VSV/NDV HYBRID VIRUSES FOR ONCOLYTIC THERAPY OF CANCER

Abstract

The present invention relates to recombinant oncolytic viruses comprising a vesicular stomatitis virus (VSV), wherein the glycoprotein (G protein) of VSV is deleted; and which comprises a modified fusion protein (F protein) of Newcastle disease virus (NDV); and the hemagglutinin neuraminidase (HN) protein of NDV. The present invention further relates to nucleic acids encoding for the recombinant oncolytic virus and vectors comprising the nucleic acids. The present invention further relates to pharmaceutical compositions comprising the rVSV of the invention, the nucleic acid or the vector, further to uses as gene delivery tool and/or for tumor detection. The present invention further relates to the recombinant oncolytic vesicular stomatitis virus (VSV) for use in medicine, in particular for the diagnosis, prevention and/or treatment of cancer.

Claims

1. A method for producing a recombinant oncolytic virus, comprising a vesicular stomatitis virus (VSV), wherein the method comprises the steps of deleting glycoprotein (G protein) of VSV, and wherein the VSV comprises a modified fusion protein (F protein) of Newcastle disease virus (NDV), and the hemagglutinin neuraminidase (HN) protein of NDV.

2. The method of claim 1, wherein the modified fusion protein (F protein) of NDV is a F3aa-modified F protein, and/or wherein the modified fusion protein comprises at least one amino acid substitution in the protease cleavage site, and/or wherein the G protein of VSV is replaced by the modified fusion protein and the HN protein of NDV.

3. The method of claim 1, wherein the modified fusion protein (F protein) of NDV comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NOs: 10 or 12, and/or wherein the HN protein of NDV comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6.

4. A recombinant oncolytic virus, comprising a vesicular stomatitis virus (VSV), wherein the glycoprotein (G protein) of VSV is deleted, and which comprises a modified fusion protein (F protein) of Newcastle disease virus (NDV); and the hemagglutinin neuraminidase (HN) protein of NDV, wherein the modified fusion protein (F protein) of NDV comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NOs: 10 or 12, and/or wherein the HN protein of NDV comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6.

5. A nucleic acid encoding a recombinant oncolytic virus according to claim 4.

6. A vector comprising a nucleic acid of claim 5.

7. A pharmaceutical composition, comprising: (a) (i) a recombinant oncolytic virus produced by a method for producing a recombinant oncolytic virus, comprising a vesicular stomatitis virus (VSV), wherein the method comprises the steps of deleting glycoprotein (G protein) of VSV, and wherein the VSV comprises a modified fusion protein (F protein) of Newcastle disease virus (NDV), and the hemagglutinin neuraminidase (HN) protein of NDV; or (ii) a recombinant oncolytic virus, comprising a vesicular stomatitis virus (VSV), wherein the glycoprotein (G protein) of VSV is deleted, and which comprises a modified fusion protein (F protein) of Newcastle disease virus (NDV); and the hemagglutinin neuraminidase (HN) protein of NDV, wherein the modified fusion protein (F protein) of NDV comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NOs: 10 or 12, and/or wherein the HN protein of NDV comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6; or (iii) a nucleic acid encoding a recombinant oncolytic virus according to claim 5; and (b) pharmaceutically acceptable carrier(s) and/or excipient(s).

8. The pharmaceutical composition of claim 7, further comprising one or more compounds selected from chemotherapeutic agents, radiotherapeutic agents, tumor vaccines, immune checkpoint inhibitors, cell carrier systems, small molecule inhibitors, embolization agents, and shielding polymers.

9. The pharmaceutical composition of claim 7, formulated for delivery via a route selected from intravenous administration or intra-arterial administration, intradermal, subcutaneous, intramuscular, intravenous, intratumoral, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavernous, intracerebral, intracerebroventricular and intravitreal injection.

10. A method for oncolytic therapy wherein said method comprises the step of administering to a patient a therapeutically effective amount of: a recombinant oncolytic virus produced by a method for producing a recombinant oncolytic virus, comprising a vesicular stomatitis virus (VSV), wherein the method comprises the steps of deleting glycoprotein (G protein) of VSV, and wherein the VSV comprises a modified fusion protein (F protein) of Newcastle disease virus (NDV), and the hemagglutinin neuraminidase (HN) protein of NDV; or a nucleic acid encoding the recombinant oncolytic virus according to claim 5.

11. The method according to claim 10, further comprising the step of administering one or more additional agents selected from cell carrier systems, immunotherapies, and standard tumor therapies, to said patient.

12. A method of treatment of cancer comprising the step of administering to a subject in need thereof a therapeutically effective amount of: a recombinant oncolytic virus produced by a method for producing a recombinant oncolytic virus, comprising a vesicular stomatitis virus (VSV), wherein the method comprises the steps of deleting glycoprotein (G protein) of VSV, and wherein the VSV comprises a modified fusion protein (F protein) of Newcastle disease virus (NDV), and the hemagglutinin neuraminidase (HN) protein of NDV; or a nucleic acid encoding the recombinant oncolytic virus according to claim 5.

13. The method according to claim 12, wherein the cell carrier system is selected from T cells, dendritic cells, NK cells, and mesenchymal stem cells; the immunotherapy is selected from tumor vaccines and immune checkpoint inhibitors; and the standard tumor therapy is selected from radiofrequency ablation, chemotherapy, embolization, and small molecule inhibitors.

14. The vector according to claim 6, further comprising one or more reporter genes and/or genes to be delivered to a target cell or tissue.

15. The vector according to claim 14, wherein the reporter gene is selected from HSV1-sr39TK, the sodium iodide symporter (NIS), somatostatin receptor 2 (SSTR2), luciferase, green fluorescence protein (GFP), lacZ, and tyrosinase; and the gene to be delivered to a target cell or tissue is selected from immune stimulating genes, immune checkpoint inhibitory antibodies, and tumor associated antigens (TAA).

16. A method of delivering genes, of imaging of virus biodistribution and/or for tumor detection, wherein the vector of claim 14 is administered to a patient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0152] FIG. 1. Recombinant pseudotyped VSV construct expressing the glycoprotein of NDV. The endogenous glycoprotein of VSV was deleted from a plasmid encoding the full-length VSV genome. The NDV glycoprotein, comprising a modified fusion protein (NDV/F(L289A)) and hemagglutinin-neuraminidase (NDV/HN), was inserted as discrete transcription units between the VSV matrix (M) and large polymerase (L) genes. The respective pseudotyped VSV vector was rescued using an established reverse-genetics system.

[0153] FIGS. 2A-2D. rVSV-NDV can replicate in HCC cell lines and cause complete cytotoxicity. Human HCC cell lines Huh7 (2A, 2B) and HepG2 (2C, 2D) were infected with a multiplicity of infection (MOI) of 0.01 of rVSV, rNDV, or rVSV-NDV. After a 1 hour infection, the cells were washed and fresh medium was added to the cells. At various time-points post-infection aliquots of the supernatant were collected for cytotoxicity measurements by LDH assay (2B, 2D) and cell monolayers were lysed for measurements of intracellular titers by TCID50 assay (2A, 2C). Experiments were performed in triplicate, and data are presented as mean+/−standard deviation.

[0154] FIG. 3. rVSV-NDV infection leads to rapid syncytia formation in HCC cells. In order to assess the ability of the pseudotyped rVSV-NDV vector to induce syncytia formation in tumor cells, various HCC cell lines were infected with rVSV-NDV, rNDV, or rVSV at an MOI of 0.01, and observed microscopically at various time-points post-infection. Additional cells were treated with PBS as a control. Huh7 cells are shown as a representative human HCC cell line, and representative images were captured under 200× magnification.

[0155] FIG. 4. Pseudotyping VSV with NDV envelope proteins does not alter the sensitivity of the vector to the antiviral actions of IFN. To assess the sensitivity of rVSV-NDV to type I IFN, an IFN-sensitive cell line (A549) was infected with rVSV-NDV, rVSV, and rNDV at an MOI of 0.01. Cells were lysed at 48 hours post-infection, and intracellular viral titers were measured by TCID50 assay. Experiments were performed in triplicate, and mean values+/−standard deviation are shown.

[0156] FIGS. 5A-5B. Replication and cytotoxicity of rVSV-NDV is substantially diminished in primary human hepatocytes. Primary human hepatocytes were infected at an MOI of 0.01 with rVSV, rNDV, or rVSV-NDV. Cell lysates were subjected to TCID50 analysis of intracellular virus titers at various timepoints. Additionally, aliquots of supernatant were collected at various timepoints for cytotoxicity measurements by LDH assay. Experiments were performed in duplicate, and means+/−standard deviation are shown.

[0157] FIGS. 6A-6B. Replication and cytotoxicity of rVSV-NDV is substantially diminished in primary mouse neurons. Primary mouse neurons were infected at an MOI of 0.01 with rVSV, rNDV, or rVSV-NDV. Cell lysates were subjected to TCID50 analysis of intracellular virus titers at various timepoints. Additional wells were assayed for cell viability using a standard MTS assay. Experiments were performed in duplicate, and means+/−standard deviation are shown.

[0158] FIG. 7. The pseudotyped rVSV-NDV vector causes immunogenic cell death. Huh7 cells were infected with rVSV, rNDV, or rVSV-NDV at an MOI of 0.01 or mock-infected for 48 hours. The conditioned media were concentrated, and 10 μg of protein were subjected to Western blot analysis for detection of released HMGB1, Hsp70, and Hsp90.

[0159] FIG. 8. Pseudotyped rVSV-NDV vector demonstrates enhanced safety compared to rVSV in immune-deficient mice. Immune-deficient male NOD-SCID mice were treated by tail vein injection with rVSV-NDV or rVSV-GFP (referred to as rVSV in the figure for simplicity) at a dose of 10.sup.6 TCID50. Mice were monitored daily and euthanized at humane endpoints. Body weight changes were plotted over time with respect to the injection (left); Viral titers in blood were measured on day 1 and 7 by TCID50 analysis (center); The survival proportions were plotted by Kaplan-Maier survival curve (left).

[0160] FIG. 9. Mice treated with 10.sup.6 TCID50 rVSV revealed pathological changes in the liver and brain. H/E staining of liver revealed small group necrosis of hepatocytes after rVSV treatment, marked by hepatocellular degeneration with karyolysis (top left panel). Acute necrosis in the brain stem after rVSV application was observed with degenerating glial cells exhibiting pyknosis and karyorrhexis (top right panel). Degeneration of glial cells could be further confirmed by immunohistochemical staining for caspase-3 (bottom right). Representative images are shown; scale bars equal 50 μm. Viral titers were quantified from brain and liver tissue lysate from mice receiving rVSV after demonstrating signs of toxicity. Means+SEM are shown.

EXAMPLES

1. Material and Methods

1.1 Viruses

[0161] Recombinant VSV expressing the GFP reporter (referred to herein as “rVSV”) was engineered and rescued as previously described (Huang et al., 2003). Recombinant NDV harboring the F3aa(L289A) mutations and expressing the GFP reporter gene (referred to herein as “rNDV”) was engineered and rescued as previously described (Altomonte et al., 2010).

[0162] Recombinant rVSV-NDV was produced by first modifying a plasmid encoding for the full-length VSV genome (pVSV-XN2) and expressing the F3aa(L289A)-modified fusion protein of NDV (Ebert et al., 2004) as an additional transcription unit between the G and L genes. The endogenous VSV glycoprotein (G) was deleted by digestion with MluI and XhoI restriction enzymes, which recognize the unique restriction sites in the 5′ and 3′ noncoding regions of the G, respectively. Following self-ligation of the G-deleted plasmid, a short oligonucleotide linker was inserted at the unique NheI restriction site following the NDV F gene, to create a multiple cloning site for insertion of the HN gene. The HN gene was amplified by PCR from a plasmid encoding the full-length NDV genome, utilizing primers to introduce PacI and PmeI restriction sites at the 5′ and 3′ ends of the PCR product, respectively, for insertion into the newly incorporated restriction sites in the G-deleted VSV-NDV/F3aa(L289A) plasmid. The resulting plasmid was subjected to sequence analysis to confirm the fidelity of the PCR insert, as well as the intergenic transcription start and stop sequences and the gene order. Finally, the infectious virus, referred to here as “rVSV-NDV”, was rescued using the established reverse genetics system for rescuing negative-strand RNA viruses (Lawson et al., 1995).

[0163] See also FIG. 1.

1.2 Cell Lines

[0164] Two human HCC cell lines (HepG2 and Huh-7) were obtained from Dr. Ulrich Lauer (University Hospital Tübingen, Germany) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine (200 mM), 1% Penicillin/streptomycin, 1% non-essential amino acids and 1% sodium pyruvate. A549 cells were obtained from the ATCC (Rockville, Md.) and cultured in the same medium as the HCC cell lines. Primary human hepatocytes were derived from patients (negative for hepatitis B and C virus and human immunodeficiency virus) who had undergone surgical resection of liver tumors, in accordance with the guidelines of the charitable state-controlled Human Tissue and Cell Research (HTCR) foundation (Regensburg, Germany). The hepatocytes were maintained in HepatoZYME-SFM medium (Gibco-Invitrogen, Karlsruhe, Germany). Primary embryonic primary cortical neurons were dissociated from E16.5 mouse cortex and provided by the laboratory of Stefan Lichtenthaler (DZNE, Munich, Germany). Neuronal cultures were maintained in Neurobasal medium (Gibco) supplemented with B27 (2%), 0.5 mM glutamine, and 1% penicillin/streptomycin. All cell lines and primary cells were maintained in the 37° C. humidified incubator with 5% CO.sub.2

1.3 Microscopic Analysis

[0165] The human HCC cell lines, Huh7 and HepG2, were plated at approximately 90% confluency in 6-well dishes and infected with either rVSV, rNDV, or rVSV-NDV at an MOI of 0.01 or mock-infected. Cells were visualized at 200× magnification on an Axiovert 40CFL microscope (Zeiss) at 16-, 24- and 48-hours post-infection, and representative images were captured with a Canon Powershot A620 camera attached to the microscope.

1.4 IFN Dose Response Assay

[0166] Interferon-sensitive A549 cells were plated in 24-well dishes at a density of 10.sup.5 cells per well and cultured overnight. The following evening they were pre-treated with different concentrations (0, 100, 500, and 1000 IU/ml) of Universal type I Interferon added directly to the culture medium. After overnight incubation, the cells were infected with either rVSV, rNDV or rVSV-NDV at a multiplicity of infection (MOI) of 0.01. 48 hours post-infection, cells were collected in 100 μl of PBS and lysed by three freeze-thaw cycles. The intratumoral virus titer was determined by TCID.sub.50 analysis of the cell lysates.

1.5 Growth Curves (TCID50 Assay)

[0167] Viral growth curves were performed in HCC cell lines (Huh7 and HepG2), as well as in primary human hepatocytes and primary mouse neurons.

HCC cell lines were plated in 6-well dishes at a density of 3.5×10.sup.5 cells per well, while PHH and neurons were seeded in collagen-coated 24-well dishes at a density of 10.sup.5 cells per well. Each cell line was infected with rVSV, rNDV and rVSV-NDV at a multiplicity of infection (MOI) of 0.01. The infections were performed in 1 ml of PBS (6-well dishes) or 250 μl of PBS (24-well dishes) at 37° C. for 1 hour. After incubation, cells were washed three times with PBS and fresh medium was added. Cell lysate was collected at 0, 16, 24, 48 and 72 hours post-infection for TCID.sub.50 analysis of intracellular virus titers.

1.6 Cytotoxicity Assays (LDH or MTS Assay)

[0168] Cell viability of infected HCC cell lines (Huh7 and HepG2) and primary human hepatocytes was analyzed by measuring released Lactate Dehyrogenase (LDH) from cell culture supernatant. The cells were plated, infected and washed as in the growth curve experiments. At 24, 48 and 72 hours post-infection, aliquots of supernatant were collected, and LDH-release was quantified using the CytoTox 96 Non-Radioactive Cytotoxicity Assay protocol (Promega). For each time point, LDH-release following virus infection was calculated as a percentage of the maximum LDH-release control. Baseline LDH levels detected in the supernatant of mock-treated cells were subtracted from the values obtained from the experimental wells.

[0169] Cell viability of neurons was analyzed by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carbooxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay using the CellTiter96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, Wis.). Neurons were seeded in collagen coated 96-well dishes at a density of 5×10.sup.4 cells/well and mock-treated or infected with rVSV, rNDV or rVSV-NDV at an MOI of 0.01. At 24, 48 and 72 hours post-infection, cell viability was measured according to the manufacturer's protocol. Cytotoxicity was calculated as difference in cell viability of the experimental samples compared to the uninfected controls.

1.7 Western Blots

[0170] Huh7 cells were plated in 6-well plates at approximately 90% confluence and infected with rVSV, rNDV, or rVSV-NDV at an MOI of 0.01 or mock-infected for 48 hours. The conditioned media were collected and concentrated to about 200 μl using Amicon Ultra Centrifugal filters with a 10 kD cutoff (Merck Millipore, Billerica, Mass.). Protein concentrations were quantified using the Pierce BCA Protein Assay (Thermo Fisher Scientific, Waltham, Mass.), and 10 μg of each sample was loaded onto a 7.5% denaturing SDS-PAGE gel, followed by transfer onto a nitrocellulose membrane. Protein bands were detected using specific antibodies against HMGB1 and Hsp90 (Cell Signaling Technology, Danvers, Mass.) and Hsp70 (Santa Cruz Biotechnology, Dallas, Tex.) and the appropriate secondary antibody conjugated with horseradish peroxidase. Bands were visualized using Amersham ECL Prime Western Blot Detection Reagent (GE Healthcare Life Sciences, Pittsburgh, Pa.).

2. Results

[0171] The recombinant VSV-NDV vector (FIG. 1) has been characterized in vitro for replication and cytotoxicity in tumor cells, as well as in healthy hepatocytes and neurons. We used two human hepatocellular carcinoma (HCC) cell lines as representative tumor cells, and compared the rVSV-NDV with rVSV and rNDV in terms of its relative ability to replicate and kill the cells. Although rVSV-NDV replication was a bit delayed compared to the wildtype vectors, it was able to reach similar titers at about 72 hours post-infection, which resulted in complete cell killing in vitro (FIG. 2).

[0172] In order to observe virus-induced syncytial formation, additional cells were infected with rVSV-NDV, as well as the parental rVSV and rNDV, for photomicroscopy. Microscopic analysis of the tumor cells revealed multiple foci of syncytia in the wells infected with rVSV-NDV by 16 hours post-infection, while it was significantly delayed in those infected with rNDV. As expected, cells that were treated with rVSV did not form syncytia; however, they were highly susceptible to the cytopathic effect (CPE), which is classic of VSV infection and occurred earlier than 16 hours post-infection (FIG. 3).

[0173] In order to rule out that the glycoprotein exchange inadvertently resulted in a loss of sensitivity of the vector to the antiviral actions of type I interferon (IFN), an IFN dose response was performed. The exquisite sensitivity of VSV to type I IFN is a key mechanism of tumor specificity, as tumor cells are often defective in their IFN signaling pathways, while healthy cells can efficiently clear the virus via IFN responsive genes. Although this assay revealed a relative insensitivity of rNDV to type I IFN, the rVSV-NDV vector was rapidly attenuated by the addition of IFN and reduced to levels similar to those observed for rVSV (FIG. 4).

[0174] We next performed growth curves and cytotoxicity assays in normal primary human hepatocytes and mouse neurons in order to assess the safety of rVSV-NDV. Very little replication of the pseudotyped vector could be observed over time, and titers were approximately 5 logs lower than the control VSV vector at 48 hours post-infection and 3 logs lower than rNDV at the same time-point in primary hepatocytes (FIG. 5). Although nearly all hepatocytes were dead by 72 hours post-infection with rVSV, no cytotoxicity could be observed by LDH assay in cells infected with rVSV-NDV (FIG. 5). Similarly, titers of rVSV-NDV were significantly lower than the control VSV vector in primary mouse neurons at all time-points investigated, which corresponded to similar levels of cell viability as those observed in PBS-treat neurons (FIG. 6). Taken together, rVSV-NDV showed little evidence of replication in primary healthy cells and resulted in little to no cytotoxicity in vitro, indicating that it is a substantially safer virus than both rVSV and rNDV.

[0175] To determine whether the pseudotyped rVSV vector would induce an immunogenic cell death, as has been shown for rNDV through syncytia formation, we investigated the release of high mobility group box 1 (HMGB1) and heat-shock proteins 70 and 90 from infected Huh7 cells. After a 48 hour infection, we observed relatively low levels of HMGB1, Hsp70, and Hsp90 released into the supernatant of rVSV-infected cells. However, infection with both rNDV and rVSV-NDV resulted in high levels of all three secreted markers for immunogenic cell death (FIG. 7). These results indicate that, in addition to the potent direct cytotoxicity caused by infection with the pseudotyped rVSV-NDV vector, in vivo treatment with this virus could result in substantial immune responses directed against the tumor.

[0176] In order to assess the safety of the pseudotyped rVSV-NDV vector in vivo, immune-deficient male NOD-SCID mice approximately 8 weeks of age were treated by tail vein injection with either rVSV-NDV or the control rVSV-GFP virus (N=6) at a dose of 10.sup.6 TCID50 per mouse. Mice were monitored daily for body weight and overall physical appearance, and they were euthanized at humane endpoints. Blood was sampled on day 1, 3, 7, 14, and at the time of euthanization for analysis serum chemistry and circulating virus titers. Two mice receiving rVSV-GFP rapidly began losing weight during the first week after treatment, and all six died acutely or were euthanized due to extreme body weight loss, dehydration, signs of distress (changes in posture, impaired movement, isolation, etc.), and/or signs of neurotoxicity (limb paralysis and circling) between 11 and 17 days post-treatment (FIG. 8). Additionally, infectious virus titers could be recovered from the blood on day 1 and 7 post-treatment (FIG. 8, center). In contrast, the mice who received rVSV-NDV lost only negligible amounts of weight, appeared healthy and exhibited normal behavior throughout the study. Three of these mice were euthanized at 21 days post-treatment for histological analysis of major organs. while the remaining animals were monitored for 60 days post-treatment, at which time they were euthanized for pathological analysis. No infectious virus titers could be detected in the blood of mice treated with rVSV-NDV at any time-point analyzed. Plasma measurements of liver function (GPT) and kidney function (BUN and Creatinine) revealed no abnormal values for either treatment group (data not shown).

[0177] Tissue sections were examined by a pathologist who was blinded to the treatment groups of the specimens. Histological analysis revealed no major pathological findings in tissue excised from mice treated with rVSV-NDV, either euthanized on day 21 or day 60. Furthermore, no detectable titers within the brain or liver tissue could be observed in mice treated with rVSV-NDV (data not shown). In stark contrast, mice that received rVSV-GFP at the same dose exhibited heavy intrasinusoidal edema, moderate acute hepatitis with single cell and small group necrosis, and apoptosis of hepatic tissue (FIG. 9). Furthermore, acute necrosis in the brain stem, with degenerating glial cells exhibiting pyknosis and karyorhexis could be observed. Degeneration of glial cells was further confirmed by immunohistochemical staining for caspase-3. TCID50 analysis of tissue lysates revealed quantifiable levels of infectious VSV in the liver and brain at the time of necropsy.

[0178] The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

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