Modified extracellular enveloped virus

Abstract

The present disclosure provides modified oncolytic poxviruses (e.g., vaccinia viruses) that can contain modifications in the viral genome that enhance the production of extracellular enveloped forms of the virus. The modified oncolytic poxviruses can be used as vectors for systemic delivery. Methods for using the modified oncolytic poxviruses are also provided. For example, the present invention provides a modified oncolytic poxvirus that contains a nucleic acid encoding an A34R protein or a fragment thereof that contains at least two mutations, wherein the at least two mutations are at positions corresponding to Lys119 and Lys151 of the wild-type vaccinia virus A43R protein (SEQ ID NO: 4).

Description

[Technical field]

[0001] cross reference This application claims priority to U.S. Provisional Application No. 62 / 916,035, filed October 16, 2019, which is incorporated by reference in its entirety. Incorporation by Reference

[0002] All publications, patents, patent applications, and NCBI accession numbers mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference and was set forth in its entirety. In the event of a discrepancy between a term described herein and a term defined in an incorporated reference, the definition in this disclosure shall prevail. Summary of the Invention [Means for solving the problem]

[0003] Abstract One embodiment provides a modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising at least two mutations, wherein the at least two mutations are at positions corresponding to positions Lys119 and Lys151 of a wild-type vaccinia virus A43R protein (SEQ ID NO: 4). In some embodiments, the mutation at the position corresponding to position Lys119 is Lys119Glu. In some embodiments, the mutation at the position corresponding to position Lys151 is Lys151Glu. In some embodiments, the at least two mutations at the positions corresponding to positions Lys119 and Lys151 of a wild-type vaccinia virus A43R protein (SEQ ID NO: 4) are Lys119Glu and Lys151Glu, respectively.

[0004] One embodiment provides a modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising at least two non-naturally occurring mutations in amino acid residues that are positively charged at pH 5 in the wild-type A34R protein (SEQ ID NO: 4).

[0005] Another embodiment provides a modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising at least two non-naturally occurring mutations not at position 110 of the wild-type A34R protein (SEQ ID NO: 4).

[0006] Another embodiment provides a modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising at least two non-naturally occurring mutations at no aspartic acid residue in the wild-type A34R protein (SEQ ID NO: 4).

[0007] Another embodiment provides a modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof, comprising at least two non-naturally occurring mutations, independently, in alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residues in said wild-type A34R protein (SEQ ID NO: 4).

[0008] Another embodiment provides a modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising a non-naturally occurring mutation in a lysine residue at a position other than Lys151 of the wild-type A34R protein (SEQ ID NO: 4).

[0009] Another embodiment provides a modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof, comprising a non-naturally occurring mutation at position Lys119 of said wild-type A34R protein (SEQ ID NO: 4).

[0010] Another embodiment provides a modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising at least two non-naturally occurring mutations in amino acid residues that are positively charged at pH 5 in said wild-type A34R protein (SEQ ID NO: 4), wherein said modified oncolytic poxvirus produces an increased number of comet tail-shaped plaques in a viral plaque formation assay compared to an otherwise identical oncolytic virus that does not comprise said at least two non-naturally occurring mutations.

[0011] Another embodiment provides a modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising at least two non-naturally occurring mutations, wherein if any of the non-naturally occurring mutations is at position 110 in the wild-type A34R protein (SEQ ID NO: 4), the encoded amino acid is not an asparagine residue.

[0012] Another embodiment provides a modified oncolytic poxvirus that exhibits increased resistance to neutralizing antibodies compared to a wild-type strain of oncolytic poxvirus, wherein said increased resistance is measured by the number of plaques produced by said modified oncolytic poxvirus or said wild-type strain in a viral plaque assay following treatment with an anti-L1 NR-45114 antibody or an anti-VIG antibody, and wherein said modified oncolytic poxvirus produces at least about 55,000 plaque forming units / mL.

[0013] Another embodiment provides a modified oncolytic poxvirus that produces at least about 55,000 plaque forming units / mL in a viral plaque assay following treatment with a neutralizing antibody.

[0014] In some embodiments, the neutralizing antibody is an anti-L1 NR-45114 antibody or an anti-VIG antibody. In some embodiments, the A34R protein or fragment thereof further comprises a mutation at position Lys151 of the wild-type A34R protein (SEQ ID NO: 4). In some embodiments, the amino acid residue that is positively charged at pH 5 is a lysine residue. In some embodiments, the nucleic acid comprises a nucleotide sequence or a fragment thereof that is at least about 80% homologous to the coding sequence in the viral gene VACWR157. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least about 80% homologous to the nucleotide sequence set forth as SEQ ID NO: 3. In some embodiments, at least one of the two non-naturally occurring mutations is at position Lys119 of the wild-type A34R protein (SEQ ID NO: 4). In some embodiments, the non-naturally occurring mutation is at position Lys119 of the wild-type A34R protein (SEQ ID NO: 4). In some embodiments, the mutation at position Lys119 of the wild-type A34R protein (SEQ ID NO: 4) is Lys119Glu. In some embodiments, at least one of the two non-naturally occurring mutations is at position Lys151 of the wild-type A34R protein (SEQ ID NO: 4). In some embodiments, the mutation at position Lys151 of the wild-type A34R protein (SEQ ID NO: 4) is Lys151Glu.

[0015] One embodiment provides a modified oncolytic poxvirus expressing an A34R protein containing the mutations Lys119Glu and Lys151Glu.

[0016] In some embodiments, positions 305-307 of SEQ ID NO:3 comprise the nucleotides GAA or GAG. In some embodiments, positions 451-453 of SEQ ID NO:3 comprise the nucleotides GAA or GAG. In some embodiments, the modified oncolytic poxvirus produces a greater amount of extracellular enveloped virus forms than intracellular mature virus forms compared to an otherwise identical oncolytic virus that does not comprise the at least two non-naturally occurring mutations. In some embodiments, the modified oncolytic poxvirus produces a greater amount of extracellular enveloped virus forms than intracellular mature virus forms compared to an otherwise identical oncolytic virus that does not comprise the non-naturally occurring mutations. In some embodiments, the modified oncolytic poxvirus further encodes an exogenous nucleic acid encoding at least one therapeutic or diagnostic protein. In some embodiments, the exogenous nucleic acid may encode at least one of a chemokine receptor, a membrane-associated protein, a microbial protein capable of degrading hyaluronan, a microbial protein, SOCS3, PH-20, HMGB1, PIAS3, IL15, IL15-Rα, LIGHT, ITAC, fractalkine, CCL5, N1L, an immune checkpoint modulator, a metabolic regulatory protein, or any combination thereof, such as a fusion protein comprising any combination of the above (e.g., a metabolic regulatory protein and a cytokine). In some embodiments, the exogenous nucleic acid encodes a chemokine receptor, wherein the chemokine receptor comprises at least one of CXCR4 and CCR2. In some embodiments, the exogenous nucleic acid encodes the membrane-associated protein. In some embodiments, the membrane-associated protein comprises a membrane-associated hyaluronidase. In some embodiments, the membrane-associated hyaluronidase comprises PH-20. In some embodiments, the PH-20 is GPI-anchored.In some embodiments, the exogenous nucleic acid encodes a microbial protein capable of degrading hyaluronan, wherein the microbial protein comprises a secreted hyaluronidase. In some embodiments, the secreted hyaluronidase comprises at least one of HysA, lin, sko, and rv, or any combination thereof. In some embodiments, the exogenous nucleic acid encodes a microbial protein. In some embodiments, the microbial protein comprises HysA. In some embodiments, the modified oncolytic poxvirus further comprises a modification in the genome of the virus, wherein the modification comprises a mutation or deletion of the B5R gene. In some embodiments, the modification in the genome of the virus, wherein the modification comprises a mutation or deletion in an SCR region of the B5R gene, wherein the SCR region comprises SCR1, SCR3, SCR4, or any combination thereof, and wherein the SCR region does not comprise SCR2. In some embodiments, the modified oncolytic poxvirus comprises a thymidine kinase (TK), B8R, B18R, B15R, K7R, C6L, K4L, F8L, F9L, F10L, F17R, E1L, E4L, E6R, E8R, E10R, E11L, O2L, I1L, I2L, I3L, I5L, I7L, I8R, G1L, G3L, G4L, G5.5R, G7L, G9R, L1R, L3L, L4R, L5R, J1R, J4R, J6R, H1L, H2R, H3L, H4L, H5R, H6R, In some embodiments, the modified oncolytic poxvirus further comprises a mutation or deletion in a viral gene selected from the group consisting of D1R, D2L, D3R, D6R, D7R, D8L, D11L, D12L, D13L, A2.5L, A3L, A4L, A5R, A6L, A7L, A9L, A10L, A13L, A14L, A15L, A16L, A17L, A18R, A21L, A24R, A25L, A26L, A27L, A28L, A29L, A30L, A31R, A34R, A42R, A45R, A46R, A52R, and any combination thereof. ...In some embodiments, the modified oncolytic poxvirus comprises: (i) the exogenous nucleic acid encoding a chemokine receptor, wherein the chemokine receptor comprises at least one of CXCR4 and CCR2; (ii) the exogenous nucleic acid encoding PIAS3; (iii) a mutation or deletion in the thymidine kinase gene; and (iv) a mutation or deletion in the A52R gene. In some embodiments, the virus is suitable for systemic delivery. In some embodiments, the virus is capable of immune evasion. In some embodiments, the systemic delivery comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof. In some embodiments, the parenteral administration comprises intravenous injection. In some embodiments, the virus is suitable for intratumoral delivery. In some embodiments, the poxvirus is a vaccinia virus.

[0017] One embodiment provides a process for engineering an oncolytic poxvirus comprising: (i) obtaining an oncolytic poxvirus DNA backbone vector, the oncolytic poxvirus DNA backbone vector comprising one or more modifications according to any one of the preceding claims; (ii) further modifying the oncolytic virus DNA vector to generate an engineered DNA vector; (iii) transfecting a mammalian cell with the engineered DNA vector; (iv) culturing the mammalian cell under conditions suitable for viral replication; and (v) recovering the viral particles.

[0018] In some embodiments, the mammalian cells comprise HeLa cells, 293 cells, A549 cells, or Vero cells.

[0019] One embodiment provides a kit comprising the oncolytic poxvirus, a container; and instructions for administering the oncolytic virus to a subject to treat a disorder associated with pathological angiogenesis.

[0020] One embodiment provides a method of treating a tumor, the method comprising administering to a subject a therapeutically effective amount of the oncolytic poxvirus according to.

[0021] One embodiment provides a method of treating a tumor, the method comprising administering to a subject a composition comprising white blood cells from a patient infected with a modified oncolytic poxvirus expressing an A34R protein comprising mutations at positions 119 and 151 of the wild-type A34 protein (SEQ ID NO: 4), wherein the modified oncolytic poxvirus generates a population of viral particles in a tumor microenvironment. In some embodiments, the white blood cells from the patient comprise macrophages. In some embodiments, the white blood cells from the patient comprise tumor-targeting T cells. In some embodiments, at least about 10% to at least about 90% of the population of viral particles are EEV particles as measured by a viral plaque assay. In some embodiments, the method further comprises recovering the EEV particles from the tumor microenvironment and administering the EEV particles intravenously to the subject. In some embodiments, the modified oncolytic poxvirus is a modified oncolytic vaccinia virus.

[0022] One embodiment provides a process comprising infecting a culture of host cells with a population of a modified oncolytic poxvirus comprising at least about 10% to at least about 90% EEV particles, wherein said modified oncolytic poxvirus expresses an A34R protein comprising mutations at positions 119 and 151 of said wild-type A34 protein (SEQ ID NO: 4). In some embodiments, said modified oncolytic poxvirus is a modified oncolytic vaccinia virus.

[0023] One embodiment provides a method of treating cancer, comprising administering to a patient a modified oncolytic virus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising at least two mutations, wherein the at least two mutations are at positions corresponding to positions Lys119 and Lys151 of wild-type vaccinia virus A43R protein (SEQ ID NO: 4). In some embodiments, the at least two mutations at positions corresponding to positions Lys119 and Lys151 of wild-type vaccinia virus A43R protein (SEQ ID NO: 4) are Lys119Glu and Lys151Glu, respectively.

[0024] One embodiment provides a method of treating a tumor, the method comprising administering to a patient a modified oncolytic virus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising at least two mutations, wherein the at least two mutations are at positions corresponding to positions Lys119 and Lys151 of the wild-type vaccinia virus A43R protein (SEQ ID NO: 4). In some embodiments, the at least two mutations at positions corresponding to positions Lys119 and Lys151 of the wild-type vaccinia virus A43R protein (SEQ ID NO: 4) are Lys119Glu and Lys151Glu, respectively.

[0025] In some embodiments, the administration is by intratumoral injection, intravenous injection, or a combination thereof. In some embodiments, the administration is by intratumoral injection, intravenous injection, or a combination thereof.

[0026] In some embodiments, the method further comprises administering an additional therapy in combination with the oncolytic poxvirus, wherein the additional therapy comprises at least one of chemotherapy, radiation therapy, oncolytic virus therapy with an additional virus, treatment with an immunomodulatory protein, CAR T cell therapy, an anti-cancer agent, an immunomodulatory agent, or any combination thereof.

[0027] In some embodiments, the additional therapy comprises an immunomodulatory agent selected from the group consisting of an anti-CD33 antibody or antigen-binding fragment thereof, an anti-CD11b antibody or antigen-binding fragment thereof, a COX2 inhibitor, a cytokine, a chemokine, an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, and a TLR agonist.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments. [Brief description of the drawings]

[0029] [Figure 1] FIG. 1 shows an exemplary assembly scheme for generating DNA for creating a recombinant viral library.

[0030] [Diagram 2] FIG. 2 shows a comparison of viral plaque comet tails formed by different vaccinia virus strains.

[0031] [Figure 3-1] Figures 3A-3B show the results of neutralization assays performed using different vaccinia virus strains (Figure 3A shows the results after treatment with anti-L1 NR-45114 antibody, and Figure 3B shows the results after treatment with anti-L1R and anti-VIG antibodies). [Figure 3-2] Same as above.

[0032] [Figure 4] FIG. 4 shows the results of cell viability after infection with different vaccinia virus strains (upper panel: MC38 cells; lower panel: HCT116 cells).

[0033] [Diagram 5] FIG. 5 shows the results of a viral replication assay in cancer cells for different vaccinia virus strains (upper panel: HCT116 cells; lower panel: MC38 cells). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Detailed Description Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are shown for illustrative purposes only. Numerous variations, modifications, and variances will be readily apparent to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in the practice of the present disclosure. The following claims define the scope of the disclosure, and it is intended that methods and structures within the scope of the claims and their equivalents be covered therein. A specific definition

[0035] The terms used herein are for the purpose of describing particular cases only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" can include the plural forms unless the context clearly dictates otherwise. Furthermore, when the terms "contains," "containing," "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, these terms are intended to be inclusive in a similar manner to the term "comprising."

[0036] The term "about" or "approximately" may mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined (e.g., the limitations of the measurement system). For example, "about" may mean within one standard deviation or within more than one standard deviation, according to the practice of given values. When a particular value is described in this application and claims, unless otherwise stated, the term "about" should be considered to mean an acceptable error range for that particular value, for example, ±10% of the value modified by the term "about."

[0037] The terms "individual," "patient," or "subject" may be used interchangeably. None of these terms require or are limited to situations characterized by the supervision (e.g., constant or intermittent) of a medical professional (e.g., a physician, registered nurse, nurse practitioner, medical assistant, janitor, or hospice worker). In some embodiments, a patient, subject, or individual may be under the supervision of a medical professional.

[0038] With respect to a particular virus, the term "heterologous nucleic acid sequence," or "foreign nucleic acid sequence," or "transgene," as used herein, can refer to a nucleic acid sequence derived from a source other than that particular virus.

[0039] As used herein, the term "mutation" can refer to a deletion, an insertion of a heterologous nucleic acid, an inversion, or a substitution (including an open reading frame ablating mutation as generally understood in the art).

[0040] The term "gene", as used herein, may refer to a segment of nucleic acid (also called a "coding sequence" or "coding region") that encodes a particular protein or RNA, optionally together with associated control regions (e.g., promoters, operators, terminators, etc.) that may be located upstream or downstream of the coding sequence.

[0041] The terms "mutant virus" and "modified virus," as used interchangeably herein, may refer to a virus that contains one or more mutations in its genome, including but not limited to deletions, insertions of heterologous nucleic acid, inversions, substitutions, or combinations thereof.

[0042] The term "naturally occurring" as used herein in reference to a virus may refer to the virus being capable of being found in nature, i.e., the virus can be isolated from a natural source and has not been intentionally modified (e.g., a wild-type virus).

[0043] The term "non-naturally occurring" as used herein in reference to one or more mutations in a viral nucleic acid sequence or in the amino acid sequence of a viral protein can refer to the fact that a viral strain containing said one or more mutations cannot be found in nature, i.e., the viral strain cannot be isolated from a natural source, but instead has been intentionally altered.

[0044] The terms "inhibit," "reduce," or "prevention," or any variation of these terms, as referred to herein, can include any measurable decrease, or complete inhibition, to achieve a desired result.

[0045] A "promoter," as used herein, may be a control sequence (a region of a nucleic acid sequence that controls the initiation and rate of transcription). In certain embodiments, a promoter may include genetic elements to which regulatory proteins and molecules may bind RNA polymerase and other transcription factors. The terms "operably positioned," "operably linked," "under control," and "under transcriptional control" may mean that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence to control transcription initiation and / or expression of that sequence. In certain embodiments, a promoter may or may not be used with an "enhancer" (referring to a cis-acting control sequence involved in the transcriptional activation of a nucleic acid sequence).

[0046] The term "homology" as used herein may be a calculation of "homology" or "percent homology" between two or more nucleotide or amino acid sequences that can be determined by aligning the sequences for optimal comparison (e.g., gaps can be inserted in the sequence of the first sequence). The nucleotides at the corresponding positions can then be compared, and the percent identity between the two sequences can be a function of the number of matching positions common to the sequences (i.e., % homology = number of matching positions / total number of positions x 100). For example, a position in a first sequence can be occupied by the same nucleotide as the corresponding position in a second sequence, in which case the molecules are identical at that position. The percent homology between two sequences can be a function of the number of matching positions common to the sequences, taking into account the number of gaps that need to be inserted for optimal alignment of the two sequences, and the length of each gap. In some embodiments, the length of the aligned sequences for comparison purposes may be at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 95% of the length of the reference sequence. The homology between two sequences may be determined by a BLAST® search. The homology may be between the full length of the two sequences or between a portion of the full length of the two sequences. The two sequences may be genes, nucleotide sequences, protein sequences, peptide sequences, amino acid sequences, or fragments thereof. The actual comparison of the two sequences may be accomplished by well-known methods, for example, using a mathematical algorithm. A non-limiting example of such a mathematical algorithm may be found in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90-5873-5877 (1993). Such an algorithm may be incorporated into the NBLAST and XBLAST programs (version 2.0) as described in Altschul, S. et al., Nucleic Acids Res., 25:3389-3402 (1997).When using BLAST and Gapped BLAST, any suitable parameters of each program (e.g., NBLAST) can be used. For example, parameters for sequence comparison can be set at score=100, word length=12, or can be changed (e.g., W=5 or W=20). Other examples include the algorithm of Myers and Miller, CABIOS (1989), ADVANCE, ADAM, BLAT, and FASTA. In another embodiment, the percent identity between two amino acid sequences can be determined, for example, using the GAP program in the GCG software package (Accelrys, Cambridge, UK).

[0047] The term "subject" may refer to an animal, including, but not limited to, a primate (e.g., a human), cow, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms "subject" and "patient" are used interchangeably herein, for example, with respect to a mammalian subject (e.g., a human subject).

[0048] The terms "treat", "treating" and "treatment" may be intended to include reducing or eliminating a disorder, disease or condition; or one or more symptoms associated with said disorder, disease or condition; or reducing or eradicating the cause(s) of said disorder, disease or condition itself. Desirable effects of treatment may include, but are not limited to, preventing the onset or recurrence of a disease, reducing symptoms, reducing the direct or indirect pathological consequences of said disease, preventing metastasis, slowing the rate of disease progression, improving or palliating the disease state, and remission or improved prognosis.

[0049] The term "therapeutically effective amount" may refer to an amount of a compound that, when administered, may be sufficient to prevent or to alleviate to some extent one or more symptoms of the disorder, disease, or condition being treated. The term "therapeutically effective amount" may also refer to an amount of a compound sufficient to elicit the biological or medical response of a cell, tissue, system, animal, or human that is desired by a researcher, veterinarian, physician, or clinician.

[0050] The terms "pharmaceutical acceptable carrier", "pharmaceutical acceptable excipient", "physiologically acceptable carrier", or "physiologically acceptable excipient" may refer to a pharmaceutically acceptable material, composition, or vehicle (e.g., a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, etc.). A component may be "pharmaceutically acceptable" in that it is compatible with other ingredients of a pharmaceutical formulation. It may also be suitable for use in contact with tissues or organs of humans and animals without more toxicity, irritation, allergic response, immunogenicity, or other problem or complications commensurate with a reasonable benefit / risk ratio. Remington: The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th Edition; Rowe et al., eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash, eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson, ed., CRC Press LLC: Boca Raton, FL, 2004).

[0051] The term "pharmaceutical composition" may refer to a mixture of the compounds disclosed herein with other chemical components, such as diluents or carriers. The pharmaceutical composition may facilitate administration of the compounds to an organism. Numerous techniques for administering compounds exist in the art, including but not limited to oral, injection, aerosol, parenteral, and topical administration. Pharmaceutical compositions may also be obtained by reacting the compounds with inorganic or organic acids, such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

[0052] "Anti-cancer agent" as used herein may refer to an agent or therapy that can negatively affect cancer in a subject, for example, by killing cancer cells (including apoptosis in cancer cells), slowing the rate of proliferation of cancer cells, reducing the frequency or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting immune responses to tumors or cancer cells, preventing or inhibiting the progression of cancer, or extending the lifespan of a subject with cancer. Non-limiting examples of anti-cancer agents may include biological agents (biological therapy), chemotherapy agents, and radiation therapy agents.

[0053] The term "oncolytic" as used herein may refer to the killing of cancer or tumor cells by agents (e.g., oncolytic viruses, oncolytic poxviruses, oncolytic vaccinia viruses, etc.), for example, by direct lysis of the cells, stimulating immune response against the cells, apoptosis, expression of toxic proteins, autophagy, and stopping protein synthesis, inducing anti-tumor immunity, or any combination thereof. The direct lysis of the cancer or tumor cells infected with agents (e.g., oncolytic vaccinia viruses, etc.) may be the result of the virus replicating in the cells. In certain instances, the term "oncolytic" may refer to the killing of cancer or tumor cells without lysing the cells.

[0054] The term "oncolytic virus" as used herein may refer to a virus that selectively infects and kills tumor cells. It is understood that under certain non-limiting circumstances, oncolytic viruses can induce anti-tumor responses by a dual mechanism that relies not only on selectively killing tumor cells but also on stimulating the host's anti-tumor immune response.

[0055] In some embodiments, the oncolytic virus may include, but is not limited to, (i) viruses that naturally replicate selectively in cancer cells and are non-pathogenic in humans (often due to their high sensitivity to innate antiviral signaling pathways or their dependency on oncogenic signaling pathways); and (ii) viruses that have been genetically engineered for use. In some embodiments, the oncolytic virus may include herpes simplex virus (HSV). In some embodiments, the oncolytic virus may include a poxvirus. In some embodiments, the poxvirus may include a leporipoxvirus or a vaccinia virus. In some embodiments, the vaccinia virus may include the Ankara, Western Reserve (WR), or Copenhagen strains of vaccinia virus. In some embodiments, the vaccinia virus may include the Lister, Wyeth, New York City Board of Health, Tian Tan, Tash Kent, or USSR strains. In some embodiments, the leporipoxvirus may include a myxoma virus. In some embodiments, the oncolytic virus may be modified.

[0056] The term "modified oncolytic virus" as used herein may refer to an oncolytic virus that includes modifications to its components, such as, but not limited to, modifications in the native genome ("backbone") of the virus (such as, for example, mutation or deletion of viral genes, introduction of foreign nucleic acid, chemical modification of viral nucleic acid or viral protein, and introduction of foreign or modified viral protein into viral capsid). In general, oncolytic viruses can be modified (also known as "engineered") to improve therapeutic effect on tumor cells. In certain embodiments, the modified oncolytic virus can be a modified poxvirus. In certain embodiments, the modified oncolytic virus can be a modified poxvirus.

[0057] The terms "systemic delivery" and "systemic administration" are used interchangeably herein and may, in some cases, refer to the route of administration of a medical drug, oncolytic virus, or other substance into the circulatory system. The systemic administration may include oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof. Oncolytic vaccinia virus in the form of an extracellular enveloped virus

[0058] Poxviruses (such as vaccinia virus) can exist in several forms, including IMV (Intracellular Mature Virus; which is highly antigenic but stable and may be important for host-to-host transmission of poxviruses), and EEV (Extracellular Enveloped Virus; which may be unstable outside the host but may be capable of enhanced transmission within the host due to an outer envelope derived from the host cell that encapsulates the virus; as such, the EEV form may be useful for systemic transmission of the vaccinia virus (e.g., of the oncolytic vaccinia virus within a host).

[0059] Different vaccinia strains are known to produce different ratios of IMV and EEV particles after infection of susceptible cells, with the Western Reserve (WR) strain being a low EEV producer and the International Health Department (IHD)-J strain of vaccinia (IHD-J) being a high EEV producer. One example of a point mutation in a vaccinia gene that is present in IHD-J but not in WR is in the A34R protein (K151E), which is encoded by the vaccinia virus gene VACWR157. A strain derived from WR containing this mutation (the WI strain, which is a WR virus in which the A34R gene from IHD-J has been recombined at the site of the WR A34R gene) has been shown to have increased EEV production (see Blasco, R. et al., 1993, J Virol. Jun; 67(6):3319-25).

[0060] In some embodiments of the present disclosure, modified oncolytic poxviruses (e.g., modified oncolytic vaccinia virus strains) are provided that may contain modifications (such as a non-naturally occurring mutation in a viral glycoprotein (e.g., A34R protein; wild-type sequence shown in UniProt Accession No. P24761; SEQ ID NO: 4)) that increase the ratio of extracellular enveloped virus (EEV) to intracellular mature virus (IMV) forms of the virus. For example, the modified oncolytic vaccinia virus strains that contain the non-naturally occurring mutation in the viral glycoprotein (e.g., A34R protein) may release a higher amount of EEV particles compared to IMV particles.

[0061] In some embodiments, modified oncolytic vaccinia virus strains are provided that may contain two or more non-naturally occurring mutations in the viral glycoprotein (such as A34R). An exemplary amino acid sequence for the mutant A34R protein (also referred to herein as "WO34") is shown in SEQ ID NO:5.

[0062] In some embodiments, the two or more non-naturally occurring mutations can be at a positively charged amino acid residue (e.g., lysine) in the wild-type A34R protein (SEQ ID NO: 4) or fragment thereof, where the positively charged amino acid residue is positively charged at pH 5. In some embodiments, at least one of the two or more non-naturally occurring mutations can be at position 110 in the wild-type A34R protein (SEQ ID NO: 4) or fragment thereof. In some examples, when at least one of the two or more mutations is at position 110 of the wild-type A34R protein (SEQ ID NO: 4), the mutated amino acid at that position is not asparagine.

[0063] In some embodiments, the two or more non-naturally occurring mutations are not at aspartic acid residues in the wild-type A34R protein (SEQ ID NO: 4) or fragment thereof. In some embodiments, the two or more non-naturally occurring mutations may be independently at alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residues in the wild-type A34R protein (SEQ ID NO: 4) or fragment thereof. In some embodiments, the A34R protein or fragment thereof expressed by the modified oncolytic poxvirus may comprise a non-naturally occurring mutation at a lysine residue, wherein the lysine residue may be at a position other than Lys151 of the wild-type A34R protein (SEQ ID NO: 4) (or fragment thereof). In some embodiments, the A34R protein or fragment thereof expressed by the modified oncolytic poxvirus may comprise a non-naturally occurring mutation at position Lys119.

[0064] In some embodiments, the two or more non-naturally occurring mutations can be at residues 119 and 151 of the wild-type A34R protein (SEQ ID NO:4) or fragment thereof. In some embodiments, the mutation at position 119 can be Lys119Glu (K119E). In some embodiments, the mutation at position 151 can be Lys151Glu (K151E).

[0065] In some embodiments, modified oncolytic poxvirus strains are provided that may contain mutations in viral proteins such as haemaglutinin, neuraminidase, Spike (S) glycoprotein, E1, E2, gp120, gp160, gp41, gp1, gp2, E (dimer), E1, or E2.

[0066] In some cases, the two or more non-naturally occurring mutations in the A34R protein of the modified oncolytic poxvirus may result in an increased ratio of extracellular enveloped virus (EEV) to intracellular mature virus (IMV) forms of the virus.

[0067] In some embodiments, the two or more non-naturally occurring mutations in the A34R protein of the modified oncolytic poxvirus may result in an increased ratio of EEV to IMV forms of the virus compared to an otherwise identical poxvirus strain that does not contain the two or more non-naturally occurring mutations in the A34R protein.

[0068] In some embodiments, the modified poxvirus strains provided herein that contain at least two non-naturally occurring mutations in the A34 protein release higher levels of EEV particles (as measured by more comet formation in tissue culture) compared to the large, round plaques formed in tissue culture by poxvirus strains that release lower levels of EEV particles in tissue culture (e.g., vaccinia virus strains that do not contain at least two non-naturally occurring mutations in the A34R protein).

[0069] The EEV particles released by modified poxviruses of the present disclosure that contain at least two non-naturally occurring mutations in the A34R protein are, in some embodiments, resistant to neutralization by antibodies (neutralizing antibodies) and complement toxicity, whereas the IMV particles are not, and thus can mediate long-term spread in vitro and in vivo.

[0070] The EEV particles may also have a higher infection specificity (as determined by a lower particle / PFU ratio) compared to IMV particles. Thus, the modified poxvirus strains releasing higher levels of EEV particles may be improved viruses for therapeutic applications.

[0071] In some embodiments, certain host cell proteins may co-localize with EEV preparations but not with IMV, and the amount of cell proteins may depend on the host cell line and the virus strain. For example, studies have shown that WR EEV contains more cell proteins compared to VV IHD-J strain (see van Eijl H, Hollinshead M, Smith GL. The vaccinia virus A36R protein is a type lb membrane protein present on intracellular but not extracellular enveloped virus particles. Virology 2000;271:26-36). In some cases, host cell proteins may modify the biological effect of EEV particles. As an example, the incorporation of the host membrane protein CD55 into the surface of the EEV particles released by the WR vaccinia virus strain containing at least two non-naturally occurring mutations in the A34R protein may render them resistant to complement toxicity.

[0072] For the Western Reserve (WR) strain of vaccinia virus, about 1% of the viral particles are usually EEV and are released into the culture supernatant before oncolysis of the producer cells. Some studies have shown that 50-fold more EEV particles are released from the IHD-J strain of vaccinia (see Blasco R, Sisler JR, Moss B. Dissociation of progeny vaccinia virus from the cell membrane is regulated by a viral envelope glycoprotein: effect of a point mutation in the lectin homology domain of the A34R gene. J Virol 1993;67:3319-25; see also Mcintosh AA, Smith GL. Vaccinia virus glycoprotein A34R is required for infectivity of extracellular enveloped virus. J Virol 1996;70:272-81).

[0073] The modified poxvirus (e.g., vaccinia virus) strains of the present disclosure can, in some instances, release about 10-fold to about 200-fold higher levels of EEV particles compared to an otherwise identical poxvirus strain that does not contain the A34R protein containing at least two non-naturally occurring mutations (e.g., K151E and K119E).

[0074] In some embodiments, the modified poxvirus strains of the present disclosure have a nucleotide sequence that is about 10-fold to about 15-fold, about 15-fold to about 20-fold, about 20-fold to about 25-fold, about 25-fold to about 30-fold, about 30-fold to about 35-fold, about 35-fold to about 40-fold, or about 40-fold to about 50-fold, or about 50-fold to about 60-fold, or about 60-fold to about 70-fold, or about 70-fold to about 80-fold, or about 80-fold to about 90-fold, or about 90-fold to about 100-fold, or about 100-fold to about 150-fold, or about 150-fold to about 200-fold, or about 200-fold to about 250-fold, or about 250-fold to about 300-fold, or about 300-fold to about 350-fold, or about 350-fold to about 400-fold, or about 100-fold to about 150-fold, or about 150-fold to about 200-fold, or about 150-fold to about 200-fold, or about 150-fold to about 300-fold, or about 150-fold to about 400-fold, or about 150-fold to about 5 ...500-fold, or about 150-fold to about 200-fold, or about 150-fold It may release 0 times, about 40 times to about 45 times, about 45 times to about 50 times, about 50 times to about 55 times, about 55 times to about 60 times, about 60 times to about 65 times, about 65 times to about 70 times, about 75 times to about 80 times, about 85 times to about 90 times, about 95 times to about 100 times, about 100 times to about 120 times, about 120 times to about 140 times, about 140 times to about 160 times, about 160 times to about 180 times, or about 180 times to about 200 times more EEV particles.

[0075] In some embodiments, the modified vaccinia virus strain of the present disclosure can be a WR strain in which the A34R protein comprises the K119E and K151E mutations, and can be about 10-fold to about 15-fold, about 15-fold to about 20-fold, about 20-fold to about 25-fold, about 25-fold to about 30-fold, about 30-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, about 90-fold to about 100-fold, about 100-fold to about 150-fold, about 150-fold to about 200-fold, about 200-fold to about 250-fold, about 250-fold to about 300-fold, about 30 ...50-fold to about 200-fold, about 150-fold to about 300-fold, about 150-fold to about 200-fold, about 15 fold to about 35 times, about 35 times to about 40 times, about 40 times to about 45 times, about 45 times to about 50 times, about 50 times to about 55 times, about 55 times to about 60 times, about 60 times to about 65 times, about 65 times to about 70 times, about 75 times to about 80 times, about 85 times to about 90 times, about 95 times to about 100 times, about 100 times to about 120 times, about 120 times to about 140 times, about 140 times to about 160 times, about 160 times to about 180 times, or about 180 times to about 200 times more EEV particles can be released.

[0076] In some embodiments, the modified vaccinia virus strain of the present disclosure may be a WR strain in which the A34R protein comprises the mutations K119E and K151E (WO34), and is about 10-fold to about 15-fold, about 15-fold to about 20-fold, about 20-fold to about 25-fold, about 25-fold to about 30-fold, about 30-fold to about 35-fold, about 35-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, about 90-fold to about 100-fold, about 90-fold to about 120-fold, about 90-fold to about 130-fold, about 90-fold to about 140-fold, about 90-fold to about 150-fold, about 90-fold to about 160-fold, about 90-fold to about 170-fold, about 90-fold to about 180-fold, about 90-fold to about 190-fold, about 100-fold to about 200-fold, about 100-fold to about 200-fold, about 100-fold to about 200-fold, about 100-fold to about 250-fold, about 250-fold to about 300-fold, about 300-fold to about 350-fold, about 350-fold to about 250-fold, about 100-fold to about 250-fold, about 100-fold to about 250-fold, about 100-fold to about fold to about 45 times, about 45 times to about 50 times, about 50 times to about 55 times, about 55 times to about 60 times, about 60 times to about 65 times, about 65 times to about 70 times, about 75 times to about 80 times, about 85 times to about 90 times, about 95 times to about 100 times, about 100 times to about 120 times, about 120 times to about 140 times, about 140 times to about 160 times, about 160 times to about 180 times, or about 180 times to about 200 times more EEV particles can be released.

[0077] In some cases, increased EEV particle release by the modified poxvirus of the present disclosure compared to IMV particles can be determined by performing a viral plaque assay, in which an increased number of comet tail formations can be observed in a poxvirus comprising said two or more non-naturally occurring mutations in the A34R protein compared to an otherwise identical poxvirus that does not comprise said two or more mutations in the A34R protein.

[0078] In some cases, the increase in EEV particle release compared to IMV particles can be determined by performing a neutralization assay, in which cells infected with a modified poxvirus of the present disclosure (e.g., a vaccinia virus strain) and exposed to a neutralizing antibody (e.g., an anti-L1 NR-45114 antibody or a VIG antibody) can be tested in a viral plaque assay, and viral plaque formation (e.g., by PFU / mL) can be compared to a suitable control virus (e.g., a vaccinia virus strain that does not contain at least two non-naturally occurring mutations in the A34R protein). In some cases, anti-L1 can neutralize and block IMV infection. In some cases, VIG antibody can block VV infection.

[0079] In some cases, an increase in EEV particle release compared to IMV particles can be determined by observing an increase in comet tail formation. In some cases, observing an increase in comet tail formation can include counting the number of colonies on a plate that have the appearance of a comet tail generated from a known amount of virus plated and comparing it to the number of colonies on a plate with another equivalent amount of virus plated. In some cases, an increase in comet formation can indicate an increase in the amount of EEV compared to the IMV form of the virus strain.

[0080] In some cases, the modified poxvirus strains of the present disclosure may contain one or more additional mutations within regions of the viral genome that encode a phospholipase, a kinase, a phosphoprotein, a polymerase, a membrane protein, a virion core protein, a glutaredoxin, a DNA binding protein, an RNA binding protein, an IMV protein, a proteinase, a helicase, a metalloproteinase, a virion structural protein, a myristyl protein, a phosphatase, a heparin binding protein, a glycoprotein, an ATPase, a capping enzyme, a transcription factor, a precursor protein, a subunit protein, a DNA helicase, a palmityl protein, or a receptor.

[0081] In some cases, the one or more additional mutations are in a poxvirus gene, e.g., TK (thymidine kinase), B8R, B18R, B15R, K7R, C6L, K4L, F8L, F9L, F10L, F17R, E1L, E4L, E6R, E8R, E10R, E11L, O2L, I1L, I2L, I3L, I5L, I7L, I8R, G1L, G3L, G4L, G5.5R, G7L, G9R, L1R, L3L, L4R, L5R, J1R, J4R, J 6R, H1L, H2R, H3L, H4L, H5R, H6R, D1R, D2L, D3R, D6R, D7R, D8L, D11L, D12L, D13L, A2.5L, A3L, A4L, A5R, A6L, A7L, A9L, A10L, A13L, A14L, A15L, A16L, A17L, A18R, A21L, A24R, A25L, A26L, A27L, A28L, A29L, A30L, A31R, A34R, A42R, A45R, A46R, A52R gene, and the like. Also provided herein in some embodiments are modified oncolytic poxviruses (e.g., vaccinia virus strains that may contain non-naturally occurring mutations that increase the EEV morphology of the virus and may further contain an exogenous nucleic acid that may encode a non-viral protein (e.g., a therapeutic or diagnostic protein, etc.). Non-limiting examples of proteins encoded by the exogenous nucleic acid include SOC3, PH-20, HMGB1, PIAS3, IL15, IL15-Rα, LIGHT, ITAC, fractalkine, CXCR4, CCR2, CCL5, N1L, immune checkpoint modulators (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA4 antibodies), GM-CSF, IL-12, IL-2, INS, EPO, env, F8, GCG, IFNG, IGHG1, F9, GH1, IL-2, CSF2, TNFRSF1B, ALB, PLAU, IFNB1, CSF3, IFNA2, FSHB, botulinum toxin type A, alefacept, pancrelipase, antithrombin alfa, arcitumomab, anti-rhesus (rh) immunoglobulin G, antithymocyte globulin, alemtuzumab, abciximab, alglucosidase alfa, abatacept, pegademase, apcitide, human serum albumin,Rasburicase, bevacizumab, botulinum toxin type B, bivalirudin, chorionic gonadotropin alpha, pegfilgrastim, collagenase Clostridium histiolyticum, filgrastim, CroFab (crotalidae polyvalent immune Fab), sargramostim, dornase alfa, denileukin diftitox, digoxin immune Fab, epoetin alfa, darbepoetin alfa, eculizumab, enfuvirtide, exenatide, efalizumab, palifermin, coagulation factor IX, antihemophilic factor, thyrotropin alfa, follitropin beta, recombinant human coagulation factor VIIa, gemtuzumab ozogamicin, galsulfase, pegvisomant, imiglucerase, somatropin, glucagon, agalsidase beta, alglucerase, hyaluronidase, histrelin, hepatitis C antigen, HIV antigen, hepatitis B surface antigen, HPV vaccine, hyaluronidase, interferon alfa-2b, interferon gamma-1b, insulin, laronidase, ibritumomab tiuki cetane, insulin, infliximab, interferon beta-1b, oprelvekin, idursulfase, panitumumab, human immunoglobulin, cetuximab, adalimumab, pegaspargase, daclizumab, asparaginase, interferon alfacon-1, interferon alfa-n3, lutropin alfa, lepirudin, lactase, muromonab, mecasermin, natalizumab, nofetumomab, nesiritide, octreotide, ospA lipoprotein, tenecteplase, pramlintide, papain, urokinase, anistreplase, drotrecogin alfa, reteplase, becaplermin, palivizumab, alteplase, ranibizumab, recombinant human bone morphogenetic protein 7 human bone morphogenic protein 7 (rhBMP7), recombinant purified protein derivative (DPPD), streptokinase, calcitonin, sermorelin, secretin, α1 proteinase inhibitors, satumomab pendetide, technetium fanolesomab, teriparatide, trypsin, etanercept,and functional domains or fragments or variants thereof, or any combination thereof.

[0082] Hyaluronan (HA) is an important structural component of the ECM and is a high molecular weight linear glycosaminoglycan composed of repeating disaccharide units. Hyaluronan (HA) can be widely distributed throughout connective, epithelial, and neural tissues, and its expression level can be significantly elevated in many types of tumors. Hyaluronidases are a family of enzymes that catalyze the degradation of HA. At least five functional hyaluronidases have been identified in humans to date (i.e., HYAL1, HYAL2, HYAL3, HYAL4, and HYAL5 (also known as PH-20 or SPAM1)), and of these, only one, PH-20, is known to function at a relatively neutral pH. In some embodiments of the present disclosure, hyaluronidase can be combined with other tumor-targeting therapeutic agents, such as transgenes (also referred to herein as exogenous nucleic acids), to enhance the therapeutic effect of the modified oncolytic virus by at least reducing the ECM and facilitating the transport of the therapeutic agent within and between tumors.

[0083] Some embodiments herein disclose modified oncolytic viruses that may contain exogenous nucleic acids encoding membrane-bound proteins (such as hyaluronidase) capable of degrading hyaluronan. It should be noted that the term "hyaluronidase" as used herein may refer to any enzyme or fragment thereof that catalyzes the degradation of HA in tumors, including but not limited to PH-20 and homologs from other species, as well as other engineered / designed proteins with similar enzymatic functions. As used herein, hyaluronidase may refer to a class of hyaluronan degrading enzymes.

[0084] In some embodiments, the modified oncolytic virus comprises an exogenous nucleic acid that can encode a chemokine receptor that is a chimeric protein. At least a portion of its extracellular domain can be derived from a chemokine receptor that can facilitate the tumor-targeted delivery of the virus, and at least a portion of its intracellular domain can be derived from a chemokine receptor that can facilitate the tumor-specific replication, inhibit immunosuppressive activity, or convey some other beneficial effect, or vice versa. For example, the modified oncolytic virus can comprise a nucleic acid that encodes a protein having an intracellular GTPase domain of CCR5 and an extracellular chemokine-binding domain of CXCR4 or CCR2. In some cases, further improvement of the therapeutic ability of the modified oncolytic virus can be realized by combining domains with different functionalities. In one embodiment of the present disclosure, the modified oncolytic virus can comprise an exogenous nucleic acid that can encode at least one chemokine receptor. In some cases, the modified oncolytic virus can comprise an exogenous nucleic acid that can encode two or more different chemokine receptors that can be simultaneously expressed by the virus. Exemplary chemokine receptors that can be simultaneously expressed from the modified oncolytic viruses described herein can include CXCR4 and CCR2. In modified oncolytic viruses expressing more than one chemokine receptor, a combinatorial or synergistic effect on tumor cells can be achieved with respect to clinical application of the oncolytic virus.

[0085] In certain embodiments, the modified oncolytic virus comprises an exogenous CXCR4-expressing nucleic acid. In certain embodiments, the modified oncolytic virus comprises an exogenous CCR2-expressing nucleic acid. Certain embodiments disclose modified oncolytic viruses that comprise both CXCR4 and CCR2, and both chemokines are expressed from the same virus. In certain circumstances, CXCL12 and / or CCL2, which are typically expressed in the tumor microenvironment, can attract lymphocytes expressing CXCR4 and / or CCR2 or other migratory cells infected with the modified oncolytic virus, thereby enhancing the tumor-targeted delivery of the modified oncolytic virus.

[0086] In certain embodiments, the modified viruses described herein may contain one or more exogenous nucleic acid sequences (alternatively referred to as transgenes), which may generate mRNAs encoding agents capable of modulating the activity of STAT3, and thus may also modulate the activation of genes controlled by STAT3. Thus, certain examples provided herein provide oncolytic vaccinia viruses comprising exogenous nucleic acid sequences encoding agents capable of modulating STAT-3-mediated gene activation. The phrase "modulating STAT3-mediated gene activation" as used herein may refer to the process by which STAT3 activity is modulated, and thus the activation of one or more genes controlled by STAT3 is also modulated.

[0087] In certain embodiments, the agent that can regulate STAT3-mediated gene activation can be a protein or a fragment thereof. In certain embodiments, the protein or the fragment thereof can inhibit, reduce, or minimize STAT3 activity and STAT3-mediated gene activation. The protein or the fragment thereof that inhibits, reduces, and / or minimizes STAT3 activity and STAT3-mediated gene activation can, for example, block the binding of STAT3 to the DNA binding sequence in the promoter region of STAT3-responsive genes. In additional examples, the protein or the fragment thereof that inhibits, reduces, or minimizes STAT3 activity and STAT3-mediated gene activation can directly bind the STAT3 protein, for example, with the SH2 domain. In certain embodiments, the protein that inhibits, reduces, and / or minimizes STAT3 activity blocks, inhibits, reduces, and / or minimizes the phosphorylation of STAT3 and / or dephosphorylates STAT3. In certain non-limiting embodiments, the proteins that regulate STAT3 activity may include phosphotyrosine phosphatases (PTPs), protein inhibitors of activated STATs (PIAS, e.g., PIAS3), and suppressor of cytokine signaling (SOCS) proteins (e.g., SOC3). cancer target

[0088] In an embodiment of the present disclosure, a method of treatment for hyperproliferative diseases (such as cancer or tumor) is provided by delivery of the modified oncolytic poxvirus described herein.Cancers that can be treated by the modified oncolytic poxvirus described herein can include, but are not limited to, melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, renal cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, ovarian epithelial carcinoma, acute lymphoblastic lymphoma, myeloproliferative neoplasm, and sarcoma.

[0089] Cancer cells that may be treated by the methods of the present disclosure may include cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. Furthermore, the cancers may include, but are not limited to, the following histological types of cancer: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial adenomatous polyposis; solid carcinoma; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma (branchio-alveolar adenocarcinoma), pulmonary adenocarcinoma, ... chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; carcinoma);skin adnexal carcinoma;apocrine gland carcinoma;sebaceous gland carcinoma;ear canal adenocarcinoma;mucoepidermoid carcinoma;cystadenocarcinoma;papillary cystadenocarcinoma;papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma;mucinous adenocarcinoma;signet ring cell carcinoma;invasive ductal carcinoma;medullary carcinoma;lobular carcinoma;inflammatory carcinoma;Paget's disease, breast;acinic cell carcinoma;adenosquamous carcinoma;adenocarcinoma with squamous metaplasia;thymoma, malignant;ovarian stromal tumor, malignant;theca cell tumor, malignant;granulosa cell tumor, malignant;androblastoma, malignant;Sertoli cell tumor;Leydig cell tumor, malignant;lipid cell tumor tumors), malignant;paraganglioma, malignant;extramammary paraganglioma, malignant;pheochromocytoma;glomus angiosarcoma;malignant melanoma;amelanotic melanoma;superficial spreading melanoma;malignant melanoma in giant pigmented nevus;epithelioid cell melanoma;blue nevus, malignant;sarcoma;fibrosarcoma;fibrous histiocytoma, malignant;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;mixed tumor, malignant;mu Lary duct mixed tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma;Mesenchymoma, malignant;Brenner tumor, malignant;Phyllodes tumor, malignant;Synovial sarcoma;Mesothelioma, malignant;Dysgerminoma;Embryonal carcinoma;Teratoma, malignant;Ovarian goiter, malignant;Choriocarcinoma;Mesonephroma, malignant;Hemangiosarcoma;Hemangioendothelioma, malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Parosteal osteosarcoma;Chondrosarcoma;Chondrosarcoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastic odontosarcoma;Ameloblastoma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrous astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Anaplastic neuroectodermal tumor;Cerebellar sarcoma;Ganglioblastoma;Neuroblastoma;Retinoblastoma;Olfactory neurogenic tumor;Meningioma, malignant;Neurofibrosarcoma;Schwannoma, malignant;Granular cell tumor, malignant;Lymphoma The tumor may be: lymphoma; Hodgkin's disease; Hodgkin; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specific non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. In some cases, solid tumors that are metastatic may be treated using the modified oncolytic viruses of the present disclosure, such as modified oncolytic poxviruses that are advantageous for systemic delivery. In some cases, solid cancers that are inaccessible or difficult to access (e.g., for intratumoral delivery of therapeutic agents) can be treated using the modified oncolytic poxviruses of the present disclosure, which are favorable for systemic delivery. Cancers associated with increased expression of free fatty acids can be treated using the modified oncolytic poxviruses of the present disclosure, which in some instances are favorable for systemic delivery and increase the amount of EEV formation.

[0090] The present disclosure also contemplates a method for inhibiting or preventing the local invasion or metastasis, or both, of any type of primary cancer. For example, the primary cancer may be melanoma, non-small cell lung, small cell lung, lung, hepatocellular carcinoma, retinoblastoma, astrocytoma, glioblastoma, gum, tongue, leukemia, neuroblastoma, head, neck, breast, pancreas, prostate, kidney, bone, testicular, ovarian, mesothelioma, cervical, gastrointestinal, lymphoma, brain, colon, or bladder. In certain embodiments, the primary cancer may be lung cancer. For example, the lung cancer may be non-small cell lung cancer. Furthermore, the present disclosure may be used to prevent cancer or to treat pre-cancer or pre-malignant cells, including metaplasia, dysplasia, and hyperplasia. It may also be used to inhibit unwanted but benign cells, such as squamous metaplasia, dysplasia, benign prostatic hyperplasia cells, hyperplastic lesions, etc. In some embodiments, cancer, or the progression to more severe forms of cancer, may be halted, interrupted, or slowed by the methods of the present disclosure involving the modified oncolytic poxviruses described herein.

[0091] Furthermore, the modified oncolytic poxviruses disclosed herein can be administered for the treatment of tumors with high bioavailability of free fatty acids in the tumor microenvironment. In some instances, free fatty acids released by adipocytes in tumors in obese patients can nourish and promote the replication of modified oncolytic poxviruses in the tumor and the formation of EEV forms of the virus. This advantage can also be realized in non-obese patients, particularly those with peritoneal cancer. For example, peritoneal cancers can be a target for therapy using the modified oncolytic viruses of the present disclosure, since some peritoneal cancers tend to grow in the omental wall and can be nourished by adipocytes, and as described above, free fatty acids released by adipocytes in tumors can nourish and promote the replication of modified oncolytic viruses in the tumor. The modified oncolytic poxviruses disclosed herein can form increased titers of extracellular enveloped viruses (EEVs) in tumors with high bioavailability of free fatty acids.

[0092] In some embodiments, a method of treating tumors is provided by administering cells infected with the modified poxvirus disclosed herein. The infected cells can be administered to a subject (e.g., intratumorally), whereby the modified poxvirus produces a population of virus particles in situ (e.g., within a tumor or tumor microenvironment) that includes a high percentage of EEV particles (e.g., at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more than about 90%). The EEV particles can then be collected from the subject (e.g., from a biological sample isolated from the subject) and used for subsequent systemic delivery (e.g., intravenous delivery) to the subject. In some cases, this can enhance systemic spread of the modified oncolytic virus in the subject, improving treatment outcomes. Methods of treatment, and efficacy and pharmacokinetic assays

[0093] The present disclosure provides, in some embodiments, methods for treating a subject by administering a modified oncolytic poxvirus disclosed herein.

[0094] A method for producing a toxic effect in a cancer cell is provided, comprising administering a therapeutically effective amount of the modified oncolytic poxvirus described above, or a pharmaceutical composition containing the same, to the cancer cell. The present disclosure further provides a method for inhibiting at least one of the growth or proliferation of a second cancer cell, comprising administering the modified oncolytic poxvirus described above to a first cancer cell, such that the first cancer cell is infected with the virus. Thus, in some embodiments of the method disclosed herein, it is intended that upon administration of an effective amount of the modified oncolytic poxvirus described herein, or a pharmaceutical composition containing the same, not all cancer or tumor cells are infected, and the growth of uninfected cells can be inhibited without direct infection.

[0095] In some instances, cancer cells or tumors may be contacted with a therapeutically effective amount of the exemplary modified oncolytic poxviruses described herein or pharmaceutical compositions containing same, using the methods and compositions of the present disclosure, to induce oncolysis, kill cells, inhibit growth, inhibit metastasis, reduce tumor size, and otherwise reverse or reduce the malignant phenotype of tumor cells. In certain embodiments, an effective amount of the modified oncolytic poxviruses of the present disclosure, or pharmaceutical compositions thereof, may include an amount sufficient to cause oncolysis, destruction or lysis of cancer cells, or inhibit or reduce the growth or size of cancer cells. The reduction in the growth of cancer cells may be manifested, for example, by cell death, or a lower replication or proliferation rate of tumors containing said cells, or an increased survival time of subjects containing said cancer cells.

[0096] In some embodiments, a method of treating a subject having cancer or a tumor is provided, comprising administering to the subject an effective amount of the modified virus described above. In such a method, the effective amount may include an amount that reduces the growth rate or metastasis of the cancer or extends the survival time of the subject. The present disclosure provides a method of reducing tumor growth, comprising administering to the tumor an effective amount of the modified oncolytic poxvirus described above. In certain embodiments, an effective amount of the modified oncolytic poxvirus, or a pharmaceutical composition thereof, may include an amount sufficient to induce a slowdown, inhibition, or reduction in tumor growth or size, and may also include an amount sufficient to induce the eradication of the tumor. A reduction in tumor growth may be manifested, for example, by a reduction in growth rate or an extension in the survival time of the subject containing the tumor.

[0097] The present disclosure also provides a method for determining the infectivity or anti-tumor activity of a modified oncolytic poxvirus described herein, or the amount of tumor-specific viral replication, which may include: (i) administering to a subject a therapeutically effective amount of a modified oncolytic poxvirus or pharmaceutical composition according to the present disclosure, which further expresses a luciferase reporter gene, alone or in combination with an additional therapy; (ii) collecting a first biological sample from the subject immediately after administering the virus and determining the level of luciferase reporter in the first biological sample; (iii) collecting a second biological sample from the subject following administration in step (ii), and (iii) detecting the level of luciferase reporter in the second biological sample, wherein if the level of luciferase is higher in step (iii) than in step (ii), the modified oncolytic poxvirus is determined to be infectious, exhibits anti-tumor activity, or exhibits tumor-specific viral replication. The second biological sample is collected about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 15 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 1 month, to about 2 months after administration in step (i). In some embodiments, the above method may further comprise, in steps (i) and (iii), detecting one or more assay cytokine levels (e.g., IL-2, IL-7, IL-8, IL-10, IFN-γ, GM-CSF, TNF-α, IL-6, IL-4, IL-5, and IL-13) in a plasma sample collected from the subject after administering a therapeutically effective amount of a modified oncolytic poxvirus of the present disclosure (e.g., a modified oncolytic poxvirus described herein) or a pharmaceutical composition comprising same.In some embodiments of the present disclosure, the increase in luciferase bioluminescence between steps (ii) and (iv) above is greater for the modified oncolytic poxviruses described herein compared to an otherwise identical virus that does not contain the modifications in the modified oncolytic poxvirus. Other exemplary techniques for detecting and monitoring viral load following administration of the modified oncolytic poxvirus include real-time quantitative PCR.

[0098] Further provided herein is a method for monitoring pharmacokinetics after administration of a therapeutically effective amount of a modified oncolytic poxvirus according to the present disclosure or a pharmaceutical composition containing said poxvirus. An exemplary method for monitoring pharmacokinetics includes the following steps: (i) administering to said subject a therapeutically effective amount of a modified oncolytic poxvirus or a pharmaceutical composition comprising said poxvirus (alone or in combination with an additional therapy); (ii) collecting pharmacokinetic data from said subject at about 15, 30, 45, 60, 75, 90, 120, 180, and 240 minutes, about 5, 6, 7, 8, 9, 10, 12, 15, 16, 20, 22, 24, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 100, 110, 120, 130, 140, 150, 160, 170, 182, 190, It may include collecting biological samples at one or more time points selected from 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 1 month, to about 2 months; and (iii) detecting the amount of the viral genome (or the reporter gene (e.g., luciferase) inserted into the viral genome) in the biological samples collected at the above-mentioned time points. In some examples, the viral genome copies / mL may be maximum in the sample collected at 15 minutes, and the sample collected at 240 minutes may not contain a detectable amount of the viral genome. Thus, in some examples, a viral peak may be observed about 15 minutes after administration, and the virus may be largely cleared from the subject's system after about 240 minutes (in other words, 4 hours). In some instances, a first viral peak may be observed about 15 minutes after administration, and a second viral peak may be observed in the biological sample collected at a later time point (e.g., about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes). The biological sample may be blood in an exemplary embodiment, and the amount of viral genomes / mL may be determined by quantitative PCR or other suitable techniques.In some instances, a first viral peak may be observed about 15 minutes after administration, and a second viral peak may be observed about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 15 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 1 month, to about 2 months after administration of a modified oncolytic virus of the disclosure (e.g., an oncolytic poxvirus described herein).

[0099] In some examples, tumor-selective replication of the modified oncolytic poxvirus can be measured by using a reporter gene (e.g., a luciferase gene). In some embodiments, the luciferase gene can be inserted into the genome of the virus and tumor cells can be infected with the virus. Bioluminescence in the infected tumor cells can be measured to monitor tumor-selective replication. Some examples show increased bioluminescence of a luciferase reporter in the modified oncolytic poxvirus of the present disclosure compared to an otherwise identical oncolytic poxvirus that does not contain a modification in the modified oncolytic virus. Delivery of engineered oncolytic viruses

[0100] In some embodiments, the amount of modified oncolytic poxvirus of the present disclosure administered to a subject is about 10 3 ~10 12 Infectious viral particles or plaque forming units (PFU), or approximately 10 5 ~10 10 PFU, or about 10 5 ~10 8 PFU, or about 10 8 ~10 10 In some embodiments, the amount of the modified oncolytic poxvirus of the present disclosure administered to a subject is about 10 PFU. 3 ~10 12Viral particles or plaque forming units (PFU), or approximately 10 5 ~10 10 PFU, or about 10 5 ~10 8 PFU, or about 10 8 ~10 10 In some embodiments, the modified oncolytic poxvirus of the present disclosure can be about 10 PFU. 3 PFU / dose~about 10 4 PFU / dose, approximately 10 4 PFU / dose~about 10 5 PFU / dose, approximately 10 5 PFU / dose~about 10 6 PFU / dose, approximately 10 7 PFU / dose~about 10 8 PFU / dose, approximately 10 9 PFU / dose~about 10 10 PFU / dose, approximately 10 10 PFU / dose~about 10 11 PFU / dose, approximately 10 11 PFU / dose~about 10 12 PFU / dose, approximately 10 12 PFU / dose~about 10 13 PFU / dose, approximately 10 13 PFU / dose~about 10 14 PFU / dose, or approximately 10 14 PFU / dose~about 10 15 In some embodiments, the modified oncolytic poxvirus of the present disclosure may be administered at a dose that may include about 2×10 PFU / dose. 3 PFU / dose, 3×10 3 PFU / dose, 4×10 3 PFU / dose, 5×10 3 PFU / dose, 6×10 3 PFU / dose, 7×10 3 PFU / dose, 8×10 3 PFU / dose, 9×10 3 PFU / dose, approximately 10 4 PFU / dose, approximately 2 x 10 4 PFU / dose, approximately 3 x 10 4 PFU / dose, approximately 4 x 10 4 PFU / dose, approximately 5 x 10 4PFU / dosage, approximately 6×10 4 PFU / dosage, approximately 7×10 4 PFU / dosage, approximately 8×10 4 PFU / dosage, approximately 9×10 4 PFU / dosage, approximately 10 5 PFU / dosage, 2×10 5 PFU / dosage, 3×10 5 PFU / dosage, 4×10 5 PFU / dosage, 5×10 5 PFU / dosage, 6×10 5 PFU / dosage, 7×10 5 PFU / dosage, 8×10 5 PFU / dosage, 9×10 5 PFU / dosage, approximately 10 6 PFU / dosage, approximately 2×10 6 PFU / dosage, approximately 3×10 6 PFU / dosage, approximately 4×10 6 PFU / dosage, approximately 5×10 6 PFU / dosage, approximately 6×10 6 PFU / dosage, approximately 7×10 6 PFU / dosage, approximately 8×10 6 PFU / dosage, approximately 9×10 6 PFU / dosage, approximately 10 7 PFU / dosage, approximately 2×10 7 PFU / dosage, approximately 3×10 7 PFU / dosage, approximately 4×10 7 PFU / dosage, approximately 5×10 7 PFU / dosage, approximately 6×10 7 PFU / dosage, approximately 7×10 7 PFU / dosage, approximately 8×10 7 PFU / dosage, approximately 9×10 7 PFU / dosage, approximately 10 8 PFU / dosage, approximately 2×10 8 PFU / dosage, approximately 3×10 8 PFU / dosage, approximately 4×10 8 PFU / dosage, approximately 5×10 8 PFU / dosage, approximately 6×10 8 PFU / dosage, approximately 7×10 8 PFU / dosage, approximately 8×10 8 PFU / dosage, approximately 9×10 8 PFU / dosage, approximately 109 PFU / dose, approximately 2 x 10 9 PFU / dose, approximately 3 x 10 9 PFU / dose, approximately 4 x 10 9 PFU / dose, approximately 5 x 10 9 PFU / dose, approximately 6 x 10 9 PFU / dose, approximately 7 x 10 9 PFU / dose, approximately 8 x 10 9 PFU / dose, approximately 9 x 10 9 PFU / dose, approximately 10 10 PFU / dose, approximately 2 x 10 10 PFU / dose, approximately 3 x 10 10 PFU / dose, approximately 4 x 10 10 PFU / dose, approximately 5 x 10 10 PFU / dose, approximately 6 x 10 10 PFU / dose, approximately 7 x 10 10 PFU / dose, approximately 8 x 10 10 PFU / dose, approximately 9 x 10 10 PFU / dose, approximately 10 10 PFU / dose, approximately 2 x 10 10 PFU / dose, approximately 3 x 10 10 PFU / dose, approximately 4 x 10 10 PFU / dose, approximately 5 x 10 10 PFU / dose, approximately 6 x 10 10 PFU / dose, approximately 7 x 10 10 PFU / dose, approximately 8 x 10 10 PFU / dose, approximately 9 x 10 10 PFU / dose, approximately 10 11 PFU / dose, approximately 2 x 10 11 PFU / dose, approximately 3 x 10 11 PFU / dose, approximately 4 x 10 11 PFU / dose, approximately 5 x 10 11 PFU / dose, approximately 6 x 10 11 PFU / dose, approximately 7 x 10 11 PFU / dose, approximately 8 x 10 11 PFU / dose, approximately 9 x 10 11 PFU / dose, or approximately 10 12 PFU / dose, approximately 10 12 PFU / dose~about 10 13 PFU / dose, approximately 10 13 PFU / dose~about 10 14PFU / dose, or approximately 10 14 PFU / dose~about 10 15 In some embodiments, the modified oncolytic poxvirus of the present disclosure may be administered at a dose that may include 5×10 PFU / dose. 9 In some embodiments, the modified oncolytic poxvirus of the present disclosure may be administered in a dose that may include up to 5×10 PFU / dose. 9 It may be administered in doses that may include PFU / dose.

[0101] In some embodiments, the modified oncolytic poxvirus of the present disclosure is 3 Virus particles / dose ~ approx. 10 4 Viral particles / dose, approximately 10 4 Virus particles / dose ~ approx. 10 5 Viral particles / dose, approximately 10 5 Virus particles / dose ~ approx. 10 6 Viral particles / dose, approximately 10 7 Virus particles / dose ~ approx. 10 8 Viral particles / dose, approximately 10 9 Virus particles / dose ~ approx. 10 10 Viral particles / dose, approximately 10 10 Virus particles / dose ~ approx. 10 11 Viral particles / dose, approximately 10 11 Virus particles / dose ~ approx. 10 12 Viral particles / dose, approximately 10 12 Virus particles / dose ~ approx. 10 13 Viral particles / dose, approximately 10 13 Virus particles / dose ~ approx. 10 14 viral particles / dose, or approximately 10 14 Virus particles / dose ~ approx. 10 15 It may be administered in a dose that may contain viral particles / dose.

[0102] In some embodiments, the modified oncolytic poxvirus of the present disclosure is 3 PFU / kg ~ approx. 10 4 PFU / kg, approximately 10 4 PFU / kg ~ approx. 10 5 PFU / kg, approximately 105 PFU / kg ~ approx. 10 6 PFU / kg, approximately 10 7 PFU / kg ~ approx. 10 8 PFU / kg, approximately 10 9 PFU / kg ~ approx. 10 10 PFU / kg, approximately 10 10 PFU / kg ~ approx. 10 11 PFU / kg, approximately 10 11 PFU / kg ~ approx. 10 12 PFU / kg, approximately 10 12 PFU / kg ~ approx. 10 13 PFU / kg, approximately 10 13 PFU / kg ~ approx. 10 14 PFU / kg, or approximately 10 14 PFU / kg ~ approx. 10 15 In some embodiments, the modified oncolytic poxvirus of the present disclosure may be administered at a dose that may include about 2×10 PFU / kg. 3 PFU / kg, 3×10 3 PFU / kg, 4×10 3 PFU / kg, 5×10 3 PFU / kg, 6×10 3 PFU / kg, 7×10 3 PFU / kg, 8 x 10 3 PFU / kg, 9×10 3 PFU / kg, approximately 10 4 PFU / kg, approximately 2×10 4 PFU / kg, approximately 3×10 4 PFU / kg, approximately 4×10 4 PFU / kg, approximately 5×10 4 PFU / kg, approximately 6×10 4 PFU / kg, approximately 7×10 4 PFU / kg, approximately 8×10 4 PFU / kg, approximately 9×10 4 PFU / kg, approximately 10 5 PFU / kg, 2×10 5 PFU / kg, 3×10 5 PFU / kg, 4×10 5 PFU / kg, 5×10 5 PFU / kg, 6×10 5 PFU / kg, 7×10 5PFU / kg, 8×10 5 PFU / kg, 9×10 5 PFU / kg, approximately 10 6 PFU / kg, approximately 2×10 6 PFU / kg, approximately 3×10 6 PFU / kg, approximately 4×10 6 PFU / kg, approximately 5×10 6 PFU / kg, approximately 6×10 6 PFU / kg, approximately 7×10 6 PFU / kg, approximately 8×10 6 PFU / kg, approximately 9×10 6 PFU / kg, approximately 10 7 PFU / kg, approximately 2×10 7 PFU / kg, approximately 3×10 7 PFU / kg, approximately 4×10 7 PFU / kg, approximately 5×10 7 PFU / kg, approximately 6×10 7 PFU / kg, approximately 7×10 7 PFU / kg, approximately 8×10 7 PFU / kg, approximately 9×10 7 PFU / kg, approximately 10 8 PFU / kg, approximately 2×10 8 PFU / kg, approximately 3×10 8 PFU / kg, approximately 4×10 8 PFU / kg, approximately 5×10 8 PFU / kg, approximately 6×10 8 PFU / kg, approximately 7×10 8 PFU / kg, approximately 8×10 8 PFU / kg, approximately 9×10 8 PFU / kg, approximately 10 9 PFU / kg, approximately 2×10 9 PFU / kg, approximately 3×10 9 PFU / kg, approximately 4×10 9 PFU / kg, approximately 5×10 9 PFU / kg, approximately 6×10 9 PFU / kg, approximately 7×10 9 PFU / kg, approximately 8×10 9 PFU / kg, approximately 9×10 9 PFU / kg, approximately 10 10 PFU / kg, approximately 2×10 10 PFU / kg, approximately 3×1010 PFU / kg, approximately 4×10 10 PFU / kg, approximately 5×10 10 PFU / kg, approximately 6×10 10 PFU / kg, approximately 7×10 10 PFU / kg, approximately 8×10 10 PFU / kg, approximately 9×10 10 PFU / kg, approximately 10 10 PFU / kg, approximately 2×10 10 PFU / kg, approximately 3×10 10 PFU / kg, approximately 4×10 10 PFU / kg, approximately 5×10 10 PFU / kg, approximately 6×10 10 PFU / kg, approximately 7×10 10 PFU / kg, approximately 8×10 10 PFU / kg, approximately 9×10 10 PFU / kg, approximately 10 11 PFU / kg, approximately 2×10 11 PFU / kg, approximately 3×10 11 PFU / kg, approximately 4×10 11 PFU / kg, approximately 5×10 11 PFU / kg, approximately 6×10 11 PFU / kg, approximately 7×10 11 PFU / kg, approximately 8×10 11 PFU / kg, approximately 9×10 11 PFU / kg, or approximately 10 12 PFU / kg, approximately 10 12 PFU / kg ~ approx. 10 13 PFU / kg, approximately 10 13 PFU / kg ~ approx. 10 14 PFU / kg, or approximately 10 14 PFU / kg ~ approx. 10 15 In some embodiments, the modified oncolytic poxvirus of the present disclosure may be administered at a dose that may include 5×10 PFU / kg. 9 In some embodiments, the modified oncolytic poxvirus of the present disclosure may be administered at a dose that may include up to 5×10 PFU / kg. 9 It may be administered in a dose that may contain PFU / kg.

[0103] In some embodiments, the modified oncolytic poxvirus of the present disclosure is 3 Virus particles / kg ~ approx. 10 4 Virus particles / kg, approximately 10 4 Virus particles / kg ~ approx. 10 5 Virus particles / kg, approximately 10 5 Virus particles / kg ~ approx. 10 6 Virus particles / kg, approximately 10 7 Virus particles / kg ~ approx. 10 8 Virus particles / kg, approximately 10 9 Virus particles / kg ~ approx. 10 10 Virus particles / kg, approximately 10 10 Virus particles / kg ~ approx. 10 11 Virus particles / kg, approximately 10 11 Virus particles / kg ~ approx. 10 12 Virus particles / kg, approximately 10 12 Virus particles / kg ~ approx. 10 13 Virus particles / kg, approximately 10 13 Virus particles / kg ~ approx. 10 14 Virus particles / kg, or approximately 10 14 Virus particles / kg ~ approx. 10 15 It may be administered in a dose that may contain viral particles / kg.

[0104] In certain embodiments, the liquid dosage form of the modified oncolytic poxvirus described herein is about 10 3 PFU / mL~about 10 4 PFU / mL, approximately 10 4 PFU / mL~about 10 5 PFU / mL, approximately 10 5 PFU / mL~about 10 6 PFU / mL, approximately 10 7 PFU / mL~about 10 8 PFU / mL, approximately 10 9 PFU / mL~about 10 10 PFU / mL, approximately 10 10 PFU / mL~about 10 11 PFU / mL, approximately 10 11 PFU / mL~about 10 12 PFU / mL, approximately 10 12PFU / mL~about 10 13 PFU / mL, approximately 10 13 PFU / mL~about 10 14 PFU / mL, or approximately 10 14 PFU / mL~about 10 15 In some embodiments, the modified oncolytic poxvirus of the present disclosure may comprise a viral dose of about 2×10 PFU / mL. 3 PFU / mL, 3×10 3 PFU / mL, 4×10 3 PFU / mL, 5×10 3 PFU / mL, 6×10 3 PFU / mL, 7×10 3 PFU / mL, 8×10 3 PFU / mL, 9×10 3 PFU / mL, approximately 10 4 PFU / mL, approximately 2×10 4 PFU / mL, approximately 3×10 4 PFU / mL, approximately 4×10 4 PFU / mL, approximately 5×10 4 PFU / mL, approximately 6×10 4 PFU / mL, approximately 7×10 4 PFU / mL, approximately 8×10 4 PFU / mL, approximately 9×10 4 PFU / mL, approximately 10 5 PFU / mL, 2×10 5 PFU / mL, 3×10 5 PFU / mL, 4×10 5 PFU / mL, 5×10 5 PFU / mL, 6×10 5 PFU / mL, 7×10 5 PFU / mL, 8×10 5 PFU / mL, 9×10 5 PFU / mL, approximately 10 6 PFU / mL, approximately 2×10 6 PFU / mL, approximately 3×10 6 PFU / mL, approximately 4×10 6 PFU / mL, approximately 5×10 6 PFU / mL, approximately 6×10 6 PFU / mL, approximately 7×10 6 PFU / mL, approximately 8×10 6PFU / mL, approximately 9×10 6 PFU / mL, approximately 10 7 PFU / mL, approximately 2×10 7 PFU / mL, approximately 3×10 7 PFU / mL, approximately 4×10 7 PFU / mL, approximately 5×10 7 PFU / mL, approximately 6×10 7 PFU / mL, approximately 7×10 7 PFU / mL, approximately 8×10 7 PFU / mL, approximately 9×10 7 PFU / mL, approximately 10 8 PFU / mL, approximately 2×10 8 PFU / mL, approximately 3×10 8 PFU / mL, approximately 4×10 8 PFU / mL, approximately 5×10 8 PFU / mL, approximately 6×10 8 PFU / mL, approximately 7×10 8 PFU / mL, approximately 8×10 8 PFU / mL, approximately 9×10 8 PFU / mL, approximately 10 9 PFU / mL, approximately 2×10 9 PFU / mL, approximately 3×10 9 PFU / mL, approximately 4×10 9 PFU / mL, approximately 5×10 9 PFU / mL, approximately 6×10 9 PFU / mL, approximately 7×10 9 PFU / mL, approximately 8×10 9 PFU / mL, approximately 9×10 9 PFU / mL, approximately 10 10 PFU / mL, approximately 2×10 10 PFU / mL, approximately 3×10 10 PFU / mL, approximately 4×10 10 PFU / mL, approximately 5×10 10 PFU / mL, approximately 6×10 10 PFU / mL, approximately 7×10 10 PFU / mL, approximately 8×10 10 PFU / mL, approximately 9×10 10 PFU / mL, approximately 10 10 PFU / mL, approximately 2×10 10 PFU / mL, approximately 3×10 10PFU / mL, approximately 4×10 10 PFU / mL, approximately 5×10 10 PFU / mL, approximately 6×10 10 PFU / mL, approximately 7×10 10 PFU / mL, approximately 8×10 10 PFU / mL, approximately 9×10 10 PFU / mL, approximately 10 11 PFU / mL, approximately 2×10 11 PFU / mL, approximately 3×10 11 PFU / mL, approximately 4×10 11 PFU / mL, approximately 5×10 11 PFU / mL, approximately 6×10 11 PFU / mL, approximately 7×10 11 PFU / mL, approximately 8×10 11 PFU / mL, approximately 9×10 11 PFU / mL, or approximately 10 12 PFU / mL, approximately 10 12 PFU / mL~about 10 13 PFU / mL, approximately 10 13 PFU / mL~about 10 14 PFU / mL, or approximately 10 14 PFU / mL~about 10 15 In some embodiments, the modified oncolytic poxvirus of the present disclosure may be administered at a dose that may include 5×10 PFU / mL. 9 In some embodiments, the modified oncolytic poxvirus of the present disclosure may be administered at a dose that may include up to 5×10 PFU / mL. 9 It may be administered in a dose that may contain PFU / mL.

[0105] In some instances, the modified oncolytic poxvirus may be administered by injection, the dosage is about 10 3 Viral particles / injection, 10 4 Viral particles / injection, 10 5 Viral particles / injection, 10 6 Viral particles / injection, 10 7 Viral particles / injection, 10 8 Viral particles / injection, 10 9 Viral particles / injection, 10 10 Viral particles / injection, 1011 Viral particles / injection, 10 12 Viral particles / injection, 2 x 10 12 Viral particles / injection, 10 13 Viral particles / injection, 10 14 viral particles / injection, or 10 15 In a further example where the modified oncolytic poxvirus is administered by injection, the dosage may comprise about 10 3 Infectious viral particles / injection, 10 4 Infectious viral particles / injection, 10 5 Infectious viral particles / injection, 10 6 Infectious viral particles / injection, 10 7 Infectious viral particles / injection, 10 8 Infectious viral particles / injection, 10 9 Infectious viral particles / injection, 10 10 Infectious viral particles / injection, 10 11 Infectious viral particles / injection, 10 12 Infectious viral particles / injection, 2 x 10 12 Infectious viral particles / injection, 10 13 Infectious viral particles / injection, 10 14 infectious viral particles / injection, or 10 15 In an additional embodiment, the modified oncolytic poxvirus of the present disclosure may comprise about 10 infectious viral particles / injection. 3 Tissue Culture Inhibitor Dose 50% (TCID 50 ) / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 3×10 8 TCID 50 / kg, 4×10 8 TCID 50 / kg, 5×10 8 TCID 50 / kg, 3×10 9 TCID 50 / kg, 4×10 9 TCID 50 / kg, 5×10 9 TCID 50 / kg, 3×10 10 TCID 50 / kg, 4×10 10 TCID 50 / kg, or 4 × 10 10 TCID 50 The present invention relates to a method for administering a medicament for the treatment of a patient having a disease, the method comprising administering the medicament for a period of time in a dose range of 10 to 100 mg / kg. ×It should be noted that, instead, 1 eX is expressed as 1 eX. In certain embodiments, the modified oncolytic poxvirus may be administered in one or more doses. In certain embodiments, the virus may be administered in an amount sufficient to induce oncolysis in at least about 20% of the cells in the tumor, in at least about 30% of the cells in the tumor, in at least about 40% of the cells in the tumor, in at least about 50% of the cells in the tumor, in at least about 60% of the cells in the tumor, in at least about 70% of the cells in the tumor, in at least about 80% of the cells in the tumor, or in at least about 90% of the cells in the tumor. In certain embodiments, a dose of virus may refer to an amount administered to a subject or tumor over a period of 1, 2, 5, 10, 15, 20, or 24 hours. In certain embodiments, the doses may be separated over time or by separate injections. In certain embodiments, multiple doses (e.g., 2, 3, 4, 5, 6, or more doses) of the poxvirus may be administered to the subject, for example, where the second treatment may occur within 1, 2, 3, 4, 5, 6, 7 days, or weeks of the first treatment. In certain embodiments, the modified oncolytic v may be administered to the subject over a period of 1, 2, 3, 4, 5, 6, 7 days, or weeks. In certain embodiments, an oncolytic vaccinia virus or pharmaceutical composition described herein may be administered for about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks, or about 52 weeks, or longer. The frequency of administration of the oncolytic poxvirus or pharmaceutical composition described herein can, in certain instances, be once daily, twice daily, once weekly, once every 3 weeks, once every 4 weeks (or once monthly), once every 8 weeks (or once every 2 months), once every 12 weeks (or once every 3 months), or once every 24 weeks (or once every 6 months).In some embodiments of the methods disclosed herein, the oncolytic poxvirus or the pharmaceutical composition may be administered, independently, at an initial dose for a first period, at an intermediate dose for a second period, and at a high dose for a third period. In some embodiments, the initial dose may be lower than the intermediate dose, and the intermediate dose may be lower than the high dose. In some embodiments, the first, second, and third periods may be, independently, about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks, or about 52 weeks, or longer.

[0106] In some examples, the subject may be placed on a reduced carbohydrate diet (e.g., a ketogenic diet) prior to, concurrently with, and after administration of a modified oncolytic poxvirus described herein according to any of the methods of treatment described herein. In certain embodiments, the subject may be fed a diet that may comprise carbohydrate consumption of less than 500 grams per day, less than 450 grams per day, less than 450 grams per day, less than 400 grams per day, less than 350 grams per day, less than 300 grams per day, less than 250 grams per day, less than 200 grams per day, less than 150 grams per day, less than 100 grams per day, less than 90 grams per day, less than 80 grams per day, less than 70 grams per day, less than 60 grams per day, less than 50 grams per day, less than 40 grams per day, less than 30 grams per day, less than 20 grams per day, less than 10 grams per day.

[0107] An exemplary method for delivery of the modified oncolytic poxvirus of the present disclosure or pharmaceutical compositions comprising the same to cancer or tumor cells may be by intratumoral injection. However, alternative administration methods may also be used, such as intravenous, by infusion, parenteral, intravenous, intradermal, intramuscular, transdermal, rectal, intraurethral, ​​intravaginal, intranasal, intrathecal, or intraperitoneal administration. The route of administration may vary depending on the location and nature of the tumor. In certain embodiments, the route of administration may be intradental, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, local (e.g., in the vicinity of a tumor (especially one having a vasculature) or in the vasculature adjacent to a tumor), transdermal, intrathecal, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, by inhalation, perfusion, lavage, or orally. The administrable dose of the modified oncolytic poxvirus may be administered as a bolus injection or as a slow infusion. In certain embodiments, the modified oncolytic poxvirus may be administered to a patient from a source implanted in the patient. In certain embodiments, the administration of the modified oncolytic poxvirus may be by continuous infusion over a selected period of time. In some examples, the oncolytic poxvirus described herein, or a pharmaceutical composition containing the same, may be administered by infusion over a therapeutically effective dose of about 15 minutes, about 30 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 100 minutes, or about 120 minutes or longer. The modified oncolytic poxvirus or pharmaceutical composition of the present disclosure may be administered as a liquid dosage form, wherein the total volume administered is about 1 mL to about 5 mL, about 5 mL to about 10 mL, about 15 mL to about 20 mL, about 25 mL to about 30 mL, about 30 mL to about 50 mL, about 50 mL to about 100 mL, about 100 mL to about 150 mL, about 150 mL to about 200 mL, about 200 mL to about 250 mL, about 250 mL to about 300 mL, about 300 mL to about 350 mL, about 350 mL to about 400 mL, about 400 mL to about 450 mL, about 450 mL to about 500 mL, about 500 mL to about 750 mL, or about 750 mL to about 1000 mL. Pharmaceutical Compositions

[0108] The pharmaceutical compositions containing the modified oncolytic poxviruses described herein may be prepared as solutions, dispersions in glycerol, liquid polyethylene glycols, and any combination thereof in oils, as solid dosage forms, as inhalable dosage forms, as intranasal dosage forms, as liposomal formulations, dosage forms including nanoparticles, dosage forms including microparticles, polymeric dosage forms, or any combination thereof. In some embodiments, the pharmaceutical compositions described herein may include stabilizers and buffers. In some embodiments, the pharmaceutical compositions described herein may include solubilizing agents (e.g., sterile water, Tris buffer). In some embodiments, the pharmaceutical compositions described herein may include excipients. The excipients may be those excipients described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986). Non-limiting examples of suitable excipients may include buffers, preservatives, stabilizers, binders, compression agents, lubricants, chelating agents, dispersion enhancers, disintegrants, flavoring agents, sweetening agents, and coloring agents.

[0109] In some embodiments, the excipient may be a buffer. Non-limiting examples of suitable buffers include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate. As buffers, sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide, and other calcium salts, or combinations thereof, may be used in pharmaceutical preparations.

[0110] In some embodiments, the excipient may include a preservative. Non-limiting examples of suitable preservatives may include antioxidants (such as alpha-tocopherol and ascorbic acid) and antimicrobials (such as parabens, chlorobutanol, and phenol). Antioxidants may further include, but are not limited to, EDTA, citric acid, ascorbic acid, butyl hydroxytoluene (BHT), butyl hydroxyanisole (BHA), sodium sulfite, paraaminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol, and N-acetylcysteine. In some examples, the preservative may include validamycin A, TL-3, sodium orthovanadate, sodium fluoride, N-α-tosyl-Phe-chloromethylketone, N-α-tosyl-Lys-chloromethylketone, aprotinin, phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, kinase inhibitors, phosphatase inhibitors, caspase inhibitors, granzyme inhibitors, cell adhesion inhibitors, cell division inhibitors, cell cycle inhibitors, lipid signaling inhibitors, protease inhibitors, reducing agents, alkylating agents, antimicrobial agents, oxidase inhibitors, or other inhibitors.

[0111] In some embodiments, the pharmaceutical compositions described herein may include a binder as an excipient. Non-limiting examples of suitable binders include starch, pregelatinized starch, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C 12 -C 18It may comprise fatty acid alcohol, polyethylene glycol, polyol, saccharide, oligosaccharide, and combinations thereof.The binder that may be used in pharmaceutical preparations may be selected from starch, such as potato starch, corn starch, wheat starch, etc.; sugar, such as sucrose, glucose, dextrose, lactose, maltodextrin, etc.; natural and synthetic gums; gelatin; cellulose derivatives, such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, etc.; polyvinylpyrrolidone (povidone); polyethylene glycol (PEG); wax; calcium carbonate; calcium phosphate; alcohol, such as sorbitol, xylitol, mannitol, etc., and water, or combinations thereof.

[0112] In some embodiments, the pharmaceutical compositions described herein may include a lubricant as an excipient. Non-limiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oil, sterotex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil. Lubricants that may be used in pharmaceutical formulations may be selected from metal stearates (e.g., magnesium stearate, calcium stearate, aluminum stearate, etc.), fatty acid esters (e.g., sodium stearyl fumarate, etc.), fatty acids (e.g., stearic acid, etc.), fatty alcohols, glyceryl behenate, mineral oil, paraffin, hydrogenated vegetable oil, leucine, polyethylene glycol (PEG), metal lauryl sulfates (e.g., sodium lauryl sulfate, magnesium lauryl sulfate, etc.), sodium chloride, sodium benzoate, sodium acetate, and talc, or combinations thereof. In some embodiments, the pharmaceutical formulation may include a dispersion enhancer as an excipient. Non-limiting examples of suitable dispersing agents may include starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, refined wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high HLB emulsifier surfactants.

[0113] In some embodiments, the pharmaceutical compositions described herein may include a disintegrant as an excipient. In some embodiments, the disintegrant may be a non-foaming disintegrant. Non-limiting examples of suitable non-foaming disintegrants may include starches (e.g., corn starch, potato starch, pregelatinized starches thereof, etc.), sweeteners, clays (e.g., bentonite, etc.), microcrystalline cellulose, alginates, sodium starch glycolate, gums (e.g., agar, guar, locust bean, karaya, pectin, and tragacanth, etc.). In some embodiments, the disintegrant may be a foaming disintegrant. Non-limiting examples of suitable foaming disintegrants may include a combination of citric acid and sodium bicarbonate, and a combination of sodium bicarbonate and tartaric acid.

[0114] In some embodiments, the excipient may include flavoring agent.The flavoring agent incorporated in the outer layer may be selected from synthetic flavor oils and flavor aromatics; natural oils; extracts from plants, leaves, flowers, and fruits; and combinations thereof.In some embodiments, the flavoring agent may be selected from the group consisting of cinnamon oil; wintergreen oil; peppermint oil; clover oil; hay oil; anise oil; eucalyptus; vanilla; citrus oil (e.g., lemon oil, orange oil, grape and grapefruit oil, etc.); and fruit essence (including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, and apricot).

[0115] In some embodiments, excipient may include sweetener.Non-limiting examples of suitable sweetener may include glucose (corn syrup), dextrose, invert sugar, fructose, and their mixtures (when not used as carrier); saccharin and its various salts (e.g., sodium salt); dipeptide sweetener (e.g., aspartame, etc.); dihydrochalcone compounds, glycyrrhizin; Stevia rebaudiana (stevioside); chlorine derivatives of sucrose (e.g., sucralose, etc.); and sugar alcohols (e.g., sorbitol, mannitol, sylitol, etc.).

[0116] In some examples, the pharmaceutical compositions described herein may include colorants. Non-limiting examples of suitable colorants may include Food, Drug, and Cosmetic Colors (FD&C), Drug and Cosmetic Colors (D&C), and External Drug and Cosmetic Colors (Ext. D&C). Colorants may be used as dyes or their corresponding lakes.

[0117] In some cases, the pharmaceutical compositions described herein may include a chelating agent. In some cases, the chelating agent may be a fungicidal chelating agent. Examples include, but are not limited to, ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA); disodium, trisodium, tetrasodium, dipotassium, tripotassium, dilithium, and diammonium salts of EDTA; barium, calcium, cobalt, copper, dysprosium, europium, iron, indium, lanthanum, magnesium, manganese, nickel, samarium, strontium, or zinc chelates of EDTA; trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid. acid monohydrate;N,N-bis(2-hydroxyethyl)glycine;1,3-diamino-2-hydroxypropane-N,N,N',N'-tetraacetic acid;1,3-diaminopropane-N,N,N',N'-tetraacetic acid;Ethylenediamine-N,N'-diacetic acid;Ethylenediamine-N,N'-dipropionic acid dihydrochloride;Ethylenediamine-N,N'-bis(methylenephosphonic acid) hemihydrate;N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid;Ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic acid) acid);O,O'-bis(2-aminoethyl)ethylene glycol-N,N,N',N'-tetraacetic acid;N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid;1,6-hexamethylenediamine-N,N,N',N'-tetraacetic acid;N-(2-hydroxyethyl)iminodiacetic acid;iminodiacetic acid;1,2-diaminopropane-N,N,N',N'-tetraacetic acid;nitrilotriacetic acid;nitrilotripropionic acid;trisodium salt of nitrilotris(methylene phosphoric acid);7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo[11,11,11]pentatriacontane hexahydrobromide;ortriethylenetetramine-N,N,N',N'',N''',N'''-hexaacetic acid.

[0118] Also contemplated are combination products comprising one or more modified oncolytic viruses disclosed herein and one or more other antimicrobial and antifungal agents (e.g., polyenes (e.g., amphotericin B, amphotericin B lipid complex (ABCD), liposomal amphotericin B (L-AMB), and liposomal nystatin), azoles and triazoles (e.g., voriconazole, fluconazole, ketoconazole, itraconazole, pozaconazole, and the like); glucan synthase inhibitors (e.g., caspofungin, micafungin (FK463), and V-echinocandin (LY303366)); griseofulvin; allylamines (e.g., terbinafine, and the like); flucytosine, or other antifungal agents, including those described herein). It is further contemplated that the peptides may be combined with topical antifungal agents (e.g., ciclopirox olamine, haloprogin, tolnaftate, undecylenate, topical nystatin, amorolfine, butenafine, naftifine, terbinafine, and other topical agents). In some instances, the pharmaceutical composition may include an additional agent. In some cases, the additional agent may be present in the pharmaceutical composition in a therapeutically effective amount.

[0119] Under ordinary conditions of storage and use, the pharmaceutical compositions described herein may contain a preservative to inhibit the growth of microorganisms. In certain instances, the pharmaceutical compositions described herein may be free of preservatives. Pharmaceutical forms suitable for injectable use may include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The pharmaceutical compositions may contain a carrier that is a solvent or dispersion medium, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and / or a vegetable oil, or any combination thereof. The proper fluidity can be maintained, for example, by the use of a coating (for example, lecithin), by maintaining the required particle size (in the case of dispersions), and by the use of surfactants. The activity of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to incorporate isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0120] For parenteral administration in aqueous solution, for example, the liquid dosage form may be suitably buffered, if necessary, and the liquid diluent may be made isotonic with sufficient saline or glucose. The liquid dosage form is particularly suitable for intravenous, intramuscular, subcutaneous, intratumoral, and intraperitoneal administration. In this regard, sterile aqueous media that may be used will be known to those of skill in the art in light of this disclosure. For example, a single dosage may be dissolved in 1 mL to 20 mL of isotonic NaCl solution and added to 100 mL to 1000 mL of liquid (e.g., sodium bicarbonate buffered saline) or injected into the proposed site of injection.

[0121] In certain embodiments, a sterile injectable solution can be prepared by blending the modified oncolytic poxvirus described herein or a pharmaceutical composition containing it in the required amount in a suitable solvent with various other ingredients as listed above, as needed, followed by filtered sterilization. In general, dispersions are prepared by blending various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the required other ingredients as listed above. The compositions disclosed herein can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. Upon formulation, the pharmaceutical compositions can be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective.

[0122] In certain embodiments, the pharmaceutical composition of the present disclosure may comprise an effective amount of the modified oncolytic poxvirus disclosed herein in combination with a pharma- ceutically acceptable carrier. "Pharmaceutically acceptable," as used herein, includes any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and / or is not toxic to the patient receiving administration. Non-limiting examples of suitable pharmaceutical carriers may include phosphate buffered saline solution, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, and sterile solutions. Additional non-limiting examples of pharma- ceutical compatible carriers may include gels, bioabsorbable matrix materials, implant elements containing the modified oncolytic virus, or any other suitable vehicle, delivery or dosing means or material. Such carriers may be formulated by conventional methods and administered to the subject in an effective amount. Manufacturing method

[0123] The modified oncolytic poxvirus of the present disclosure can be produced by methods known to those skilled in the art. In certain embodiments, the modified oncolytic virus can be grown in suitable host cells (e.g., HeLa, 293, or Vero cells), isolated from the host cells, and stored under conditions that promote the stability and integrity of the virus so as to minimize loss of infectivity over time. In some examples, the modified oncolytic virus that produces a high percentage of EEV particles can serve as an improved inoculum in the process for producing the oncolytic virus. In certain exemplary methods, the modified oncolytic poxvirus is grown in host cells using cell stacks, roller bottles, or perfusion bioreactors. In some examples, downstream methods for purifying the modified oncolytic virus can include filtration (e.g., depth filtration, tangential flow filtration, or a combination thereof), ultracentrifugation, or chromatographic capture. The modified oncolytic virus can be stored, for example, frozen or dried (e.g., by lyophilization). In certain embodiments, prior to administration, the stored modified oncolytic poxvirus may be reconstituted (if dried for storage) and diluted in a pharma- ceutically acceptable carrier for administration.

[0124] In some embodiments, the modified oncolytic poxviruses described herein exhibit higher titers in HeLa cells and 293 cells compared to an otherwise identical virus that does not include the modifications in the modified oncolytic virus. In certain instances, higher titers in HeLa cells and 293 cells are seen in the modified oncolytic poxviruses. Combination therapy

[0125] In certain embodiments, the method of the present disclosure comprises administering a modified oncolytic poxvirus disclosed herein or a pharmaceutical composition containing the same, followed by, preceded by, or combined with one or more additional therapies. Examples of the additional therapies may include, but are not limited to, chemotherapy, radiation, oncolytic virus therapy with additional viruses, treatment with immunomodulatory proteins, anti-cancer agents, or any combination thereof. The additional therapies may be given simultaneously or subsequent to the administration of the modified poxvirus (such as an oncolytic vaccinia virus). In certain embodiments, the method of the present disclosure may comprise administering a modified oncolytic virus disclosed herein, followed by, preceded by, or combined with one or more anti-cancer agents or cancer therapies. Anti-cancer agents may include, but are not limited to, chemotherapeutic agents, radiotherapeutic agents, cytokines, immune checkpoint inhibitors, anti-angiogenic agents, apoptosis inducers, anti-cancer antibodies, and / or anti-cyclin-dependent kinase agents. In certain embodiments, the cancer therapy may include chemotherapy, biological therapy, radiation therapy, immunotherapy, hormonal therapy, antivascular therapy, cryotherapy, toxin therapy, and / or surgery, or a combination thereof. In certain embodiments, the method of the present disclosure may include administering a modified virus disclosed herein, followed by, preceded by, or combined with a modified oncolytic virus of the present disclosure. The combination of the modified oncolytic poxvirus (such as the modified vaccinia virus) and chemotherapy achieves a synergistic effect not seen in modified oncolytic viruses that do not include the modifications in the modified oncolytic virus. The synergistic effect of the combination may be advantageously used to reduce the dose of chemotherapy (such as Taxol®). Thus, the treatment method disclosed herein (with the modified virus) may reduce the toxicity associated with chemotherapy (e.g., patients who respond to chemotherapy but suffer from side effects at therapeutic doses). The synergistic effect may result in reduced tumor growth in certain cases compared to chemotherapy alone or oncolytic vaccinia virus alone.The reduction in tumor growth can be, for example, about 2% to about 50% (eg, about 5%, about 10%, about 20%, about 25%, about 35%, about 45%, or about 50%, etc.).

[0126] In certain embodiments, treatment with modified oncolytic poxviruses (such as vaccinia viruses) can be used alone or in combination with one or more immunomodulatory agents. Immunomodulatory agents can include any compound, molecule, or substance capable of suppressing antiviral immunity associated with tumors or cancer. In certain embodiments, the immunomodulatory agent can suppress innate or adaptive immunity against the modified virus. Non-limiting examples of immunomodulatory agents include anti-CD33 antibodies or variable regions thereof (also referred to herein as antigen-binding fragments thereof), anti-CD11b antibodies or variable regions thereof (also referred to herein as antigen-binding fragments thereof), COX2 inhibitors (e.g., celecoxib), cytokines (e.g., IL-12, GM-CSF, IL-2, IFN3, and IFNγ, etc.), and chemokines (e.g., MIP-1, MCP-1, and IL-8, etc.). In certain embodiments, the immunomodulatory agent may include immune checkpoint modulators, such as, but not limited to, anti-CTLA4, anti-PD-1, and anti-PD-L1, and TLR agonists (e.g., poly I:C). In some examples, the immunomodulatory agent may include immune checkpoint inhibitors, such as, for example, antagonists of PD-1 (e.g., antagonist antibodies that bind to PD-1), antagonists of PD-L1 (e.g., antagonist antibodies that bind to PD-L1), antagonists of CTLA-4 (e.g., antagonist antibodies that bind to CTLA-4), antagonists of A2AR (e.g., antagonist antibodies that bind to A2AR), antagonists of B7-H3 (e.g., antagonist antibodies that bind to B7-H3), and the like. ), antagonists of B7-H4 (e.g., an antagonist antibody that binds to B7-H4), antagonists of BTLA (e.g., an antagonist antibody that binds to BTLA), antagonists of IDO (e.g., an antagonist antibody that binds to IDO), antagonists of KIR (e.g., an antagonist antibody that binds to KIR), antagonists of LAG3 (e.g., an antagonist antibody that binds to LAG3), antagonists of TIM-3 (e.g., an antagonist antibody that binds to TIM3), and the like.In some embodiments, the additional therapy may include administering an immune checkpoint regulator. In one example, the immune checkpoint regulator may be TGN1412. In one example, the immune checkpoint regulator may be NKTR-214. In one example, the immune checkpoint regulator may be MEDI0562. In one example, the immune checkpoint regulator may be MEDI6469. In one example, the immune checkpoint regulator may be MEDI6383. In one example, the immune checkpoint regulator may be JTX-2011. In one example, the immune checkpoint regulator may be Keytruda (pembrolizumab). In one example, the immune checkpoint regulator may be Opdivo (nivolumab). In one example, the immune checkpoint regulator may be Yervoy (ipilimumab). In one example, the immune checkpoint regulator may be tremelimumab. In one example, the immune checkpoint regulator can be Tecentriq (atezolizumab). In one example, the immune checkpoint regulator can be MGA271. In one example, the immune checkpoint regulator can be indoximod. In one example, the immune checkpoint regulator can be epacadostat. In one example, the immune checkpoint regulator can be lirilumab. In one example, the immune checkpoint regulator can be BMS-986016. In one example, the immune checkpoint regulator can be MPDL3280A. In one example, the immune checkpoint regulator can be avelumab. In one example, the immune checkpoint regulator can be durvalumab. In one example, the immune checkpoint regulator can be MEDI4736. In one example, the immune checkpoint regulator can be MEDI4737. In one example, the immune checkpoint regulator can be TRX518.In one example, the immune checkpoint regulator can be MK-4166. In one example, the immune checkpoint regulator can be urelumab (BMS-663513). In one example, the immune checkpoint regulator can be PF-05082566 (PF-2566).

[0127] In certain instances, when the additional therapy is radiation, an exemplary dose is 5,000 rads (50 Gy) to 100,000 rads (1000 Gy), or 50,000 rads (500 Gy), or other suitable doses within the ranges. Alternatively, the dose may be about 30 to 60 Gy, about 40 to about 50 Gy, about 40 to 48 Gy, or about 44 Gy, or other suitable doses within the ranges, the dose being determined, for example, by the dosimetry methods described above. As used herein, "Gy" may refer to a unit for a specific absorbed dose of radiation, equal to 100 rads. Gy is an abbreviation for "Gray."

[0128] In certain instances, when the additional therapy is chemotherapy, exemplary chemotherapeutic agents may include, but are not limited to, alkylating agents (e.g., nitrogen mustard derivatives, ethyleneimines, alkylsulfonates, hydrazines and triazines, nitrosureas, and metal salts), plant alkaloids (e.g., vinca alkaloids, taxanes, podophyllotoxins, and camptothecan analogs), antitumor antibiotics (e.g., anthracyclines, chromomycins, etc.), antimetabolites (e.g., folate antagonists, pyrimidine antagonists, purine antagonists, and adenosine deaminase inhibitors), topoisomerase I inhibitors, topoisomerase II inhibitors, and various antineoplastic agents (e.g., ribonucleotide reductase inhibitors, corticosteroid inhibitors, enzymes, microtubule inhibitors, and retinoids). Exemplary chemotherapeutic agents include, but are not limited to, anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (Bi CNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyte®), dacarbazine (DTIC-Dome®), dactinomycin (ActinomycinD, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposomal injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Bepcid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®), ), Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycitidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), (Trademark), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Prinetol®), methotrexate (Folex®), mitoxantrone (Novantron®), Mylotarg, paclitaxel (Taxol®), phoenix (Yttrium-90 / MX-DTPA), pentostatin, carmustine implant (Gliadel®), polypheprosan 20, citrate These may include tamoxifen (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®), ibrutinib, idelalisib, and brentuximab vedotin.

[0129] Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, and triazenes), i.e., uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil Nitrogen Mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxa), , Procytox, Revimmune™), ifosfamide (Mitoxana), melphalan (Alkeran), chlorambucil (Leukeran), pipobroman (Amedel, Vercyte), triethylenemelamine (Hemel, Hexalen, Hexastat), triethylenethiophosphoramine, temozolomide (Temodar), thiotepa (Thioplex), busulfan (Busilvex, Myleran), carmustine (BiCNU), lomustine (CeeNU), streptozocin (Zanosar), and dacarbazine (DTIC-Dome).Additional exemplary alkylating agents include, but are not limited to, oxaliplatin (Eloxatin®); temozolomide (Temodar® and Temodar®); dactinomycin (Actinomycin D, also known as Cosmegen®); melphalan (L-PAM, L-melphalan, and phenylalanine mustard, also known as Alkeran®); altretamine (hexamethylmelamine (HMM), also known as Hexalen®) known as); carmustine (BiCNU®); bendamustine (Treanda®); busulfan (Busulfex® and Myleran®); carboplatin (Paraplatin®); lomustine (also known as CCNU, CeeNU®); cisplatin (also known as CDDP, Platinol®, and Platinol®-AQ); chlorambucil (Leukeran®); cyclophosphamide (Citroxane®); Sun® and Neosar®; dacarbazine (also known as DTIC, DIC, and imidazole carboxamide, DTIC-Dome®); altretamine (also known as hexamethylmelamine (HMM), Hexalen®); ifosfamide (IFEX®); Prednumustine; procarbazine (Matulane®); mechlorethamine (nitrogen mustard, mustine, and mechlorethamine hydrochloride ( mechloroethamine), also known as Mustargen®; streptozocin (Zanosar®); thiotepa (thiophosphoamide, TESPA, and TSPA, also known as Thioplex®); cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and bendamustine hydrochloride (Treanda®).

[0130] Exemplary anthracyclines may include, but are not limited to, for example, doxorubicin (Adriamycin® and Rubex®); bleomycin (Lenoxane®); daunorubicin (daunorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposome (daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (idamycin®, idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and desacetyl ravidomycin.

[0131] Exemplary vinca alkaloids may include, but are not limited to, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®), and vindesine (Eldisine®); vinblastine (also known as vinblastine sulfate, vincaleukoblastine, and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®).

[0132] Exemplary proteasome inhibitors include, but are not limited to, bortezomib (VELCADE®); carfilzomib (PX-171-007, (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenyl butanamide)-pentanamide); marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[0133] "In combination with" as used herein means that the modified poxvirus (e.g., an oncolytic vaccinia virus described herein) or pharmaceutical composition comprising the same and the additional therapy (e.g., an additional therapy comprising one or more drugs) are given to a subject as part of a treatment regimen or plan. In certain embodiments, being used in combination does not require that the modified oncolytic virus and the one or more drugs are physically combined prior to administration or that they are administered at the same time. For example, but not limited to, the modified oncolytic virus and the one or more drugs may be administered together to the subject undergoing treatment, or may be administered simultaneously or sequentially in any order or at different times.

[0134] The additional therapy may be administered in various embodiments in a liquid dosage form, a solid dosage form, a suppository, an inhalable dosage form, an intranasal dosage form, a liposomal formulation, a nanoparticle-containing dosage form, a microparticle-containing dosage form, a polymeric dosage form, or any combination thereof. In certain embodiments, the additional therapy may be administered for about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks, or about 52 weeks, or longer. The frequency of administration of the additional therapy may be, in certain instances, once a day, twice a day, once a week, once every 3 weeks, once every 4 weeks (or once a month), once every 8 weeks (or once every 2 months), once every 12 weeks (or once every 3 months), or once every 24 weeks (or once every 6 months). In certain embodiments, the method of treating a subject with cancer may comprise administering to the subject an effective amount of a modified oncolytic poxvirus (such as a modified oncolytic vaccinia virus of the present disclosure). In certain embodiments, the method of the present disclosure may further comprise administering to the subject an effective amount of one or more agents. For example, but not limited to, the agent may be an anti-cancer agent, an immunomodulatory agent, or any combination thereof, as described above. kit

[0135] In an embodiment, the present disclosure provides a kit for administering a modified oncolytic poxvirus (such as a modified oncolytic vaccinia virus described herein). In certain embodiments, the kit of the present disclosure may include a modified oncolytic poxvirus (such as a modified oncolytic vaccinia virus described herein or a pharmaceutical composition comprising a modified oncolytic vaccinia virus). In certain embodiments, the kit of the present disclosure may further include one or more components, such as instructions for use, devices and additional reagents, and components such as tubes, containers, and syringes for carrying out the methods disclosed above. In certain embodiments, the kit of the present disclosure may further include one or more agents (such as at least one of an anti-cancer agent, an immunomodulator, or any combination thereof) that can be administered in combination with the modified virus.

[0136] In certain embodiments, the kit of the present disclosure may include one or more containers containing the modified viruses disclosed herein.For example, but not limited to, the kit of the present disclosure may include one or more containers containing the modified oncolytic viruses of the present disclosure.

[0137] In certain embodiments, the kit of the present disclosure may include instructions for use, a device for administering the modified oncolytic virus to a subject, or a device for administering additional drugs or compounds to a subject.For example, but not limited to, the instructions may include a description of the modified oncolytic virus, and optionally other components included in the kit, and a method for administration, including a method for determining the appropriate condition of the subject, the appropriate dosage, and the appropriate administration method for administering the modified virus.The instructions may also include guidance for monitoring the subject over the duration of treatment.

[0138] In certain embodiments, the kit of the present disclosure may include a device for administering the modified oncolytic virus to a subject. Any of a variety of devices known in the art for administering medical drugs and pharmaceutical compositions may be included in the kit described herein. Such devices include, but are not limited to, hypodermic needles, intravenous needles, catheters, needleless injection devices, inhalers, and liquid dispensers (e.g., eye droppers, etc.). In certain embodiments, the modified oncolytic virus that is delivered systemically (e.g., by intravenous injection, intratumoral injection, intraperitoneal injection) may be included in the kit along with a hypodermic needle and a syringe. EXAMPLES

[0139] The following examples further illustrate the described embodiments, but do not limit the scope of the disclosure.

[0140] Example 1: Preparation and characterization of modified oncolytic vaccinia viruses, including WO34

[0141] This study identified and characterized a new mutation in vaccinia virus (WR strain) that increases the release of EEV particles from the virus, which may increase the oncolytic potential of the virus (e.g., by delivery and spread through the bloodstream) and may also provide advantages for other therapeutic uses of vaccinia (e.g., as a vaccine).

[0142] Random mutagenesis of the WR A34R gene was performed, and the resulting vaccinia virus strains (containing various mutations in the A34R gene) were screened for EEV particle production to identify strains containing mutations that enhance high-level EEV production / release. Strains containing the K151E mutation along with an additional point mutation (K119E) in A34R were identified. In the case of strains containing double mutations (K119E and K151E) in A34R (the WO34), a significant increase in the level of EEV particle production was observed. Additional studies performed showed that oncolytic strains containing the WO34 (A34R double mutations K119E and K151E) exhibited enhanced therapeutic effects in cancer treatment in mouse models. Screening method for identifying WO34

[0143] Random mutations in the viral gene VACWR157 (herein also referred to as the A34R gene encoding the A34R protein) were performed using PCR. The A34R open reading frame (ORF) was amplified using Taq polymerase in the presence of the nucleotide analogs 8-oxo-dGTP and dPTP. The 5' and 3' regions of VACWR157 were also amplified directly by PCR. The latter was assembled with a GFP reporter (GFP at the 5' end), also by PCR. Finally, the 5' mutated VACWR157 and GFP-3' fragments were all assembled by PCR, taking advantage of the short complementary region between the fragments, which was added by the PCR primers. A simplified assembly scheme is shown in Figure 1 (overhanging promoters and primers are not shown).

[0144] The fully assembled fragment containing the mutated VACWR157 and GFP flanked by the 5' and 3' regions of vaccinia virus DNA was purified and used to transfect 143B human osteosarcoma cells. The transfected cells were then infected with vaccinia virus and induced by recombination to generate the mutated VACWR157 virus library. To preferentially select for mutants with enhanced EEV production, medium from flasks infected with the virus pool was added to uninfected flasks. Clonal plaques were isolated after several rounds of successive infections, and mutations in A34R that enhanced EEV production were characterized by Sanger sequencing.

[0145] In all isolated clones, two missense mutations, K119E and K151E, and one silent mutation at F129 (SEQ ID NO:2) were commonly observed. This mutant protein was designated WO34 (SEQ ID NO:2) and one corresponding clone was assigned UID WO0064R.002. The GFP reporter in this initial cohort of clones is not flanked by loxP sites and cannot be removed. A codon-scrambled ORF encoding WO34 was synthesized and used to clone a transfer vector (pWR157-KE.R) containing loxP-flanked GFP. New recombinant viruses encoding WO34 were selected from fluorescent plaques and then used to infect Cre recombinase-expressing cells. Clones lacking the reporter were selected after Cre treatment. Table 1: A34R gene and protein sequence [Table 1-1] [Table 1-2] Viral plaque comet tails formed by EEV-enhanced vaccinia viruses containing WO34

[0146] The pre-titered EEV-enhanced vaccinia virus was diluted and plated on confluent BS-C-40 cells in 6-well plates. The plates were incubated and left undisturbed for 48 hours, and then the cells were stained with crystal violet to examine differences in comet tail formation due to abnormal EEV production. The results are shown in Figure 2. As shown in Figure 2, vaccinia virus strains containing WO34 showed enhanced comet tail formation compared to WI strain (A34R single mutation in WR strain) and WR strain (non-mutated A34R). The comet tail formation of vaccinia virus strains containing WO34 was visibly increased. Neutralization of EEV-enhanced vaccinia viruses, including WO34

[0147] In two separate experiments using different strains of vaccinia virus (WO34-containing vaccinia virus, WI strain, IHD-J strain, and WR strain), HeLa cells were grown to confluence in 6-well plates and infected with 1 MOI (multiplicity of infection) of vaccinia virus strains. After 24 hours, 1 mL of culture medium was withdrawn and centrifuged at 800 g. Approximately 500 μl of the supernatant was then removed and used for viral plaque assay. During the serial dilution, the samples were treated with anti-L1 NR-45114 antibody or anti-VIG antibody and incubated at 37° C. for 1 hour. The dilutions were then added to confluent 6-well plates of BS-C-40 cells for plaque assay. After 1.5 hours, the medium was replaced with CM10 with 3% CMC. After 48 hours, cells were stained with crystal violet and viral plaques were counted to determine the viral titer in the HeLa cell supernatant and the blocking capacity of neutralizing antibodies, as shown in Figures 3A-3B (Figure 3A shows viral plaque titers following treatment with the anti-L1 NR-45114 antibody, and Figure 3B similarly shows viral plaque titers following treatment with the anti-L1R and VIG antibodies). Cell viability following infection with EEV-enhanced vaccinia virus containing WO34

[0148] In 96-well plates, HCT116 or MC38 cell lines were seeded and grown to 90% confluence. The cells were then infected with different vaccinia virus strains (WR, IHD-J, WI, and WO34-containing strains) at 1 MOI. Every 24 hours, cell viability was examined with the CellTiter 96 AQueous Non-Radioactive Cell Proliferation kit (Promega). Relative viability was calculated by subtracting the average blank value from all wells, calculating the average value of the uninfected control group, and then calculating the relative value of each infected well as (A490 / average of uninfected cells). The results are shown in Figure 4 (upper panel: MC38 cells; lower panel: HCT116 cells). Viral replication assay of EEV-enhanced vaccinia virus containing WO34 in cancer cells

[0149] In a separate experiment, HCT116 or MC38 cells were grown on 12-well plates to 90% confluence, and one plate was used per day in a 3-day replicate assay. All plates were infected with different vaccinia virus strains (WR, IHD-J, WI, WO34-containing strains) at 1 MOI on the same day. Every 24 hours after infection, one plate was frozen in a -80°C freezer. Plates were freeze-thawed twice to disrupt the cells, and the lysates were used for viral plaque assay on BS-C-40 cells. Plaque-forming units / ml are shown in Figure 5 (upper panel: HCT116 cells; lower panel: MC38 cells). In certain embodiments, for example, the following items are provided: (Item 1) A modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising at least two mutations, wherein the at least two mutations are at positions corresponding to Lys119 and Lys151 of the wild-type vaccinia virus A43R protein (sequence number 4). (Item 2) 2. The modified oncolytic poxvirus of item 1, wherein the mutation at the position corresponding to position Lys119 is Lys119Glu. (Item 3) 3. The modified oncolytic virus of item 1 or 2, wherein the mutation at the position corresponding to position Lys151 is Lys151Glu. (Item 4) 2. The modified oncolytic virus of item 1, wherein the at least two mutations at positions corresponding to positions Lys119 and Lys151 of the wild-type vaccinia virus A43R protein (SEQ ID NO: 4) are Lys119Glu and Lys151Glu, respectively. (Item 5) A modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising at least two non-naturally occurring mutations in amino acid residues that are positively charged at pH 5 in the wild-type A34R protein (sequence number 4). (Item 6) A modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof, the A34R protein comprising at least two non-naturally occurring mutations not located at position 110 of the wild-type A34R protein (SEQ ID NO: 4). (Item 7) A modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof, the A34R protein comprising at least two non-naturally occurring mutations at no aspartic acid residue in the wild-type A34R protein (SEQ ID NO: 4). (Item 8) A modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof, the A34R protein or a fragment thereof comprising at least two non-naturally occurring mutations in alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residues in the wild-type A34R protein (SEQ ID NO: 4), independently. (Item 9) A modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof, comprising a non-naturally occurring mutation in a lysine residue at a position other than Lys151 of the wild-type A34R protein (SEQ ID NO: 4). (Item 10) 13. A modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof, comprising a non-naturally occurring mutation at position Lys119 of the wild-type A34R protein (SEQ ID NO: 4). (Item 11) A modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising at least two non-naturally occurring mutations in amino acid residues that are positively charged at pH 5 in the wild-type A34R protein (sequence number 4), wherein the modified oncolytic poxvirus produces an increased number of comet tail-shaped plaques in a viral plaque formation assay compared to an otherwise identical oncolytic virus that does not comprise the at least two non-naturally occurring mutations. (Item 12) A modified oncolytic poxvirus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising at least two non-naturally occurring mutations, wherein when any of the non-naturally occurring mutations is at position 110 in the wild-type A34R protein (sequence number 4), the encoded amino acid is not an asparagine residue. (Item 13) A modified oncolytic poxvirus that exhibits increased resistance to neutralizing antibodies compared to a wild-type strain of oncolytic poxvirus, wherein the increased resistance is measured by the number of plaques produced by the modified oncolytic poxvirus or the wild-type strain in a viral plaque assay following treatment with an anti-L1 NR-45114 antibody or an anti-VIG antibody, producing at least about 55,000 plaque forming units / mL. (Item 14) A modified oncolytic poxvirus that produces at least about 55,000 plaque forming units / mL in a viral plaque assay following treatment with a neutralizing antibody. (Item 15) 11. The modified oncolytic poxvirus of item 10, wherein the neutralizing antibody is an anti-L1 NR-45114 antibody or an anti-VIG antibody. (Item 16) 11. The modified oncolytic poxvirus of item 9 or 10, wherein the A34R protein or fragment thereof further comprises a mutation at position Lys151 of the wild-type A34R protein (SEQ ID NO: 4). (Item 17) 2. The modified oncolytic poxvirus of item 1, wherein the amino acid residue that is positively charged at pH 5 is a lysine residue. (Item 18) 18. The modified oncolytic poxvirus of any one of items 5 to 12 and 16 to 17, wherein the nucleic acid comprises a nucleotide sequence or a fragment thereof that is at least about 80% homologous to the coding sequence in the viral gene VACWR157. (Item 19) 18. The modified oncolytic poxvirus of any one of items 5 to 12 and 16 to 17, wherein the nucleic acid comprises a nucleotide sequence that is at least about 80% homologous to the nucleotide sequence set forth as SEQ ID NO:3. (Item 20) 20. The modified oncolytic poxvirus of any one of items 5 to 8, 11 to 12, and 17 to 19, wherein at least one of the two non-naturally occurring mutations is at position Lys119 of the wild-type A34R protein (SEQ ID NO: 4). (Item 21) 20. The modified oncolytic poxvirus of any one of items 9 and 18 to 19, wherein the non-naturally occurring mutation is at position Lys119 of the wild-type A34R protein (SEQ ID NO: 4). (Item 22) 22. The modified oncolytic poxvirus of item 10 or 21, wherein the mutation at position Lys119 of the wild-type A34R protein (SEQ ID NO: 4) is Lys119Glu. (Item 23) 23. The modified oncolytic poxvirus of any one of items 5 to 8, 11 to 12, 17 to 19, and 20 to 22, wherein at least one of the two non-naturally occurring mutations is at position Lys151 of the wild-type A34R protein (SEQ ID NO: 4). (Item 24) 24. The modified oncolytic poxvirus of item 23, wherein the mutation at position Lys151 of the wild-type A34R protein (SEQ ID NO: 4) is Lys151Glu. (Item 25) An engineered oncolytic poxvirus expressing the A34R protein containing the mutations Lys119Glu and Lys151Glu. (Item 26) 24. The modified oncolytic poxvirus of any one of items 19 to 23, wherein positions 305-307 of SEQ ID NO: 3 comprise the nucleotides GAA or GAG. (Item 27) 27. The modified oncolytic poxvirus of any one of items 19 to 23 and 26, wherein positions 451-453 of SEQ ID NO:3 comprise the nucleotides GAA or GAG. (Item 28) 28. The modified oncolytic poxvirus of any one of items 5 to 8, 11 to 12, and 17 to 27, wherein said modified oncolytic poxvirus produces a greater amount of extracellular enveloped virus forms than intracellular mature virus forms compared to an otherwise identical oncolytic virus not comprising said at least two non-naturally occurring mutations. (Item 29) 23. The modified oncolytic virus of any one of items 9-10, 16, and 21-22, wherein the modified oncolytic poxvirus produces a greater amount of extracellular enveloped viral form than intracellular mature viral form compared to an otherwise identical oncolytic virus that does not contain the non-naturally occurring mutation. (Item 30) 30. The modified oncolytic poxvirus of any one of items 1 to 29, further comprising an exogenous nucleic acid encoding at least one therapeutic or diagnostic protein. (Item 31) 31. The modified oncolytic poxvirus of item 30, wherein the exogenous nucleic acid can encode at least one of a chemokine receptor, a membrane-associated protein, a microbial protein capable of degrading hyaluronan, a microbial protein, SOCS3, PH-20, HMGB1, PIAS3, IL15, IL15-Rα, LIGHT, ITAC, fractalkine, CCL5, N1L, an immune checkpoint modulator, a metabolic regulatory protein, or any combination thereof. (Item 32) 32. The modified oncolytic poxvirus of item 31, comprising the exogenous nucleic acid encoding a chemokine receptor, the chemokine receptor comprising at least one of CXCR4 and CCR2. (Item 33) 33. The modified oncolytic poxvirus of item 31 or 32, comprising said exogenous nucleic acid encoding said membrane-associated protein. (Item 34) 34. The modified oncolytic poxvirus of item 33, wherein the membrane-associated protein comprises a membrane-associated hyaluronidase. (Item 35) 35. The modified oncolytic poxvirus of item 34, wherein the membrane-bound hyaluronidase comprises PH-20. (Item 36) 36. The modified oncolytic poxvirus of item 35, wherein the PH-20 is a GPI anchor. (Item 37) 37. The modified oncolytic virus of any one of items 31 to 36, comprising the exogenous nucleic acid encoding the microbial protein capable of degrading hyaluronan, wherein the microbial protein comprises a secreted hyaluronidase. (Item 38) 38. The modified oncolytic poxvirus of item 37, wherein the secreted hyaluronidase comprises at least one of HysA, lin, sko, and rv, or any combination thereof. (Item 39) 39. The modified oncolytic poxvirus of any one of items 31 to 38, comprising said exogenous nucleic acid encoding said microbial protein. (Item 40) 44. The modified oncolytic poxvirus of item 43, wherein the microbial protein comprises HysA. (Item 41) 41. The modified oncolytic poxvirus of any one of items 1 to 40, further comprising a modification in the genome of the virus, wherein the modification comprises a mutation or deletion of the B5R gene. (Item 42) 42. The modified oncolytic poxvirus of item 41, comprising the modification in the genome of the virus, wherein the modification comprises a mutation or deletion in an SCR region of the B5R gene, wherein the SCR region comprises SCR1, SCR3, SCR4, or any combination thereof, and wherein the SCR region does not comprise SCR2. (Item 43) Thymidine kinase (TK), B8R, B18R, B15R, K7R, C6L, K4L, F8L, F9L, F10L, F17R, E1L, E4L, E6R, E8R, E10R, E11L, O2L, I1L, I2L, I3L, I5L, I7L, I8R , G1L, G3L, G4L, G5.5R, G7L, G9R, L1R, L3L, L4R, L5R, J1R, J4R, J6R, H1L, H2R, H3L, H4L, H5R, H6R, D1R, D2L, D3R, D6R, D7R, D8L, D11L, D12 43. The modified oncolytic poxvirus of any one of items 1 to 42, further comprising a mutation or deletion in a viral gene selected from the group consisting of: L, D13L, A2.5L, A3L, A4L, A5R, A6L, A7L, A9L, A10L, A13L, A14L, A15L, A16L, A17L, A18R, A21L, A24R, A25L, A26L, A27L, A28L, A29L, A30L, A31R, A34R, A42R, A45R, A46R, A52R, and any combination thereof. (Item 44) 44. The modified oncolytic poxvirus of item 43, comprising a mutation or deletion of the viral gene A52R. (Item 45) 45. The modified oncolytic poxvirus according to any one of items 31 to 44, comprising: (i) the exogenous nucleic acid encoding a chemokine receptor, the chemokine receptor comprising at least one of CXCR4 and CCR2; (ii) the exogenous nucleic acid encoding PIAS3; (iii) a mutation or deletion in the thymidine kinase gene; and (iv) a mutation or deletion in the A52R gene. (Item 46) 46. ​​The modified oncolytic poxvirus of any one of items 1 to 45, wherein the virus is suitable for systemic delivery. (Item 47) 47. The modified oncolytic poxvirus of any one of items 1 to 46, wherein the virus is capable of immune evasion. (Item 48) 48. The modified oncolytic poxvirus of item 46 or 47, wherein the systemic delivery comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof. (Item 49) 50. The modified oncolytic poxvirus of item 48, wherein said parenteral administration comprises intravenous injection. (Item 50) 50. The modified oncolytic poxvirus of any one of items 1 to 49, wherein the virus is suitable for intratumoral delivery. (Item 51) 51. The modified oncolytic poxvirus of any one of items 1 to 50, wherein said poxvirus is a vaccinia virus. (Item 52) A process for engineering an oncolytic poxvirus, comprising: (i) obtaining an oncolytic poxvirus DNA backbone vector, the oncolytic poxvirus DNA backbone vector comprising one or more modifications as described in any one of the preceding items; (ii) further modifying the oncolytic virus DNA vector to generate an engineered DNA vector; (iii) transfecting a mammalian cell with the engineered DNA vector; (iv) culturing the mammalian cell under conditions suitable for viral replication; and (v) recovering the viral particles. (Item 53) 53. The process of item 52, wherein the mammalian cells comprise HeLa cells, 293 cells, A549 cells, or Vero cells. (Item 54) 54. A kit comprising the oncolytic poxvirus according to any one of items 1 to 53, a container; and instructions for administering the oncolytic virus to a subject to treat a disorder associated with pathological angiogenesis. (Item 55) 55. A method for treating a tumor, comprising administering to a subject a therapeutically effective amount of an oncolytic poxvirus according to any one of items 1 to 54. (Item 56) A method for treating a tumor, comprising administering to a subject a composition comprising white blood cells from a patient infected with a modified oncolytic poxvirus expressing an A34R protein containing mutations at positions 119 and 151 of the wild-type A34 protein (sequence number 4), wherein the modified oncolytic poxvirus generates a population of viral particles in the tumor microenvironment. (Item 57) 57. The method of item 56, wherein the patient-derived white blood cells comprise macrophages. (Item 58) 57. The method of item 56, wherein the patient-derived white blood cells comprise tumor-targeting T cells. (Item 59) 57. The method of item 56, wherein at least about 10% to at least about 90% of the population of viral particles are EEV particles as measured by viral plaque assay. (Item 60) 60. The method of any one of items 56 to 59, further comprising recovering the EEV particles from the tumor microenvironment and intravenously administering the EEV particles to the subject. (Item 61) 61. The method of any one of items 56 to 60, wherein the modified oncolytic poxvirus is a modified oncolytic vaccinia virus. (Item 62) A process comprising infecting a culture of host cells with a population of a modified oncolytic poxvirus comprising at least about 10% to at least about 90% EEV particles, wherein the modified oncolytic poxvirus expresses an A34R protein comprising mutations at positions 119 and 151 of the wild-type A34 protein (SEQ ID NO: 4). (Item 63) 63. The process of item 62, wherein the modified oncolytic poxvirus is a modified oncolytic vaccinia virus. (Item 64) A method for treating cancer, comprising administering to a patient a modified oncolytic virus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising at least two mutations, the at least two mutations being at positions corresponding to Lys119 and Lys151 of the wild-type vaccinia virus A43R protein (sequence number 4). (Item 65) 65. The method of item 64, wherein the at least two mutations at positions corresponding to positions Lys119 and Lys151 of the wild-type vaccinia virus A43R protein (SEQ ID NO: 4) are Lys119Glu and Lys151Glu, respectively. (Item 66) A method for treating a tumor, comprising administering to a patient a modified oncolytic virus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising at least two mutations, the at least two mutations being at positions corresponding to Lys119 and Lys151 of the wild-type vaccinia virus A43R protein (sequence number 4). (Item 67) 67. The method of item 66, wherein the at least two mutations at positions corresponding to positions Lys119 and Lys151 of the wild-type vaccinia virus A43R protein (SEQ ID NO: 4) are Lys119Glu and Lys151Glu, respectively. (Item 68) 68. The method of any one of items 64 to 67, wherein the administration is by intratumoral injection, intravenous injection, or a combination thereof. (Item 69) 62. The method of any one of items 55-61, wherein said administering is by intratumoral injection, intravenous injection, or a combination thereof. (Item 70) 70. The method of item 68 or 69, comprising administering an additional therapy in combination with said oncolytic poxvirus, said additional therapy comprising at least one of chemotherapy, radiation therapy, oncolytic virus therapy with an additional virus, treatment with an immunomodulatory protein, CAR T cell therapy, an anticancer agent, an immunomodulatory agent, or any combination thereof. (Item 71) 71. The method of item 70, wherein the additional therapy comprises the immunomodulatory agent selected from the group consisting of an anti-CD33 antibody or antigen-binding fragment thereof, an anti-CD11b antibody or antigen-binding fragment thereof, a COX2 inhibitor, a cytokine, a chemokine, an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, and a TLR agonist.

Claims

1. An oncolytic virus comprising a nucleic acid encoding an A34R protein or a fragment thereof comprising at least two mutations in amino acid residues that are positively charged at pH 5, wherein the A34R protein is mutated compared to a sequence in a wild-type A34R protein (SEQ ID NO: 4), and the at least two mutations are at positions corresponding to positions Lys119 and Lys151 in the wild-type vaccinia virus A34R protein (SEQ ID NO: 4), and are Lys119Glu and Lys151Glu, respectively.

2. The oncolytic virus of claim 1, wherein the oncolytic virus produces a greater amount of extracellular enveloped virus forms than intracellular mature virus forms compared to an otherwise identical oncolytic virus that does not contain the at least two mutations.

3. The oncolytic virus of claim 1 or claim 2, further comprising an exogenous nucleic acid, wherein the exogenous nucleic acid may encode at least one of CXCR4, CCR2, hyaluronidase, SOCS3, PH-20, HMGB1, PIAS3, IL15, IL15-Rα, LIGHT, ITAC, fractalkine, CCL5, N1L, an immune checkpoint modulator, or any combination thereof.

4. The oncolytic virus of any one of claims 1 to 3, further comprising a mutation or deletion of the viral gene A52R.

5. The oncolytic virus according to any one of claims 1 to 4, which is a poxvirus.

6. The oncolytic virus of claim 5 , wherein the poxvirus is a vaccinia virus.

7. A composition comprising an oncolytic virus according to any one of claims 1 to 6 for the treatment of cancer.

8. The composition of claim 7 , wherein a therapeutically effective amount of the composition is administered to a subject.

9. The composition of claim 7 or 8, which is administered by intratumoral injection, intravenous injection, or a combination thereof.

10. 10. The composition of any one of claims 7-9, wherein the composition is administered in combination with an additional therapy, wherein the additional therapy comprises at least one of chemotherapy, radiation therapy, oncolytic virotherapy with an additional virus, treatment with an immunomodulatory protein, CAR T cell therapy, an anti-cancer agent, an immunomodulatory agent, or any combination thereof.

11. 11. The composition of claim 10, wherein the additional therapy comprises the immunomodulatory agent selected from the group consisting of an anti-CD33 antibody or antigen-binding fragment thereof, an anti-CD11b antibody or antigen-binding fragment thereof, a COX2 inhibitor, a cytokine, a chemokine, an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, and a TLR agonist.

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