum or plasma and aliquots were stored at -80°C until use.

[0156] Neutralizing antibody (nAb) assay: Analysis of pre-immune sera collected prior to first treatment on D-4 and serum samples taken 10 days after each treatment cycle for the presence of nAbs against VSV-AG SEAP GP virus or viruses pseudotyped with different glycoprotein variants. SEAP activity in the supernatant of infected BHK21 Cl.13 cells was measured by conversion of p-nitro-phenylphosphate (pNPP) to p-nitrophenol, which has a yellow color in solution by the virus encoded SEAP. The colorimetric reaction was read in a conventional microplate reader at 405 nm and serves as an indirect measure of the infection rate of the SEAP expressing virus. Comparison of serum and serum-free treated control samples, the nAb assay indicates whether a serum sample contains neutralizing antibodies. In the absence of nAbs, the virus is not neutralized and is able to infect BHK21 Cl.13 cells leading to expression of SEAP. In contrast, if the virus is neutralized by antibodies in the serum, the virus is not able to infect the cells and thus does not express SEAP.

[0157] One day prior to virus neutralization, a multichannel dispensing pipette was used to seed 1 x 105 4 Cells BHK21 Cl.13 cells were seeded in 96-well cell culture plates. To ensure low inter-assay variation, cells were counted using a Luna cell counter (Logos Biosystems). Any other cell counting device based on photographic analysis of cell numbers (e.g. Tecan reader) can be used. The use of a CASY counter (OLS OMNI Life Science) or a Kovar Glasstic slide is not recommended for counting cells. Cells were incubated overnight at 37°C, 6% CO2, 95% humidity. Cell culture plates were kept in a humidified chamber to prevent evaporation of the wells at the edge of the plate.

[0158] The following day, serial 1 :5 serum dilutions were prepared starting with the lowest dilution of 1 :5. Briefly, a multi-channel dispensing pipette was used to add 200 μl cGMEM per well into a 96 well plate. 50 μl of KL25 monoclonal ΔLCMV GP antibody (inter-assay control) at a concentration of 100 μg / ml or heat inactivated (1 h, 56°C) serum sample was added to 200 μl GMEM in the wells of column C of the 96 well plate and pipetted up and down several times to mix. A multi-channel pipette was used to perform a five-fold serial dilution starting with column C and ending with column H of the 96 well plate: 50 μl from the wells of column C was transferred to the wells of the next column and mixed by pipetting up and down several times. Then 50 μl from the wells of column D was added to the wells of the next column and so on until the end of column H. A multi-channel pipette was used to transfer 175 μl of serum dilution per well to a clean 96 well plate. The serum dilutions were kept on ice. Columns A and B of the 96 well plate were used as no infection control (column A) or no serum control (column B).

[0159] One day prior to inoculation of 1 x 10 4 BHK21 Cl.13 cells, dilutions of pseudotyped VSVAG SEAP virus were prepared in cGMEM at a MOI of 0.1 or 1 corresponding to 1 x 10 3 or 1 x 10 4 Infectious particles. The amount of virus needed for each of the three replicates in the nAb assay was calculated according to the following formula:

[0160]

[0161] and

[0162] Volume of cGMEM [μl] = (175 μl x number of wells) - volume of virus stock needed [μl]

[0163] Subsequently, 175 μl of virus solution was added to each well of B-H rows containing 175 μl of serial serum dilutions to prepare 350 μl serum / virus samples. Using a multi-channel pipette, the virus / serum samples were mixed by pipetting up and down once. 175 μl of cGMEM was added to the wells of column A (no infection control). The serum / virus was incubated on ice for 1 h.

[0164] After incubation, 100 μl of serum / virus sample was added in triplicate to BHK21 Cl.13 inoculated the day before in a 96 well plate. Cells should be 80-90% confluent. BHK21 Cl.13 was incubated for another 24 h at 37°C, 6% CO2 and 95% humidity.

[0165] The next day, nAb activity was measured as a function of SEAP activity. Briefly, a p-nitro-phenylphosphate (pNPP) substrate solution was prepared according to the manufacturer's recommendations (SIGMA-FAST, Sigma Aldrich). For each 96-well cell culture, a pNPP substrate solution was prepared by dissolving one tablet of buffer and one tablet of pNPP substrate in 21 ml sterile H20. Using a multichannel pipette, 40 μΐ of cell culture supernatant from the cells infected the previous day was transferred to a new 96-well plate. Subsequently, 200 μΐ of pNNP substrate solution was added to the cell culture supernatant using a multichannel dispensing pipette. The plate was incubated at room temperature in the dark for at least 1 h. The OD of the wells was read in a conventional microplate reader at 405 nm. If the OD of the serum-free control was below 1.0, the incubation time was extended for another hour or up to 4 h before repeating the OD measurement. The measured OD values were plotted against the serum dilution. EC50values were calculated by non-linear curve fitting using GraphPad Prism 5 software version 5.03 (GraphPad, San Diego, California USA). 405 Low

[0166] Example 6: Treatment of Calu6 lung tumor xenograft model Figure 11

[0167] Recipient mice: Eight-week-old female NMRI-Nude mice were purchased from Janvier (France). The animals were housed in individually ventilated cages in groups of 8 animals. For identification purposes, the mice were ear-cipped. The mice were acclimatized for at least one week prior to tumor transplantation.

[0168] Tumor cells: The Calu6 lung carcinoma cell line was obtained from Dr. Edith Lorenz, OncoTyrol (Department of Internal Medicine, Hematology and Oncology, AG Zwierzina, Innsbruck). The cells were grown in DMEM medium containing 10% fetal calf serum, 2 mM L-glutamine and 1 % penicillin / streptomycin. At 80% confluency, the cells were subcultured every 2-3 days using EDTA-trypsin 0.05%. The cells were seeded at low cell number. Twenty-four hours prior to transplantation into NRMI nude mice, the cells were harvested, pooled and subsequently passaged in a 1 :2 ratio in T175 flasks.

[0169] ​On the day of implantation, cells were isolated using EDTA-trypsin 0.05%, added complete medium and counted in KOVA Glasstic slides (Fisher Scientific). To determine the cell number, 10 μl of cell suspension was mixed with 90 μl of trypan blue solution by repeated pipetting in a 96-well plate. 20 μl of the counting mixture was transferred onto a KOVA Glasstic slide and cells in 3 squares (a, b, c) were counted (including the top and right grid lines), excluding the blue marked dead cells. The cell number per ml was determined by the following equation: ((count(a) + count(b) + count(c)) / 3) x 10 4 = cell number per ml. 5 x 10 6 cells per animal were transferred into 50 ml Falcon tubes and washed with PBS. Cells were resuspended in PBS to obtain a final concentration of 1 x 10 8 cells per ml. Subsequently, the cell suspension was divided into 1 ml aliquots and stored on ice for up to 30 minutes before injection.

[0170] Calu6 cell implantation into NMRI-Nude recipient mice: Calu6 cells were prepared as described above and resuspended by gently flicking the tube. Mice were anesthetized by inhalation of isoflurane and the injection site was cleaned using a sterile alcohol swab. 5 x 10 6 cells in 50 μl were administered s.c. in the right flank using a 0.5 ml syringe with a 27G needle. After injection of tumor cells, animals were checked for palpable lesions every two weeks. After detection of a lesion, animals were weighed and tumor size was measured. Tumor size was measured using a caliper and the approximate tumor volume (cm 3 ) was calculated using the values for length (cm) and width (cm) of the tumor using the following formula: volume = length x (width)2x 0.4. Length was defined independent of the orientation of the animal axis of the longer dimension.

[0171] Treatment by recombinant VSV vectors: Tumors were treated when the average size of the tumors reached a volume of 0.05 to 0.07 cm 3 . Eight mice were assigned to one cage per group. The assignment of the cages was done to obtain similar mean values and distribution of tumor sizes between the different groups. For the negative control group, 30 μl of PBS was injected i.t. For the other three treatment groups, the final concentration of VSV-GP, VSV-GP(x)DANDV and VSV-GP(x)MOPV was 3.3 x 10 8 TCID 50ml. Virus was diluted in PBS. Prior to injection, mice were anesthetized by isoflurane inhalation and the injection site was cleaned using a sterile alcohol swab. 30 μl of 1 x 10 7 TCID 50 VSV-GP, VSV-GP(x)DANV and VSV-GP(x)MOPV. Treatments were repeated twice four days apart.

[0172] Monitoring of tumor growth: After treatment, animals were checked and tumor sizes were measured at least twice, with a maximum interval of 4 days. Tumors were measured as described above. If the tumor size of one animal was measured between two times for ethical reasons (e.g. just before sacrifice), all animals in the group were measured at least. Body weight of mice was determined once a week. The following endpoint criteria required euthanasia: (i) tumor volume exceeding 0.8 cm 3 (ii) mouse body weight loss >20% or (iii) tumor ulceration. The date of sacrifice was used to calculate the Kaplan-Mayer survival curves.

[0173] Example 7: Neurotoxicity of VSV-GP(x)-DANV and -MOPV

[0174] Wild-type VSV infection causes neurological symptoms when the virus enters the brain. These neurological complications include severe encephalitis that can lead to death of the infected. The advantage of using chimeric VSV-GP is that it has been shown that there is almost no neuronal infection, making the VSV-backbone a safe oncolytic agent. The reason for the attenuated phenotype is thought to be due to a change in viral tropism facilitated by the viral envelope glycoprotein. Alterations or modifications of the GP glycoprotein can affect the tropism profile of VSV-GP, therefore a neurotoxicity assessment of VSV-G(x)DANV and VSV-G(x)MOPV was performed. Both VSV-G(x)DANV and VSV-G(x)MOPV did not show any signs of neurotoxicity after direct intracranial injection in Swiss CD1 mice as shown for VSV-LCMV GP Figure 13 ). All control mice receiving VSV-G DsRed died within the first week after infection.

[0175] Mice. 8-week-old female Swiss CD-1 mice were purchased from Janvier (France). Animals were housed in individually ventilated cages in groups of 5 animals. For identification purposes, mice were ear-clipped. Animals were acclimatized for at least one week prior to the start of the experiment.

[0176] Viruses. For each experimental group, the indicated virus stock was thawed on ice. Viruses were diluted in PBS to 1 x 10 6 TCID50 / 3 μΐ. The total volume was 100 μΐ. 80 μΐ of the virus suspension was used for stereotactic injection in a group of mice (n=5). The remaining 20 μΐ was used for titration according to standard procedures. Serial 1 / 2 log dilutions of the sample ranged from 1 x 10 5 - 1 x 10 10 The virus titration was performed within one hour after the preparation of the virus dilution for stereotactic injection.

[0177] Stereotactic injection. Mice were weighed and injected i.p. with the appropriate dose of ketamine and xylazine (100 μΐ per 10 g of body weight). After the start of anesthesia, the skull between the eye line and the occipital bone was shaved appropriately and the eyes were protected with ointment (eye cream). The mice were placed in the stereotactic frame by fixing ear bars in the bilateral external auditory canal and enclosing the upper jaw with a mandibular bar. The skin was cleaned with disinfection with pholodrine in ethanol before making a midline linear skin incision along the mid-posterior axis. The periosteum was scraped from the midline with centrifugal force and the bone was dried in the same way with a cotton swab. After identifying the Bregma, the tip of the Hamilton syringe fixed in the stereotactic frame was adjusted directly above the Bregma. Then, the needle was moved to the target position 0.4 mm from the obex and 2 mm to the right of the Bregma and marked with the tip of a scalpel, then the needle was removed again. A burr hole was made at the target position using a dental drill and the bone debris and dust were removed from the lesion with a PBS wet cotton swab.

[0178] The syringe was filled with the prepared virus solution using the fast reverse mode of the automatic injector. The syringe plunger was pushed fast until a small drop of liquid formed at the tip of the needle, which was cleaned with a wet cotton swab. After positioning the tip of the needle at the burr hole 0.4 mm from the obex and 2 mm to the right of the Bregma, the needle was lowered to the desired coordinates at a depth of 3 mm. Then the automatic injection program was started. After the injection was completed, the needle was left in place for 1 minute to prevent backflow through the needle track. Then, the needle was slowly withdrawn from the brain and skull. Before closing the wound with Vetbond 3M, the lesion was washed with PBS. Then, the wound was wiped with betadine to prevent infection and the animals were transferred to a recovery cage equipped with a heating pad to prevent hypothermia.

[0179] Postoperative analgesia was provided according to FELASA requirements for postoperative analgesia in rodents. In short, ibuprofen was given orally in the drinking water at 30 mg / kg / day for 72 hours. The animals were monitored from the end of the surgery until lying down and full activity. On the first day, the animals were monitored several times and then the opening of the wound site, discharge, redness or other possible signs of infection were monitored daily.

[0180] Evaluation of neurotoxicity. After treatment, animals were examined twice a day for one week or until all VSV-G animals have been euthanized. Thereafter, animals were monitored daily until 40 days after treatment. Scoring parameters included body weight, motility, appearance and general condition, and clinical and evoked behavioral signs of neurotoxicity Figure 12

[0181] Sequence Listing

[0182] SEQ ID NO: 1

[0183]

[0184]

[0185] SEQ ID NO: 2

[0186] >Danedong NC_010248.1

[0187] MGQLITMFEALPHIIDEVINIVIIVLVIITSIKAVYNFATCGIIALISFCLLAGRSCGLYGVTGPDIYKGLYQFKSVEFNMSQLNLTMPNACSANNSHHYISMGKSGLELTFTNDSIISHNFCNLTDGFKKKTFDHTLMSIVASLHLSIRGNTNYKAVSCDFNNGITIQYNLSFSDAQSAINQCRTFRGRVLDMFRTAFGGKYMRSGYGWKGSDGKTTWCSQTSYQYLIIQNRTWENHCEYAGPFGLSRVLFAQEKTKFLTRRLAGTFTWTLSDSSGTENPGGYCLTKWMLIAAELKCFGNTAVAKCNINHDEEFCDMLRLIDYNKAALKKFKEDVESALHLFKTTVNSLISDQLLMRNHLRDLMGVPYCNYSKFWYLEHVKTGDTSVPKCWLVSNGSYLNETHFSDQIEQEADNMITEMLRKDYIKRQGSTPLALMDLLMFSTSAYLISVFLHLMKIPTHRHIKGGTCPKPHRLTSKGICSCGAFKVPGVKTVWKRR

[0188] SEQ ID NO: 3

[0189]

[0190] ​SEQ ID NO:4

[0191] >Mopeia JN_561684.1

[0192] MGQIVTFFQEVPHILEEVMNIVLMTLSILAILKGIYNVMTCGIIGLITFLFLCGRSCSSIYKDNYEFFSLDLDMSSLNATMPLSCSKNNSHHYIQVGNETGLELTLTNTSIIDHKFCNLSDAHRRNLYDKALMSILTTFHLSIPDFNQYEAMSCDFNGGKISIQYNLSHSNYVDAGNHCGTIANGIMDVFRRMYWSTSLSVASDISGTQCIQTDYKYLIIQNTSWEDHCMFSRPSPMGFLSLLSQRTRNFYISRRLLGLFTWTLSDSEGNDMPGGYCLTRSMLIGLDLKCFGNTAIAKCNQAHDEEFCDMLRLFDFNKQAISKLRSEVQQSINLINKAVNALINDQLVMRNHLRDLMGIPYCNYSKFWYLNDTRTGRTSLPKCWLVTNGSYLNETQFSTEIEQEANNMFTDMLRKEYEKRQSTTPLGLVDLFVFSTSFYLISVFLHLIKIPTHRHIKGKPCPKPHRLNHMAICSCGFYKQPGLPTQWKR

[0193] SEQ ID NO:5

[0194]

[0195]

[0196] SEQ ID NO:6

[0197] >Ippy NC_007905.1

[0198] MGQIITFFQEVPHIIEEVMNIVLITLSLLAILKGVYNVMTCGLIGLISFLLLCGKSCSLIYKDTYNFSSIELDLSHLNMTLPMSCSRNNSHHYVFFNGSGLEMTFTNDSLLNHKFCNLSDAHKKNLYDHALMGIVTTFHLSIPNFNQYEAMACDFNGGNISIQYNLSHNDRTDAMNHCGTVANGVLDAFYRFHWGRNITYIAQLPNGDGTGRWTFCYATSYKYLVIQNISWADHCQMSRPTPIGFASILSQRIRSIYISRRLMSTFTWSLSDSSGTENPGGYCLTRWMLFAADLKCFGNTAIAKCNLNHDEEFCDMLRLIDFNKQALKTFKSEVNHGLQLITKAINALINDQLIMKNHLRDLMGIPYCNYSKFWYLNDTRTGRVSLPKCWMISNGTYLNETHFSDEIEQEADNMITEMLRKEYQERQGKTPLGLVDLFIFSTSFYSITVFLHLIKIPTHRHIVGQGCPKPHRLNSRAICSCGAYKQPGLPTKWKR

[0199] SEQ ID NO: 7

[0200]

[0201] SEQ ID NO: 8

[0202] >Latino NC_010758.1

[0203] MGQVIGFFQSLPEIINEALNIALICVALLATIKGMVNIWKSGLIQLLFFLTLAGRSCSHSFTIGRFHEFQSVTVNFTQFMSYAPSSCSVNNTHHYFKGPQNTTWGLELTLTNESMINITNSMRVFTNIHHNVTNCVQNISEHEGVLKWLLETMHLSISKPGKHIAPVMCERQKGLLIEYNLTMTKDHHPNYWNQVLYGLAKLLGSSKRLWFGACNKADCQMQSDHQHIKCNYSNCKGYTSFKYLIIQNTTWENHCEYNHLNTIHLLMSSIGQSFITRRLQAFLTWTLSDALGNDLPGGYCLEQWAVVWFGIKCFDNTAMAKCNQNHDSEFCDMLRLFDYNRNAIQSLNDQSQARLNLLTNTINSLVSDNLLMKNKLRELMNVPYCNYTRFWFINDTKNGRHTLPQCWLVSDGSYLNETRFRTQWLSESNSLYTEMLTEEYEKRQGRTPLSLVDLCFWSTLFYISTLFAHLVGFPTHRHLIGEGCPKPHRLTGSGICSCGHYGIPGKPVRWTKMSR

[0204] SEQ ID NO: 9

[0205]

[0206]

[0207] SEQ ID NO: 10

[0208] >Oliveros NC_010248.1

[0209] MGQVIGFFQSLPNIINEALNIALICVALIAILKGIVNIWKSGLIQLFIFLILAGRSCSHTFQIGRNHEFQSITLNFTQFLGYAPSSCSVNNTHHYFRGPGNVSWGIELTLTNNSVINASNSLKVFTNIHHNITNCVQNIDEQDHLMKWLIETMHLQIMKPGKRLPPILCEKDKGLLIEYNLTNIASREEKHSEYWSQLLYGLSKLLGSSKSLWFDYCQRADCMMQEHSSHLKCNYSECSGHTTFKYLILQNTTWENHCEFNHLNTIHLLMSSTGQSFITRRLQAFLTWTLSDATGNDLPGGYCLEQWAIVWAGIKCFGNTAVAKCNQNHDSEFCDMLRLFDYNRNAIKSLNDQSQSRLNLLTNTINSLISDNLLMKNKLAEIMNIPYCNYTKFWYINDTRTGRHTLPQCWLISNGSYLNETKFRTQWLSESNALYTEMLTEDYDKRQGSTPLSLVDLCFWSTLFYVTTLFAHLVGFPTHRHILDGPCPKPHRLTKKGICSCGHFGIPGKPVRWVKRSR

[0210] Bibliography

[0211] [1] Sean E. Lawler, Maria-Carmela Speranza, Choi-Fong Cho et al. Oncolytic Viruses in Cancer Treatment JAMA Oncol. 2017; 3(6): 841-849.

[0212] [2] Liu BL, Robinson M, Han ZQ et al. ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties. Gene Ther. 2003; 10(4): 292-303.

[0213] [3] Guse K, Cerullo V, Hemminki A. Oncolytic vaccinia virus for the treatment of cancer. Expert Opin Biol Ther. 2011; 11(5): 595-608.

[0214] [4] Weller SK, Coen DM. Herpes simplex viruses: mechanisms of DNA replication. Cold Spring Harb Perspect Biol. 2012; 4(9): a013011.

[0215] [5] Lichty BD, Power AT, Stojdl DF, Bell JC. Vesicular stomatitis virus: re-inventing the bullet. Trends Mol Med. 2004; 10(5): 210-216.

[0216] [6] Hastie E, Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic virotherapy against cancer. J Gen Virol. 2012; 93(pt 12): 2529-2545.

[0217] [7] Russell SJ, Peng KW. Measles virus for cancer therapy. Curr Top Microbiol Immunol. 2009; 330: 213-241.

[0218] [8] Zamarin D, Palese P. Oncolytic Newcastle disease virus for cancer therapy: old challenges and new directions. Future Microbiol. 2012; 7(3): 347-367.

[0219] [9] Alexander Muik, Inna Kneiske, Marina Werbizki et al. Pseudotyping Vesicular Stomatitis Virus with Lymphocytic Choriomeningitis Virus Glycoproteins Enhances Infectivity for Glioma Cells and Minimizes Neurotropism. J Virol. June 2011; 85(11): 5679-5684.

[0220]

[10] Reinhard Tober, Zoltan Banki, Lisa Egerer et al. VSV-GP: a Potent Viral Vaccine Vector That Boosts the Immune Response upon Repeated Applications J Virol. May 2014; 88(9): 4897-4907.

[0221]

[11] R Tober, Z Banki, A Ejaz et al. The viral vector vaccine VSV-GP boosts immune response upon repeated applications. Retrovirology. 2012; 9(Suppl 2): P301. Published online 2012 Sep 13.

[0222]

[12] Palacios G, Druce J, Du L et al. A New Arenavirus in a cluster of fatal transplant-associated diseases. N Engl J Med. 2008; 358: 991-998

[0223]

[13] Georges AJ, Gonzalez JP, Abdul-Wahid S, Saluzzo JF, Meunier DM, McCormick JB. Antibodies to Lassa and Lassa-like viruses in man and mammals in the Central African Republic. Trans R Soc Trop Med Hyg. 1985;79(1):78 -9.

[0224]

[14] Digoutte, Rapport de l’Institut Pasteur de Bangui(1970), p.59.

[0225]

[15] Murphy FA, Webb PA, Johnson KM, Whitfield SG, Chappell WA. Murphy等,Arenoviruses in Vero cells: ultrastructural studies. J.Virol 6(1970), pp.507 - 518.

[0226]

[16] Bowen MD, Peters CJ, Mills JN, Nichol ST. Oliveros virus: a novel arenavirus from Argentina. Virology. 1996年3月1日;217(1):362 - 6.

[0227]

[17] Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004年3月19日;32(5):1792 - 7.

[0228]

[18] Beyer WR, Miletic H, Ostertag W, von Laer D. Recombinant expression of lymphocytic choriomeningitis virus strain WE glycoproteins: a single amino acid makes the difference. J Virol. 2

[0229]

[19] Owens RJ, Rose JK. Cytoplasmic domain requirement for incorporation of a foreign envelope protein into vesicular stomatitis virus. J Virol. 1993 Jan;67(l):360-5.

[0230]

[20] Tani H, Komoda Y, Matsuo E, Suzuki K, Hamamoto I, Yamashita T, Moriishi K, Fujiyama K, Kanto T, Hayashi N, Owsianka A, Patel AH, Whitt MA, Matsuura Y. Replication-competent recombinant vesicular stomatitis virus encoding hepatitis C virus envelope proteins. J Virol. 2007 Aug;81(16):8601-12. Epub 2007 Jun 6.

[0231]

[21] Ge J, Wen Z, Wang X, Hu S, Liu Y, Kong X, Chen H, Bu Z. Generating vesicular stomatitis virus pseudotype bearing the severe acute respiratory syndrome coronavirus spike envelope glycoprotein for rapid and safe neutralization test or cell-entry assay. Ann N Y Acad Sci. 2006 Oct;1081 :246-8.

[0232]

[22] Garbutt M, Liebscher R, Wahl-Jensen V, Jones S, P, Wagner R, Volchkov V, Klenk HD, Feldmann H, U. Garbutt et al. Properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses. J Virol. 2004 May; 78(10): 5458-65.

[0233]

[23] Miletic H, Fischer Y, Litwak S, Giroglou T, Waerzeggers Y, Winkeler A, Li H, Himmelreich U, Lange C, Stenzel W, Deckert M, Neumann H, Jacobs AH, von Laer D. Bystander killing of malignant glioma by bone marrow-derived tumor-infiltrating progenitor cells expressing a suicide gene. Mol Ther. 2007 Jul; 15(7): 1373-81.

[0234]

[24] Fischer YH, Miletic H, Giroglou T, Litwak S, Stenzel W, Neumann H, von Laer D. A retroviral packaging cell line for pseudotype vectors based on glioma-infiltrating progenitor cells. J Gene Med. 2007 May; 9(5): 335-44.

[0235]

[25] Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Ed. (1980)

[0236]

[26] McLaughlin SK 1Collis, P., Hermonat, P. L., Muzyczka, N. Adeno-associated virus general transduction vectors: analysis of proviral structures. J Virol. 1988 Jun;62(6): 1963-73.

[0237]

[27] Urbiola, C., Santer, F. R., Petersson, M., van der Pluijm, G., Horninger, W., Erlmann, P., Wollmann, G., Kimpel, J., Culig, Z., von Laer, D. Oncolytic activity of the rabdovirus VSV-GP against prostate cancer. Int J Cancer. 2018 Apr 26.

[0238]

[28] Jenks, N., Myers, R., Greiner, S. M., Thompson, J., Mader, E. K., Greenslade, A., Griesmann, G. E., Federspiel, M. J., Rakela, J., Borad, M. J., Vile, R. G., Barber, G. N., Meier, T. R., Blanco, M. C., Carlson, S. K., Russell, S. J., Peng, K. W. Safety studies on intrahepatic or intratumoral injection of oncolytic vesicular stomatitis virus expressing interferon-beta in rodents and nonhuman primates. Hum Gene Ther. 2010 Apr;21(4):451-62.

[0239]

[29] Kaku Y, Noguchi A, Marsh GA, Barr JA, Okutani A, Hotta K, Bazartseren B, Fukushi S, Broder CC, Yamada A, Inoue S, Wang LF Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase. J Virol Methods. 2012 Jan;179(l):226-32.

Claims

1. A VSV chimeric carrier, characterized in that... The vector contains a gene encoding the glycoprotein GP of Dandenong virus and lacks a functional gene encoding the envelope protein G of VSV, wherein the envelope protein G of VSV is replaced by the GP of Dandenong virus.

2. The carrier of claim 1, characterized in that... The vector further contains at least one genetic modification.

3. A pharmaceutical composition, characterized in that... The composition comprises the VSV chimeric carrier of claim 1 or 2.

4. Use of the VSV chimeric carrier of claim 1 or 2 or the pharmaceutical composition of claim 3 in the preparation of a medicament for the treatment of non-small cell lung cancer.

5. Use of the VSV chimeric carrier of claim 1 or 2 or the pharmaceutical composition of claim 3 in the preparation of a medicament for treating cancer, wherein the cancer is non-small cell lung cancer.

Images