Oncolytic vaccinia virus

Abstract

The present invention provides genetically modified vaccinia virus which may be employed as an oncolytic viral vector, wherein the genetically modified vaccinia virus comprises a nucleic acid molecule encoding a modified matrix protein from a vesicular stomatitis virus and wherein the genetically modified vaccinia virus is fusogenic.

Description

ONCOLYTIC VACCINIA VIRUS

[0001] CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 597,077 filed November 8, 2023.

[0003] FIELD

[0004] The present invention relates to a genetically modified vaccinia virus which may be employed as an oncolytic viral vector.

[0005] BACKGROUND

[0006] Oncolytic viruses are viruses that preferentially infect and / or kill cancer cells. Various approaches have been undertaken to develop oncolytic viruses for the effective targeting of cancer cells in the treatment of cancer.

[0007] Numerous viruses have been explored for their oncolytic potential. These include adenovirus, herpes simplex virus (HSV), vesicular stomatitis virus (VSV), reovirus, measles virus, picomaviruses, protoparvovirus, and vaccinia virus (Yun et al., Frontiers (2022), 13).

[0008] Furthermore, numerous approaches for the genetic modification of each of these types of oncolytic viruses with varying degrees of success.

[0009] For example, oncolytic adenoviruses have been developed incorporating numerous modifications including: insertion of E2F-binding sites in E1A; insertion of MDA- 7 / IL-24 transgene; replacement of El A by tCCNl; incorporation of FP3 transgene; replacement of HVR5 by the peptide CKS17; insertion of D24, ICOSL, and CD40 genes; and replacing the Ad5 knob in an Ad5 adenoviral vector with Ad3 (Apolonio et al., World J. Virol. (2021), 10(5):229-255).

[0010] In another example, oncolytic HSV have been developed incorporating numerous modifications including: insertion of GM-CSF and deletion of gamma34.5 and US12; insertion of UL53, UL54, and deletion of UL43, UL49.5, UL55, UL56, and LAT; incorporation of the HSV-1 TK gene and deletion of alphaO, alpha4, gamma34.5, UL56, and UL24; and insertion of lacZ and deletion of gamma34.5 (Apolonio et al., World J. Virol. (2021), 10(5):229-255).

[0011] In another example, oncolytic VSV have been developed incorporating numerous modifications including: mutation of the matrix (M) protein; 3’ shift of VSV genes by insertionof a 5’ transgene; insertion of FMDV IRES and rhinovirus type 2 IRES elements upstream of VSV M; pseudotyping of the glycoprotein; insertion of a human p53 transgene; and insertion of cytokine transgenes (Felt, S. and Grdzelishvili, V., J. Gen. Virol. (2017), 98(12):2895-2911; WO 2004 / 085658).

[0012] In another example, oncolytic vaccinia viruses have been developed incorporating numerous modifications including: expression of GM-CSF and lacZ transgenes; insertion of immune modulatory or checkpoint inhibitory antibody transgenes; insertion of co-stimulatory transgenes; and deletion of various endogenous genes including A26R, A56R, B21R, C10L, C9L, C4L, MIL, A51R, A52R, A55R, B13R, B14R, J2R, C11R, F4L, VGF, OIL, F1L, NIL, M2L, K1L, K7R, C2L, N2L, K2L, K3L, F3L, B16R, B19R, K4L, K5L, K6L, F2L, B15R, B17L, B18R, and B20R, among others (US 2022 / 0313761; US 2020 / 0385758).

[0013] Despite the wide array of genetic modifications that have been explored, research into the provision of optimized and enhanced oncolytic viruses with improved efficacy in the treatment of cancer remains ongoing.

[0014] SUMMARY

[0015] The present inventors disclose herein a genetically modified vaccinia virus with improved oncolytic activity.

[0016] In an embodiment, there is provided a genetically modified vaccinia virus, comprising a nucleic acid molecule encoding a modified matrix protein from a vesicular stomatitis virus (VSV), wherein the genetically modified vaccinia virus is fusogenic.

[0017] In an embodiment, there is provided a pharmaceutical composition comprising the genetically modified vaccinia virus as described herein and a pharmaceutically acceptable carrier.

[0018] In an embodiment, there is provided a method of treating cancer in a subject, comprising administering the genetically modified vaccinia virus or the pharmaceutical composition as described herein to the subject.

[0019] In an embodiment, there is provided a use of the genetically modified vaccinia virus or the pharmaceutical composition as described herein for treating cancer in a subject.

[0020] In an embodiment, there is provided a use of the genetically modified vaccinia virus as described herein in the preparation of a medicament for treating cancer in a subject.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Embodiments will be described, by way of example only, with reference to the accompanying figures.

[0023] FIG. 1: Map of pMA-deltaTK-mCherry vector. Common sequencing primer Ml 3 fwd 293-309; complementary region to J1R 369-808; mCherry fluorescent reported gene 815- 1531; promoter pSEL 1543-1585; complementary region to J3R 1596-1808; origin of replication ori 2046-2634; ampicillin resistance gene AmpR 2805-3665; AmpR promoter 3666-3770.

[0024] FIG. 2: Map of pSC65-deltaTK-delta51M-eGFP vector. Complementary region to J1R 1-773; eGFP fluorescent reporter 879-1598; promoter pSEL 1624-1670; promoter p7.5 1676-1804; modified matrix protein from VSV dM51 (delta51M) 1815-2504; complementary region to J3R 2523-3513; origin of replication ori 3532-4120; ampicillin resistance gene AmpR 4291-5151; AmpR promoter 5152-5256.

[0025] FIG. 3: Map of pUC57-deltaTK-Nivo-Atezo vector. Common sequencing primer M13 fwd 379-395; complementary region to J1R 396-836; light chain (LC) of atezolizumab 856- 1557; promoter pSEL 1563-1604; promoter pSEL 1613-1654; heavy chain (HC) of atezolizumab 1660-3063; heavy chain (HC) of nivolumab 3095-4474; promoter pSEL 4480-4521; promoter pSEL 4526-4567; light chain (LC) of nivolumab 4573-5274; complementary region to J3R 5293-5736; common sequencing primer M13 rev 5749-5765; replication origin ori 6151-6739; ampicillin resistance gene AmpR 6910-7770; AmpR promoter 7771-7875.

[0026] FIG. 4: Map of pUC57-LS-deltaC12-delta51M vector. Common sequencing primer M13 379-395; complementary region to C12L 401-894; modified matrix protein from VSV dM51 (delta51M) 1036-1725; complementary region to C12R; common sequencing primer M13 rev 2262-2278; origin of replication ori 2664-3252; ampicillin resistance gene AmpR 3423- 4283; AmpR promoter 4284-4388.

[0027] FIG. 5: Map of pUC57-deltaTK-atezo vector. Common sequencing primer M13 379-395; complementary region to J1R 396-836; light chain (LC) of atezolizumab 856-1557; promoter pSEL 1563-1604; promoter pSEL 1613-1654; heavy chain (HC) of atezolizumab 1660- 3063; complementary region to J3R 3083-3526; common sequencing primer M13 rev 3539-3555; origin of replication ori 3941-4529; ampicillin resistance gene AmpR 4700-5560; AmpR promoter 5561-5665.

[0028] FIG. 6: Images of cell cultures with non infected control NIC (A); infected with Pl 600 virus (B); infected with Pl 602 virus (C); infected with Pl 605 virus (D); infected with Pl 607 virus (E); infected with P1615 virus (F); infected with P1616 virus (G); or infected with non-modified vaccinia strains Western Reserve, Lister, Wyeth, and Copenhagen (H). Images taken at 70hrs post infection.

[0029] FIG. 7 : Images of 96-well plates containing cells infected with virus and stained for viability. (A) Illustration of non infected wells (NIC) and MOI of infected wells in 96-well plates. (B) Images of stained cells in 96-well plates 3 days post infection, infected with unmodified Lister (WT), P1600, P1602, P1605, P1607, P1615, or P1616.

[0030] FIG. 8: Tumor growth in CT26LacZ inoculated mice treated with modified viruses. (A) Average tumor volume for each treatment. (B) Tumor growth in each individual mouse for each treatment group. Y-axis: tumor volume (mm3); X-axis: days post tumor implantation.

[0031] DETAILED DESCRIPTION

[0032] The present inventors have developed genetically modified vaccinia viruses with surprisingly improved oncolytic activity. As shown in FIG. 8A, a genetically modified vaccinia virus of the present invention (P1616), comprising both a modified matrix protein from a VSV and exhibiting a fusogenic phenotype, was significantly more effective at inhibiting tumor growth in a CT26LacZ tumor model as compared to vaccinia viruses comprising only a modified matrix protein from a VSV (Pl 602) or only exhibiting a fusogenic phenotype (Pl 605). This synergistic effect from the combination of a modified matrix protein from a VSV and a fusogenic phenotype resulted in an unexpectedly improved control of tumor growth.

[0033] Genetic Modification

[0034] As used herein, the term “genetically modified” or “genetically engineered” refers to altering genetic material using molecular biology techniques known in the art such as, but not limited to, molecular cloning, recombinant DNA methods, and gene editing by known techniques such as with the use of restriction endonucleases, Gibson assembly, zinc finger nucleases, TALENs, and Crispr-Cas systems such as Crispr-Cas9. Genetic modification includesaddition, deletion, substitution, modification, and / or mutation of genetic material. Numerous techniques for the genetic modification of vaccinia virus are known and routinely used in the art (Mackett et al., J. Virol. (1984), 49(3):857-864; Falkner, F. and Moss, B., J. Virol. (1990), 64(6):3108-3111; Domi, A., and Moss, B., PNAS (2002), 99(19): 12415-12420; Yuan et al., J. Virol. (2015), 89(9):5176-5179). As used herein, the term “modified” may refer to a nucleic acid molecule or a polypeptide that contains one or more changes in the nucleotide or amino acid sequence compared to the un-modified wild-type version of said nucleic acid molecule or polypeptide.

[0035] As used herein, the term “wild-type” carries the ordinary meaning in the art of an organism, nucleic acid molecule, or polypeptide that can be found naturally occurring in the absence of a modification as described herein. A naturally occurring, wild-type nucleic acid molecule or polypeptide can be modified to differ in sequence, structure, and / or biological properties as compared to the un-modified wild-type version of the nucleic acid molecule or polypeptide.

[0036] As used herein, the term “nucleic acid molecule” may refer to a large sequence of nucleotides encoding multiple cassettes, operons, genes, and / or reading frames (e.g. a chromosomes, a plasmid, an artificial chromosome, a viral genome) but may also refer to a smaller sequence of nucleotides encoding one or a small number (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of cassettes, operons, genes and / or reading frames (e.g. a transgene or a cassette) wherein the smaller sequence of nucleic acids may be isolated or may be comprised in a larger sequence of nucleotides (e.g. . a chromosomes, a plasmid, an artificial chromosome, a viral genome). Further, as used herein, the term "nucleic acid molecule" is intended to include unmodified DNA or RNA or modified DNA or RNA. The nucleic acid molecules of the disclosure may contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus "nucleic acid molecule" embraces chemically, enzymatically, or metabolically modified forms.

[0037] As used herein, the term “exogenous” refers to an element that has been introduced into a nucleic acid molecule, such as a viral genome. An exogenous nucleic acid molecule is introduced into a genome by a method of genetic modification. An exogenous nucleic acid molecule may code for the expression of an RNA and / or a protein. An exogenous nucleic acid molecule may have been derived from the same species (homologous) or from adifferent species (heterologous). An exogenous nucleic acid molecule may comprise a homologous sequence that is altered such that it is introduced into a genome in a form that is not naturally found in the genome. For example, an exogenous nucleic acid molecule that is homologous may contain mutations or be integrated into a different region of the genome, relative to the endogenous version of the nucleic acid molecule.

[0038] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two nucleic acid (polynucleotide) or two amino acid (polypeptide) sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions / total number of positions multiplied by 100%). The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin, S. and Altschul, S., PNAS (1990), 87(6):2264-2268, modified in Karlin, S. and Altschul, S., PNAS (1993), 90(12):5873-5877. Such an algorithm is incorporated into the BLAST programs. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a given nucleic acid molecule. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score=50, wordlength=3 to obtain amino acid sequences homologous to a given polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. (1997), 25(17):3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers, E. and Miller, W., Bioinformatics (1988), 4(1): 11-17. Such an algorithm is incorporated in the ALIGN programwhich is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0039] As used herein, the term “functional inactivation” refers to any modification to a nucleic acid molecule or to a polypeptide that eliminates the activity of said nucleic acid molecule or said polypeptide. In some embodiments, a nucleic acid molecule (e.g. a gene) is functionally inactivated such that the nucleic acid molecule is incapable of expressing a functional polypeptide, such as by preventing transcription of the nucleic acid molecule into mRNA, preventing proper translation of the mRNA encoded by the nucleic acid molecule, or by causing a mutation in the nucleic acid molecule that results in translation of a non-functional polypeptide. A nucleic acid molecule (e.g. a gene) can be functionally inactivated by known methods including, but not limited to: complete deletion, truncation, frameshift mutation, or a mutation providing an early stop codon.

[0040] As used herein, a “genetically modified virus” refers to a virus that is genetically modified relative to a wild-type or parental strain of said virus. A genetically modified virus may contain modification to one or more endogenous genes or regulatory regions in its genome. A genetically modified virus may contain insertion of one or more exogenous (either homologous or heterologous) nucleic acid molecules into its genome. Exogenous nucleic acid molecules may be inserted into existing gene loci (i.e. replacing the existing gene) or be inserted into intergenic regions.

[0041] Vaccinia Virus

[0042] The present invention relates to a genetically modified vaccinia virus. Vaccinia virus is a member of the orthopoxvirus or Poxviridae family. Vaccinia is a double-stranded DNA virus with a genome of about 190 kb and encoding for approximately 250 genes, including C12L which encodes a interleukin- 18 binding protein; C2L and F3L which each encode a kel ch-like protein; NIL which encodes a BCL-2 inhibitor; N2L which encodes a TLR signaling inhibitor; MIL, B18R, and B20R which each encode an Ankyrin repeat protein; M2L, K1L and K7R which each encode aNF-KB inhibitor; K2L which encodes a serine protease inhibitor; K3L which encodes a PKR inhibitor; K4L which encodes a phospholipase-D; K5L and K6L which each encode a monoglyceride lipase; F1L which encodes a caspase-9 inhibitor; F2L whichencodes a dUTPase; B16R which encodes an IL- 1 -beta-inhibitor; B19R which encodes a interferon-alpha / beta-receptor-like secreted glycoprotein; A56R which encodes a glycoprotein; J2R which encodes the viral thymidine kinase (TK); A28L, H2R, A21L, L5R, G3L, O3L, A16L, G9R, J5L, L1R, and F9L which encode proteins of the entry / fusion complex; and A26L, A27L, D8L, H3L which encode attachment proteins. Vaccinia virus replicates only in the cytoplasm of the host cell, and the large genome codes for various enzymes and proteins needed for viral DNA replication (Greseth, M. and Traktman, P., Annu. Rev. Virol. (2022), 9:239-259).

[0043] Many strains of vaccinia virus are known in the art and may be used in the present invention. In some embodiments, the genetically modified vaccinia virus of the present invention is a genetically modified LISTER strain, MVA strain, Copenhagen strain, Wyeth strain, Western Reserve strain, or Tian Tan strain of vaccinia virus. In some embodiments, the genetically modified vaccinia virus of the present invention is a genetically modified LISTER strain of vaccinia virus.

[0044] In some embodiments, the thymidine kinase (TK) gene in the genetically modified vaccinia virus is functionally inactivated. TK may be functionally inactivated by known methods and as described herein including, but not limited to: complete deletion, truncation, frameshift mutation, or a mutation providing an early stop codon. In some embodiments, TK is functionally inactivated by replacing the TK gene with a transgene encoding a polypeptide, optionally a modified matrix protein from a vesicular stomatitis virus (VSV) or a checkpoint inhibitor such as an antibody or antigen binding fragment thereof. Functional inactivation of TK may lead to attenuation, as the vaccinia virus becomes dependent upon the activity of cellular thymidine kinase for DNA replication and, thus, viral propagation. Cellular TK is expressed at a low level in most normal tissues and at elevated levels in many highly replicating cells such as cancer cells.

[0045] In some embodiments, the C12L gene in the genetically modified vaccinia virus is functionally inactivated. C12L may be functionally inactivated by known methods and as described herein including, but not limited to: complete deletion, truncation, frameshift mutation, or a mutation providing an early stop codon. In some embodiments, C12L is functionally inactivated by replacing the C12L gene with a transgene encoding a polypeptide, optionally a modified matrix protein from a vesicular stomatitis virus (VSV) or a checkpoint inhibitor such as an antibody or antigen binding fragment thereof. The C12L gene encodes an interleukin- 18 (IL-18) binding protein that interferes with IL-18 ’s interaction with its natural receptor IL-18R. C12L is expressed in the early phase of infection to neutralize an IL-18 response.

[0046] VSV Matrix Protein

[0047] The present invention relates to a genetically modified vaccinia virus comprising a nucleic acid molecule encoding a modified matrix protein from a vesicular stomatitis virus (VSV).

[0048] VSV is a member of the Vesiculoviridae. VSV is a negative sense RNA virus with a genome encoding five proteins: nucleocapsid (N), phosphoprotein (P), matrix (M) protein, glycoprotein (G), and large (L) viral polymerase. A number of different strains of VSV are known in the art, the matrix proteins of which are suitable for use in the present invention to provide a modified matrix protein. Examples include, but are not limited to, the Indiana and New Jersey strains of VSV. The skilled person will understand that the matrix protein from other strains of VSV may be used in the present invention to provide modified matrix proteins.

[0049] The VSV matrix protein plays multiple roles in VSV infection, and is responsible for the majority of the cytopathic effects observed in VSV-infected cells (Redondo et al. PLoS One (2015), 10(6):e0131137). VSV matrix protein functions include virion assembly, budding, disorganization of the cytoskeleton, and inhibition of host gene expression. Inhibition of host gene expression by VSV matrix protein is due to inhibition of the nuclear transport of both proteins and mRNAs into and out of the host nucleus.

[0050] In a genetically modified vaccinia virus of the present invention, the matrix protein from a VSV may be modified to reduce or prevent the ability of the matrix protein to block or inhibit nuclear transport of mRNA or protein, as compared to the wild-type VSV matrix protein. In some embodiments, the matrix protein from a VSV is modified to reduce or prevent the ability of the matrix protein to block or inhibit the production of one or more cytokines in infected cells, as compared to the wild-type VSV matrix protein. While the modified matrix protein from a VSV will retain one or more cytopathic effects in cells infected with a genetically modified vaccinia virus of the present invention, the modified matrix protein from a VSV may have a decreased ability to block or inhibit the induction of antiviral immunity that results from the production of cytokines (such as interferon, e.g. interferon alpha, interferon beta, and / or interferon gamma) by infected cells. In many cancers, tumor cells exhibit a loss of inflammatory responses that may improve tumor growth and survival at the expense of increased susceptibilityto viral infection. A genetically modified vaccinia virus of the present invention may therefore employ cytopathic effects of a modified VSV matrix protein in infected cells, maintaining the ability to induce inflammatory responses in normal cells (such as interferon, e.g. interferon alpha, interferon beta, and / or interferon gamma) while preferentially infecting and killing tumor cells lacking normal inflammatory responses.

[0051] The VSV matrix protein for use in the present invention may be modified by deletion and / or substitutions of one or more amino acids. In some embodiments, the modified matrix protein from a VSV comprises one or more modifications as disclosed in WO 2004 / 085658. In some embodiments, the modified matrix protein from a VSV comprises a deletion or a substitution of the methionine at position 51. In some embodiments, the modified matrix protein from a VSV comprises an M51 deletion, M51-54 deletion, M51-54 deletion, M51-57 deletion, M51X substitution, M51R substitution, M51A substitution, M51-54A substitution, V221-S226 deletion, V221X substitution, V221F substitution, S226X substitution, S226R substitution, or combinations thereof, wherein X denotes any amino acid. In some embodiments, the modified matrix protein from a VSV comprises modifications selected from M51R and V221F substitutions; M51A and V221F substitutions; M51-54A and V221F substitutions; M51 deletion and V221F substitution; M51-54 deletion and V221F substitution; M51-57 deletion and V221F substitution; M51R and S226R substitutions; M51A and S226R substitutions; M51-54A and S226R substitutions; M51 deletion and S226R substitution; M51-54 deletion and S226R substitution; M51-57 deletion and S226R substitution; M51R, V221F and S226R substitutions; M51A, V221F and S226R substitutions; M51-54A, V221F and S226R substitutions; M51 deletion and V221F and S226R substitutions; M51-54 deletion and V221F and S226R substitutions; and M51-57 deletion and V221F and S226R substitutions. The amino acid positions above are relative to the amino acid sequence of VSV Indiana matrix protein of SEQ ID NO: 1. The skilled person will understand that the amino acid sequence of a matrix protein from other VSV strains may be differ from that of the Indiana VSV matrix protein due to the presence or absence of some amino acids resulting in slightly different numbering of corresponding amino acids. It is within the routine skill in the art to align the amino acid sequence of matrix proteins from other VSV strains with the amino acid sequence of VSV Indiana matrix proteins to identify the corresponding amino acid positions for modification as described herein. Such alignment can be readily carried out using standard techniques as known in the art and as described herein.

[0052] In some embodiments, the modified matrix protein is a modified version of the matrix protein from VSV Indiana or VSV New Jersey. The amino acid sequence of the VSV Indiana matrix protein is shown in SEQ ID NO: 1 and the amino acid sequence of the VSV New Jersey matrix protein is shown in SEQ ID NO: 2. In some embodiments, the modified matrix protein from a VSV comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 1 or 2 and optionally comprises an M51 deletion, M51-54 deletion, M51-54 deletion, M51-57 deletion, M51X substitution, M51R substitution, M51A substitution, M51-54A substitution, V221-S226 deletion, V221X substitution, V221F substitution, S226X substitution, S226R substitution, or combinations thereof, wherein X denotes any amino acid. In some embodiments, the modified matrix protein from a VSV comprises the amino acid sequence of SEQ ID NO: 3.

[0053] Fusogenic

[0054] The present invention relates to a genetically modified vaccinia virus that is fusogenic. By “fusogenic” it is meant that the virus is capable of inducing fusion of an infected cell with other cells. A genetically modified vaccinia virus of the present invention comprises one or more modifications to become fusogenic, as compared to a wild-type vaccinia virus. The fusion of infected cells (e.g. cancer cells) may improve transmissibility of the genetically modified vaccinia virus as well as the killing of infected cancer cells.

[0055] In some embodiments, the genetically modified vaccinia virus is made fusogenic by a modification as disclosed in US 2022 / 0313761. In some embodiments, the genetically modified vaccinia virus is made fusogenic by functional inactivation of one or more vaccinia virus genes. In some embodiments, the genetically modified vaccinia virus is made fusogenic by functional inactivation of the A56R gene and / or functional inactivation of the K2L gene. In some embodiments, the genetically modified vaccinia virus is made fusogenic by functional inactivation of a gene that encodes a protein that is comprised in the vaccinia virus entry / fusion complex or functional inactivation of a gene that encodes an attachment protein. The vaccinia virus entry / fusion complex comprises 11 proteins (A28L, H2R, A21L, L5R, G3L, O3L, A16L, G9R, J5L, L1R, F9L) and mediates a post-attachment step of virus entry, in addition to four attachment proteins (A26L, A27L, D8L, H3L) (Bernard Moss, Semin. Cell Dev. Biol. (2016), 60:89-96). The A16-G9 subcomplex of the entry / fusion complex interacts with the complex of A56R and K2L to prevent superinfection and cell-cell fusion.

[0056] In some embodiments, the genetically modified vaccinia virus is made fusogenic by functional inactivation of A56R and / or functional inactivation of K2L. Functional inactivation of the A56R or the K2L genes, either alone or in combination, in the vaccinia virus genome induces a fusogenic phenotype. A56R and / or K2L may be functionally inactivated by known methods and as described herein including, but not limited to: complete deletion, truncation, frameshift mutation, or a mutation providing an early stop codon.

[0057] In some embodiments, the genetically modified vaccinia virus is made fusogenic by functional inactivation of one or more of A16L, A21L, A25L, A26L, A28L, G3L, G9R, H2R, J5L, or L5R. The A16L, A21L, A25L, A26L, A28L, G3L, G9R, H2R, J5L, or L5R genes. These genes may be functionally inactivated, either alone or in combination, by known methods and as described herein including, but not limited to: complete deletion, truncation, frameshift mutation, or a mutation providing an early stop codon. Functional inactivation of one or more of A16L, A21L, A25L, A26L, A28L, G3L, G9R, H2R, J5L, or L5R. The A16L, A21L, A25L, A26L, A28L, G3L, G9R, H2R, J5L, or L5R in the genetically modified vaccinia virus may be in addition to a functional inactivation of A56R and / or a functional inactivation of K2L.

[0058] In some embodiments, the genetically modified vaccinia virus is made fusogenic by the insertion of a nucleic acid molecule encoding a fusion protein from another virus. In some embodiments, the genetically modified vaccinia virus is made fusogenic by the insertion of an exogenous nucleic acid molecule encoding measles virus H (hemagglutinin) protein (SEQ ID NO: 4) and the F (fusion) protein (SEQ ID NO: 5); reovirus p!4 FAST protein (SEQ ID NO: 6); or Newcastle disease virus F protein (SEQ ID NO: 7). In some embodiments, the genetically modified vaccinia virus comprises a nucleic acid molecule encoding an amino acid with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 4, 5, 6, or 7.

[0059] Checkpoint Inhibitors

[0060] In some embodiments, the genetically modified vaccinia virus comprises a nucleic acid molecule encoding a checkpoint inhibitor.

[0061] As used herein, a “checkpoint inhibitor” refers to a molecule that totally or partially reduces, inhibits, interferes with or modulates one or more checkpoint proteins. Checkpoint proteins regulate cell activation or function. Numerous checkpoint proteins are known, such as for example CTLA-4 and its ligands CD80 and CD86; PD-1 and its ligands PD- L1 and PD-L2; CD27; CD28; CD40; CD122; CD137; CD137 / 4-1BB; ICOS; IL-10; 0X40TGF-beta; TOR receptor; glucocorticoid-induced TNFR-related protein GITR; killer-cell immunoglobulin-like receptor (KIR); lymphocyte activation gene-3 (LAG3); V-domain Ig suppressor of T cell activation (VISTA) T-cell immunoglobulin domain and mucin domain 3 (TIM-3); and indoleamine 2,3-dioxygenase (IDO), as well as their ligands and / or receptors.

[0062] In some embodiments, the checkpoint inhibitor is an antibody or an antigen binding fragment thereof that is an inhibitor of Programmed Death-Ligand 1 (PD-L1, also known as B7-H1, CD274), Programmed Death 1 (PD-1, CD279), CTLA-4 (CD154), PD-L2 (B7-DC, CD273), LAG3 (CD223), TIM3 (HAVCR2, CD366), 41BB (CD137), 2B4, A2aR, B7H1, B7H3, B7H4, B- and T-lymphocyte attenuator (BTLA), CD2, CD27, CD28, CD30, CD33, CD40, CD70, CD80, CD86, CD160, CD226, CD276, DR3, GAL9, GITR, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulator), Killer inhibitory receptor (KIR), LAG-3, LAIR1, LIGHT, MARCO (macrophage receptor with collageneous structure), phosphatidylserine (PS), OX-40, Siglec-5, Siglec-7, Siglec-9, Siglec-11, SLAM, TIGIT, TIM3, TNF-a, VISTA, VTCN1, or any combination thereof.

[0063] In some embodiments, the immune response checkpoint inhibitor is an inhibitor of PD-L1 or PD-1. In an embodiment, the inhibitor of PD-L1 or PD-1 may be an anti -PD-1 or anti-PD-Ll antibody or an antigen binding fragment thereof, such as but not limited to those disclosed in WO 2015 / 103602. In some embodiments, the anti-PD-1 antibody or anti-PD-Ll antibody is selected from nivolumab, pembrolizumab, pidilizumab, atezolizumab, or durvalumab.

[0064] In some embodiments, the immune response checkpoint inhibitor is an inhibitor of CTLA-4. In an embodiment, the inhibitor of CTLA-4 may be an anti-CTLA-4 antibody or an antigen binding fragment thereof, such as but not limited to ipilimumab and tremelimumab.

[0065] An “antibody” refers to a polypeptide or protein that consists of or comprises antibody domains, which are understood as constant and / or variable domains of the heavy and / or light chains of immunoglobulins, with or without a linker sequence. The term “antibody” refers to an intact antibody. In an embodiment, an “antibody” may comprise a full-length immunoglobulin molecule, including e.g. polyclonal, monoclonal, chimeric, humanized and / or human versions having full length heavy and / or light chains. The term “antibody” encompasses any and all isotypes and subclasses, including without limitation the major classes of IgA, IgD, IgE, IgG and IgM, and the subclasses IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The term“chimeric antibody” as used herein refers to a recombinant protein that contains the variable domains (including the complementarity determining regions (CDRs)) of an antibody derived from one species, such for example a rodent, while the constant domains of the antibody are derived from a different species, such as a human. A “humanized antibody” as used herein refers to a recombinant protein in which the CDRs from an antibody from one species (e.g. a rodent) are transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains, including human framework region (FR) sequences. The constant domains of the humanized antibody are likewise derived from a human antibody. As used herein, the term “antigen binding fragment” or “functional fragment”, used interchangeably, with respect to an antibody, refers to an antigen binding portion of an antibody. In this context, by “functional” it is meant that the fragment maintains its ability to bind to the target antigen. The binding affinity of the antigen binding fragment may be equivalent to, or greater than, that of parent antibody, or may be less than the parent antibody, but nevertheless the antigen binding fragment maintains a specificity and / or selectivity for the target antigen. Antigen binding fragments of antibodies include, without limitation, a portion of an antibody such as a F(ab')2, a F(ab)2, a Fab', a Fab, a Fab2, a Faba. a single domain antibody. Regardless of structure, an antigen binding fragment of an antibody binds with the same antigen that is recognized by the full antibody. The term “antigen binding fragment” or “functional fragment”, used interchangeably, in relation to antibodies, also includes isolated fragments consisting of the variable regions, such as the “Fv” fragments consisting of the variable regions of the heavy and light chains and recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker (“scFv proteins”). As used herein, the term “antigen binding fragment” or "functional fragment" does not include fragments such as Fc fragments that do not contain antigen binding site.

[0066] Pharmaceutical Compositions

[0067] In some embodiments, a genetically modified vaccinia virus of the present invention is comprised in a pharmaceutical composition.

[0068] Pharmaceutical compositions comprising the genetically modified vaccinia virus of the present invention can be prepared using methods known in the art using physiologically acceptable carriers, excipients, and / or or stabilizers (Remington's Pharmaceutical Sciences 23rd Edition, Adejare, A. Ed. (2020) and in a desired form such as a solution, an emulsion, an aerosol, a capsule, a tablet, or a lyophilized formulation.

[0069] As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical substances is well known in the art. Supplementary active ingredients can also be incorporated into the pharmaceutical compositions. The phrase “pharmaceutically acceptable” refers to carriers and compositions that do not result in unnecessary allergy or toxicity when administered to a subject.

[0070] As used herein, an “excipient” is a molecule or substance that is included in a composition to improve the non-therapeutic properties of a composition such as its stability, pH, tonicity, shelf-life, color, melting point, viscosity, or other non-therapeutic properties. Many pharmaceutical excipients are known in the art and may be used in compositions of the present invention (Pramanick et al., Pharma Times (2013), 45(3):65-77). In some embodiments, the excipient comprises a bulking agent, a cytoprotectant, a buffering agent, a tonicity agent, a preservative, a surfactant, an antioxidant, or a polymer.

[0071] For parenteral administration in an aqueous solution, for example, the solution may be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. Aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral, and intraperitoneal administration. Sterile aqueous solutions that can be employed will be known to those of skill in the art.

[0072] Treatment

[0073] The present invention relates to genetically modified vaccinia virus that exhibits oncolytic activity useful for the treatment of cancer in a subject.

[0074] “Treating” or “treatment of’ as used herein, refers to an approach for obtaining beneficial or desired results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilisation of the state of disease, prevention of development of disease, prevention of spread of disease, delay or slowing of disease progression, delay or slowing of disease onset, and amelioration or palliation of the disease state. “Treating” can also mean prolonging survival of a patient beyond that expected in the absence of treatment and can also mean inhibiting the progression of disease temporarily or preventing the occurrence of disease. “Treating” may also refer to a reduction in the size of a tumor mass, reduction in tumor aggressiveness, etc.

[0075] As used herein, a "therapeutically effective amount" means an amount of the pharmaceutical composition or genetically modified vaccinia virus effective to provide a therapeutic benefit to a subject. In some embodiments, a therapeutically effective amount of the composition is an amount capable of inducing a clinical response in a subject in the treatment of a particular disease or disorder such as cancer. Determination of a therapeutically effective amount of the composition is well within the capability of those skilled in the art. The therapeutically effective amount may vary according to a variety of factors such as the subject’s condition, weight, sex and age.

[0076] As used herein, the terms “cancer”, “cancer cells”, “tumor” and “tumor cells” may be used interchangeably to refer to cells or masses of cells that exhibit abnormal growth, characterized by a significant loss of control of cell proliferation or cells that have been immortalized. The term “cancer” or “tumor” includes metastatic as well as non-metastatic cancer or tumors. As used herein, a “solid tumor” refers to a tumor that does not contain cysts of liquid areas. A cancer may be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor.

[0077] As used herein, the term “oncolytic,” refers to the capacity of virus, such as a genetically modified vaccinia virus of the present invention, to target cancer cells, inhibit cancer cell growth, destroy cancer cells and / or aid in the destruction of cancer cells by the immune system. As used herein "oncolytic” activity refers to inhibition or suppression of tumor and / or malignant and / or cancerous cell growth; regression of tumor and / or malignant and / or cancerous cell growth; cell death of tumor and / or malignant and / or cancerous cells or prevention of the occurrence of additional tumor and / or malignant and / or cancerous cells. As used herein, "inhibiting or suppressing tumor growth" refers to reducing the rate of growth of a tumor, halting tumor growth completely, causing a regression in the size of an existing tumor, eradicating an existing tumor and / or preventing the occurrence of additional tumors.

[0078] The genetically modified vaccinia virus of the present invention may be used for treating cancer in a subject and / or in the preparation of a medicament for treating cancer in a subject. In some embodiments, the cancer comprises one or more solid tumors. In some embodiments, the cancer is selected from ovarian cancer, renal cancer, lung cancer, pancreatic cancer, skin cancer, stomach cancer, liver cancer, hepatic cell cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, uterine cancer, breast cancer, cervical cancer, ovariancancer, bladder cancer, prostate cancer, testicular cancer, head and neck cancer, brain cancer, thymic cancer, sarcoma, melanoma, and bone cancer.

[0079] In some embodiments, the cancer is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumors, burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube cancer, fibrous histiocytoma of bone, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brain stem glioma, hairy cell leukemia, hepatocellular cancer, langerhans cell histiocytosis, hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, wilms tumor and other childhood kidney tumors, langerhans cell histiocytosis, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, multiple endocrine neoplasia syndromes, multiple myeloma / plasma cell neoplasm, myelodysplastic syndromes, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, kaposi sarcoma, rhabdomyosarcoma, sezary syndrome, small intestine cancer, soft tissue sarcoma, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenstrom macroglobulinemia.

[0080] The genetically modified vaccinia virus of the present invention may be delivered directly or in pharmaceutical compositions containing carriers, excipients, and / or stabilizers, as is known in the art. The genetically modified vaccinia virus or pharmaceutical composition of the present invention may be administered to a subject by conventional techniques, such as intravenously, intramuscularly, intraperitoneally, intra-cerebrally, subcutaneously, intraarticularly, intrasynovially, intrathecally, transdermally, intranasally, inhalation, or intra- tumorally. In some embodiments, the genetically modified vaccinia virus or pharmaceutical composition of the present invention is administered adjacent to a tumor or directly into a tumor, optionally by injection.

[0081] Intratumoral injection, or injection directly into the tumor vasculature may be provided for discrete, solid, accessible tumors. Local, regional or systemic administration also may be used to expose tumor cells to the genetically modified vaccinia virus. The genetically modified vaccinia virus may be administered in multiple injections to the tumor or adjacent to the tumor. Continuous administration also may be applied where appropriate, for example, by implanting a catheter into a tumor or into tumor vasculature. Generally, the dose of the genetically modified vaccinia virus or pharmaceutical composition via continuous perfusion may be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs.

[0082] In some embodiments, the genetically modified vaccinia virus is formulated for administration at a dose of from about 1x105to about 5x109plaque forming units (PFU) per administration. In some embodiments, the dose is from about IxlO7to about 5xl09PFU per administration. In some embodiments, the dose is about 9x108PFU per administration. In some embodiments, a dose of the genetically modified vaccinia virus is administered in up to 9 doses, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9 doses, by means of administration known in the art or as described herein, such as but not limited to intratumoral injection or injection adjacent to a tumor.

[0083] As used herein, the term “comprising” and its derivatives are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence ofthe stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps.

[0084] Particular embodiments of the disclosure include, without limitation, the following:1. A genetically modified vaccinia virus, comprising a nucleic acid molecule encoding a modified matrix protein from a vesicular stomatitis virus (VSV), wherein the genetically modified vaccinia virus is fusogenic.2. The genetically modified vaccinia virus of embodiment 1, wherein the modified matrix protein from a VSV exhibits reduced inhibition of nuclear export of mRNA or protein compared to a wild-type matrix protein from a VSV.3. The genetically modified vaccinia virus of embodiment 1 or 2, wherein the modified matrix protein from a VSV exhibits reduced inhibition of the production of one or more cytokines in an infected cell compared to a wild-type matrix protein from a VSV.4. The genetically modified vaccinia virus of embodiment 3, wherein the one or more cytokines comprises an interferon, optionally interferon alpha, interferon beta, and / or interferon gamma.5. The genetically modified vaccinia virus of any one of embodiments 1 to 4, wherein the modified matrix protein from a vesicular stomatitis virus comprises an M51 deletion.6. The genetically modified vaccinia virus of any one of embodiments 1 to 4, wherein the modified matrix protein from a vesicular stomatitis virus comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 1 or 2 and comprises an M51 deletion.7. The genetically modified vaccinia virus of embodiment 5 or 6, wherein the modified matrix protein from a vesicular stomatitis virus further comprises an M51-54 deletion or an M51-57 deletion.8. The genetically modified vaccinia virus of any one of embodiments 5 to 7, wherein the modified matrix protein from a vesicular stomatitis virus further comprises a V221-S226 deletion or further comprises a V221 substitution and / or a S226 substitution.9. The genetically modified vaccinia virus of embodiment 8, wherein the V221 substitution is V221F and / or wherein the S226 substitution is S226R.10. The genetically modified vaccinia virus of any one of embodiments 1 to 4, wherein the modified matrix protein from a vesicular stomatitis virus comprises the amino acid sequence of SEQ ID NO: 3.11. The genetically modified vaccinia virus of any one of embodiments 1 to 10, wherein the A56R gene in the genetically modified vaccinia virus is functionally inactivated.12. The genetically modified vaccinia virus of any one of embodiments 1 to 11, wherein the K2L gene in the genetically modified vaccinia virus is functionally inactivated.13. The genetically modified vaccinia virus of any one of embodiments 1 to 12, wherein one or more of the A16L, A21L, A25L, A26L, A28L, G3L, G9R, H2R, J5L, or L5R. The A16L, A21L, A25L, A26L, A28L, G3L, G9R, H2R, J5L, and / or L5R gene in the genetically modified vaccinia virus is functionally inactivated.14. The genetically modified vaccinia virus of any one of embodiments 1 to 13, comprising a nucleic acid molecule encoding measles virus H protein (SEQ ID NO: 4) and measles virus F protein (SEQ ID NO: 5), reovirus p!4 FAST protein (SEQ ID NO: 6), or Newcastle disease virus F protein (SEQ ID NO: 7).15. The genetically modified vaccinia virus of any one of embodiments 1 to 14, wherein the thymidine kinase (TK) gene in the genetically modified vaccinia virus is functionally inactivated.16. The genetically modified vaccinia virus of any one of embodiments 1 to 15, wherein the C12L gene in the genetically modified vaccinia virus is functionally inactivated.17. The genetically modified vaccinia virus of any one of embodiments 1 to 16, comprising a nucleic acid molecule encoding a checkpoint inhibitor.18. The genetically modified vaccinia virus of embodiment 17, wherein the checkpoint inhibitor is an antibody or an antigen binding fragment thereof.19. The genetically modified vaccinia virus of embodiment 18, wherein the antibody or antigen binding fragment thereof binds to PD-1, PD-L1, or CTLA-4.20. The genetically modified vaccinia virus of embodiment 18, wherein the checkpoint inhibitor is atezolizumab and / or nivolumab.21. The genetically modified vaccinia virus of any one of embodiments 1 to 20, which is a genetically modified LISTER strain, MVA strain, Copenhagen strain, Wyeth strain, Western Reserve strain, or Tian Tan strain of vaccinia virus.22. The genetically modified vaccinia virus of any one of embodiments 1 to 21, which is a genetically modified LISTER strain of vaccinia virus.23. A pharmaceutical composition comprising the genetically modified vaccinia virus of any one of embodiments 1 to 22 and a pharmaceutically acceptable carrier.24. A method of treating cancer in a subject, comprising administering the genetically modified vaccinia virus of any one of embodiments 1 to 22 or the pharmaceutical composition of embodiment 23 to the subject.25. Use of the genetically modified vaccinia virus of any one of embodiments 1 to 22 or the pharmaceutical composition of embodiment 23 for treating cancer in a subject.26. Use of the genetically modified vaccinia virus of any one of embodiments 1 to 22 in the preparation of a medicament for treating cancer in a subject.27. The method of embodiment 24 or the use of embodiment 25 or 26, wherein the cancer comprises a solid tumor.28. The method of embodiment 24 or the use of embodiment 25 or 26, wherein the cancer is selected from ovarian cancer, lung cancer, pancreatic cancer, skin cancer, stomach cancer, liver cancer, hepatic cell cancer, colorectal cancer, esophageal cancer, uterine cancer, breast cancer, cervical cancer, ovarian cancer, bladder cancer, prostate cancer, testicular cancer, head and neck cancer, brain cancer, thymic cancer, lymphoma, leukemia, and bone cancer / osteo sarcoma.

[0085] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude thepresence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps.

[0086] All publications and patents cited herein are incorporated by reference in their entirety as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In the case of any conflict between a definition of a term in the present disclosure and a definition in a cited publication or patent, the definition provided in the present disclosure is to be used in describing the present invention.

[0087] The present invention will now be described by way of non-limiting examples having regard to the appended drawings.

[0088] EXAMPLE 1

[0089] Modifications

[0090] Genetically modified vaccinia virus was derived from vaccinia Lister strainATCC-VR-1549. The experimental vaccinia viruses produced are summarized below in Table 1.

[0091] Table 1

[0092] Wild-type vaccinia virus Lister strain was amplified first on human sarcoma cell line U2-OS (ATCC HTB-96). A thymidine kinase (TK, also known as J2R) deleted recombinant Lister virus, Pl 600, was generated by recombination of the WT virus with pMA-dTK-mCherry (FIG. 1, SEQ ID NO: 8) to insert an mCherry transgene at the TK locus.

[0093] Pl 602 was constructed by recombination of Pl 600 with the vector pSC65- deltaTK-delta51M-eGFP (FIG. 2, SEQ ID NO: 9) to insert a transgene encoding a modified matrix (M) protein of VSV Indiana with M51 deletion (dM51; SEQ ID NO: 3) and an eGFP transgene at the TK locus.

[0094] Pl 605 was constructed by inactivating the A56R gene in Pl 600 by insertion of a‘g’ nucleotide at position 73, causing a frameshift mutation that provided two sequential stop codons at position 82.

[0095] P1607 was constructed by recombination of P1602 with the vector pUC57- deltaTK-Nivo-Atezo (FIG. 3, SEQ ID NO: 10) comprising sequences encoding the light and heavy chains of two antibodies, nivolumab (anti-PD-1, SEQ ID NOs: 13 and 14) and atezolizumab (anti-PD-Ll, SEQ ID NOs: 15 and 16), to insert the antibody transgenes into the TK locus.

[0096] P1615 was constructed by recombination of P1607 with the vector pUC57-LS- deltaC12-delta51M (FIG. 4, SEQ ID NO: 11) to insert a transgene encoding a modified matrix (M) protein of VSV Indiana with M51 deletion (dM51; SEQ ID NO: 3) at the C12L locus.

[0097] P1616 was constructed by a first recombination of P1605 with the vector pUC57- deltaTK-atezo (FIG. 5, SEQ ID NO: 12) comprising sequences encoding the light and heavy chains of atezolizumab (anti-PD-Ll, SEQ ID NOs: 15 and 16) to insert the antibody transgenes into the TK locus, and then a second recombination of the resulting mutant with the vectorpUC57-LS-deltaC12-delta51M (FIG. 4, SEQ ID NO: 11) to insert a transgene encoding a modified matrix (M) protein of VSV Indiana with M51 deletion (dM51; SEQ ID NO: 3) at the C12L locus.

[0098] Imaging of in vitro infection

[0099] The cytotoxicity of the modified viruses was tested by in vitro infection of human sarcoma cells (U2OS) and murine colorectal cancer cells (CT26LacZ). 8X105U2OS and CT26LacZ cells were plated in 6-well plate and allowed to attach overnight. A sufficient amount of virus to achieve MOI (multiplicity of infection) of 0.1 was diluted in ImL of DMEM medium. The cell culture medium was replaced with the viral infection medium for 2 hours before being replaced with regular growth medium. Non-infected control (NIC) cells were kept in culture medium. FIG. 6 shows images taken 3 days post infection.

[0100] FIG. 6A: NIC cells remained healthy and confluent.

[0101] FIG. 6B: P1600 virus induced noticeable death of U2OS cells, but CT26LacZ cells remained confluent and viable.

[0102] FIG. 6C: P1602 virus induced greater death of U2OS cells compared to P1600, but CT26LacZ cells remained confluent and viable.

[0103] FIG. 6D: P1605 virus caused near-complete death of U2OS, with some noticeable death of CT26LacZ cells.

[0104] FIG. 6E: P1607 virus caused similar death of U2OS cells at P1600, with some noticeable death of CT26LacZ cells.

[0105] FIG. 6F: P1615 virus caused increased death of U2OS cells and CT26LacZ cells.

[0106] FIG. 6G: P1616 virus caused increased death of U2OS cells and CT26LacZ cells.

[0107] FIG. 6H: Infection of U2OS and CT26LacZ cells with control, unmodified strains of vaccinia: Western Reserve, Lister, Wyeth, and Copenhagen strains.

[0108] Cell death assay

[0109] The cytotoxicity of the modified viruses was tested by in vitro infection of U2OS cells. U2OS cells were diluted to 3X105 / mL, and lOOpL of the diluted cells were plated in each well of 96-well flat bottom plates using muti-channel pipet and sterile reservoirs. Afterincubating at 37°C, 5% CO2 for 4 hours to let cells attach, viral dilutions series were prepared in 50pL and added to columns of the 96-well plates. Columns 2 & 7 MOI 1.0; columns 3 & 8 MOI 0.1; columns 4 & 9 MOI 0.01; columns 5 & 10 MOI 0.005. Rows A and H, columns 1, 6, 11 and 12 were left as non infected control (NIC). At 3 days post infection, culture medium was aspirated, wells were washed with 200 pL PBS, and then 150 pL 0.1% Crystal Violet (CV) in 80% Methanol was added to each well. After incubation at room temperature for 10-15 minutes to stain and fix the cells, the wells were rinsed with water and dried before imaging.

[0110] FIG. 7 A illustrates the NIC and MOI of the wells in each plate. As shown in FIG. 7B, Pl 605 and P1616 virus achieved the greatest killing of U2OS cells at low MOI.

[0111] In vivo tumor control

[0112] The modified viruses were tested for their ability to control tumor growth in vivo. Female BALB / c mice (8 weeks old) were inoculated with 1X106CT26LacZ cancer cells (in lOOpL) in the right flank to form tumors. Modified viruses were administered on Days 11, 13 and 16 post inoculation with 5X107PFU of virus, administered in 100 pL intratumorally. Tumor size measurement was performed 3 times per week. Mice were sacrificed if tumor size reached 2000mm3.

[0113] As shown in FIG. 8A, the P1616 virus according to the present invention, comprising both a modified matrix protein from a VSV and exhibiting a fusogenic phenotype, was significantly more effective at inhibiting tumor growth compared to vaccinia viruses comprising only a modified matrix protein from a VSV (Pl 602) or only exhibiting a fusogenic phenotype (Pl 605). This synergistic effect resulted in an unexpectedly improved control of tumor growth.

Claims

CLAIMS:

1. A genetically modified vaccinia virus, comprising a nucleic acid molecule encoding a modified matrix protein from a vesicular stomatitis virus, wherein the genetically modified vaccinia virus is fusogenic.

2. The genetically modified vaccinia virus of claim 1, wherein the modified matrix protein from a vesicular stomatitis virus exhibits reduced inhibition of nuclear export of mRNA or protein compared to a wild-type matrix protein from a vesicular stomatitis virus.

3. The genetically modified vaccinia virus of claim 1 or 2, wherein the modified matrix protein from a vesicular stomatitis virus exhibits reduced inhibition of the production of one or more cytokines in an infected cell compared to a wild-type matrix protein from a vesicular stomatitis virus.

4. The genetically modified vaccinia virus of claim 3, wherein the one or more cytokines comprises an interferon, optionally interferon alpha, interferon beta, and / or interferon gamma.

5. The genetically modified vaccinia virus of any one of claims 1 to 4, wherein the modified matrix protein from a vesicular stomatitis virus comprises an M51 deletion.

6. The genetically modified vaccinia virus of any one of claims 1 to 4, wherein the modified matrix protein from a vesicular stomatitis virus comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 1 or 2 and comprises an M51 deletion.

7. The genetically modified vaccinia virus of claim 5 or 6, wherein the modified matrix protein from a vesicular stomatitis virus further comprises an M51-54 deletion or an M51-57 deletion.

8. The genetically modified vaccinia virus of any one of claims 5 to 7, wherein the modified matrix protein from a vesicular stomatitis virus further comprises a V221-S226 deletion or further comprises a V221 substitution and / or a S226 substitution.

9. The genetically modified vaccinia virus of claim 8, wherein the V221 substitution is V221F and / or wherein the S226 substitution is S226R.

10. The genetically modified vaccinia virus of any one of claims 1 to 4, wherein the modified matrix protein from a vesicular stomatitis virus comprises the amino acid sequence of SEQ ID NO: 3.

11. The genetically modified vaccinia virus of any one of claims 1 to 10, wherein the A56R gene in the genetically modified vaccinia virus is functionally inactivated.

12. The genetically modified vaccinia virus of any one of claims 1 to 11, wherein the K2L gene in the genetically modified vaccinia virus is functionally inactivated.

13. The genetically modified vaccinia virus of any one of claims 1 to 12, wherein one or more of the A16L, A21L, A25L, A26L, A28L, G3L, G9R, H2R, J5L, or L5R. The A16L, A21L, A25L, A26L, A28L, G3L, G9R, H2R, J5L, and / or L5R gene in the genetically modified vaccinia virus is functionally inactivated.

14. The genetically modified vaccinia virus of any one of claims 1 to 13, comprising a nucleic acid molecule encoding measles virus H protein (SEQ ID NO: 4) and measles virus F protein (SEQ ID NO: 5); reovirus p!4 FAST protein (SEQ ID NO: 6); or Newcastle disease virus F protein (SEQ ID NO: 7).

15. The genetically modified vaccinia virus of any one of claims 1 to 14, wherein the thymidine kinase (TK) gene in the genetically modified vaccinia virus is functionally inactivated.

16. The genetically modified vaccinia virus of any one of claims 1 to 15, wherein the C12L gene in the genetically modified vaccinia virus is functionally inactivated.

17. The genetically modified vaccinia virus of any one of claims 1 to 16, further comprising a nucleic acid molecule encoding a checkpoint inhibitor.

18. The genetically modified vaccinia virus of claim 17, wherein the checkpoint inhibitor is an antibody or an antigen binding fragment thereof.

19. The genetically modified vaccinia virus of claim 18, wherein the antibody or antigen binding fragment thereof binds to PD-1, PD-L1, or CTLA-4.

20. The genetically modified vaccinia virus of claim 18, wherein the checkpoint inhibitor is atezolizumab and / or ni vol umab.

21. The genetically modified vaccinia virus of any one of claims 1 to 20, which is a genetically modified LISTER strain, MVA strain, Copenhagen strain, Wyeth strain, Western Reserve strain, or Tian Tan strain of vaccinia virus.

22. The genetically modified vaccinia virus of any one of claims 1 to 21, which is a genetically modified LISTER strain of vaccinia virus.

23. A pharmaceutical composition comprising the genetically modified vaccinia virus of any one of claims 1 to 22 and a pharmaceutically acceptable carrier.

24. A method of treating cancer in a subject, comprising administering the genetically modified vaccinia virus of any one of claims 1 to 22 or the pharmaceutical composition of claim 23 to the subject.

25. Use of the genetically modified vaccinia virus of any one of claims 1 to 22 or the pharmaceutical composition of claim 23 for treating cancer in a subject.

26. Use of the genetically modified vaccinia virus of any one of claims 1 to 22 in the preparation of a medicament for treating cancer in a subject.

27. The method of claim 24 or the use of claim 25 or 26, wherein the cancer comprises a solid tumor.

28. The method of claim 24 or the use of claim 25 or 26, wherein the cancer is selected from ovarian cancer, lung cancer, pancreatic cancer, skin cancer, stomach cancer, liver cancer, hepatic cell cancer, colorectal cancer, esophageal cancer, uterine cancer, breast cancer, cervical cancer, ovarian cancer, bladder cancer, prostate cancer, testicular cancer, head and neck cancer, brain cancer, thymic cancer, lymphoma, leukemia, and bone cancer / osteo sarcoma.

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