Modified extracellular enveloped viruses

Abstract

The present disclosure provides modified oncolytic poxviruses, such as vaccinia viruses, which may contain modifications in the viral genome that increase the production of the extracellular envelope form of the virus. The modified oncolytic poxviruses can be used as vectors for systemic delivery. Methods of using the modified oncolytic poxviruses are also provided.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 916,035, filed October 16, 2019, which is incorporated herein by reference in its entirety.

[0003] Incorporated by Reference

[0004] All publications, patents, patent applications, and NCBI accession numbers mentioned in this specification are incorporated herein 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 set forth in its entirety. In the event of a conflict between a term used herein and a term defined in an incorporated reference, the definitions in the present disclosure shall control.

[0005] Overview

[0006] 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 Lys119 and Lys151 of the wild-type vaccinia virus A34R 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 positions corresponding to positions Lys119 and Lys151 of the wild-type vaccinia virus A34R protein (SEQ ID NO.4) are Lys119Glu and Lys151Glu, respectively.

[0007] 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 located in amino acid residues that are positively charged at pH 5 within the 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 at least two non-naturally occurring mutations, wherein the at least two non-naturally occurring mutations are not at position 110 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 at least two non-naturally occurring mutations, wherein the at least two non-naturally occurring mutations are not at aspartic acid residues within the 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, wherein the at least two non-naturally occurring mutations are independently located at alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine residues within the wild-type A34R protein (SEQ ID No. 4).

[0011] 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 a lysine residue other than position Lys151 of the wild-type A34R protein (SEQ ID No. 4).

[0012] 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 the wild-type A34R protein (SEQ ID No. 4).

[0013] 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 the at least two non-naturally occurring mutations are located in amino acid residues that are positively charged at pH 5 within the wild-type A34R protein (SEQ ID No. 4), wherein the modified oncolytic poxvirus produces an increased number of comet tail 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.

[0014] 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 within the wild-type A34R protein (SEQ ID No. 4), the encoded amino acid is not an asparagine residue.

[0015] Another embodiment provides a modified oncolytic poxvirus that exhibits increased resistance to neutralizing antibodies compared to a wild-type strain of an 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 after treatment with an anti-L1 NR-45114 antibody or an anti-VIG antibody, and wherein the modified oncolytic poxvirus produces at least about 55,000 plaque forming units / mL.

[0016] Another embodiment provides a modified oncolytic poxvirus that produces at least about 55,000 plaque forming units / mL in a viral plaque assay after treatment with a neutralizing antibody.

[0017] 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 a 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 within 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 listed as SEQ ID No. 3. In some embodiments, at least one of the two non-naturally occurring mutations is located 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 located 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.

[0018] One embodiment provides a modified oncolytic poxvirus that expresses an A34R protein comprising mutations Lys119Glu and Lys151Glu.

[0019] In some embodiments, positions 305-307 of SEQ ID No.3 comprise nucleotides GAA or GAG. In some embodiments, positions 451-453 of SEQ ID No.3 comprise nucleotides GAA or GAG. In some embodiments, compared to an otherwise identical oncolytic virus that does not comprise at least two non-naturally occurring mutations, the modified oncolytic poxvirus produces a greater amount of extracellular enveloped virus forms than the intracellular mature virus form. In some embodiments, compared to an otherwise identical oncolytic virus that does not comprise a non-naturally occurring mutation, the modified oncolytic poxvirus produces a greater amount of extracellular enveloped virus forms than the intracellular mature virus form. In some embodiments, the modified oncolytic poxvirus further comprises an exogenous nucleic acid encoding at least one of a therapeutic protein or a diagnostic protein. In some embodiments, exogenous nucleic acid can encode at least one of the following: chemokine receptors, membrane-associated proteins, microbial proteins capable of degrading hyaluronic acid, microbial proteins, SOCS3, PH-20, HMGB1, PIAS3, IL15, IL15-Rα, LIGHT, ITAC, fractal chemokines, CCL5, N1L, immune checkpoint regulators, metabolic regulatory proteins, or any combination thereof, such as fusion proteins (such as metabolic regulatory proteins and cytokines) comprising any of the above combinations. In some embodiments, exogenous nucleic acid encodes chemokine receptors, wherein the chemokine receptors include at least one of CXCR4 and CCR2. In some embodiments, exogenous nucleic acid encodes membrane-associated proteins. In some embodiments, membrane-associated proteins include membrane-associated hyaluronidase. In some embodiments, membrane-associated hyaluronidase includes PH-20. In some embodiments, PH-20 is GPI-anchored. In some embodiments, exogenous nucleic acid encodes microbial proteins capable of degrading hyaluronic acid, wherein microbial proteins include 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 viral genome, wherein the modification comprises a mutation or deletion of the B5R gene. In some embodiments, the modified oncolytic poxvirus further comprises a modification in the viral genome, wherein the modification comprises a mutation or deletion in the 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 further comprises a mutation or deletion in a viral gene selected from the group consisting of: 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, In some embodiments, the modified oncolytic poxvirus comprises a mutation or deletion in the viral gene A52R. In some embodiments, the modified oncolytic poxvirus comprises (i) an exogenous nucleic acid encoding a chemokine receptor, wherein the chemokine receptor includes at least one of CXCR4 and CCR2; (ii) an exogenous nucleic acid encoding PIAS3; (iii) a mutation or deletion of a thymidine kinase gene; (iv) a mutation or deletion of an 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, systemic delivery includes oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof. In some embodiments, parenteral administration includes intravenous injection. In some embodiments, the virus is suitable for intratumoral delivery. In some embodiments, the poxvirus is a vaccinia virus.

[0020] One embodiment provides a method for engineering an oncolytic poxvirus, comprising: (i) obtaining an oncolytic poxvirus DNA backbone vector comprising one or more modifications described above; (ii) further modifying the oncolytic virus DNA vector to produce an engineered DNA vector; (iii) transfecting mammalian cells with the engineered DNA vector; (iv) culturing the mammalian cells under conditions suitable for viral replication; and (v) harvesting viral particles.

[0021] In some embodiments, the mammalian cells include HeLa cells, 293 cells, A549 cells, or Vero cells.

[0022] One embodiment provides a kit comprising: an oncolytic poxvirus, a container; and instructions for administering the oncolytic virus to a subject to treat a disorder associated with pathological angiogenesis.

[0023] One embodiment provides a method of treating a tumor, comprising administering to a subject a therapeutically effective amount of an oncolytic poxvirus.

[0024] One embodiment provides a method for treating a tumor, the method comprising administering to a subject a composition comprising patient-derived leukocytes infected with a modified oncolytic poxvirus, the modified oncolytic poxvirus expressing a mutated A34R protein comprising positions 119 and 151 of a wild-type A34R protein (SEQ ID No. 4), wherein the modified oncolytic poxvirus produces a population of viral particles in the tumor microenvironment. In some embodiments, the patient-derived leukocytes include macrophages. In some embodiments, the patient-derived leukocytes include tumor-targeted T cells. In some embodiments, at least about 10% to at least about 90% of the viral particle population are EEV particles, as measured in a viral plaque assay. In some embodiments, the method further comprises harvesting 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.

[0025] One embodiment provides a method comprising infecting a host cell culture with a modified oncolytic poxvirus population 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 A34R protein (SEQ ID No. 4). In some embodiments, the modified oncolytic poxvirus is a modified oncolytic vaccinia virus.

[0026] One embodiment provides 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, wherein the at least two mutations are located at positions corresponding to positions Lys119 and Lys151 of the wild-type vaccinia virus A34R 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 A34R protein (SEQ ID NO. 4) are Lys119Glu and Lys151Glu, respectively.

[0027] One embodiment provides 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, wherein the at least two mutations are located at positions corresponding to Lys119 and Lys151 of the wild-type vaccinia virus A34R protein (SEQ ID NO. 4). In some embodiments, the at least two mutations at positions corresponding to Lys119 and Lys151 of the wild-type vaccinia virus A34R protein (SEQ ID NO. 4) are Lys119Glu and Lys151Glu, respectively.

[0028] In some embodiments, administration is via intratumoral injection, intravenous injection, or a combination thereof. In some embodiments, administration is via intratumoral injection, intravenous injection, or a combination thereof.

[0029] 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 another virus, treatment with an immunomodulatory protein, CAR T cell therapy, an anticancer agent, an immunomodulatory agent, or any combination thereof.

[0030] In some embodiments, the additional therapy includes an immunomodulator selected from the group consisting of an anti-CD33 antibody or an antigen-binding fragment thereof, an anti-CD11b antibody or an antigen-binding fragment thereof, a COX2 inhibitor, a cytokine, a chemokine, an anti-CTLA4 antibody or an antigen-binding fragment thereof, an anti-PD-1 antibody or an antigen-binding fragment thereof, an anti-PD-L1 antibody or an antigen-binding fragment thereof, and a TLR agonist. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] 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 which illustrates illustrative embodiments utilizing the principles of the present disclosure, and the accompanying drawings, in which:

[0033] Figure 1 An exemplary assembly scheme for generating DNA for making a recombinant virus library is shown.

[0034] Figure 2 Shown is a comparison of viral plaque comet tails formed by different vaccinia virus strains.

[0035] Figure 3A-3B The results of neutralization assays using different vaccinia virus strains are shown ( Figure 3A The results after treatment with anti-L1 NR-45114 antibody are shown, and Figure 3BResults after treatment with anti-L1R and VIG antibodies are shown).

[0036] Figure 4 The results of cell viability after infection with different vaccinia virus strains are shown (upper panel: MC38 cells; lower panel: HCT116 cells).

[0037] Figure 5 Shown are the results of viral replication assays of different vaccinia virus strains in cancer cells (upper panel: HCT116 cells; lower panel: MC38 cells).

[0038] Detailed description

[0039] Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, changes, and substitutions will now occur 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 implementing the present disclosure. It is intended that the scope of the present disclosure be defined by the following claims, and that methods and structures within the scope of these claims and their equivalents be encompassed thereby.

[0040] Certain definitions

[0041] The terms used herein are for descriptive purposes only and are not intended to be limiting. Unless the context clearly dictates otherwise, as used herein, the singular forms "a," "an," and "the" may include the plural forms. Furthermore, to the extent that the terms "contains," "containing," "including," "includes," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0042] The term "about" or "approximately" can mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art (this will depend in part on how the value is measured or determined), for example, limitations of the measurement system. For example, depending on the practice of a given value, "about" can mean within 1 or more than 1 standard deviation. Where particular values are described in this application and claims, unless otherwise indicated, the term "about" should be considered to mean an acceptable error range for that particular value, such as ±10% of the value modified by the term "about."

[0043] The terms "individual," "patient," or "subject" are used interchangeably. None of these terms require or are limited to situations characterized by the supervision (e.g., continuous or intermittent) of a health care worker (e.g., a physician, registered nurse, nurse practitioner, physician assistant, orderlies, or hospice staff). In some embodiments, a patient, subject, or individual may be under the supervision of a health care worker.

[0044] As used herein, the terms "heterologous nucleic acid sequence" or "exogenous nucleic acid sequence" or "transgene" in relation to a particular virus can refer to a nucleic acid sequence that is derived from a source other than the specified virus.

[0045] As used herein, the term "mutation" may refer to deletion, insertion of a heterologous nucleic acid, inversion or substitution, including mutations that eliminate an open reading frame as generally understood in the art.

[0046] As used herein, the term "gene" may refer to a nucleic acid segment encoding a single protein or RNA (also referred to as a "coding sequence" or "coding region"), optionally together with associated regulatory regions such as a promoter, operator, terminator, etc., which may be located upstream or downstream of the coding sequence.

[0047] As used interchangeably herein, the terms "mutant virus" and "modified virus" may refer to a virus that comprises one or more mutations in its genome, including but not limited to deletions, insertions of heterologous nucleic acids, inversions, substitutions, or combinations thereof.

[0048] The term "naturally occurring" as used herein with respect to a virus may mean that the virus can be found in nature, ie, it can be isolated from a source in nature and has not been intentionally modified, eg, a wild-type virus.

[0049] As used herein with respect to one or more mutations in a viral nucleic acid sequence or in the amino acid sequence of a viral protein, the term "non-naturally occurring" can indicate that the viral strain contains one or more mutations that cannot be found in nature, i.e., it cannot be isolated from a source in nature and has been intentionally modified.

[0050] Reference herein to the terms "inhibit," "reduce," or "prevent," or any variation of these terms, may include any measurable decrease or complete inhibition to achieve the desired result.

[0051] As used herein, a "promoter" can be a control sequence in a nucleic acid sequence region that controls transcription initiation and transcription rate. In certain embodiments, a promoter can contain genetic elements that regulate proteins and molecules, such as RNA polymerase and other transcription factors, that can bind. The terms "operably positioned," "operably connected," "under control," and "under transcriptional control" can mean that a promoter is in the correct functional position and / or orientation relative to a nucleic acid sequence to control transcription initiation and / or expression of the sequence. In certain embodiments, a promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence that participates in the transcriptional activation of a nucleic acid sequence.

[0052] As used herein, the term "homology" can be a calculation of "homology" or "homology percentage" between two or more nucleotide or amino acid sequences, which can be determined by comparing sequences for optimal comparison purposes (e.g., introducing a gap in the sequence of the first sequence). The nucleotides at the corresponding positions can then be compared, and the identity percentage between the two sequences can be a function of the number of identical positions that the sequences have (i.e., the total number of positions x 100 of the number / position of % homology=identical positions). For example, if the position in the first sequence can be occupied by the nucleotides identical to the corresponding positions in the second sequence, these molecules are identical at this position. The homology percentage between the two sequences can be a function of the number of identical positions that the sequences have, wherein considering the number of gaps that need to be introduced for the optimal comparison of the two sequences and the length of each gap. In some embodiments, the length of the sequences aligned for comparison purposes can be at least about: 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the length of the reference sequence. The search can determine the homology between two sequences. Homology can be between the entire length of the two sequences or between portions of the entire length of the two sequences. The two sequences can be genes, nucleotide sequences, protein sequences, peptide sequences, amino acid sequences, or fragments thereof. The actual comparison of the two sequences can be accomplished by known methods, for example, using a mathematical algorithm. Non-limiting examples of such mathematical algorithms can be described in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90-5873-5877 (1993). As described in Altschul, S. et al., Nucleic Acids Res., 25: 3389-3402 (1997), such algorithms can be incorporated into NBLAST and XBLAST programs (version 2.0). When using BLAST and Gapped BLAST programs, any relevant parameters of the corresponding programs (e.g., NBLAST) can be used. For example, the parameters for sequence comparison can be set to score = 100, word length = 12, or can be varied (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 accomplished using, for example, the GAP program in the GCG software package (Accelrys, Cambridge, UK).

[0053] The term "subject" may refer to an animal, including but not limited to a primate (e.g., human), cow, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms "subject" and "patient" are used interchangeably herein when referring to, for example, a mammalian subject such as a human subject.

[0054] The terms "treat," "treating," and "treatment" may be intended to include alleviating or eliminating a disorder, disease, or condition, or one or more symptoms associated with the disorder, disease, or condition; or alleviating or eradicating the cause of the disorder, disease, or condition itself. Desirable therapeutic effects may include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis.

[0055] The term "therapeutically effective amount" may refer to an amount of a compound that, when administered, is sufficient to prevent the development of, or 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 that is sufficient to elicit the biological or medical response of a cell, tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor, or clinician.

[0056] The terms "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," "physiologically acceptable carrier," or "physiologically acceptable excipient" may refer to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. A component may be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It may also be suitable for use in contact with tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See Remington: The Science and Practice of Pharmacy, 21st ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th ed.; Rowe et al., eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash, eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson, ed., CRC Press LLC: Boca Raton, FL, 2004.

[0057] The term "pharmaceutical composition" may refer to a mixture of a compound disclosed herein with other chemical components such as a diluent or carrier. A pharmaceutical composition can facilitate administration of the compound to an organism. Various techniques for administering compounds exist in the art, including but not limited to oral, injection, aerosol, parenteral, and topical administration. Pharmaceutical compositions can also be obtained by reacting a compound with an inorganic or organic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

[0058] As used herein, "anticancer agent" may refer to a drug or therapy that can negatively affect cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cell, promoting an immune response against a cancer cell or tumor, preventing or inhibiting the progression of cancer, or extending the lifespan of a subject with cancer. Non-limiting examples of anticancer agents may include biologics (biotherapy), chemotherapeutic agents, and radiotherapeutic agents.

[0059] As used herein, the term "oncolysis" may refer to the killing of cancer cells or tumor cells by a pathogen, such as an oncolytic virus, such as an oncolytic poxvirus, such as an oncolytic vaccinia virus, for example, by stimulating an immune response to the cell, apoptosis, expression of toxic proteins, autophagy and shutdown of protein synthesis, induction of anti-tumor immunity, or any combination thereof. Direct lysis of cancer cells or tumor cells infected by pathogens such as oncolytic vaccinia viruses can be the result of the virus replicating within the cell. In certain instances, the term "oncolysis" may refer to the killing of cancer cells or tumor cells without lysing the cell.

[0060] As used herein, the term "oncolytic virus" may refer to a virus that preferentially infects and kills tumor cells. In certain non-limiting cases, it is understood that oncolytic viruses can promote anti-tumor responses through a dual mechanism that relies not only on the selective killing of tumor cells but also on the stimulation of the host anti-tumor immune response.

[0061] In some embodiments, oncolytic viruses may include, but are not limited to: (i) viruses that naturally replicate preferentially in cancer cells and are generally non-pathogenic in humans due to increased sensitivity to innate antiviral signal transduction or dependence on oncogenic signal transduction pathways; and (ii) viruses that have been genetically manipulated for use. In some embodiments, oncolytic viruses may include herpes simplex virus (HSV). In some embodiments, oncolytic viruses may include poxviruses. In some embodiments, poxviruses may include rabbit poxviruses or vaccinia viruses. In some embodiments, vaccinia viruses may include vaccinia viruses of Ankara strains, Western Reserve strains (WR), or Copenhagen strains. In some embodiments, vaccinia viruses may include Lister, Wyeth, New York City Board of Health, Tian Tan, Tash Kent, or USSR strains. In some embodiments, rabbit poxviruses may include myxoma viruses. In some embodiments, oncolytic viruses may be modified.

[0062] As used herein, the term "modified oncolytic virus" may refer to an oncolytic virus comprising modifications to its components, such as, but not limited to, modifications in the natural genome ("skeleton") of the virus, such as mutations or deletions of viral genes, the introduction of exogenous nucleic acids, chemical modifications of viral nucleic acids or viral proteins, and the introduction of exogenous proteins or modified viral proteins into viral capsids. Typically, oncolytic viruses can be modified (also referred to as "engineering") to obtain improved therapeutic effects for tumor cells. In certain embodiments, the modified oncolytic virus may be a modified poxvirus. In certain embodiments, the modified oncolytic virus may be a modified poxvirus.

[0063] The terms "systemic delivery" and "systemic administration," used interchangeably herein, may in some cases refer to a route of administering a 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.

[0064] Oncolytic vaccinia virus in the form of an extracellular enveloped virus

[0065] Poxviruses, such as vaccinia virus, can exist in several forms, including IMV (intracellular mature virus; it is highly antigenic but stable and may be important for the spread of poxviruses between hosts) and EEV (extracellular enveloped virus; it may be unstable outside the host but may enhance spread inside the host due to the host cell-derived outer envelope that conceals the virus; thus, the EEV form may facilitate systemic spread of vaccinia viruses, such as oncolytic vaccinia viruses, within the host).

[0066] Different vaccinia strains are known to produce different ratios of IMV and EEV particles after infecting 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. An example of a vaccinia gene point mutation present in IHD-J but not in WR is in the A34R protein (K151E), which is encoded by the vaccinia virus gene VACWR157. WR-derived strains containing this mutation (WI strain, which is a WR virus with the A34R gene of IHD-J recombined into the A34R gene locus of WR) show increased EEV production (see Blasco, R., et al. 1993 J Virol. Jun; 67(6):3319-25).

[0067] In some embodiments of the present disclosure, modified oncolytic poxviruses (e.g., modified oncolytic vaccinia virus strains) are provided that can include modifications such as non-naturally occurring mutations in viral glycoproteins (e.g., A34R protein; the wild-type sequence is provided in UniProt Accession No. P24761; SEQ ID No. 4) that enhance the ratio of extracellular enveloped virus (EEV) to intracellular mature virus (IMV) forms of the virus. For example, modified oncolytic vaccinia virus strains comprising non-naturally occurring mutations in viral glycoproteins (e.g., A34R protein) can release higher amounts of EEV particles than IMV particles.

[0068] In some embodiments, a modified oncolytic vaccinia virus strain is provided that can comprise two or more non-naturally occurring mutations in a viral glycoprotein, such as A34R. An exemplary amino acid sequence of a mutant A34R protein (also referred to herein as "WO34") is provided in SEQ ID No. 5.

[0069] In some embodiments, the two or more non-naturally occurring mutations may be at a positively charged amino acid residue (e.g., lysine) within the wild-type A34R protein (SEQ ID No. 4) or a fragment thereof, wherein 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 may be at position 110 within the wild-type A34R protein (SEQ ID No. 4) or a fragment thereof. In some examples, if 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.

[0070] In some embodiments, two or more non-natural mutations are not located at aspartic acid residues within wild-type A34R protein (SEQ ID No.4) or its fragment. In some embodiments, two or more non-natural mutations can be independently located at alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine residues within wild-type A34R protein (SEQ ID No.4) or its fragment. In some embodiments, the A34R protein or its fragment expressed by the modified oncolytic poxvirus may include a non-natural mutation in a lysine residue, wherein the lysine residue is at a position other than Lys151 of the wild-type A34R protein (SEQ ID No.4) or its fragment. In some embodiments, the A34R protein or its fragment expressed by the modified oncolytic poxvirus may include a non-natural mutation at position Lys119.

[0071] In some embodiments, the two or more non-naturally occurring mutations may be at residues 119 and 151 of the wild-type A34R protein (SEQ ID No. 4) or a fragment thereof. In some embodiments, the mutation at position 119 may be Lys119Glu (K119E). In some embodiments, the mutation at position 151 may be Lys151Glu (K151E).

[0072] In some embodiments, modified oncolytic poxvirus strains are provided that may comprise mutations in viral proteins such as hemagglutinin, neuraminidase, spike (S) glycoprotein, E1, E2, gp120, gp160, gp41, gp1, gp2, E (dimer), E1, or E2.

[0073] In some cases, two or more non-naturally occurring mutations in the modified oncolytic poxvirus A34R protein can result in an increase in the ratio of the extracellular enveloped virus (EEV) form to the intracellular mature virus (IMV) form of the virus.

[0074] In some embodiments, the two or more non-naturally occurring mutations in the modified oncolytic poxvirus A34R protein can result in an increased ratio of the EEV form to the IMV form of the virus compared to an otherwise identical poxvirus strain that does not comprise the two or more non-naturally occurring mutations in the A34R protein.

[0075] In some embodiments, the modified poxvirus strains provided herein that comprise at least two non-naturally occurring mutations in the A34R protein release high levels of EEV particles, as measured by increased comet formation in tissue culture, compared to 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 comprise at least two non-naturally occurring mutations in the A34R protein).

[0076] EEV particles released by the modified poxviruses of the present disclosure comprising 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 IMV particles are not resistant. Thus, EEV particles can mediate long-distance dissemination in vitro and in vivo.

[0077] EEV particles may also have a higher specific infectivity (as determined by a lower particle / pfu ratio) than IMV particles. Thus, modified poxvirus strains that release higher levels of EEV particles may be improved viruses for therapeutic use.

[0078] In some embodiments, certain host cell-derived proteins can colocalize with EEV preparations but not with IMVs, and the amount of cell-derived proteins can depend on the host cell line and viral strain. For example, studies have shown that WR EEVs contain more cell-derived proteins than VV IHD-J strains (see van Eijl H, Hollinshead M, Smith GL. The vaccinia virus A36R protein is a type 1b membrane protein present on intracellular but not extracellular enveloped virus particles. Virology 2000; 271: 26-36). In some cases, host cell-derived proteins can alter the biological effects of EEV particles. For example, incorporation of the host membrane protein CD55 into the surface of EEV particles released by WR vaccinia virus strains containing at least two non-naturally occurring mutations in the A34R protein can render them resistant to complement toxicity.

[0079] For the Western Reserve (WR) strain of vaccinia virus, approximately 1% of the viral particles are normally EEV and are released into the culture supernatant before cell oncolysis occurs. Some studies have shown that EEV particles released from the IHD-J strain of vaccinia can be 50 times more (see Blasco R, Sisler JR, Moss B. Dissociation of progenyvaccinia 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 envelope virus. J Virol 1996;70:272-81).

[0080] In some examples, the modified poxvirus (e.g., vaccinia virus) strains of the present disclosure can release about 10-fold to about 200-fold higher levels of EEV particles compared to an otherwise identical poxvirus strain that does not comprise an A34R protein comprising at least two non-naturally occurring mutations (e.g., K151E and K119E).

[0081] In some embodiments, the modified poxvirus strains of the present disclosure can release 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 45-fold, or about 40-fold to about 45-fold more virus than an otherwise identical poxvirus strain that does not comprise an A34R protein comprising at least two non-naturally occurring mutations (e.g., K151E and K119E). 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, about 180 times to about 200 times more EEV particles.

[0082] In some embodiments, the modified vaccinia virus strain of the present disclosure can be a WR strain in which the A34R protein comprises the mutations K119E and K151E, and can release 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 45-fold to about 50-fold more than an otherwise identical WR vaccinia virus strain that does not comprise the A34R protein comprising the mutations K119E and K151E. 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, about 180 times to about 200 times more EEV particles.

[0083] In some embodiments, the modified vaccinia virus strain of the present disclosure can be a WR strain in which the A34R protein comprises mutations K119E and K151E (WO34), and can release 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 45-fold more than the WI vaccinia virus strain. 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, and about 180 times to about 200 times more EEV particles.

[0084] In some cases, the increase in release of EEV particles compared to IMV particles by the modified poxviruses of the disclosure can be determined by performing a viral plaque assay, wherein a greater number of comet tail formation can be observed in a poxvirus comprising two or more non-naturally occurring mutations in the A34R protein compared to an otherwise identical poxvirus that does not comprise the two or more mutations in the A34R protein.

[0085] In some cases, the increase in EEV particle release compared to IMV particles can be determined by performing a neutralization assay, wherein cells infected with a modified poxvirus of the present disclosure (e.g., a vaccinia virus strain) and exposed to a neutralizing antibody (such as an anti-L1 NR-45114 antibody or a VIG antibody) can be tested in a viral plaque assay, and viral plaque formation (e.g., in PFU / mL) can be compared to an appropriate 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 antibodies can block VV infection.

[0086] In some cases, an increase in the release of EEV particles compared to IMV particles can be determined by observing an increase in comet tail formation. In some cases, observing for an increase in comet tail formation can include counting the number of comet-tail-appearing colonies on a plate produced by a known amount of virus plated, and comparing the number to that on a plate plated with another equivalent amount of virus. In some cases, an increase in comet formation can indicate an increase in the amount of EEV relative to the IMV form of the viral strain.

[0087] In some cases, the modified poxvirus strains of the present disclosure may comprise one or more additional mutations in regions of the viral genome encoding 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 protease, a helicase, a metalloprotease, 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 palmitoyl protein, or a receptor.

[0088] In some cases, one or more additional mutations may be in a poxvirus gene, such as 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, J6R, H1L, L, H2R, H3L, H4L, H5R, H6R, D1R, D2L, D3R, D6R, D7R, D8L, D11L, D12L, D13L, A2.5L, A3L, A4L, A5R, A6L, A7L, A9L, A10L, A13 L, A14L, A15L, A16L, A17L, A18R, A21L, A24R, A25L, A26L, A27L, A28L, A29L, A30L, A31R, A34R, A42R, A45R, A46R, A52R genes. In some embodiments, the present invention also provides a modified oncolytic poxvirus (e.g., vaccinia virus) strain that can include a non-natural mutation that increases the EEV form of the virus and further includes an exogenous nucleic acid that can encode a non-viral protein (such as a therapeutic protein or a diagnostic protein). Non-limiting examples of proteins encoded by exogenous nucleic acids can include SOC3, PH-20, HMGB1, PIAS3, IL15, IL15-Rα, LIGHT, ITAC, fractal chemokines, CXCR4, CCR2, CCL5, N1L, immune checkpoint regulators (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, G H1, IL-2, CSF2, TNFRSF1B, ALB, PLAU, IFNB1, CSF3, IFNA2, FSHB, botulinum toxin type A, Alefacept, pancreatic lipase, antithrombin alfa, arcitumomab, anti-rhesus (rh) immunoglobulin G, antithymocyte globulin, alemtuzumab, abciximab, alglucosidase alfaalfa), abatacept, pegademase, apcitide, human serum albumin, rasburicase, bevacizumab, botulinum toxin type B, bivalirudin, chorionic gonadotropin alfa, pegfilgrastim, Clostridium histolyticum collagenase, filgrastim, crotalidae multivalent immune Fab, sargramostim, dornase alfa, denileukin diftitox, digoxin immune Fab, epoetin alfa, darbepoetin alfa alfa), eculizumab, enfuvirtide, exenatide, efalizumab, palifermin, coagulation factor IX, antihemophilic factor, thyrotropin alfa, follicle-stimulating hormone beta, recombinant human coagulation factor VIIa, gemtuzumab ozogamicin, galsulfase, pegvisomant, imiglucerase, somatropin, glucagon, recombinant alglucosidase beta beta), alglucerase, hyaluronidase, histrelin, hepatitis C antigen, HIV antigen, hepatitis B surface antigen, HPV vaccine, hyaluronidase, interferon alpha-2b, interferon gamma-1b, insulin, laronidase, ibritumomabtiuxetan), insulin, infliximab, interferon beta-1b, oprelvekin, idursulfase, panitumumab, human immunoglobulin, cetuximab, adalimumab, pegaspargase, daclizumab, asparaginase, interferon alfacon-1, interferon alpha-n3, luteinizing hormone alfa, lepidomide Lepirudin, lactase, muromonab, mecasermin, natalizumab, nofetumomab, nesiritide, octreotide, ospA lipoprotein, tenecteplase, pramlintide, papain, urokinase, anistreplase, drotrecogin alfa alfa), reteplase, becaplermin, palivizumab, alteplase, ranibizumab, recombinant human bone morphogenetic protein 7 (rhBMP7), recombinant purified protein derivative (DPPD), streptokinase, calcitonin, sermorelin, secretin, alpha-1-proteinase inhibitor, satumomab pendetide, technetium fanolesomab, teriparatide, trypsin, etanercept, and functional domains, fragments, or variants thereof, or any combination thereof.

[0089] Hyaluronic acid (HA) is an important structural element of ECM. It is a high molecular weight straight-chain glycosaminoglycan composed of repeated disaccharide units. It can be widely distributed in connective tissue, epithelial tissue and neural tissue, and its expression level can be significantly increased in many types of tumors. Hyaluronidase is an enzyme family that catalyzes the degradation of HA. At least five functional hyaluronidases have been identified in humans so far: HYAL1, HYAL2, HYAL3, HYAL4 and HYAL5 (also known as PH-20 or SPAM1), wherein PH-20 is the only enzyme known to date that works at relatively neutral pH. In some embodiments of the present disclosure, hyaluronidase is combined with other tumor-targeted therapeutic agents (such as transgenic, also referred to as exogenous nucleic acids in this article) to promote the therapeutic effect of modified oncolytic viruses at least by reducing ECM and enhancing the transport of therapeutic agents within and between tumors.

[0090] Some embodiments herein disclose modified oncolytic viruses that may include exogenous nucleic acids encoding membrane-bound proteins (such as hyaluronidase) capable of degrading hyaluronic acid. 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 its homologues from other species, and other engineered / designed proteins with similar enzyme functions. As used herein, hyaluronidase may refer to a class of hyaluronan degrading enzymes.

[0091] In some embodiments, the modified oncolytic virus includes 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 from a chemokine receptor that promotes tumor-targeted delivery of the virus, and at least a portion of its intracellular domain can be from a chemokine receptor that promotes tumor-specific replication, suppresses immunosuppressive activity, or transmits some other beneficial effects, or vice versa. For example, the modified oncolytic virus can include nucleic acids encoding proteins with an intracellular GTPase domain of CCR5 and an extracellular chemokine binding domain of CXCR4 or CCR2. In some cases, by combining domains with different functions, it is possible to achieve further improvements in the therapeutic properties of the modified oncolytic virus. One embodiment of the present disclosure is that the modified oncolytic virus can include an exogenous nucleic acid that can encode at least one chemokine receptor. In some cases, the modified oncolytic virus can include an exogenous nucleic acid that can encode two or more different chemokine receptors, which can be expressed simultaneously by the virus. Exemplary chemokine receptors that can be simultaneously expressed by the modified oncolytic viruses described herein include CXCR4 and CCR2. In modified oncolytic viruses that express more than one chemokine receptor, therapeutic applications of oncolytic viruses can achieve combined or synergistic effects on tumor cells.

[0092] In certain embodiments, the modified oncolytic virus comprises an exogenously expressed nucleic acid of CXCR4. In certain embodiments, the modified oncolytic virus comprises an exogenously expressed nucleic acid of CCR2. Certain embodiments disclose a modified oncolytic virus, wherein the modified oncolytic poxvirus comprises an exogenous nucleic acid encoding both CXCR4 and CCR2, and both chemokines are expressed from the same virus. In some cases, CXCL12 and / or CCL2, which are normally expressed in the tumor microenvironment, can attract lymphocytes or other migratory cells expressing CXCR4 and / or CCR2 infected by the modified oncolytic virus, thereby enhancing the tumor-targeted delivery of the modified oncolytic virus.

[0093] In certain embodiments, the modified viruses described herein may include one or more exogenous nucleic acid sequences, alternatively referred to as transgenes, which may produce mRNA encoding an agent that can modulate STAT3 activity, and therefore also modulate the activation of genes regulated by STAT3. Therefore, certain examples provided herein provide oncolytic vaccinia viruses containing exogenous nucleic acid sequences that may encode an agent that can modulate STAT-3 mediated gene activation. As used herein, the phrase "modulate STAT3 mediated gene activation" may refer to a process in which STAT3 activity is modulated, and as a result, the activation of one or more genes regulated by STAT3 is also modulated.

[0094] In certain embodiments, the agent that can regulate the gene activation of STAT3 mediation can be albumen or its fragment.In certain embodiments, this albumen or its fragment can suppress, reduce or minimize the gene activation of STAT3 activity and STAT3 mediation.The albumen or its fragment that suppresses, reduces and / or minimizes the gene activation of STAT3 activity and STAT3 mediation can for example block the combination of STAT3 and the DNA binding sequence in STAT3 response gene promoter region.In another example, the albumen or its fragment that suppresses, reduces or minimizes the gene activation of STAT3 activity and STAT3 mediation can directly bind to STAT3 protein, for example, at SH2 domain.In certain embodiments, the albumen that suppresses, reduces and / or minimizes STAT3 activity blocks, prevents, reduces and / or minimizes the phosphorylation of STAT3 and / or dephosphorylates STAT3.In certain non-limiting embodiments, the albumen that regulates STAT3 activity can include protein inhibitor (PIAS, for example, PIAS3) and cytokine signaling inhibitor (SOCS) albumen (for example, SOC3) of phosphotyrosine phosphatase (PTP), activated STAT.

[0095] Cancer Targets

[0096] In an embodiment of the present disclosure, a method for treating a hyperproliferative disease such as a cancer or a tumor by delivering a modified oncolytic poxvirus as described herein is provided. Cancers that can be treated by a modified oncolytic poxvirus as described herein 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 epithelial cancer, hepatocellular carcinoma, bile duct sarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasms, and sarcoma.

[0097] Cancer cells that can be treated by the methods of the present disclosure can include cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicles, tongue, or uterus. In addition, the cancer can specifically be of the following histological types, but is not limited to these histological types: malignant neoplasms; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; bile duct epithelial carcinoma; hepatocellular carcinoma; mixed hepatocellular and bile duct epithelial carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma in familial polyposis coli; solid carcinoma; malignant carcinoid tumor; bronchiolar acinar Adenocarcinoma; Papillary adenocarcinoma; Chromophobe cell carcinoma; Oncocytic cell carcinoma; Oncocytic adenocarcinoma; Basophilic cell carcinoma; Clear cell adenocarcinoma; Granular cell carcinoma; Follicular adenocarcinoma; Papillary and follicular adenocarcinomas; Non-encapsulated sclerosing carcinoma; Adrenocortical carcinoma; Endometrioid carcinoma; Carcinoma of the skin adnexa; Apocrine adenocarcinoma; Sebaceous adenocarcinoma; Cerebellar gland carcinoma; 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 of the breast disease); acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; malignant androblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomus sarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma within giant nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; Müllerian mixed tumor; Wilms' tumor; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner's tumor malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant goiter; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma;Protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's; paragranuloma; malignant small lymphocytic lymphoma; malignant leukemia Diffuse large cell lymphoma; malignant follicular lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative enteropathy; leukemia; lymphoid 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, the modified oncolytic virus of the present disclosure, such as the modified oncolytic poxvirus that is conducive to systemic delivery, can be used to treat metastatic solid cancer. In some cases, the modified oncolytic poxvirus of the present disclosure that is conducive to systemic delivery can be used to treat solid cancers that are inaccessible or difficult to access for the purpose of intratumoral delivery of therapeutic agents. In some examples, modified oncolytic poxviruses of the present disclosure that facilitate systemic delivery and formation of increased amounts of EEV can be used to treat cancers associated with increased free fatty acid expression.

[0098] The present disclosure also contemplates methods for suppressing or preventing local invasion or metastasis or both of any type of primary cancer. For example, the primary cancer can be melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, liver cancer, retinoblastoma, astrocytoma, glioblastoma, gum cancer, tongue cancer, leukemia, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, brain cancer, colon cancer, or bladder cancer. In certain embodiments, the primary cancer can be lung cancer. For example, lung cancer can be non-small cell lung cancer. In addition, the present disclosure can be used to prevent cancer or treat precancerous lesions or pre-malignant cells, including metaplasia, dysplasia, and hyperplasia. It can also be used to suppress undesirable but benign cells, such as squamous metaplasia, dysplasia, benign prostatic hyperplasia cells, hyperplastic lesions, etc. In some embodiments, progression to cancer or more severe forms of cancer can be stopped, disrupted, or delayed by the methods of the disclosure involving the modified oncolytic poxviruses discussed herein.

[0099] In addition, the modified oncolytic poxvirus disclosed herein can be administered to treat tumors with high bioavailability of free fatty acids in the tumor microenvironment. In some cases, the free fatty acids released by the fat cells in the tumor of obese patients can feed (feed) the modified oncolytic poxvirus in the tumor and enhance its replication and the formation of viruses in the form of EEV. In non-obese patients, especially patients with peritoneal cancer, this advantage can also be achieved. For example, several peritoneal cancers can be targets for the therapy of the modified oncolytic virus using the present disclosure, because these cancers tend to grow in the omentum wall and can be supplied by fat cells, and as described above, the free fatty acids released by the fat cells in the tumor can feed the modified oncolytic virus in the tumor and enhance its replication. The modified oncolytic poxvirus disclosed herein can form an extracellular enveloped virus (EEV) with increased titer in tumors with high bioavailability of free fatty acids.

[0100] In some embodiments, a method for treating a tumor by administering cells infected with a modified poxvirus as disclosed herein is provided. Infected cells can be administered to a subject (e.g., intratumorally), whereby the modified poxvirus produces a population of viral particles containing 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 greater) in situ (such as in a tumor or tumor microenvironment). EEV particles can then be harvested from a subject (such as a biological sample isolated from a subject) and used for subsequent systemic delivery (e.g., intravenous delivery) to the subject. In some cases, this can enhance the systemic spread of the modified oncolytic virus in the subject and improve treatment outcomes.

[0101] Treatment approaches and efficacy and pharmacokinetic determinations

[0102] In some embodiments, the present disclosure provides methods of treating a subject by administering a modified oncolytic poxvirus as disclosed herein.

[0103] A method for producing a toxic effect in a cancer cell is provided, the method comprising administering to the cancer cell a therapeutically effective amount of a modified oncolytic poxvirus as described above or a pharmaceutical composition containing the modified oncolytic poxvirus. The present disclosure also provides a method for inhibiting at least one of the growth and proliferation of a second cancer cell, the method comprising administering to a first cancer cell a modified oncolytic poxvirus as described above, such that the first cancer cell is infected with the virus. Thus, in some embodiments of the methods disclosed herein, it is contemplated that not every cancer cell or tumor cell is infected after administration of a therapeutically effective amount of a modified oncolytic poxvirus as described herein or a pharmaceutical composition containing a modified oncolytic poxvirus, and that the growth of uninfected cells can be inhibited without direct infection.

[0104] In some examples, in order to use 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, cancer cells or tumors can be contacted with a therapeutically effective dose of an exemplary modified oncolytic poxvirus as described herein or a pharmaceutical composition containing the modified oncolytic poxvirus. In certain embodiments, an effective amount of a modified oncolytic poxvirus of the present disclosure or a pharmaceutical composition thereof may include an amount sufficient to induce oncolysis, destruction or lysis of cancer cells, or to inhibit or reduce the growth or size of cancer cells. For example, a reduction in the growth of cancer cells may be manifested as cell death, or a reduction in the replication rate or growth rate of a tumor containing the cells, or an extension of the survival of a subject containing cancer cells.

[0105] In some embodiments, a method for treating a subject having cancer or a tumor is provided, the method comprising administering to the subject an effective amount of a modified virus as described above. The effective amount in such a method may include an amount that slows the growth rate or spread of the cancer, or prolongs the survival of the subject. The present disclosure provides a method for slowing tumor growth, which may include administering to the tumor an effective amount of a modified oncolytic poxvirus as described above. In certain embodiments, an effective amount of a modified oncolytic poxvirus or a pharmaceutical composition thereof may include an amount sufficient to induce a slowing, inhibition, or reduction in tumor growth or size, and may include eradicating the tumor. For example, a slowing of tumor growth may be manifested as a reduction in growth rate or an extension of the survival of a subject containing a tumor.

[0106] The present disclosure also provides a method for determining the infectivity or anti-tumor activity or tumor-specific viral replication amount of a modified oncolytic poxvirus as described herein, 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, alone or in combination with another therapy, which also expresses a luciferase reporter gene; (ii) immediately after administering the virus, collecting a first biological sample from the subject and determining the level of the luciferase reporter gene in the first biological sample; (iii) collecting a second biological sample from the subject after administration in step (ii); and (iv) detecting the level of the luciferase reporter gene in the second biological sample, wherein if the level of luciferase in step (iii) is higher than that in step (ii), the modified oncolytic poxvirus is determined to be infectious, exhibit anti-tumor activity, and exhibit 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 the administration in step (i). In some embodiments, the above methods may also include detecting in steps (i) and (iii) one or more of the levels of cytokines (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, such as a modified oncolytic poxvirus as disclosed herein, or a pharmaceutical composition comprising the virus. In some embodiments of the present disclosure, the increase in luciferase bioluminescence between steps (ii) and (iv) of the modified oncolytic poxvirus as described herein is higher than that of the modified virus that is otherwise identical but does not comprise the modified oncolytic poxvirus. Other exemplary techniques for detecting and monitoring viral load after administering a modified oncolytic poxvirus include real-time quantitative PCR.

[0107] Also provided are methods for monitoring pharmacokinetics after administering a therapeutically effective amount of a modified oncolytic poxvirus according to the present disclosure or a pharmaceutical composition containing the poxvirus. Exemplary methods for monitoring pharmacokinetics may include the following steps: (i) administering to a subject a therapeutically effective amount of a modified oncolytic poxvirus or a pharmaceutical composition containing the modified oncolytic poxvirus, alone or in combination with another therapy; (ii) administering to a subject a therapeutically effective amount of a modified oncolytic poxvirus or a pharmaceutical composition containing the modified oncolytic poxvirus, alone or in combination with another therapy; and (iii) administering to a subject a therapeutically effective amount of a modified oncolytic poxvirus or a pharmaceutical composition containing the modified oncolytic poxvirus, alone or in combination with another therapy; and (iv) administering to a subject a therapeutically effective amount of a modified oncolytic poxvirus or a pharmaceutical composition containing the modified oncolytic poxvirus, alone or in combination with another therapy; and (v) administering to a subject a therapeutically effective amount of a modified oncolytic poxvirus or a pharmaceutical composition containing the modified oncolytic poxvirus, alone or in combination with another therapy; and (v) administering to a subject a therapeutically effective amount of a modified oncolytic poxvirus or a pharmaceutical composition containing the modified oncolytic poxvirus, alone or in combination with another therapy; and (v) administering to a subject a therapeutically effective amount of a modified oncolytic poxvirus or a pharmaceutical composition containing the modified oncolytic poxvirus, alone or in combination with another therapy; and (vii ... In some cases, the amount of viral genome (or reporter gene inserted into the viral genome, such as luciferase) in the biological sample collected at the above time points can be measured. In some cases, viral genome copies / mL can be the highest in the sample collected at the 15 minute time point, and further, the sample collected at the 240 minute time point may not include a detectable amount of viral genome. Therefore, in some cases, a viral peak can be observed at about 15 minutes after administration, and most of the virus can be cleared from the subject's system after about 240 minutes (or 4 hours). In some cases, a first viral peak can be observed at about 15 minutes after administration, and a second viral peak can be observed in a biological sample collected at a later time point (e.g., at about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes). In an exemplary embodiment, the biological sample can be blood, and the amount of viral genomes per mL can be determined by quantitative PCR or other suitable techniques. In some examples, a first viral peak can be observed at about 15 minutes after administration, and a second viral peak can be observed after 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 present disclosure, such as an oncolytic poxvirus as described herein.

[0108] In some cases, the tumor selective replication of the modified oncolytic poxvirus can be measured by using a reporter gene such as a luciferase gene. In some embodiments, the luciferase gene can be inserted into the genome of the virus, and the virus can be used to infect tumor cells. Bioluminescence in infected tumor cells can be measured to monitor tumor selective replication. Some examples show that the luciferase reporter molecule bioluminescence in the modified oncolytic poxvirus of the present disclosure is increased compared to an oncolytic poxvirus that is otherwise identical but does not contain the modification in the modified oncolytic virus.

[0109] Delivery of modified oncolytic viruses

[0110] In some embodiments, the amount of a modified oncolytic poxvirus of the present disclosure administered to a subject can be about 10 3 and 10 12 Infectious virus particles or plaque forming units (PFU), or about 10 5 and 10 10 PFU, or about 10 5 and 10 8 PFU, or about 10 8 and 10 10 In some embodiments, the amount of the modified oncolytic poxvirus of the present disclosure administered to a subject can be between about 10 3 and 10 12 between 10 virus particles or plaque forming units (PFU), or about 10 5 and 10 10 PFU, or about 10 5 and 10 8 PFU, or about 10 8 and 10 10 In some embodiments, the modified oncolytic poxvirus of the present disclosure may be administered at a dose that may include about 10 3 PFU / dose is about 10 4 PFU / dose, about 10 4 PFU / dose is about 10 5 PFU / dose, about 10 5 PFU / dose is about 10 6 PFU / dose, about 10 7 PFU / dose is about 10 8 PFU / dose, about 10 9 PFU / dose is about 10 10 PFU / dose, about 10 10 PFU / dose is about 10 11 PFU / dose, about 10 11 PFU / dose is about 1012 PFU / dose, about 10 12 PFU / dose is about 10 13 PFU / dose, about 10 13 PFU / dose is about 10 14 PFU / dose or about 10 14 PFU / dose is about 10 15 PFU / dose. In some embodiments, the modified oncolytic poxviruses of the present disclosure may be administered at a dose that may include about 2 x 10 3 PFU / dose, 3x10 3 PFU / dose, 4x10 3 PFU / dose, 5x10 3 PFU / dose, 6x10 3 PFU / dose, 7x10 3 PFU / dose, 8x10 3 PFU / dose, 9x10 3 PFU / dose, about 10 4 PFU / dose, about 2x10 4 PFU / dose, about 3x10 4 PFU / dose, about 4x10 4 PFU / dose, about 5x10 4 PFU / dose, about 6x10 4 PFU / dose, about 7x10 4 PFU / dose, about 8x10 4 PFU / dose, about 9x10 4 PFU / dose, about 10 5 PFU / dose, 2x10 5 PFU / dose, 3x10 5 PFU / dose, 4x10 5 PFU / dose, 5x10 5 PFU / dose, 6x10 5 PFU / dose, 7x10 5 PFU / dose, 8x10 5 PFU / dose, 9x10 5 PFU / dose, about 10 6 PFU / dose, about 2x10 6 PFU / dose, about 3x10 6 PFU / dose, about 4x10 6 PFU / dose, about 5x10 6 PFU / dose, about 6x10 6 PFU / dose, about 7x10 6 PFU / dose, about 8x10 6 PFU / dose, about 9x106 PFU / dose, about 10 7 PFU / dose, about 2x10 7 PFU / dose, about 3x10 7 PFU / dose, about 4x10 7 PFU / dose, about 5x10 7 PFU / dose, about 6x10 7 PFU / dose, about 7x10 7 PFU / dose, about 8x10 7 PFU / dose, about 9x10 7 PFU / dose, about 10 8 PFU / dose, about 2x10 8 PFU / dose, about 3x10 8 PFU / dose, about 4x10 8 PFU / dose, about 5x10 8 PFU / dose, about 6x10 8 PFU / dose, about 7x10 8 PFU / dose, about 8x10 8 PFU / dose, about 9x10 8 PFU / dose, about 10 9 PFU / dose, about 2x10 9 PFU / dose, about 3x10 9 PFU / dose, about 4x10 9 PFU / dose, about 5x10 9 PFU / dose, about 6x10 9 PFU / dose, about 7x10 9 PFU / dose, about 8x10 9 PFU / dose, about 9x10 9 PFU / dose, about 10 10 PFU / dose, about 2x10 10 PFU / dose, about 3x10 10 PFU / dose, about 4x10 10 PFU / dose, about 5x10 10 PFU / dose, about 6x10 10 PFU / dose, about 7x10 10 PFU / dose, about 8x10 10 PFU / dose, about 9x10 10 PFU / dose, about 10 10 PFU / dose, about 2x10 10 PFU / dose, about 3x10 10 PFU / dose, about 4x10 10 PFU / dose, about 5x10 10 PFU / dose, about 6x1010 PFU / dose, about 7x10 10 PFU / dose, about 8x10 10 PFU / dose, about 9x10 10 PFU / dose, about 10 11 PFU / dose, about 2x10 11 PFU / dose, about 3x10 11 PFU / dose, about 4x10 11 PFU / dose, about 5x10 11 PFU / dose, about 6x10 11 PFU / dose, about 7x10 11 PFU / dose, about 8x10 11 PFU / dose, about 9x10 11 PFU / dose or about 10 12 PFU / dose, about 10 12 PFU / dose is about 10 13 PFU / dose, about 10 13 PFU / dose is about 10 14 PFU / dose or about 10 14 PFU / dose is about 10 15 PFU / dose. In some embodiments, the modified oncolytic poxviruses of the present disclosure may be administered at a dose that may include 5x10 9 PFU / dose. In some embodiments, the modified oncolytic poxviruses of the present disclosure may be administered in a dose that may include up to 5x10 9 PFU / dose.

[0111] In some embodiments, the modified oncolytic poxviruses of the present disclosure may be administered in a dose that may include about 10 3 Virus particles / dose to about 10 4 Virus particles / dose, about 10 4 Virus particles / dose to about 10 5 Virus particles / dose, about 10 5 Virus particles / dose to about 10 6 Virus particles / dose, about 10 7 Virus particles / dose to about 10 8 Virus particles / dose, about 10 9 Virus particles / dose to about 10 10 Virus particles / dose, about 10 10 Virus particles / dose to about 10 11 Virus particles / dose, about 10 11 Virus particles / dose to about 10 12 Virus particles / dose, about 1012 Virus particles / dose to about 10 13 Virus particles / dose, about 10 13 Virus particles / dose to about 10 14 Virus particles / dose, or 10 14 Virus particles / dose to about 10 15 Virus particles / dose.

[0112] In some embodiments, the modified oncolytic poxviruses of the present disclosure may be administered in a dose that may include about 10 3 PFU / kg is about 10 4 PFU / kg, about 10 4 PFU / kg is about 10 5 PFU / kg, about 10 5 PFU / kg is about 10 6 PFU / kg, about 10 7 PFU / kg is about 10 8 PFU / kg, about 10 9 PFU / kg is about 10 10 PFU / kg, about 10 10 PFU / kg is about 10 11 PFU / kg, about 10 11 PFU / kg is about 10 12 PFU / kg, about 10 12 PFU / kg is about 10 13 PFU / kg, about 10 13 PFU / kg is about 10 14 PFU / kg or about 10 14 PFU / kg is about 10 15 PFU / kg. In some embodiments, the modified oncolytic poxviruses of the present disclosure may be administered at a dose that may include about 2×10 3 PFU / kg, 3x10 3 PFU / kg, 4x10 3 PFU / kg, 5x10 3 PFU / kg, 6x10 3 PFU / kg, 7x10 3 PFU / kg, 8x10 3 PFU / kg, 9x10 3 PFU / kg, about 10 4 PFU / kg, about 2x10 4 PFU / kg, about 3x10 4 PFU / kg, about 4x10 4PFU / kg, about 5x10 4 PFU / kg, about 6x10 4 PFU / kg, about 7x10 4 PFU / kg, about 8x10 4 PFU / kg, about 9x10 4 PFU / kg, about 10 5 PFU / kg, 2x10 5 PFU / kg, 3x10 5 PFU / kg, 4x10 5 PFU / kg, 5x10 5 PFU / kg, 6x10 5 PFU / kg, 7x10 5 PFU / kg, 8x10 5 PFU / kg, 9x10 5 PFU / kg, about 10 6 PFU / kg, about 2x10 6 PFU / kg, about 3x10 6 PFU / kg, about 4x10 6 PFU / kg, about 5x10 6 PFU / kg, about 6x10 6 PFU / kg, about 7x10 6 PFU / kg, about 8x10 6 PFU / kg, about 9x10 6 PFU / kg, about 10 7 PFU / kg, about 2x10 7 PFU / kg, about 3x10 7 PFU / kg, about 4x10 7 PFU / kg, about 5x10 7 PFU / kg, about 6x10 7 PFU / kg, about 7x10 7 PFU / kg, about 8x10 7 PFU / kg, about 9x10 7 PFU / kg, about 10 8 PFU / kg, about 2x10 8 PFU / kg, about 3x10 8 PFU / kg, about 4x10 8 PFU / kg, about 5x10 8 PFU / kg, about 6x10 8 PFU / kg, about 7x10 8 PFU / kg, about 8x10 8 PFU / kg, about 9x108 PFU / kg, about 10 9 PFU / kg, about 2x10 9 PFU / kg, about 3x10 9 PFU / kg, about 4x10 9 PFU / kg, about 5x10 9 PFU / kg, about 6x10 9 PFU / kg, about 7x10 9 PFU / kg, about 8x10 9 PFU / kg, about 9x10 9 PFU / kg, about 10 10 PFU / kg, about 2x10 10 PFU / kg, about 3x10 10 PFU / kg, about 4x10 10 PFU / kg, about 5x10 10 PFU / kg, about 6x10 10 PFU / kg, about 7x10 10 PFU / kg, about 8x10 10 PFU / kg, about 9x10 10 PFU / kg, about 10 10 PFU / kg, about 2x10 10 PFU / kg, about 3x10 10 PFU / kg, about 4x10 10 PFU / kg, about 5x10 10 PFU / kg, about 6x10 10 PFU / kg, about 7x10 10 PFU / kg, about 8x10 10 PFU / kg, about 9x10 10 PFU / kg, about 10 11 PFU / kg, about 2x10 11 PFU / kg, about 3x10 11 PFU / kg, about 4x10 11 PFU / kg, about 5x10 11 PFU / kg, about 6x10 11 PFU / kg, about 7x10 11 PFU / kg, about 8x10 11 PFU / kg, about 9x10 11 PFU / kg or about 10 12 PFU / kg, about 10 12 PFU / kg is about 10 13 PFU / kg, about 10 13PFU / kg is about 10 14 PFU / kg or about 10 14 PFU / kg is about 10 15 PFU / kg. In some embodiments, the modified oncolytic poxviruses of the present disclosure may be administered at a dose that may include 5×10 9 PFU / kg. In some embodiments, the modified oncolytic poxviruses of the present disclosure may be administered at a dose that may include up to 5x10 9 PFU / kg.

[0113] In some embodiments, the modified oncolytic poxviruses of the present disclosure may be administered in a dose that may include about 10 3 Virus particles / kg to about 10 4 Virus particles / kg, about 10 4 Virus particles / kg to about 10 5 Virus particles / kg, about 10 5 Virus particles / kg to about 10 6 Virus particles / kg, about 10 7 Virus particles / kg to about 10 8 Virus particles / kg, about 10 9 Virus particles / kg to about 10 10 Virus particles / kg, about 10 10 Virus particles / kg to about 10 11 Virus particles / kg, about 10 11 Virus particles / kg to about 10 12 Virus particles / kg, about 10 12 Virus particles / kg to about 10 13 Virus particles / kg, about 10 13 Virus particles / kg to about 10 14 virus particles / kg or about 10 14 Virus particles / kg to about 10 15 Virus particles / kg.

[0114] In certain embodiments, a liquid dosage form of a modified oncolytic poxvirus as described herein may include about 10 3 PFU / mL is about 10 4 PFU / mL, about 10 4 PFU / mL is about 10 5 PFU / mL, about 10 5 PFU / mL is about 10 6 PFU / mL, about 10 7 PFU / mL is about 108 PFU / mL, about 10 9 PFU / mL is about 10 10 PFU / mL, about 10 10 PFU / mL is about 10 11 PFU / mL, about 10 11 PFU / mL is about 10 12 PFU / mL, about 10 12 PFU / mL is about 10 13 PFU / mL, about 10 13 PFU / mL is about 10 14 PFU / mL or about 10 14 PFU / mL is about 10 15 In some embodiments, the modified oncolytic poxviruses of the present disclosure may be administered at a dose that may include about 2 x 10 3 PFU / mL, 3x10 3 PFU / mL, 4x10 3 PFU / mL, 5x10 3 PFU / mL, 6x10 3 PFU / mL, 7x10 3 PFU / mL, 8x10 3 PFU / mL, 9x10 3 PFU / mL, about 10 4 PFU / mL, about 2x10 4 PFU / mL, about 3x10 4 PFU / mL, about 4x10 4 PFU / mL, about 5x10 4 PFU / mL, about 6x10 4 PFU / mL, about 7x10 4 PFU / mL, about 8x10 4 PFU / mL, about 9x10 4 PFU / mL, about 10 5 PFU / mL, 2x10 5 PFU / mL, 3x10 5 PFU / mL, 4x10 5 PFU / mL, 5x10 5 PFU / mL, 6x10 5 PFU / mL, 7x10 5 PFU / mL, 8x10 5 PFU / mL, 9x10 5 PFU / mL, about 10 6 PFU / mL, about 2x106 PFU / mL, about 3x10 6 PFU / mL, about 4x10 6 PFU / mL, about 5x10 6 PFU / mL, about 6x10 6 PFU / mL, about 7x10 6 PFU / mL, about 8x10 6 PFU / mL, about 9x10 6 PFU / mL, about 10 7 PFU / mL, about 2x10 7 PFU / mL, about 3x10 7 PFU / mL, about 4x10 7 PFU / mL, about 5x10 7 PFU / mL, about 6x10 7 PFU / mL, about 7x10 7 PFU / mL, about 8x10 7 PFU / mL, about 9x10 7 PFU / mL, about 10 8 PFU / mL, about 2x10 8 PFU / mL, about 3x10 8 PFU / mL, about 4x10 8 PFU / mL, about 5x10 8 PFU / mL, about 6x10 8 PFU / mL, about 7x10 8 PFU / mL, about 8x10 8 PFU / mL, about 9x10 8 PFU / mL, about 10 9 PFU / mL, about 2x10 9 PFU / mL, about 3x10 9 PFU / mL, about 4x10 9 PFU / mL, about 5x10 9 PFU / mL, about 6x10 9 PFU / mL, about 7x10 9 PFU / mL, about 8x10 9 PFU / mL, about 9x10 9 PFU / mL, about 10 10 PFU / mL, about 2x10 10 PFU / mL, about 3x10 10 PFU / mL, about 4x10 10 PFU / mL, about 5x10 10 PFU / mL, about 6x10 10PFU / mL, about 7x10 10 PFU / mL, about 8x10 10 PFU / mL, about 9x10 10 PFU / mL, about 10 10 PFU / mL, about 2x10 10 PFU / mL, about 3x10 10 PFU / mL, about 4x10 10 PFU / mL, about 5x10 10 PFU / mL, about 6x10 10 PFU / mL, about 7x10 10 PFU / mL, about 8x10 10 PFU / mL, about 9x10 10 PFU / mL, about 10 11 PFU / mL, about 2x10 11 PFU / mL, about 3x10 11 PFU / mL, about 4x10 11 PFU / mL, about 5x10 11 PFU / mL, about 6x10 11 PFU / mL, about 7x10 11 PFU / mL, about 8x10 11 PFU / mL, about 9x10 11 PFU / mL or about 10 12 PFU / mL, about 10 12 PFU / mL is about 10 13 PFU / mL, about 10 13 PFU / mL is about 10 14 PFU / mL or about 10 14 PFU / mL is about 10 15 PFU / mL. In some embodiments, the modified oncolytic poxviruses of the present disclosure may be administered at a dose that may include 5×10 9 PFU / mL. In some embodiments, the modified oncolytic poxviruses of the present disclosure may be administered at a dose that may include up to 5x10 9 PFU / mL.

[0115] In some cases, when the modified oncolytic poxvirus is administered by injection, the dosage may include about 10 3 Virus particles, 10 per injection 4 Virus particles, 10 per injection 5 Virus particles, 10 per injection 6 Virus particles, 10 per injection 7Virus particles, 10 per injection 8 Virus particles, 10 per injection 9 Virus particles, 10 per injection 10 Virus particles, 10 per injection 11 Virus particles, 10 per injection 12 Virus particles, 2x10 per injection 12 Virus particles, 10 per injection 13 Virus particles, 10 per injection 14 Virus particles or 10 per injection 15 In other cases, when the modified oncolytic poxvirus is administered by injection, the dosage may include about 10 3 Infectious virus particles, 10 per injection 4 Infectious virus particles, 10 per injection 5 Infectious virus particles, 10 per injection 6 Infectious virus particles, 10 per injection 7 Infectious virus particles, 10 per injection 8 Infectious virus particles, 10 per injection 9 Infectious virus particles, 10 per injection 10 Infectious virus particles, 10 per injection 11 Infectious virus particles, 10 per injection 12 Infectious virus particles, 2 x 10 per injection 12 Infectious virus particles, 10 per injection 13 Infectious virus particles, 10 per injection 14 infectious virus particles or 10 15 In other embodiments, the modified oncolytic poxvirus of the present disclosure may be administered at a dose of about 10 3 Tissue culture inhibitor dose 50% (TCID 50 ) / kg、3x10 4 TCID 50 / kg, 4x10 4 TCID 50 / kg, 5x10 4 TCID 50 / kg, 3x10 5 TCID 50 / kg, 4x10 5 TCID 50 / kg, 5x10 5 TCID 50 / kg, 3x10 6TCID 50 / kg, 4x10 6 TCID 50 / kg, 5x10 6 TCID 50 / kg, 3x10 7 TCID 50 / kg, 4x10 7 TCID 50 / kg, 5x10 7 TCID 50 / kg, 3x10 8 TCID 50 / kg, 4x10 8 TCID 50 / kg, 5x10 8 TCID 50 / kg, 3x10 9 TCID 50 / kg, 4x10 9 TCID 50 / kg, 5x10 9 TCID 50 / kg, 3x10 10 TCID 50 / kg, 4x10 10 TCID 50 / kg or 5x10 10 TCID 50 / kg。 Note, text in 10 xAlternatively expressed as 1eX. In certain embodiments, the modified oncolytic poxvirus can be administered in one or more doses. In certain embodiments, the virus can be administered in an amount sufficient to induce oncolysis in at least about 20% of the cells in the tumor, at least about 30% of the cells in the tumor, at least about 40% of the cells in the tumor, at least about 50% of the cells in the tumor, at least about 60% of the cells in the tumor, at least about 70% of the cells in the tumor, at least about 80% of the cells in the tumor, or at least about 90% of the cells in the tumor. In certain embodiments, a single dose of virus may refer to an amount administered to a subject or tumor within a time period of 1 hour, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, or 24 hours. In certain embodiments, the dosage can be extended over time or by a separate injection. In certain embodiments, more than one dose (e.g., 2, 3, 4, 5, 6 or more doses) of poxvirus can be administered to the subject, for example, wherein the second treatment can be performed in 1, 2, 3, 4, 5, 6, 7 days or weeks after the first treatment. In certain embodiments, more than one dose of modified oncolytic virus can be administered to the subject in a time period of 1, 2, 3, 4, 5, 6, 7 or more days or weeks. In certain embodiments, oncolytic vaccinia virus or pharmaceutical composition as described herein can be administered in 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 time period. In some cases, the frequency of administration of an oncolytic poxvirus or pharmaceutical composition as described herein may be once a day, twice a day, once a week, once every three weeks, once every four 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 (once every 6 months). In some embodiments of the methods disclosed herein, an oncolytic poxvirus or pharmaceutical composition may be independently administered with an initial dose for the first time period, an intermediate dose for the second time period, and a high dose for the third time 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 time periods can independently be 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.

[0116] In some examples, according to any of the treatment methods described herein, the subject can be fed a reduced carbohydrate diet, e.g., a ketogenic diet, before, concurrently, and after administration of a modified oncolytic poxvirus as described herein or a pharmaceutical composition comprising the modified oncolytic poxvirus. In certain embodiments, the subject is fed a diet that can include consuming less than 500 grams of carbohydrates per day, less than 450 grams of carbohydrates per day, less than 450 grams of carbohydrates per day, less than 400 grams of carbohydrates per day, less than 350 grams of carbohydrates per day, less than 300 grams of carbohydrates per day, less than 250 grams of carbohydrates per day, less than 200 grams of carbohydrates per day, less than 150 grams of carbohydrates per day, less than 100 grams of carbohydrates per day, less than 90 grams of carbohydrates per day, less than 80 grams of carbohydrates per day, less than 70 grams of carbohydrates per day, less than 60 grams of carbohydrates per day, less than 50 grams of carbohydrates per day, less than 40 grams of carbohydrates per day, less than 30 grams of carbohydrates per day, less than 20 grams of carbohydrates per day, less than 10 grams of carbohydrates per day.

[0117] The exemplary method for delivering the modified oncolytic poxvirus of the present disclosure or the pharmaceutical composition comprising the modified oncolytic poxvirus to cancer cells or tumor cells can be via intratumoral injection. However, alternative methods of administration can also be used, for example, intravenously, via infusion, parenteral, intravenously, intradermally, intramuscularly, transdermally, rectal, intraurethral, intravaginal, intranasal, intrathecal or intraperitoneal. The route of administration can be different with the position and nature of the tumor. In certain embodiments, the route of administration can be intradental, transdermal, parenteral, intravenously, intramuscularly, intranasal, subcutaneous, local (for example, near the tumor, particularly the blood vessels or adjacent blood vessels of the tumor), percutaneous, intrathecal, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhaled, perfused, by lavage or oral. The modified oncolytic poxvirus of an injectable dose can be used as a bolus injection or as a slow infusion. In certain embodiments, the modified oncolytic poxvirus can be administered to the patient from a source implanted in the patient. In certain embodiments, administration of the modified oncolytic poxvirus can be performed by continuous infusion over a selected period of time. In some cases, an oncolytic poxvirus as described herein or a pharmaceutical composition containing the oncolytic poxvirus can be administered at a therapeutically effective dose by infusion over a period 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 can be administered in a liquid dose wherein the total volume administered is about 1 mL to about 5 mL, about 5 mL to 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 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 500 mL, about 500 mL to 750 mL, or about 750 mL to 1000 mL.

[0118] Pharmaceutical composition

[0119] Pharmaceutical compositions containing modified oncolytic poxviruses as described herein can be prepared as solutions or dispersions in glycerol, liquid polyethylene glycol, and any combination thereof, in oil, or in solid dosage forms; prepared as inhalable dosage forms, intranasal dosage forms, liposomal formulations, dosage forms comprising nanoparticles, dosage forms comprising microparticles, polymeric dosage forms, or any combination thereof. In some embodiments, pharmaceutical compositions as described herein may include stabilizers and buffers. In some embodiments, pharmaceutical compositions as described herein may include solubilizers such as sterile water or Tris buffer. In some embodiments, pharmaceutical compositions as described herein may include excipients. Excipients may be excipients described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986). Non-limiting examples of suitable excipients may include buffers, preservatives, stabilizers, binders, compacting agents, lubricants, chelating agents, dispersion enhancers, disintegrants, flavorings, sweeteners, and colorants.

[0120] In some embodiments, the excipient can be a buffer. The limiting examples of suitable buffer can include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate and calcium bicarbonate. As buffer, 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 their combination can be used in pharmaceutical preparations.

[0121] In some embodiments, the excipient may include a preservative. Non-limiting examples of suitable preservatives may include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol and phenol. Antioxidants may also include, but are not limited to, EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (butylatedhydroxytoluene) (BHT), butylated hydroxy anisole (butylated hydroxy anisole) (BHA), sodium sulfite, p-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol and N-acetylcysteine. In some cases, the preservative may include validamycin A, TL-3, sodium orthovanadate, sodium fluoride, Na-tosyl-Phe-chloromethylketone, Na-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.

[0122] In some embodiments, the pharmaceutical compositions described herein may include a binder as an excipient. Non-limiting examples of suitable binders may include starch, pregelatinized starch, gelatin, polyvinyl pyrrolidone, cellulose, methyl cellulose, sodium carboxymethyl cellulose, ethyl cellulose, polyacrylamide, polyvinyloxazolidone, polyvinyl alcohol, C 12 -C 18 Fatty acid alcohols, polyethylene glycols, polyols, sugars, oligosaccharides and combinations thereof. Binders that can be used in pharmaceutical preparations can be selected from starches such as potato starch, corn starch, wheat starch; sugars such as sucrose, glucose, dextrose, lactose, maltodextrin; natural and synthetic gums; gelatin; cellulose derivatives such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose; polyvinyl pyrrolidone (povidone); polyethylene glycol (PEG); waxes; calcium carbonate; calcium phosphate; alcohols such as sorbitol, xylitol, mannitol, and water, or combinations thereof.

[0123] In some embodiments, pharmaceutical compositions as described herein may include lubricants as excipients. Non-limiting examples of suitable lubricants may include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils (hydrogenated vegetable oils), refined hydrogenated vegetable oils (sterotex), polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate and light mineral oil. Lubricants that can be used for pharmaceutical preparations may be selected from metal stearates (such as magnesium stearate, calcium stearate, aluminum stearate), fatty acid esters (such as sodium stearyl fumarate), fatty acids (such as stearic acid), fatty alcohols, glyceryl behenate, mineral oil, paraffin, hydrogenated vegetable oils, leucine, polyethylene glycol (PEG), metal lauryl sulfate (such as sodium lauryl sulfate, magnesium lauryl sulfate), sodium chloride, sodium benzoate, sodium acetate and talc, or a combination thereof. In some embodiments, pharmaceutical preparations may include dispersion enhancers as excipients. Non-limiting examples of suitable dispersants may include starch, alginic acid, polyvinyl pyrrolidone, guar gum, kaolin, bentonite, purified lignocellulose, sodium starch glycolate, isomorphous silicates, and microcrystalline cellulose as a high HLB emulsifier surfactant.

[0124] In some embodiments, the pharmaceutical composition as described herein may include a disintegrant as an excipient. In some embodiments, the disintegrant may be a non-effervescent disintegrant. Non-limiting examples of suitable non-effervescent disintegrants may include starches such as corn starch, potato starch, their pregelatinized and modified starches, sweeteners, clays such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar gum, locust bean gum, karaya gum, pectin, and tragacanth. In some embodiments, the disintegrant may be an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants may include a combination of sodium bicarbonate and citric acid, and a combination of sodium bicarbonate and tartaric acid.

[0125] In some embodiments, the excipients may include flavoring agents. The flavoring agents incorporated into the outer layer may be selected from the group consisting of synthetic flavoring oils and flavoring aromatics; natural oils; extracts of 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 oils such as lemon oil, orange oil, grape oil, and grapefruit oil; and fruit essences including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, and apricot essences.

[0126] In some embodiments, the excipient may include a sweetener. Non-limiting examples of suitable sweeteners may include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts, such as sodium salt; dipeptide sweeteners, such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana (stevioside); chlorine derivatives of sucrose, such as sucralose; and sugar alcohols, such as sorbitol, mannitol, xylitol, and the like.

[0127] In some cases, the pharmaceutical compositions described herein may include a colorant. Non-limiting examples of suitable colorants may include food, drug, and cosmetic colorants (FD&C), drug and cosmetic colorants (D&C), and external drug and cosmetic colorants (Ext. D&C). The colorant may be used as a dye or its corresponding lake.

[0128] In some cases, a pharmaceutical composition as described herein may comprise a chelating agent. In some cases, the chelating agent may be a fungicidal chelating agent. Examples may 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 monohydrate; N,N ... (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); 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-di Aminopropane-N,N,N',N'-tetraacetic acid; nitrilotriacetic acid; nitrilotripropionic acid; trisodium salt of nitrilotris(methylenephosphonic acid); 7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo[11,11,11]pentatriacontane hexahydrobromide; or triethylenetetramine-N,N,N',N",N"',N"'-hexaacetic acid.

[0129] Also contemplated are combination products comprising one or more modified oncolytic viruses disclosed herein and one or more other antimicrobial or antifungal agents, for example, polyenes such as amphotericin B, amphotericin B lipid complex (ABCD), liposomal amphotericin B (L-AMB), and liposomal nystatin, azoles and triazoles such as voriconazole, fluconazole, ketoconazole, itraconazole, posaconazole, and the like; glucan synthase inhibitors such as caspofungin, micafungin (FK463), and V-echinocandin (LY303366); griseofulvin; allylamines such as terbinafine; flucytosine, or other antifungal agents, including those described herein. In addition, it is envisioned that the peptides can be combined with topical antifungal agents, such as ciclopirox, haloprogin, tolnaftate, undecylenate, topical nystatin, amorolfine, butenafine, naftifine, terbinafine and other topical agents. In some cases, the pharmaceutical composition may include additional agents. In some cases, the additional agents may be present in the pharmaceutical composition in a therapeutically effective amount.

[0130] Under common storage and use conditions, pharmaceutical compositions as described herein may include preservatives to prevent the growth of microorganisms. In certain instances, pharmaceutical compositions as described herein may not include preservatives. Suitable pharmaceutical forms for injection may include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. Pharmaceutical compositions may include carriers, which are solvents or dispersion media comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and / or vegetable oils, or any combination thereof. Suitable fluidity can be achieved, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersant, and by using a surfactant to maintain. Preventing the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugar or sodium chloride, are preferably included. The extended absorption of injectable compositions can be achieved by using a composition of an agent that delays absorption (e.g., aluminum monostearate and gelatin).

[0131] In some embodiments, the present invention provides the parenteral administration of a liquid dosage form. For example, for parenteral administration with an aqueous solution, if necessary, the liquid dosage form can be suitably buffered, and the liquid diluent and enough saline or glucose are isotonic. The liquid dosage form is particularly suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this respect, according to the present disclosure, operable sterile aqueous medium is well known to those skilled in the art. For example, a dosage can be dissolved in the isotonic NaCl solution of 1mL to 20mL, and added to the liquid (for example, sodium bicarbonate buffered saline) of 100mL to 1000mL, or be injected into the infusion site of suggestion.

[0132] In certain embodiments, an injectable sterile solution can be prepared by mixing a modified oncolytic poxvirus as described herein or a pharmaceutical composition comprising the modified oncolytic poxvirus into an appropriate solvent (the solvent has various other ingredients listed above as needed) in the desired amount, and then filtering and sterilizing. Typically, dispersions are prepared by mixing various sterilized active ingredients into a sterile vehicle comprising a basic dispersion medium and other ingredients required from those enumerated above. The compositions disclosed herein can be formulated in the form of a neutral or salt. Pharmaceutically acceptable salts include acid addition salts (acid addition salts formed with the free amino groups of proteins), and are acid addition salts formed with inorganic acids such as, for example, hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. The salts formed with free carboxyl groups can also be derived from inorganic bases, such as, for example, hydroxides of sodium, potassium, ammonium, calcium or iron; and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, etc. Once formulated, the pharmaceutical composition may be administered in a manner compatible with the dosage formulation, and in such amount as to be therapeutically effective.

[0133] In certain embodiments, the pharmaceutical composition of the present disclosure may include an effective amount of a modified oncolytic poxvirus disclosed herein, in combination with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" includes any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and / or is non-toxic to the patient to which it is administered. Non-limiting examples of suitable pharmaceutical carriers may include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents and sterile solutions. Other non-limiting examples of pharmaceutically compatible carriers may include gels, bioabsorbable matrix materials, implant elements comprising modified oncolytic viruses, or any other suitable vehicles, delivery or dispersion tools or materials. Such carriers can be formulated by conventional methods and can be administered to a subject in an effective amount.

[0134] Generation method

[0135] 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 bred in suitable host cells (e.g., HeLa cells, 293 cells, or Vero cells), separated from the host cells and stored under conditions that promote viral stability and integrity, so that the loss of infectivity over time is minimized. In some cases, the modified oncolytic virus producing a high percentage of EEV particles can act as an improved inoculum in the oncolytic virus manufacturing process. In certain exemplary methods, a cell stacker, a roller bottle, or a perfusion bioreactor is used to breed the modified oncolytic poxvirus in the host cell. In some instances, the downstream method for purifying the modified oncolytic virus may include filtration (e.g., deep filtration, tangential flow filtration, or a combination thereof), ultracentrifugation, or chromatographic capture. For example, the modified oncolytic virus can be stored by freezing or drying, such as by lyophilization. In certain embodiments, before administration, the stored modified oncolytic poxvirus can be reconstructed (if dry storage) and diluted in a pharmaceutically acceptable carrier for administration.

[0136] Some embodiments provide that the modified oncolytic poxviruses described herein exhibit higher titers in HeLa cells and 293 cells compared to otherwise identical viruses that do not comprise the modified oncolytic virus. In some cases, higher titers in HeLa cells and 293 cells are observed in the modified oncolytic poxviruses.

[0137] Combination therapy

[0138] In certain embodiments, the method of the present disclosure includes administering a modified oncolytic poxvirus as disclosed herein or a pharmaceutical composition containing the modified oncolytic poxvirus before, after, or in combination with one or more additional therapies. Examples of the additional therapies may include, but are not limited to, chemotherapy, radiotherapy, oncolytic virus therapy using another virus, treatment using an immunomodulatory protein, an anticancer agent, or any combination thereof. Relative to the administration of a modified poxvirus such as an oncolytic vaccinia virus, the additional therapy may be administered simultaneously or sequentially. In certain embodiments, the method of the present disclosure may include administering a modified oncolytic virus as disclosed herein before, after, or in combination with one or more anticancer agents or cancer therapies. Anticancer agents may include, but are not limited to, chemotherapeutic agents, radiotherapeutic agents, cytokines, immune checkpoint inhibitors, anti-angiogenic agents, apoptosis inducers, anticancer antibodies, and / or anti-cyclin-dependent kinase agents. In certain embodiments, cancer therapy may include chemotherapy, biotherapy, radiotherapy, immunotherapy, hormone therapy, anti-angiogenic therapy, cryotherapy, toxin therapy, and / or surgery, or a combination thereof. In certain embodiments, the methods of the present disclosure may include administering a modified virus disclosed herein before, after, or in combination with a modified oncolytic virus of the present disclosure. The combination of a modified oncolytic poxvirus, such as a modified vaccinia virus, and chemotherapy achieves a synergistic effect that is not observed in a modified oncolytic virus that does not contain the modifications in the modified oncolytic virus. The synergistic effect of the above combination can be advantageously used to reduce the risk of chemotherapy, such as Thus, the therapeutic methods disclosed herein using modified viruses can reduce toxicities associated with chemotherapy, for example, in patients who respond to chemotherapy but experience side effects at therapeutic doses. In some cases, the synergistic effect can result in reduced tumor growth compared to chemotherapy alone or oncolytic vaccinia virus alone. Exemplary reductions in tumor growth can be from about 2% to about 50%, such as about 5%, about 10%, about 20%, about 25%, about 35%, about 45%, or about 50%.

[0139] In certain embodiments, treatment using a modified oncolytic poxvirus such as vaccinia virus can be used alone or in combination with one or more immunomodulators. Immunomodulators may include any compound, molecule, or substance capable of suppressing antiviral immunity associated with a tumor or cancer. In certain embodiments, immunomodulators may be capable of suppressing innate immunity or adaptive immunity to modified viruses. Non-limiting examples of immunomodulators include anti-CD33 antibodies or their variable regions (also referred to herein as their antigen-binding fragments); anti-CD11b antibodies or their variable regions (also referred to herein as their antigen-binding fragments); COX2 inhibitors, for example, celecoxib; cytokines such as IL-12, GM-CSF, IL-2, IFN3, and IFNγ; and chemokines such as MIP-1, MCP-1, and IL-8. In certain embodiments, immunomodulators may include immune checkpoint regulators, 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 an immune checkpoint inhibitor, such as an antagonist of PD-1 (e.g., an antagonist antibody that binds to PD-1), an antagonist of PD-L1 (e.g., an antagonist antibody that binds to PD-L1), an antagonist of CTLA-4 (e.g., an antagonist antibody that binds to CTLA-4), an antagonist of A2AR (e.g., an antagonist antibody that binds to A2AR), an antagonist of B7-H3 (e.g., an antagonist antibody that binds to B7-H3), or an antagonist of PD-1 (e.g., an antagonist antibody that binds to PD-1). 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 modulator can be JTX-2011. In one example, the immune checkpoint modulator can be Keytruda (pembrolizumab). In one example, the immune checkpoint modulator can be Opdivo (nivolumab).In one example, the immune checkpoint regulator can be Yervoy (ipilimumab). In one example, the immune checkpoint regulator can 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).

[0140] In certain instances, where the additional therapy is radiation, an exemplary dose may be 5,000 Rad (50 Gy) to 100,000 Rad (1000 Gy), or 50,000 Rad (500 Gy), or other suitable doses within the range. Alternatively, the radiation 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 range, wherein the dose is determined, for example, by a dosimetric study as described above. As used herein, "Gy" may refer to a unit of specific absorbed dose of radiation equal to 100 Rads. Gy is an abbreviation for "Gray."

[0141] In certain instances, where the additional therapy is chemotherapy, exemplary chemotherapeutic agents may include, but are not limited to, alkylating agents (e.g., nitrogen mustard derivatives, ethyleneimines, alkyl sulfonates, hydrazines and triazines, nitrosureas, and metal salts), plant alkaloids (e.g., vinca alkaloids, taxanes, podophyllotoxins, and camptothecin analogs), antitumor antibiotics (e.g., anthracyclines, chromomycins, etc.), antimetabolites (e.g., folic acid antagonists, pyrimidine antagonists, purine antagonists, and adenosine deaminase inhibitors), topoisomerase I inhibitors, topoisomerase II inhibitors, and other miscellaneous antineoplastics (e.g., ribonucleotide reductase inhibitors, adrenocortical steroid inhibitors, enzymes, antimicrotubule agents, and retinoids). Exemplary chemotherapeutic agents may include, but are not limited to, anastrozole. Bicalutamide Bleomycin sulfate Busulfan Busulfan Injection Capecitabine N4-pentyloxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin Carmustine Chlorambucil Cisplatin Cladribine Cyclophosphamide ( or ), cytarabine, cytosine arabinoside Cytarabine liposome injection Dacarbazine Dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride Daunorubicin citrate liposome injection Dexamethasone, docetaxel Doxorubicin hydrochloride Etoposide Fludarabine phosphate 5-Fluorouracil Flutamide tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea Idabit Ifosfamide Irinotecan L-asparaginase Calcium folinate, melphalan 6-Mercaptopurine methotrexate Mitoxantrone Gemtuzumab (mylotarg), paclitaxel Phoenix (Yttrium90 / MX-DTPA), pentostatin, and carmustine polyphenylpropane 20 implants Tamoxifen citrate Teniposide 6-Thioguanine, thiotepa, tirapazamine Topotecan Hydrochloride for Injection Vinblastine vincristine and vinorelbine Ibrutinib, idelalisib, and brentuximab vedotin.

[0142] Exemplary alkylating agents can include, but are not limited to, nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes; uracil mustards (Aminouracil

[0143] Uracil nitrogen ), chlormethine Cyclophosphamide ( Revimmune TM ), ifosfamide Melphalan Chlorambucil Pipobrom Triethylenemelamine Triethylenethiophosphoramide, Temozolomide Thiotepa Busulfan Carmustine Lomustine Streptozotocin and dacarbazine Additional exemplary alkylating agents include, but are not limited to, oxaliplatin Temozolomide ( and ); Dactinomycin (also known as actinomycin-D, ); melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, ); Hexamethylmelamine (also known as hexamethylmelamine (HMM), ); Carmustine Bendamustine Busulfan ( and ); carboplatin Lomustine (also known as CCNU, ); Cisplatin (also known as CDDP, and -AQ); Chlorambucil Cyclophosphamide ( and ); Dacarbazine (also known as DTIC, DIC, and imidazole carboxamide, ); Hexamethylmelamine (also known as hexamethylmelamine (HMM), ); ifosfamide Prednimustine, procarbazine Dichloromethyldiethylamine (also known as nitrogen mustard, mustine, and mechloroethamine hydrochloride), ); Streptozotocin Thiotepa (also known as thiophosphoamide, TESPA, and TSPA, ); cyclophosphamide and bendamustine HCl

[0144] Exemplary anthracyclines can include, but are not limited to, for example, doxorubicin ( and ); bleomycin Daunorubicin (daunorubicin hydrochloride, daunomycin and daunomycin hydrochloride, ); daunorubicin liposomes (daunorubicin citrate liposomes, ); Mitoxantrone (DHAD, ); Epirubicin (Ellence TM ); Idabit ( Idamycin ); Mitomycin C Geldanamycin; herbimycin; ravidomycin and deacetyl ravidomycin.

[0145] Exemplary vinca alkaloids can include, but are not limited to, vinorelbine tartrate vincristine and vindesine Vinblastine (also known as vinblastine sulfate, vincaleukoblastine, and VLB, and ) and vinorelbine

[0146] Exemplary proteasome inhibitors may include, but are not limited to, bortezomib 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-phenylbutyrylamino)-pentanamide); marizomib (NPI-0 052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-serine-O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[0147] As used herein, "in combination with" means administering a modified poxvirus, such as an oncolytic vaccinia virus as described herein, or a pharmaceutical composition comprising the oncolytic vaccinia virus, to a subject with another therapy, such as another therapy comprising one or more agents, as part of a treatment regimen or plan. In certain embodiments, the combined use does not require that the modified oncolytic virus and the one or more agents be physically combined prior to administration, nor does it require that they be administered within the same timeframe. For example, without limitation, the modified oncolytic virus and the one or more agents can be administered to the subject simultaneously, or can be administered at the same time or sequentially in any order or at different time points.

[0148] In a plurality of embodiments, the other therapy can be used in liquid dosage form, solid dosage form, suppository, inhalable dosage form, intranasal dosage form, liposomal formulation, dosage form comprising nanoparticles, dosage form comprising microparticles, polymer dosage form or any combination thereof.In certain embodiments, the other therapy is used in 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 period.In some cases, the frequency of administration of the other therapy can be once a day, twice a day, once a week, once every three weeks, once every four 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 (once every 6 months). In certain embodiments, a method of treating a subject having cancer may comprise administering to the subject an effective amount of a modified oncolytic poxvirus of the present disclosure, such as a modified oncolytic vaccinia virus. 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, without limitation, the agent may be an anticancer agent, an immunomodulator, or any combination thereof as described above.

[0149] medicine box

[0150] In an embodiment, the present disclosure provides a kit for administering a modified oncolytic poxvirus, such as a modified oncolytic poxvirus as described herein. In certain embodiments, the kit of the present disclosure may include a modified oncolytic poxvirus, such as a modified oncolytic poxvirus, or a pharmaceutical composition comprising a modified oncolytic poxvirus as described above. In certain embodiments, the kit of the present disclosure may also include one or more components, such as instructions for use, devices, and additional reagents, and components for performing the methods disclosed above, such as tubes, containers, and syringes. In certain embodiments, the kit of the present disclosure may also include one or more agents, for example, at least one of an anticancer agent, an immunomodulator, or any combination thereof, which may be administered in combination with a modified virus.

[0151] In certain embodiments, the kit of the present disclosure may include one or more containers containing modified viruses disclosed herein. For example, without limitation, the kit of the present disclosure may include one or more containers containing modified oncolytic viruses of the present disclosure.

[0152] In certain embodiments, the kit of the present disclosure may include instructions for use, a device for administering a modified oncolytic virus to a subject, or a device for administering another agent or compound to a subject. For example, without limitation, the instructions may include a description of the modified oncolytic virus and optionally other components contained in the kit, and a method of administration, including a method for determining the appropriate state of the subject, an appropriate dose, and an appropriate method of administration for administering the modified virus. The instructions may also include a guide for monitoring the subject during the duration of the treatment.

[0153] In certain embodiments, the kit of the present disclosure may include a device for administering a modified oncolytic virus to a subject. Any of a variety of devices known in the art for administering drugs and pharmaceutical compositions may be included in the kit provided herein. For example, without limitation, such devices include hypodermic needles, intravenous needles, catheters, needle-free injection devices, inhalers, and liquid dispensers such as eye drops. In certain embodiments, a modified oncolytic virus that is systemically delivered, for example, by intravenous injection, intratumoral injection, or intraperitoneal injection may be included in a kit with a hypodermic needle and a syringe. Example

[0154] The following examples further illustrate the described embodiments but do not limit the scope of the disclosure.

[0155] Example 1: Preparation and characterization of modified oncolytic vaccinia viruses containing WO34

[0156] This study identified and characterized novel mutations in vaccinia virus (WR strain) that increase the release of EEV particles from the virus. EEV particles can enhance the oncolytic potential of the virus (e.g., by increasing delivery and spread through the bloodstream) and may provide benefits for other therapeutic uses of vaccinia (e.g., as a vaccine).

[0157] Random mutagenesis of the WR A34R gene was performed, and the resulting vaccinia virus strains (containing multiple mutations in the A34R gene) were screened for EEV particle production to identify strains containing mutations that enhanced high-level EEV production / release. Strains containing the K151E mutation and an additional point mutation (K119E) in A34R were identified. It was observed that when the strain contained a double mutation (K119E and K151E) in A34R (WO34), there was a significant increase in the level of EEV particle production. Additional studies were conducted that showed that oncolytic strains containing WO34 (A34R double mutants K119E and K151E) showed enhanced therapeutic effects when evaluated in the context of cancer treatment in a mouse model.

[0158] Screening method for identifying WO34

[0159] Random mutagenesis of the viral gene VACWR157 (also referred to herein as the A34R gene, which encodes the A34R protein) was 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 immediately adjacent to VACWR157 were also amplified by PCR. The 3' region was also assembled with a GFP reporter (GFP at the 5' end) by PCR. Finally, the 5'-mutagenized VACWR157 and GFP-3' fragments were assembled by PCR using the short complementary regions between the fragments (which were added by PCR primers). A simplified assembly diagram is shown in Figure 1 Depicted in (promoter and primer overhangs not shown).

[0160] A fully assembled fragment containing mutagenized VACWR157 and GFP, flanked by 5' and 3' regions of vaccinia viral DNA, was purified and used to transfect 143B human osteosarcoma cells. The transfected cells were then infected with vaccinia virus to allow recombination-driven production of a mutagenized VACWR157 viral library. To preferentially select for mutants with enhanced EEV production, medium from flasks infected with the viral pool was added to uninfected flasks. Clonal plaques were isolated after several rounds of serial infection, and the mutation in A34R that enhances EEV production was characterized by Sanger sequencing.

[0161] It was observed that all isolated clones shared two missense mutations: K119E and K151E, and a silent mutation at F129 (SEQ ID No. 2). The mutant protein was named WO34 (SEQ ID No. 2), and a corresponding clone was named UID WO0064R.002. The GFP reporter in this initial group of clones was not flanked by loxP sites and could not be removed. A codon-scrambled ORF encoding WO34 was synthesized and used to clone a transfer vector (pWR157-KE.R) containing GFP flanked by loxP. New recombinant viruses encoding WO34 were screened from fluorescent plaques and then used to infect cells expressing the cre recombinase. Clones lacking the reporter were selected after cre treatment.

[0162] Table 1: A34R gene and protein sequences

[0163]

[0164] Comet tails of viral plaques formed by vaccinia virus enhanced by EEV containing WO34

[0165] Previously titrated EEV-enhanced vaccinia virus was diluted and plated onto confluent BS-C-40 cells in 6-well plates. The plates were incubated and left undisturbed for 48 hours, and the cells were then stained with crystal violet to observe differences in comet tail formation caused by aberrant EEV production. Figure 2 As shown in Figure 2 As observed in the 2015 study, vaccinia virus strains containing WO34 showed enhanced comet tail formation compared to the WI strain (which has the A34R single mutation in the WR strain) and the WR strain (without the mutated A34R). The comet tail formation of the vaccinia virus strain containing WO34 was significantly increased.

[0166] EEV-enhanced neutralization of vaccinia virus containing WO34

[0167] In two independent experiments using different strains of vaccinia virus (vaccinia virus containing WO34, 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 removed and centrifuged at 800g. About 500 μl of supernatant was then removed and used for viral plaque assay. During serial dilution, the samples were treated with anti-L1 NR-45114 antibody or VIG and then incubated at 37°C for 1 hour. The dilutions were then added to 6-well plates of confluent BS-C-40 cells for plaque assay. After 1.5 hours, the culture medium was replaced with CM10 containing 3% CMC. After 48 hours, the cells were stained with crystal violet to count the viral plaques to determine the titer of the virus in the HeLa cell supernatant and the blocking ability of the neutralizing antibodies, as Figure 3A-3B As shown in ( Figure 3A shows the viral plaque titer after treatment with anti-L1 NR-45114 antibody, and Figure 3B (The viral plaque titers after treatment with anti-L1R antibody and VIG antibody are shown).

[0168] Enhanced cell viability after vaccinia virus infection with EEV containing WO34

[0169] In a 96-well plate, HCT116 or MC38 cell lines were inoculated and allowed to grow to 90% confluence. The cells were then infected with different vaccinia virus strains (WR strain, IHD-J strain, WI strain, strain containing WO34) at 1 MOI. Cell viability was tested daily at 24-hour intervals using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Kit from Promega. Relative viability was calculated by averaging the blank values from all wells, calculating the mean of the uninfected control group, and then calculating the relative value of each infected well as (A490 / mean of the uninfected wells). The results are shown in Table 1. Figure 4 Shown in (upper panel - MC38 cells; lower panel - HCT116 cells).

[0170] Viral replication assay of EEV-enhanced vaccinia virus containing WO34 in cancer cells

[0171] In separate experiments, HCT116 or MC38 cells were grown to 90% confluence on 12-well plates, and one plate was used each day for a 3-day replication assay. All plates were infected on the same day with 1 MOI of different vaccinia virus strains (WR strain, IHD-J strain, WI strain, strain containing WO34). Every 24 h after infection, one plate was frozen in a -80°C freezer. The plates were frozen and thawed twice to disrupt the cells, and the lysates were used for viral plaque assays on BS-C-40 cells. Plaque forming units per ml were 100 μg / ml. Figure 5 Shown in (upper panel: HCT116 cells; lower panel: MC38 cells). Sequence Listing <110> Caliwell Immunotherapy <120> Modified extracellular enveloped viruses <130> 199249.704301 <140> PCT / US2020 / 056107 <141> 2020-10-16 <150> 62 / 916,035 <151> 2019-10-16 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 507 <212> DNA <213> Vaccinia virus <400> 1 atgaaatcgc ttaatagaca aactgtaagt aggtttaaga agttgtcggt gccggccgct 60 ataatgatga tactctcaac cattattagt ggcataggaa catttctgca ttacaaagaa 120 gaactgatgc ctagtgcttg cgccaatgga tggatacaat acgataaaca ttgttattta 180 gatactaaca ttaaaatgtc tacagataat gcggtttatc agtgtcgtaa attacgagcc 240 agattgccta gaccggatac tagacatctg agagtattgt ttagtatttt ttataaagat 300 tattgggtaa gtttaaaaaa gaccaatgat aaatggttag atattaataa tgataaagat 360 atagatatta gtaaattaac aaattttaaa caactaaaca gtacgacgga tgctgaagcg 420 tgttatatat acaagtctgg aaaactggtt aaaacagtat gtaaaagtac tcaatctgta 480 ctatgtgtta aaaaattcta caagtga 507 <210> 2 <211> 507 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 2 atgaaatcgc ttaatagaca aactgtaagt aggtttaaga agttgtcggt gccggccgct 60 ataatgatga tactctcaac cattattagt ggcataggaa catttctgca ttacaaagaa 120 gaactgatgc ctagtgcttg cgccaatgga tggatacaat acgataaaca ttgttattta 180 gatactaaca ttaaaatgtc tacagataat gcggtttatc agtgtcgtaa attacgagcc 240 agattgccta gaccggatac tagacatctg agagtattgt ttagtatttt ttataaagat 300 tattgggtaa gtttaaaaaa gaccaatgat aaatggttag atattaataa tgatgaggat 360 atagatatta gtaaattaac aaatttcaaa caactaaaca gtacgacgga tgctgaagcg 420 tgttatatat acaagtctgg aaaactggtt gaaacagtat gtaaaagtac tcaatctgta 480 ctatgtgtta aaaaattcta caagtga 507 <210> 3 <211> 507<000099,6><212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 3<00,01001>atgaaatcgc ttaatagaca aactgtaagt aggtttaaga agttgtcggt gccggccgct 60 ataatgatga tactctcaac cattattagt ggcataggaa catttctgca ttacaaagaa 120 gaactgatgc ctagtgcttg cgccaatgga tggatacaat acgataaaca ttgttattta 180 gatactaaca ttaaaatgtc tacagataat gcggtttatc agtgtcgtaa attacgagcc 240 agattgccta gaccggatac tagacatctg agagtattgt ttagtatttt ttataaagat 300 tattgggtaa gtttaaaaaa gaccaatgat aaatggttag atattaataa tgatgaggat 360 atagatatta gtaaattaac aaatttcaa caactaaca gtacgacgga tgctgaagcg 420 tgttatatat acagtctgg aaaactggtt gaacagtat gtaaagtac tcaatctgta 480 ctatgtgtta aaaattcta chapter 507 <210> 4 <211> 168 <212> PRT <213> Vaccinia virus <400> 4 Met Lys Ser Leu Asn Arg Gln Thr Val Ser Arg Phe Lys Lys Leu Ser 1 5 10 15 Val Pro Only Only Only Only Only Only Thr Only Only Only Gly Only 20 25 30 Gly Thr Phe Leu His Tyr Lys Glu Glu Leu Met Pro Ser Ala Cys Ala 35 40 45 Asn Gly Trp Ile Gln Tyr Asp Lys His Cys Tyr Leu Asp Thr Asn Ile 50 55 60 Lys Met Three Asp Asn Ala Val Tyr Gln Cys Arg Lys Leu Arg Ala 65 70 75 80 Arg Leu Pro Arg Pro Asp Thr Arg His Leu Arg Val Leu Phe Ser Ile 85 90 95 Phe Tyr Lys Asp Tyr Trp Val Ser Leu Lys Lys Thr Asn Asp Lys Trp 100 105 110 Leu Asp Ile Asn Asn Asp Lys Asp Ile Asp Ile Ser Lys Leu Thr Asn 115 120 125 Phe Lys Gln Leu Asn Ser Thr Thr Asp Ala Glu Ala Cys Tyr Ile Tyr 130 135 140 Lys Ser Gly Lys Leu Val Lys Thr Val Cys Lys Ser Thr Gln Ser Val 145 150 155 160 Leu Cys Val Lys Lys Phe Tyr Lys 165 <210> 5 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 5 Met Lys Ser Leu Asn Arg Gln Thr Val Ser Arg Phe Lys Lys Leu Ser 1 5 10 15 Val Pro Ala Ala Ile Met Met Ile Leu Ser Thr Ile Ile Ser Gly Ile 20 25 30 Gly Thr Phe Leu His Tyr Lys Glu Glu Leu Met Pro Ser Ala Cys Ala 35 40 45 Asn Gly Trp Ile Gln Tyr Asp Lys His Cys Tyr Leu Asp Thr Asn Ile 50 55 60 Lys Met Three Asp Asn Ala Val Tyr Gln Cys Arg Lys Leu Arg Ala 65 70 75 80 Arg Leu Pro Arg Pro Asp Thr Arg His Leu Arg Val Leu Phe Ser Ile 85 90 95 Phe Tyr Lys Asp Tyr Trp Will Be Leu Lys Thr Asn Asp Lys Trp 100 105 110 Asp With Asn Asn Asp Glu Asp With Asp Served With Three Asns 115 120 125 Phe Lys Gln Leu Asn Serves Thr Thr Asp Ala Glu Ala Cys Tyr Ile Tyr 130 135 140 Lys Ser Gly Lys Leu Val Glu Thr Val Cys Lys Ser Thr Gln Ser Val 145 150 155 160 Leu Cys Val Lys Phe Tyr Lys 165

Claims

1. An oncolytic vaccinia virus, wherein the oncolytic vaccinia virus comprises: A nucleic acid encoding an A34R protein, wherein the A34R protein consists of a sequence that differs from the wild-type vaccinia virus A34R protein of SEQ ID NO. 4 in two mutations at positions Lys119 and Lys151 corresponding to the wild-type vaccinia virus A34R protein of SEQ ID NO. 4, and wherein the two mutations are Lys119Glu and Lys151Glu, respectively.

2. The oncolytic vaccinia virus of claim 1 , wherein the oncolytic vaccinia virus produces an increased number of comet-tail plaques in a viral plaque formation assay compared to an otherwise identical oncolytic vaccinia virus not having the two mutations.

3. The oncolytic vaccinia virus according to claim 1, wherein the oncolytic vaccinia virus is modified.

4. The oncolytic vaccinia virus of claim 1 , wherein the oncolytic vaccinia virus produces a greater amount of the extracellular enveloped virus form than the intracellular mature virus form compared to an otherwise identical oncolytic vaccinia virus that does not comprise the mutations Lys119Glu and Lys151Glu.

5. Use of the oncolytic vaccinia virus according to any one of claims 1 to 4 in the preparation of a composition for treating colon cancer, wherein when the composition is used, a therapeutically effective amount of the oncolytic vaccinia virus is administered to a subject.

6. A kit comprising: an oncolytic vaccinia virus according to any one of claims 1 to 4, a container; and instructions for administering the oncolytic vaccinia virus to a subject to treat a disorder associated with pathological angiogenesis.

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