AN IMMUNE CHECKPOINT-MODULATING VSV-NDV HYBRID VIRUS FOR ONCOLYTIC VIRUS IMMUNOTHERAPY OF CANCER

Abstract

The present invention relates to a recombinant oncolytic virus. The present invention further relates to a nucleic acid encoding a recombinant oncolytic virus. The present invention also relates to a vector comprising a nucleic acid encoding a recombinant oncolytic virus. Furthermore, the present invention relates to a pharmaceutical composition comprising a recombinant oncolytic virus. The present invention further relates a virus, a nucleic acid, a vector, and a pharmaceutical composition for use in medicine, such as for use in the prevention and/or treatment of cancer. Moreover, the present invention relates to the use of a virus, a nucleic acid, a vector, and a pharmaceutical composition as gene delivery tool, (noninvasive) imaging of virus biodistribution, and/or for tumor detection.

Claims

1. 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, further comprising soluble PD-1 (sPD-1).

2. The recombinant oncolytic virus according to claim 1, wherein said recombinant virus further comprises a Fc domain or a fragment thereof.

3. The recombinant oncolytic virus according to claim 1, wherein said Fe domain or fragment thereof is fused to said sPD-1.

4. The recombinant oncolytic virus according to claim 1, wherein said sPD-1 is a high affinity sPD-1 (HA-sPD-1).

5. The recombinant oncolytic virus according to claim 1, wherein said sPD-1 comprises a mutation at a position selected from 132 and 41 of SEQ ID NO: 2.

6. The recombinant oncolytic virus according to claim 1, wherein said sPD-1 is HA-sPD-1-A132L having a sequence of SEQ ID NO: 3.

7. The recombinant oncolytic virus according to claim 1, wherein the modified fusion protein (F protein) of NDV is the F3aa-modified F protein having a sequence of SEQ ID NO-_25, and/or comprises at least one amino acid substitution in the protease cleavage site; and/or wherein the modified fusion protein (F protein) of NDV is the F3aa-modified F protein with an amino acid substitution L289A having SEQ ID NO. 4; and/or wherein the G protein of VSV is replaced by the modified fusion protein having a sequence of SEQ ID NO: 4 and HN protein of NDV having a sequence of SEQ ID NO-_5.

8. A nucleic acid encoding a recombinant oncolytic virus of claim 1.

9. The nucleic acid according to claim 8, comprising a nucleic acid encoding a Fe domain or fragment thereof, which is fused to a nucleic acid encoding said sPD-1.

10. A vector comprising a nucleic acid of claim 8.

11. The nucleic acid according to claim 8, comprising the nucleotide sequence of SEQ ID NO: 9 or 15, or a nucleotide sequence having at least 60% sequence identity to the nucleotide sequence of SEQ ID NO: 9 or 15, or comprising the nucleotide sequence of SEQ ID NO: 16, or a nucleotide sequence having at least 60% sequence identity to the nucleotide sequence of SEQ ID NO: 16.

12. A pharmaceutical composition, comprising the recombinant oncolytic virus of claim 1, or a nucleic acid encoding the recombinant oncolytic virus of claim 1.

13. The pharmaceutical composition according to claim 12, formulated for any of systemic delivery, tumor injection, intravenous administration, and intra-arterial administration, and/or for an intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavernous, intracerebral, intracerebroventricular, or intravitreal injection.

14. (canceled)

15. A method for the prevention and/or treatment of cancer wherein said method comprises administering to a subject in need of such prevention or treatment the recombinant oncolytic virus of claim 1, or a nucleic acid encoding the virus of claim 1.

16. A method for gene delivery, imaging of virus biodistribution, and/or for tumor detection wherein said method comprises the use of the oncolytic virus of claim 1, or a nucleic acid encoding the oncolytic virus of claim 1.

17. The method according to claim 15, wherein the cancer is selected from hepatocellular carcinoma, pancreatic cancer, and melanoma.

18. The recombinant oncolytic virus according to claim 2, wherein said sPD-1 is a high affinity sPD-1 (HA-sPD-1), having an affinity to its ligands PD-L1 and PD-L2 that is at least 2-fold higher than an affinity of a wildtype sPD-1 to said ligands, and/or has an affinity with a K.sub.d of <3.2 μM with regard to PD-L1 and/or <0.1 μM with regard to PD-L2.

19. The recombinant oncolytic virus according to claim 5, wherein said sPD-1 comprises a mutation at a position selected from 132 and 41 of SEQ ID NO: 2, wherein said mutation is selected from A132L, L41I, and L41V.

20. The recombinant oncolytic virus according to claim 7, wherein the modified fusion protein (F protein) of NDV is a F3aa-modified F protein having an L289A amino acid substitution.

21. The nucleic acid according to claim 8, comprising a nucleic acid encoding a Fc domain having a sequence of SEQ ID NO: 7, which is fused to a nucleic acid encoding said sPD-1 having a sequence of SEQ ID NO: 6.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0153] The present invention is now further described by reference to the following figures.

[0154] All methods mentioned in the figure descriptions below were carried out as described in detail in the examples.

[0155] FIG. 1 Shows Interference of the PD-1/PD-L1 Interaction and a Soluble PD-1.

[0156] When exhausted T cells come into contact with tumor cells expressing PD-L1, the PD-1 on the T cell engages and becomes inactivated, allowing the tumor cell to evade immune clearance. Local expression of a soluble PD-1 competes with the PD-1 expressed by the T cells for binding to its PD-L1 ligand and interferes with the interaction, enabling the T cell to remain functional and elicit its cytotoxic effector functions against the tumor cell [5].

[0157] FIG. 2 Shows the rVSV-NDV Backbone (A) and the rVSV-NDV-sPD-1 Construct of the Present Invention (B).

[0158] A) Recombinant pseudotyped VSV construct expressing the glycoprotein of NDV (see also WO 2017/198779 Ai). 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. B) The construct of the present invention comprises a soluble human PD-1 gene, preferably the high affinity soluble human PD-1 gene (HA-sPD-1), fused with a Fe domain of human IgG. The high affinity, soluble human PD-1 gene (HA-sPD-1), fused with the Fe domain of human IgG, was cloned as an additional transcription unit between the NDV attachment protein (HN) and the VSV large polymerase (L) genes. The resultant viral genome is shown.

[0159] FIG. 3 shows that rVSV-NDV-HA-sPD-1-Fc replicates with slight attenuation in mouse melanoma cells. The mouse melanoma cell line, B16-OVA, was infected with rVSV-NDV-GFP or rVSV-NDV-HA-sPD-1-Fc at a multiplicity of infection (MOI) of 0.01. 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 measurements of viral titers by TCID50 assay. Experiments were performed in triplicate, and data are presented as mean +/− standard error of the mean.

[0160] FIG. 4 shows that rVSV-NDV-HA-sPD-1-Fc efficiently kills mouse melanoma cells. The mouse melanoma cell line, B16-OVA, was infected with rVSV-NDV-GFP or rVSV-NDV-HA-sPD-1-Fc at a multiplicity of infection (MOI) of 0.01. 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 LDH assay for cytotoxicity. Experiments were performed in triplicate, and data are presented as mean +/− standard error of the mean.

[0161] FIG. 5 shows that rVSV-NDV-HA-sPD-1-Fc causes delayed tumor growth in immune-competent mice bearing syngeneic B16-OVA melanoma cells. Male C57B1/6 mice were implanted with 2.4×10.sup.5 B16-OVA cells subcutaneously in the flanks. On days 7, 10, and 13 post-tumor implantation, PBS or 10.sup.7 TCID50 of rVSV-NDV-GFP or rVSV-NDV-HA-sPD-1-Fc was injected into the tumor in a 50 μl volume. Tumor size was monitored daily, and tumor volumes were calculated using the formula: 4/3*PI( )*((L+W)/4)3. Individual tumor growth curves for each treatment are shown. Each curve represents an individual mouse.

[0162] FIG. 6 shows that intratumoral injection of rVSV-NDV-HA-sPD-1-Fc significantly prolongs survival of B16-OVA-bearing mice. Male C57B1/6 mice were implanted with 2.4×10.sup.5 B16-OVA cells subcutaneously in the flanks. On days 7, 10, and 13 post-tumor implantation, PBS or 10.sup.7 TCID50 of rVSV-NDV-GFP or rVSV-NDV-HA-sPD-1-Fc was injected into the tumor in a 50l volume. Mice were monitored daily and euthanized when tumor diameters reached 1.5 cm or if the skin ruptured due to tumor growth. Survival times post-treatment were plotted as a Kaplan-Meier survival curves and statistical significance was determined by log-rank test. The p value for rVSV-NDV-HA-sPD-1-Fc versus PBS <0.05.

[0163] FIG. 7 shows exemplary recombinant VSV-NDV vectors encoding for soluble PD1 (sPD1). The human sPD1 gene was cloned into the VSV-NDV vector as a unique transcription unit between the hemagglutinin-neuraminidase (HN) and large polymerase (L) genes. Additional modifications, including the high affinity (HA) mutation and fusion with a human Fc fragment were additionally engineered. The resulting genomes are shown.

[0164] FIG. 8 shows that rVSV-NDV-sPD1 variants have similar growth kinetics and cytotoxic effects in B16 melanoma cells compared to the control rVSV-NDV-GFP. Mouse B16 melanoma cells were infected in vitro with rVSV-NDV-GFP, rVSV-NDV-sPD1, rVSV-NDV-HA-sPD1, rVSV-NDV-sPD1-Fe, or rVSV-NDV-HA-sPD1-Fc at an MOI of 0.01. Top panel: Representative photomicrographs were obtained at 200× magnification at 24- and 48-hours post-infection. Uninfected B16 cells served as a control. Bottom left: Cytotoxicity was quantified at various time-points post-infection using a lactate deyhydrogenase assay (LDH, Promega). Means+standard error of the mean of duplicate experiments are shown. Bottom right: Aliquots of the supernatant harvested at multiple time-points post-infection were subjected to TCID50 analysis for analysis of virus growth kinetics. Means+standard error of the mean of experiments performed in triplicate are shown.

[0165] FIG. 9 shows that recombinant sPD1-expressing VSV-NDV vectors produce and secrete human PD1. B16 mouse melanoma cells were infected with VSV-NDV-GFP or the variants of VSV-NDV expressing the soluble human PD1 (VSV-NDV-sPD1, VSV-NDV-HA-sPD1, VSV-NDV-sPD1-Fe, or VSV-NDV-HA-sPD1-Fc) at an MOI of 0.01 or mock-infected. Left: Aliquots of cell lysate and supernatant were collected at various time-points post-infection and subjected to Western blot analysis for human PD1 or GAPDH. Right: Aliquots of supernatant were collected at 24 hours and subjected to ELISA assay for quantification of released human PD1. Means+standard error of the mean are shown.

[0166] FIG. 10 shows that a treatment of immune-competent tumor-bearing mice with rVSV-NDV-HA-sPD1-Fc causes improved control of tumor growth and an enrichment of tumor-specific T cells compared to the VSV-NDV control. A) Female C57B1/6 mice were implanted with subcutaneous syngeneic B16 melanoma cells in contralateral flanks. PBS, 10.sup.7 TCID50 of rVSV-NDV, or rVSV-NDV-HA-sPD1-Fc was injected into the tumor on the right flank, while left flank tumors remained untreated. Tumors volumes were monitored daily, and blood was collected on day 15. B) Circulating OVA-specific T cells were quantified by flow cytometry using an OVA-specific tetramer to stain peripheral blood mononuclear cells (PBMCs) obtained from blood. Individual data points, means, and standard error of the mean are shown. C) The tumor volume was significantly decreased in the rVSV-NDV-HA-sPD1-Fc group compared to the rVSV-NDV and PBS groups.

[0167] In the following, reference is made to the examples, which are given to illustrate, not to limit the present invention.

EXAMPLES

Example 1: Preparation of the rVSV-NDV-sPD-1 Virus

[0168] In order to engineer the rVSV-NDV-sPD-1 virus, the signaling and extracellular domains of PD-1 were first amplified by RT-PCR from human PBMCs. The RT-PCR product generated served as a template for a round of overlapping PCR to introduce the A132L mutation for high affinity, which generated 2 overlapping PCR fragments containing the mutated base pairs. These fragments were annealed and subjected to a final elongation step to produce the full-length HA-sPD-1 gene. The HA-sPD-1 fragment was cloned into pFUSE-hIgG1-Fc1 in order to create a fusion gene of the HA sPD-1 with the human Fc fragment. In order to insert the HA-sPD-1-Fc construct into rVSV-NDV, the appropriate restriction sites were introduced using forward and reverse oligonucleotides and amplified by PCR. Finally, the insert was ligated into the full-length VSV-NDV genome as an additional transcription unit via the multi-cloning site between the NDV-HN and the VSV-L genes. The virus construct is depicted in FIG. 2B. The corresponding infectious recombinant virus was rescued using an established method of reverse genetics.

Example 2: Characterization of the rVSV-NDV-sPD-1 Virus

[0169] Characterization of the recombinant VSV-NDV-HA-sPD-1-Fc virus was carried out in the mouse melanoma cell line B16-OVA. Growth kinetics of the virus were compared to the parental rVSV-NDV virus and revealed a slight attenuation attributed to the expression of the transgene, and a peak in replication was reached at approximately 48-hours post-infection at a multiplicity of infection (MOI) of 0.01 (FIG. 3). In line with these findings, cytotoxicity assays in the same cells revealed a slight delay in tumor cell killing by infection with rVSV-NDV-HA-sPD-1-Fc, but nearly complete killing of the cell monolayer by 72-hours post-infection (FIG. 4). Accordingly, the VSV-NDV-HA-sPD-1-Fc virus provides enhanced efficiency after 72 hours post-infection compared to the virus without HA-sPD-1-Fc.

Example 3: Anticancer Effect of the rVSV-NDV-sPD-1 Virus In Vivo

[0170] In order to determine in vivo efficacy, experiments were performed in immune-competent C57B1/6 mice bearing subcutaneous B16-OVA tumors in their flanks. On days 7, 10, and 13 after tumor implantation, PBS or 10.sup.7 TCID50 of rVSV-NDV-GFP or rVSV-NDV-HA-sPD-1-Fc was injected into the tumor in a 50 μl volume. Tumors were measured daily, and tumor volumes were calculated using the following formula: V=4/3*PI( )*((L+W)/4)3. The data reveal a significant delay in tumor growth in those mice treated with rVSV-NDV-GFP, which was even more pronounced in mice receiving rVSV-NDV-HA-sPD-1-Fe, compared to PBS (FIG. 5).

Example 4: Increased Survival of Mice Treated with rVSV-NDV-HA-sPD-1-Fc

[0171] In a survival study, mice were euthanized at humane endpoints when tumors reached a diameter of 1.5 cm or if tumor growth led to skin ruptures. Survival times with respect to the first treatment dose were plotted and revealed a significant survival prolongation of mice treated with rVSV-NDV-HA-sPD-1-Fc compared to PBS, with a median survival time of 26 versus 14 days, respectively (FIG. 6). The light grey line represents survival time after treatment with PBS; medium grey represents that of rVSV-NDV-GFP treatment; black represents that of rVSV-NDV-HA-sPD-1-Fc treatment. The data demonstrate that treatment with rVSV-NDV-HA-sPD-1-Fc results in substantially delayed tumor growth, resulting in prolonged survival, compared to treatment with buffer or the parental rVSV-NDV virus.

Example 5: Recombinant VSV-NDV Vectors Encoding for Soluble PD1 (sPD1)

[0172] A panel of VSV-NDV recombinant vectors encoding for the soluble PD1, either with the human Fc fragment (sPD1-Fc) or without (sPD1) and with the high affinity mutation (HA-sPD1) or without, were engineered and rescued by the established reverse genetics system (FIG. 7). These rVSV-NDV-sPD1 variants were fully characterized in comparison with the control rVSV-NDV vector in vitro, and the rVSV-NDV-HA-sPD1-Fc vector was selected for further characterization in vivo in an immune-competent model of mouse melanoma.

Example 6: rVSV-NDV-sPD1 Variants Replicate Well and Cause Cytotoxicity in B16 Mouse Melanoma Cells

[0173] In order to characterize the panel of rVSV-NDV-sPD1 variants in vitro, a B16 mouse melanoma cell line was chosen as representative. B16 cells were infected with the various sPD1 variants or the VSV-NDV-GFP control virus at a multiplicity of infection (MOI) of 0.01. Cells were examined microscopically at various time-points post infection in order to visualize cytotoxic effects. Additionally, samples of the supernatant were collected for analysis of cell viability by lactate dehydrogenase (LDH) assay and quantification of virus replication by tissue culture infectious dose 50 (TCID50) assay. These data revealed no substantial changes in virus replication or tumor cell killing kinetics among the sPD1 variants or in comparison to rVSV-NDV (FIG. 8).

Example 7: rVSV-NDV-sPD1 Variants Produce and Release Soluble Human PD1 In Vitro

[0174] In order to confirm that the recombinant constructs produce and release the soluble human PD1, Western blots and ELISA assays were performed. B16 cells were infected with the sPD1-expressing VSV-NDV constructs or the control VSV-NDV-GFP at an MOI of 0.01 or left uninfected, and lysates and supernatants were collected at various time-points post-infection and subjected to Western blot analysis for sPD1. Additionally, GAPDH was analyzed to control for protein loading. As expected, the sPD1 virus-infected samples produced a band at the expected size, while the viruses expressing the sPD1-Fc fusion protein had a substantial shift in size corresponding to the additive size of the sPD1 and the Fc fragment (FIG. 9, left panel). Additional samples of supernatant collected at 24 hours were subjected to an ELISA assay to quantify the amount of PD1 secreted into the supernatant. As expected, only samples from cells infected with VSV-NDV expressing sPD1 produced detectable levels of PD1, while those expressing sPD1 fused with the Fc fragment (sPD1-Fc) had even higher levels than those without, supporting the hypothesis that the presence of the Fc fragment stabilizes the extracellular sPD1 (FIG. 9, right panel).

Example 8: In Vivo rVSV-NDV-HA-sPD1-Fc Treatment of Syngeneic Mouse Melanoma Results in Enhanced Tumor-Specific T Cell Responses and Delayed Tumor Growth

[0175] For in vivo analysis of VSV-NDV-mediated expression of sPD1, the inventors focused on the HA-sPD1-Fc variant, due to a potential benefit of high affinity binding via the HA mutation and stability in blood afforded by fusion of sPD1 with the Fc fragment. The inventors utilized a subcutaneous model of B16 melanoma, in which tumors were implanted into contralateral flanks, and PBS, rVSV-NDV, or rVSV-NDV-HA-sPD1-Fc at a dose of 10.sup.7 TCID50 was injected intratumorally into the lesion on the right flank on day 7, 10, and 13 after tumor-implantation (FIG. 10A). Peripheral blood mononuclear cells (PBMCs) were isolated from blood collected on day 15 and subjected to FACS analysis of CD8+ T cells specific for the model antigen, ovalbumin, that is expressed by the B16 cells. While VSV-NDV treatment resulted in a statistically significant increase in OVA-specific T cells compared to PBS, this effect was even further increased in the mice treated with rVSV-NDV-HA-sPD1-Fc (FIG. 10B).

[0176] Furthermore, analysis of tumor growth in both, the injected and contralateral uninjected lesions, revealed delayed growth in rVSV-NDV-HA-sPD1-Fc-treated animals compared to rVSV-NDV- or PBS-treated (FIG. 10C). These results indicate that rVSV-NDV-HA-sPD1-Fc allows for enhanced T cell activation, leading to immune-mediated abscopal effects.

REFERENCES

[0177] [1] Bartee MY, Dunlap KM, Bartee E. Tumor-Localized Secretion of Soluble PD1 Enhances Oncolytic Virotherapy. Cancer Res. 2017; 77(11):2952-63. [0178] [2] Lazar-Molnar E, Scandiuzzi L, Basu I, Quinn T, Sylvestre E, Palmieri E, et al. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy. EBioMedicine. 2017; 17:30-44. [0179] [3] Maute R L, Gordon S R, Mayer A T, McCracken M N, Natarajan A, et al. PD-1 variants for immunotherapy and PET imaging. PNAS. Nov 2015, 112 (47) E6506-E6514; DOI: 10.1073/pnas.1519623112. [0180] [4] Altomonte, J., S. Marozin, et al. (2010). “Engineered newcastle disease virus as an improved oncolytic agent against hepatocellular carcinoma.” Mol Ther 18(2): 275-284. [0181] [5] Malini Guha, The Pharmaceutical Journal (2014).

[0182] The features of the present invention disclosed in the specification, the claims, and/or in the accompanying figures may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof.