Nucleic acids encoding soluble PD-1 and IL-12 and uses thereof

Abstract

The present disclosure provides nucleic acids encoding soluble PD-1 variants and IL-12. Further, nucleic acids encoding chemokine receptors are provided herein. Additionally, described herein are oncolytic viruses comprising the nucleic acids described herein. The oncolytic viruses optionally comprise a mutation or deletion of a gene expressing IFN-gamma. The compositions described herein are further described for use in the treatment of cancer. Described herein are nucleic acids comprising sequences encoding at least two polypeptides, including interleukin-12 (IL-12) or a functional variant thereof and soluble PD-1 (sPD-1) or a functional variant thereof.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 417,484, filed October 19, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ST.26 xml format, which is incorporated herein by reference in its entirety. A copy of said xml, created on September 28, 2023, is named 199249-725601_SL.xml and is 112,890 bytes in size. [Background technology]

[0003] background Tumor cells express programmed cell death protein 1 ligand (PD-L1) to trigger checkpoint inhibition and evade immune responses. PD-L1 binds to PD-1 on T cells at the checkpoint and suppresses T cell responses. Blocking PD-L1 can enhance T cell function in cancer therapy. Truncated PD-1, lacking the transmembrane domain, results in soluble PD-1. Soluble PD-1 can act as a decoy receptor that binds to PD-L1 on tumor cells, blocking the PD-L1:PD-1 signaling pathway and acting as a checkpoint inhibitor. Improved cancer therapies incorporating checkpoint inhibitor intervention are described herein.

[0004] IL-12 enhances the immune response by activating T cells and NK cells. Described herein is the use of soluble PD-1 in conjunction with IL-12 for the treatment of cancer. Summary of the Invention [Means for solving the problem]

[0005] overview Described herein are nucleic acids comprising sequences encoding at least two polypeptides, including interleukin-12 (IL-12) or a functional variant thereof and soluble PD-1 (sPD-1) or a functional variant thereof.

[0006] Described herein are nucleic acids comprising a first region encoding a first polypeptide comprising a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:5 or SEQ ID NO:8, and a second region encoding a second polypeptide comprising a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:2 or SEQ ID NO:4.

[0007] Described herein are compositions comprising a vector, an exogenous nucleic acid comprising a sequence encoding a cytokine or a functional variant thereof, and an exogenous nucleic acid comprising a sequence encoding a PD-L1 receptor.

[0008] Described herein is an oncolytic virus comprising an insertion at the TK locus, the insertion comprising, in 5' to 3' order, a first promoter region, wherein the promoter is P7.5, a first region encoding IL-12, a second promoter region, wherein the promoter is P28, and a second region encoding a PD-1 variant.

[0009] Described herein are pharmaceutical compositions comprising a nucleic acid described herein or a composition described herein and a pharmaceutically acceptable excipient.

[0010] Described herein are methods for the treatment of cancer, the methods comprising administering to a subject having cancer a pharmaceutical composition described herein in an amount sufficient to treat the cancer.

[0011] Described herein are methods for activating an anti-tumor immune response, the method comprising administering to a subject with cancer a pharmaceutical composition described herein.

[0012] Described herein is a method for reducing the incidence of tumor cell growth, comprising administering to tumor cells a pharmaceutical composition described herein in an effective amount sufficient to reduce the incidence of tumor cell growth.

[0013] 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 sets forth illustrative embodiments, and the accompanying drawings, in which the principles of the disclosure are utilized. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1A is a schematic diagram of PD-1 / PD-L1 interactions between tumor cells and T cells.

[0015] Figure 1B is a schematic diagram of sPD-1 binding to PD-L1 on T cells, preventing PD-1 / PD-L1 interaction.

[0016] [Figure 2] FIG. 2 is a diagram of a transgene inserted into the TK locus, with the P7.5 promoter driving expression of a gene encoding an IL-12 polypeptide comprising the IL-12 beta and alpha subunits linked by a 22-residue glycine-rich linker, and the P28 promoter driving expression of a gene encoding a soluble PD-1 polypeptide.

[0017] [Figure 3]Figure 3A is a graph plotting the volume of Renca cell tumors induced in mice 45 days after treatment on the y-axis in the treatment groups indicated on the x-axis; treatment groups received buffer control, TK-control, or TK-virus modified to express mouse IL-12 and mouse sPD-1. Tumors in the control group measured at least 1000 mm3, while the average size in the treatment groups was approximately 200 mm3. The treatment group receiving the modified virus showed a 40% complete response (CR).

[0018] Figure 3B is a graph plotting on the y-axis the volume of B16 cell tumors induced in mice after 38 days of treatment in the treatment groups indicated on the x-axis; treatment groups received buffer control, TK-control, or TK-virus modified to express murine IL-12 and murine sPD-1. Tumors in the control group were at least 1400 mm 3 The mean size of the treatment group was below detectable levels. The treatment group receiving the modified virus showed a 90% CR.

[0019] [Figure 4] Figure 4 is a graph plotting LLC tumor volume in mice on the y-axis after 31 days of treatment in the treatment groups indicated on the x-axis; treatment groups received buffer control, TK-control, TK- / B8R-control, or TK- / BR8-virus modified to express mouse IL-12 and mouse sPD-1. Tumors in the buffer and TK-control groups were at least 1400 mm3 in size. The mean size of the TK and treatment groups was below detectable levels. The treatment group receiving the modified virus showed an 80% CR.

[0020] [Figure 5] FIG. 5 is a diagram of a transgene inserted into the A52R locus using the A52R promoter to drive expression of the CXCR3 chemokine receptor. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description Tumor cells utilize various mechanisms to evade detection and attack by the host immune system. Such mechanisms can affect the effectiveness of cancer immunotherapy. Described herein are compositions comprising a combination of immune checkpoint inhibitors and pro-inflammatory cytokines to enhance the immune response against tumor cells, either alone or in conjunction with other therapeutic modalities.

[0022] Programmed cell death protein 1 (PD-1) is an immune checkpoint protein expressed on the surface of T and B cells. Upon binding to PD-L1, a ligand found on macrophages, for example, T cell responses are suppressed. This protein helps regulate autoimmunity by suppressing T cell inflammatory responses against "self" cells. Some tumor cells exploit this immune suppression by expressing PD-L1 on their surface (Figure 1A). Binding of PD-L1 on tumor cells to PD-1 on T cells downregulates T cell activity and suppresses antitumor activity. Inhibition of PD-L1 / PD-1 interaction, or immune checkpoint blockade, can enhance T cell responses and increase antitumor activity. Described herein is a modified PD-1 protein that lacks the transmembrane and cytoplasmic domains, resulting in a soluble variant of PD-1 (sPD-1). sPD-1 can bind to PD-L1 and prevent PD-L1 / PD-1 checkpoint inhibition (Figure 1B), allowing the progression of T cell responses. In some embodiments, the sPD-1 described herein maintained its binding activity to PD-L1. Further, nucleic acids encoding sPD-1, and vectors comprising such nucleic acids, are described herein.

[0023] IL-12 activates anti-tumor cytotoxic immune responses and modulates the responses of T cells, NK cells, and antigen-presenting cells. The cytokine enhances anti-tumor immune responses and inhibits immunosuppression. Provided herein are compositions comprising nucleic acids encoding co-expression of sPD-1 and IL-12.

[0024] Compositions and uses thereof for the treatment of cancer are provided herein. The compositions described herein may comprise one or more nucleic acids encoding the polypeptides described herein. The nucleic acids provided herein may comprise DNA, RNA, nucleic acid analogs, or any combination thereof. Briefly, (1) nucleic acids encoding the expression of sPD-1 and IL-12, (2) nucleic acids encoding the expression of chemokine receptors, (3) vectors for the expression of the described nucleic acids, (4) modified oncolytic viruses, (5) treatment conditions, and (6) dosages, forms, and methods of administration of the compositions described herein are described herein.

[0025] definition The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent the terms "contains," "containing," "including," "includes," "having," "has," "with," or variations thereof, are used in either the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0026] The term "about" or "approximately" can mean within an acceptable range of error for a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. When a particular value is described in this application and claims, unless otherwise indicated, the term "about" should be assumed to mean within an acceptable range of error for that particular value, e.g., ±10% of the value modified by the term "about."

[0027] The terms "heterologous nucleic acid sequence" or "exogenous nucleic acid sequence" or "transgene" as used herein in reference to a particular virus may refer to a nucleic acid sequence that originates from a source other than the designated virus.

[0028] The term "mutation," as used herein, can refer to a deletion, insertion of a heterologous nucleic acid, inversion, or substitution, including open reading frame removal mutations as generally understood in the art.

[0029] The term "gene," as used herein, may refer to a segment of nucleic acid that encodes a particular protein or RNA (also referred to as a "coding sequence" or "coding region"), optionally along with associated regulatory regions, e.g., promoters, operators, terminators, etc., which may be located upstream or downstream of the coding sequence.

[0030] As used herein, a "promoter" may refer to a regulatory sequence, which is a region of a nucleic acid sequence that controls the initiation and rate of transcription. In certain embodiments, a promoter may include genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. The terms "operably positioned," "operably linked," "under control," and "under transcriptional control" may mean that the promoter is in the correct functional position and / or orientation relative to the nucleic acid sequence so as to control the initiation and / or expression of the transcription of the nucleic acid 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 involved in the transcriptional activation of a nucleic acid sequence.

[0031] The term "homology" as used herein can refer to the calculation of "homology" or "percent homology" between two or more nucleotide or amino acid sequences, which can be determined by aligning the sequences for optimal comparison (e.g., gaps may be introduced into the sequence of the first sequence). Nucleotides at corresponding positions can then be compared, and the percent identity between the two sequences can be a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions × 100). For example, a position in the first sequence can be occupied by the same nucleotide as the corresponding position in the second sequence, in which case the molecules are identical at that position. The percent homology between two sequences can be a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. In some embodiments, the length of sequences aligned for comparison purposes may be at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 95% of the length of the reference sequence. A BLAST® search can be used to determine homology between two sequences. Homology can be between the entire length of the two sequences or between fractions 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 well-known methods, for example, using a mathematical algorithm. When utilizing BLAST and Gapped BLAST programs, any relevant parameters of the respective programs (e.g., NBLAST) can be used. For example, parameters for sequence comparison can be set at score=100, word length=12, or may 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.

[0032] The term "subject" may refer to an animal, including, but not limited to, a primate (e.g., a human), cow, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms "subject" and "patient" are used interchangeably herein, e.g., in reference to a mammalian subject, e.g., a human subject.

[0033] The terms "treat," "treating," and "treatment" may be intended to include alleviating or arresting a disorder, disease, or condition, or one or more of the symptoms associated with a disorder, disease, or condition, or alleviating or eradicating the cause of the disorder, disease, or condition itself.

[0034] The term "therapeutically effective amount" may refer to that amount of a compound that, when administered, may be sufficient to prevent the occurrence of, or alleviate to some extent, one or more of the symptoms of the disorder, disease, or condition being treated.

[0035] The term "oncolytic" as used herein can refer to the killing of cancer or tumor cells by an agent, such as an oncolytic poxvirus, for example, an oncolytic vaccinia virus, through the direct lysis of cancer or tumor cells, for example, by stimulating an immune response against cancer or tumor cells, apoptosis, the expression of toxic proteins, the cessation of autophagy and protein synthesis, the induction of anti-tumor immunity, or any combination thereof. The direct lysis of cancer or tumor cells infected with an agent, such as an oncolytic vaccinia virus, can be the result of viral replication within the cells. In certain instances, the term "oncolytic" can refer to the killing of cancer or tumor cells without the lysis of the cells.

[0036] The term "oncolytic virus" as used herein may refer to a virus that preferentially infects and kills tumor cells. In some embodiments, oncolytic viruses can include, but are not limited to, (i) viruses that naturally replicate preferentially in cancer cells and are non-pathogenic in humans, often due to their high susceptibility to natural antiviral signaling or their dependency on tumorigenic signaling pathways, and (ii) viruses that have been genetically engineered for use. In some embodiments, oncolytic viruses can be measles viruses, polioviruses, poxviruses, vaccinia viruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, vesicular stomatitis viruses, reoviruses, Newcastle disease viruses, Seneca viruses, lentiviruses, mengoviruses, or myxoma viruses. In certain embodiments, oncolytic viruses can be poxviruses. In certain embodiments, oncolytic viruses can be vaccinia viruses.

[0037] The term "modified oncolytic virus," as used herein, may refer to an oncolytic virus that includes modifications to its components, such as, but not limited to, modifications in the virus's native genome ("backbone"), such as mutations or deletions of viral genes, introduction of exogenous nucleic acid, chemical modification of viral nucleic acid or viral proteins, and introduction of exogenous or modified viral proteins into the viral capsid. Generally, oncolytic viruses may be modified (also known as "engineered") to achieve improved therapeutic effects on tumor cells. In some embodiments, the modified oncolytic virus may be a modified poxvirus. In some embodiments, the modified oncolytic virus may be a modified poxvirus. In some embodiments, the modified oncolytic virus may be a modified vaccinia virus.

[0038] The terms "systemic delivery" and "systemic administration," used interchangeably herein, may refer, in some cases, to the route of administration of a pharmaceutical, oncolytic virus, or other substance into the circulatory system. Systemic administration may include intravenous administration, oral administration, intraperitoneal administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, intra-arterial administration, or any combination thereof.

[0039] Soluble programmed cell death protein 1 (sPD-1) Immune checkpoint inhibitors prevent checkpoint proteins from binding to their receptors, thereby allowing the immune response to proceed. The use of checkpoint inhibitors in cancer treatment circumvents the ability of cancer cells to evade attack by T cells. Native programmed cell death protein 1 (PD-1) contains a transmembrane domain that anchors the protein to the surface of expressing cells. A PD-1 variant truncated before the transmembrane domain results in a soluble version of PD-1 (sPD-1). sPD-1 can act as a dominant-negative decoy receptor, binding to and sequestering PD-L1 and blocking the PD-1:PD-L1 signaling pathway. Blocking binding to PD-L1 allows increased T cell activity and T cell-mediated killing (Figure 1B).

[0040] Provided herein are compositions comprising nucleic acids encoding PD-1 immune checkpoint inhibitors. In some embodiments, the PD-1 inhibitor prevents the interaction of PD-1 on tumor cells with PD-L1 on T cells. In some embodiments, the PD-1 inhibitor comprises modified PD-1. In some embodiments, the PD-1 is mouse PD-1. In some embodiments, the PD-1 is human PD-1.

[0041] Native PD-1 is a membrane-bound protein of 288 amino acids. Domains beginning at the amino terminus include an extracellular domain, a transmembrane domain, and a cytoplasmic domain. In mouse PD-1, the extracellular domain comprises 168 amino acids (SEQ ID NO: 2). In human PD-1, the extracellular domain comprises 170 amino acids (SEQ ID NO: 4). Exemplary sequences for inclusion in the compositions described herein are listed in Table 1, SEQ ID NOs: 1-4. Table 1. Amino acid sequence of PD-1 [Table 1-1] [Table 1-2]

[0042] Provided herein are compositions comprising a nucleic acid encoding mouse sPD-1. In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO: 2. In some embodiments, the encoded mouse sPD-1 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 2.

[0043] Provided herein are compositions comprising a nucleic acid encoding human sPD-1. In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO: 4. In some embodiments, the encoded human sPD-1 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 4.

[0044] In some embodiments, human sPD1 further comprises one or more substitutions compared to the wild-type sequence (SEQ ID NO: 3). In some embodiments, the one or more mutations result in an increase in the binding affinity between soluble PD-1 and PD-L1. In some embodiments, the one or more mutations comprise E61V, M70I, Q75F, K78W, K78L, E84F, S87W, A129H, A132L, K135M, or any combination thereof. The substitution positions are based on the positions in SEQ ID NO: 3.

[0045] IL-12 Cytokines generally regulate the growth and activity of immune system cells. IL-12 induces the differentiation of T cells into T helper 1 (Th1) cells. Th1 cells help clear pathogens from the system. Provided herein are compositions comprising a nucleic acid encoding IL-12 or a functional variant thereof. In some embodiments, the nucleic acid encodes a first polypeptide comprising interleukin 12 (IL-12) or a functional variant thereof. In some embodiments, the IL-12 comprises a heterodimer comprising subunit beta (IL-12b) and subunit alpha (IL-12a). In some embodiments, the nucleic acid encodes the murine IL-12 (mIL-12) sequence described by SEQ ID NO:5. In some cases, the encoded mIL-12 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 5. In some embodiments, the nucleic acid encodes the human IL-12 (hIL-12) sequence set forth by SEQ ID NO: 7. In some cases, the encoded hIL-12 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO:7.

[0046] Provided herein are compositions comprising a nucleic acid encoding murine IL-12 subunit alpha (IL-12a) (UniProtKB Accession ID 43431.1). In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO: 6. In some cases, the encoded IL-12a comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 6.

[0047] Provided herein are compositions comprising a nucleic acid encoding mouse IL-12 subunit beta (IL-12b) (UniProtKB Accession ID P43432.1). In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO: 7. In some cases, the encoded IL-12b comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 7.

[0048] Provided herein are compositions comprising a nucleic acid encoding human IL-12 subunit alpha (hIL-12a) (UniProtKB Accession ID P060595). In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO: 9. In some cases, the encoded hIL-12a comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 9.

[0049] Provided herein are compositions comprising a nucleic acid encoding human IL-12 subunit beta (hIL-12b) (UniProtKB Accession ID P29460). In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO: 10. In some cases, the encoded IL-12b comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 10.

[0050] Exemplary amino acid sequences of regions of IL-12 for inclusion in the compositions described herein are listed in Table 2. Table 2. IL-12 amino acid sequence. [Table 2-1] [Table 2-2]

[0051] Linker and signaling domains Compositions comprising nucleic acids encoding linkers are provided herein. In some embodiments, the nucleic acid encoding a linker is disposed between various encoded biologically functional units described herein. In some embodiments, the encoded linker is flexible or rigid. In further embodiments, the encoded linker is a cleavable linker. In further embodiments, the encoded cleavable linker comprises a disulfide bond. In further embodiments, the encoded cleavable linker comprises a protease-sensitive domain. A non-limiting list of exemplary linkers encoded by nucleic acids included in the compositions described herein is listed in Table 3. In some embodiments, the compositions described herein comprise a nucleic acid encoding a linker having a sequence set forth by SEQ ID NO:11. In some embodiments, the nucleic acid encodes a linker comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:11. Table 3. Linkers. [Table 3] Subscripts in the sequences indicate repeats. Arrows in the sequence indicate the location of the cleavage site.

[0052] Combined Nucleic Acid Sequences Compositions comprising nucleic acids encoding a combination of technical properties are provided herein. In some embodiments, the nucleic acid encodes an IL-12 dimer (IL-12) or a functional variant thereof and a soluble PD-1 (sPD-1) protein or a functional variant thereof. In some embodiments, the nucleic acid encodes a mouse IL-12 dimer (IL-12) and a mouse sPD-1 protein or a functional variant thereof. In some embodiments, the mouse IL-2 comprises the sequence of SEQ ID NO: 5. In some embodiments, the mouse sPD-1 comprises the sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid encodes a human IL-12 dimer and a human sPD-1 protein or a functional variant thereof. In some embodiments, the human IL-12 comprises the sequence of SEQ ID NO: 8. In some embodiments, the human sPD-1 comprises the sequence set forth in SEQ ID NO: 4.

[0053] In alternative embodiments, two nucleic acids are provided, wherein a first nucleic acid encodes a first polypeptide comprising IL-12 or a functional variant thereof, and a second nucleic acid encodes a second polypeptide comprising sPD-1 (sPD-1) protein or a functional variant thereof. In some embodiments, the IL-12 comprises the sequence of SEQ ID NO:5 or SEQ ID NO:8. In some embodiments, the sPD-1 protein comprises the sequence of SEQ ID NO:2 or SEQ ID NO:4.

[0054] chemokine receptors In some embodiments, provided herein is a modified oncolytic virus comprising an exogenous nucleic acid encoding a chemokine receptor, also referred to herein as a transgene. In some cases, the exogenous nucleic acid is a therapeutic transgene. In some embodiments, provided herein is a modified oncolytic virus comprising an exogenous nucleic acid encoding a membrane-associated protein that degrades hyaluronan, such as hyaluronidase. In some embodiments, provided herein is a modified oncolytic virus comprising an exogenous nucleic acid encoding both a chemokine receptor and a hyaluronidase.

[0055] Chemokines are chemotactic cytokines that regulate cell trafficking and positioning by activating seven-transmembrane chemokine receptors. Chemokines are sometimes divided into four subfamilies, including the CC, CXC, CX3C, and XC subfamilies, based on the location of their first two N-terminal cysteine ​​residues. Differential expression of chemokine receptors on leukocytes results in the selective recruitment of specific cell types under specific conditions, as needed, to provide an appropriate and efficient immune response to infectious agents or foreign damage. In addition to their pivotal role in the coordinated migration of immune cells to sites of inflammation, chemokines often also play important roles in lymphoid tissue development, immune cell maturation, and the generation and delivery of adaptive immune responses.

[0056] Tumors are increasingly recognized as complex microenvironments formed by numerous different cell types that coexist and communicate with each other in complex signaling networks. Chemokines are essential regulators of cell migration and cell-cell interactions and therefore have a profound impact on tumorigenesis. In the tumor microenvironment, tumor-associated host cells and cancer cells release a range of different chemokines, leading to the recruitment and activation of different cell types that mediate the balance between anti-tumor and pro-tumor responses. In addition to their primary role as chemoattractants, chemokines are often involved in other tumor-related processes, including tumor cell growth, angiogenesis, and metastasis.

[0057] Tumor cells have been shown to acquire the ability to produce growth-promoting chemokines. For example, melanomas have been found to express a number of chemokines, including CXCL1, CXCL2, CXCL3, CXCL8, CCL2, and CCL5, which have been implicated in tumor growth and progression. CCL2 levels can be found elevated in neuroblastoma cell lines and primary tumor cells isolated from human patients. Immunostaining studies have also suggested elevated expression levels of CXCL12 in various cancers, including breast cancer, carcinoid, cervical cancer, colorectal cancer, endometrial cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, and gastric cancer.

[0058] Chemokine receptors are cytokine receptors found on the surface of certain cells that interact with chemokines. In humans, 20 different chemokine receptors have been discovered. Each has a seven-transmembrane structure and couples to a G protein for intracellular signaling, making it a member of the large protein family of G protein-coupled receptors. After interacting with their specific chemokine ligands, chemokine receptors transduce intracellular calcium (Ca 2+ ) ions (calcium signaling), which triggers a cellular response, including the occurrence of a process known as chemotaxis, which transports cells to a desired location within an organism. Generally, the term "chemokine receptor," as used herein, can refer to a membrane-bound protein that selectively binds to and induces chemotaxis toward a chemokine ligand.

[0059] It should be understood that the chemokine receptors disclosed herein, in some cases, refer not only to naturally occurring chemokine receptors identified in the human body, but also include chemokine receptors derived from other sources, such as, but not limited to, (1) naturally occurring chemokine receptors identified in animals, e.g., pigs, dogs, cattle, and sheep, and (2) non-naturally occurring chemokine receptors, such as mutant proteins, chimeric receptors, and designed proteins having binding affinity for a particular type of chemokine. In some examples, a fragment of a naturally occurring chemokine receptor is also considered a chemokine receptor if the fragment retains the function of binding to and responding to the corresponding chemokine and inducing cellular chemotaxis. As provided herein, in some embodiments, a virus containing an exogenous nucleic acid encoding a chemokine receptor forces virus-infected cells to express the chemokine receptor because the virus hijacks the host cell's gene expression machinery.

[0060] In some cases, the modified oncolytic virus includes an exogenous nucleic acid encoding a cytokine receptor whose cognate cytokine is expressed in the tumor microenvironment (e.g., IL15-R has the cognate cytokine IL15 expressed in the tumor microenvironment). In some cases, the modified oncolytic virus encodes a chemokine receptor whose cognate chemokine is likely to be expressed on the tumor (e.g., CXCR4 has the cognate chemokine CXCL12 expressed on the tumor, and CCR2 has the target CCL2 expressed on the tumor) and is delivered systemically as a naked virus. Upon systemic delivery, after the modified oncolytic virus enters the bloodstream, the virus infects lymphocytes, e.g., B cells, and redirects the infected B cells to the tumor, resulting in a significantly increased viral load in the tumor. In certain embodiments, the increased viral load in the tumor is achieved immediately after systemic delivery. The ability to deliver the modified oncolytic viruses disclosed herein in a systemic manner provides an advantage over conventional intratumoral delivery methods of oncolytic viruses. Although intratumoral delivery is useful for treating easily accessible tumors, in some cases, it is crucial to treat inaccessible or metastatic cancers, which are considered to be the main cause of death from disease.In this situation, relying on intratumoral delivery of oncolytic viruses is ineffective, because it requires systemic dissemination after administration to distant sites.However, this dissemination is often transient and ineffective, at least in part due to the occurrence of immune response to virus infection.

[0061] Chemokine receptors are divided into different families. Non-limiting examples of chemokine receptors described herein include CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors, which correspond to the four different subfamilies of chemokines they bind to. Among CXC chemokine receptors, CXCR1 and CXCR2 are closely related, while CXCR1 binds to CXCL8 and CXCL6, CXCR2 binds to CXCL1 and CXCL7, CXCR3 binds to CXCL9, CXCL10, and CXCL11, CXCR4 binds to CXCL12 (or SDF-1), CXCR5 binds to CXCL13, and CXCR6 binds to CXCL16. Among the CC chemokine receptors, ligands for CCR1 include CCL4, CCL5, CCL6, CCL14, CCL15, CCL16, and CCL23; ligands for CCR2 include CCL2, CCL8, and CCL16; ligands for CCR3 include CCL11, CCL26, CCL7, CCL13, CCL15, CCL24, CCL5, CCL28, and CCL18; ligands for CCR4 include CCL3, CCL5, CCL17, and CCL22; and ligands for CCR5 include CCL4, CCL5, CCL17, and CCL22. Ligands for CCR6 include CCL20, CCR7 include CCL19 and CCL21, CCR8 include CCL1 and CCL16, CCR9 include CCL25, CCR10 include CCL27 and CCL28, and CCR11 include CCL19, CCL21, and CCL25. The CX3C chemokine receptor CX3CR1 has the ligand CXCL1. The XC chemokine receptor XCR1 binds to both XCL1 and XCL2.

[0062] Non-limiting embodiments of the present disclosure provide a modified oncolytic virus comprising an exogenous nucleic acid encoding a chemokine receptor. In some embodiments, the chemokine receptor is a CXC chemokine receptor, a CC chemokine receptor, a CX3C chemokine receptor, an XC chemokine receptor, or any combination thereof. In some embodiments, the chemokine receptor is CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, or any combination thereof.

[0063] In certain embodiments, the modified oncolytic virus comprises an exogenous CXCR4-expressing nucleic acid. In certain embodiments, the modified oncolytic virus comprises an exogenous CCR2-expressing nucleic acid. Certain embodiments disclose modified oncolytic viruses comprising exogenous nucleic acids encoding both CXCR4 and CCR2, wherein both chemokines are expressed from the same virus. Under certain circumstances, CXCL12 and / or CCL2, which are typically expressed in the tumor microenvironment, attract lymphocytes expressing CXCR4 and / or CCR2 or other migratory cells infected with the modified oncolytic virus, thereby enhancing tumor-targeted delivery of the modified oncolytic virus. The nucleic acid and amino acid sequences of selected chemokine receptors are listed in Table 4. Table 4. Chemokine receptor sequences. [Table 4-1] [Table 4-2] [Table 4-3]

[0064] In the compositions provided herein, the oncolytic virus gene may be mutated or replaced with a nucleic acid encoding a chemokine receptor listed in Table 4. In some embodiments, the chemokine receptor is mouse CXCR3 as set forth in SEQ ID NO: 27. In some embodiments, the encoded mouse CXCR3 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 27. In some embodiments, the chemokine receptor is human CXCR3 as set forth in SEQ ID NO: 28. In some embodiments, the encoded human CXCR3 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO:28.

[0065] promoter Provided herein are compositions comprising nucleic acids, wherein the nucleic acids encode at least one promoter region. A promoter region, or promoter, or promoter element, or regulatory region, refers to a nucleic acid sequence to which a protein binds to initiate transcription. Promoters are typically located 5', or upstream, of the DNA coding region they control. In some embodiments, the nucleic acids described herein comprise one promoter. In some embodiments, one promoter drives the transcription of all polypeptides encoded on the nucleic acid. In some embodiments, the nucleic acids described herein comprise separate promoters for each polypeptide encoded on the nucleic acid. In some embodiments, the nucleic acid comprises two promoters, each of which drives the transcription of one of the two polypeptides encoded on the nucleic acid.

[0066] The timing of expression can be modulated by the promoter structure that regulates gene expression. The number and affinity of transcription factor binding sites determine the relative timing of expression between different promoter regions. Promoters with more transcription factor binding sites and / or higher binding affinity can initiate expression earlier than promoters with fewer or lower affinity binding sites.

[0067] The application of relative temporal expression of proteins can be utilized to express specific factors from the modified viruses described herein either early or late in the infection process. Early promoters have repetitive transcription factor binding sites. Late promoters have fewer binding sites than early promoters. In some embodiments, receptors are expressed using early promoters. Expression early in infection allows for expression and processing by the cell before cellular processes are disrupted. In some embodiments, one or more cytokines are expressed using late promoters.

[0068] In some embodiments, provided herein are promoters including P7.5, P28, P135, TK promoter, A52R promoter, 454 promoter, PB8, LEO, PF11, F7L, H5R, mH5, H1L, A1L, J3R, E4L, I1L, I3L, I4L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, L4R, T7 promoter, 28kDa promoter, short synthetic promoter (SSP), or any functional variant or combination thereof. In some embodiments, the promoter is an early promoter. In some embodiments, the early promoter is A52R, PB8, mH5, I4L, LEO, PF11, I3L, P7.5, TK promoter, F7L, H5R, short synthetic promoter (SSP), or any variation or combination thereof. In some embodiments, the promoter is a late promoter. In some embodiments, the late promoter comprises SSP, P7.5, P28, P135, TK promoter, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, L4R, 28kDa promoter, or any functional variant or combination thereof. The sequences of selected promoters are listed in Table 5. Table 5. Promoter nucleic acid sequences [Table 5]

[0069] The compositions provided herein may comprise a P7.5 promoter and a P28 promoter. In some embodiments, the P7.5 promoter drives expression of a region encoding an IL-12 polypeptide. In some embodiments, the P28 promoter drives transcription of sPD-1. A schematic diagram of the promoter and transgene inserted into the TK locus is shown in Figure 2. In some embodiments, the P7.5 promoter comprises the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the P28 promoter comprises the nucleic acid sequence of SEQ ID NO: 41.

[0070] The compositions provided herein may comprise a P135 promoter and a P7.5 promoter. In some embodiments, the P135 promoter drives expression of a region encoding an IL-12 polypeptide. In some embodiments, the P7.5 promoter drives expression of a region encoding sPD-1. In some embodiments, the P7.5 promoter comprises the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the P135 promoter comprises the sequence of SEQ ID NO: 43.

[0071] Compositions comprising exogenous nucleic acids are provided herein. In some embodiments, the exogenous nucleic acid comprises RNA. In some embodiments, the exogenous nucleic acid comprises DNA. In some embodiments, the DNA comprises, in 5' to 3' order, an IL-12 beta subunit, a linker, an IL-12 alpha subunit, and sPD-1. In some embodiments, the DNA comprises, in 5' to 3' order, a P7.5 promoter (SEQ ID NO: 44), mouse IL-12b (SEQ ID NO: 49), a flexible linker (SEQ ID NO: 50), mouse IL-12a (SEQ ID NO: 51), a P28 promoter (SEQ ID NO: 41), and mouse sPD-1 (SEQ ID NO: 53). In some embodiments, the DNA comprises, in 5' to 3' order, a P7.5 promoter (SEQ ID NO: 44), mouse IL-12 (SEQ ID NO: 48), a P28 promoter (SEQ ID NO: 41), and mouse sPD-1 (SEQ ID NO: 53). In some embodiments, the DNA comprises, in 5' to 3' order, the P7.5 promoter (SEQ ID NO:44), human IL-12b (SEQ ID NO:61), a flexible linker (SEQ ID NO:50), human IL-12a (SEQ ID NO:62), the P28 promoter (SEQ ID NO:41), and human sPD-1 (SEQ ID NO:63). In some embodiments, the DNA comprises, in 5' to 3' order, the P7.5 promoter (SEQ ID NO:44), human IL-12 (SEQ ID NO:60), the P28 promoter (SEQ ID NO:41), and human sPD-1 (SEQ ID NO:60). In some embodiments, an exogenous nucleic acid described herein is integrated into a viral genome.

[0072] vector Compositions comprising vectors are provided herein. Generally, vectors are vehicles designed to deliver nucleic acids to cells. In some embodiments, vectors include plasmids, phages, viruses, cosmids, or artificial chromosomes. For purposes of this application, vectors may also include transfection agents and methods, such as liposomes, nanoparticles, electroporation, microinjection, gene guns, impalefection, hydrostatic pressure, continuous injection, sonication, or any combination thereof. Compositions comprising vectors and one or more nucleic acids described herein are provided herein. In some embodiments, the vector comprises a virus. In some embodiments, the virus is a retrovirus, lentivirus, adenovirus, adeno-associated virus, or herpes simplex virus. In some embodiments, the virus is an oncolytic virus. In some embodiments, the vector is a modified virus. In some embodiments, the virus contains mutations or deletions of one or more genes. In some embodiments, the virus contains one or more exogenous nucleic acids described herein.

[0073] Oncolytic viruses The present invention provides a composition comprising an oncolytic virus, wherein the oncolytic virus comprises a modified nucleic acid described herein. As used herein, an oncolytic virus kills cancer or tumor cells through mechanisms such as direct lysis of cancer or tumor cells by stimulating an immune response against the cells, apoptosis, expression of toxic proteins, shutting down autophagy and protein synthesis, inducing anti-tumor immunity, or any combination thereof. In some embodiments, the oncolytic viruses described herein replicate intracellularly. In some embodiments, the oncolytic viruses described herein replicate in tumor cells, immune cells, somatic cells, hematopoietic cells, or other types of cells. Exemplary oncolytic viruses that can be included in the compositions described herein include, but are not limited to, poxvirus, vaccinia virus, adeno-associated virus, adenovirus, reovirus, lentivirus, herpes simplex virus, vesicular stomatitis virus, mengovirus, myxoma virus, Newcastle disease virus, Seneca virus, retrovirus, measles virus, Maraba virus, coxsackievirus, or poliovirus.These oncolytic viruses tend to specifically target cancer cells, and during viral replication, cause significant cell death and tumor regression.In some embodiments, the oncolytic virus is vaccinia virus. Exemplary vaccinia viruses include, without limitation, the following strains, with respect to modification by inclusion of an exogenous nucleic acid as described herein: Western Reserve vaccinia virus (ATCC VR-1354), vaccinia virus Ankara (ATCC VR-1508), vaccinia virus Ankara (ATCC VR-1566), vaccinia virus strain Wyeth (ATCC VR-1536), or vaccinia virus Wyeth (ATCC VR-325). Further, in some embodiments, the recombinant vaccinia virus is a modified version of a wild-type or attenuated vaccinia virus strain.Non-limiting examples of vaccinia virus strains include the Western Reserve, Copenhagen, IHD, Wyeth (NYCBOH), Tian Tan, Lister, USSR, Ankara, NYVAC, Ankara (MVA), Paris, Bern, Temple of Heaven, Dairen, EM-63, Evans, King, Patwadangar, or Tash Kent strains of vaccinia virus.

[0074] The oncolytic virus is optionally recombinant or selected to have low toxicity and accumulate in target tissues. In some embodiments, the modification in the viral backbone / viral genome is a modification that selectively renders the virus replication-competent. The base oncolytic virus strain modified as described herein optionally contains one or more mutations or one or more deletions compared to its parent strain. In some embodiments, the modification includes a mutation or full or partial deletion in one or more of the following viral genes: A1, A2, VH1, A33, I7, A52R, TK, B15R, K7R, B14R, N1L, K1L, M2L, A49R, A46R, B8R, C12L, B18R, A52R, F3L, C4, or C16. In some embodiments, the viral backbone mutation is a complete or partial deletion of the A1 gene, a complete or partial deletion of the A2 gene, a complete or partial deletion of the VH1 gene, a complete or partial deletion of the A33 gene, a complete or partial deletion of the I7 gene, a complete or partial deletion of the A52R gene, a complete or partial deletion of the TK gene, a complete or partial deletion of the B15R gene, a complete or partial deletion of the K7R gene, a complete or partial deletion of the B14R gene, a complete or partial deletion of the N1L gene, a complete or partial deletion of the K1L gene, a complete or partial deletion of the M2L gene, a complete or partial deletion of the A49R gene, a complete or partial deletion of the A46R gene, a complete or partial deletion of the B8R gene, a complete or partial deletion of the C12L gene, a complete or partial deletion of the B18R gene, a complete or partial deletion of the A52R gene, a complete or partial deletion of the F3L gene, a complete or partial deletion of the C4 gene, or a complete or partial deletion of the C16 gene. As used herein, reference to a viral gene is made by reference to the protein encoded by the gene (e.g., the A33 gene refers to the gene encoding the A33 protein).In some embodiments, viral backbone mutations, including any combination of substitutions, insertions, and deletions, result in sequences with less than 100%, less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%, or less sequence identity to the wild-type sequence of the viral gene or viral protein encoded by that gene. In some embodiments, the viral backbone contains one, two, three, four, five, or more mutations in the amino acid sequence of a viral protein (e.g., a viral antigen). In some embodiments, the present disclosure provides recombinant oncolytic viruses that contain one or more mutations in the viral genome (viral backbone) such that the mutations increase the immune response of the T cell arm. The mutations can be additions, deletions, or substitutions of one or more nucleic acids in the viral genome (wild-type or attenuated native strain oncolytic virus). In non-limiting examples, the mutation is a full or partial deletion of a gene known to inhibit cytokines involved in Th1 immune responses. In some embodiments, the mutation is a deletion of a nucleic acid encoding B8R (an interferon gamma (IFN-g) binding protein). In some embodiments, the mutation is a deletion of a nucleic acid encoding C12L (an interleukin-18 (IL-18) binding protein). In some embodiments, the mutation is a full or partial deletion of a gene in innate immune signaling. In some embodiments, the mutation is a full or partial deletion of a nucleic acid encoding B18R (a type I interferon (IFN) binding protein). In some embodiments, the mutation is a full or partial deletion of a nucleic acid encoding A52R (a nuclear factor kappa B (NF-κB) inhibitor protein). In some embodiments, the mutation is a full or partial deletion of a nucleic acid encoding E3L (a protein kinase (PKR) inhibitor), and in some embodiments, the mutation is a full or partial deletion of a nucleic acid encoding C4 or C16 (a STING pathway inhibitor).

[0075] The oncolytic virus comprises one or more additional insertions or partial insertions of exogenous nucleic acids encoding one or more proteins, as described herein. In some embodiments, the one or more proteins comprise a chemokine receptor or a functional variant thereof, soluble PD1 or a functional variant thereof, or interleukin-12 or a functional variant thereof. In some embodiments, the one or more proteins comprise sPD-1 or a functional variant thereof and interleukin-12 or a functional variant thereof. Exemplary chemokine receptors for inclusion include, without limitation, wild-type and / or mutant CXCR3, CXCR4, CCR2, or CCL2. The oncolytic virus of the present disclosure further comprises one or more additional deletions or partial deletions of one or more genes from TK, A52R, B15R, K7R, A46R, N1L, E3L, K1L, M2L, C16, N2R, B8R, B18R, VH1, and functional domains, fragments, or variants thereof, or any combination thereof. In some cases, the oncolytic viruses provided herein comprise a complete or partial deletion of at least one of the A52R or TK viral genes and an insertion of an exogenous nucleic acid encoding one or more proteins (e.g., one or more immunomodulatory proteins). In some embodiments, the oncolytic virus further comprises a complete or partial deletion of the B8R gene.

[0076] In some embodiments, the oncolytic virus is a modified oncolytic virus that has one or more modifications that result in a superior therapeutic effect on tumor cells compared to an otherwise identical virus that does not contain the modifications. In some non-limiting examples, the superior therapeutic effect includes enhanced immune evasion of the virus, enhanced tumor-targeted systemic delivery of the virus, enhanced intratumor and intertumor spread of the virus, and enhanced tumor-specific replication of the virus, or release of immune modulators and antitumor agents into the extracellular matrix, or any combination thereof. In some cases, the modified oncolytic virus of the present disclosure is used as a platform vector for systemic delivery.

[0077] The oncolytic virus described herein comprises the exogenous nucleic acid described herein.In some embodiments, the oncolytic virus provided herein comprises the complete or partial deletion of TK gene and the insertion of the region encoding at least one of soluble PD-1 and cytokine, such as IL-12.The exemplary sequence for integration has been described herein above.

[0078] In some embodiments, the oncolytic viruses provided herein comprise a complete or partial deletion of the A52R gene and an insertion of a region encoding a chemokine receptor. In some embodiments, the chemokine receptor comprises CXCR3. In some embodiments, the region encoding a chemokine receptor comprises a sequence selected from Table 4. In some embodiments, the promoter driving expression of the chemokine receptor is an early promoter, a late promoter, a strong early promoter, a weak early promoter, a strong late promoter, a weak late promoter, or any combination thereof. In some embodiments, the A52R promoter drives expression of the chemokine receptor. In some embodiments, the A52R promoter drives expression of the region encoding CXCR3.

[0079] In some embodiments, provided herein are modified oncolytic viruses that contain modifications that enhance the immune response to tumors. Typically, oncolytic viruses are either (a) administered systemically, (b) inoculated locally against tumors, or (c) injected directly into tumors ("intratumoral delivery").

[0080] In some embodiments, provided herein are modified oncolytic viruses comprising modifications that enhance intratumoral and intertumoral spread of the virus. Enhanced intratumoral and intertumoral spread of the oncolytic virus is an effective way to boost therapeutic efficacy by increasing the number of cancer cells infected by the virus. In some embodiments, provided herein are modified oncolytic viruses comprising exogenous nucleic acid. In some embodiments, provided herein are modified oncolytic viruses comprising modifications in the viral genome. In some embodiments, provided herein are modified oncolytic viruses comprising exogenous nucleic acid and modifications in the viral genome.

[0081] In some embodiments, oncolytic viruses include, but are not limited to, (i) viruses that naturally replicate preferentially in cancer cells and are non-pathogenic in humans, often due to a high susceptibility to innate antiviral signaling or a dependency on oncogenic signaling pathways, and (ii) viruses that have been genetically engineered for use.

[0082] In some embodiments, modified oncolytic viruses are utilized. Generally, such viruses include modifications to components thereof, such as, but not limited to, modifications in the native genome ("backbone") of the virus, such as mutations or deletions of viral genes, introduction of exogenous nucleic acid, chemical modification of viral nucleic acid or viral proteins, and introduction of exogenous or modified viral proteins into the viral capsid.

[0083] In some embodiments, the modified oncolytic virus comprises a mutation or deletion in the TK gene and further comprises an exogenous nucleic acid encoding sPD-1. In some embodiments, the modified oncolytic virus comprises a mutation or deletion in the TK gene and further comprises an exogenous nucleic acid encoding sPD-1 and an exogenous nucleic acid encoding a cytokine. In some embodiments, the cytokine comprises IL-12.

[0084] In some embodiments, the modified oncolytic virus comprises a mutation or deletion of the A52R gene and further comprises an exogenous nucleic acid encoding a CXCR3 receptor. In some embodiments, the modified oncolytic virus comprises a mutation or deletion of the A52R gene (the A52R promoter is maintained) and further comprises an exogenous nucleic acid encoding a CXCR3 receptor.

[0085] In some cases, modified oncolytic virus comprises the above-mentioned full-length viral backbone gene or viral backbone protein, or a shortened version thereof, or a functional domain thereof, or a fragment thereof, or a variant thereof.In various examples, modified oncolytic virus comprises one or more mutations or deletions of viral backbone gene or viral backbone protein as described above.The mutations of viral backbone gene and viral backbone protein comprise the insertion, deletion, substitution, or modification of nucleotides in nucleic acid sequence and amino acid in protein sequence.In some cases, deletion comprises the complete or partial deletion of viral backbone gene or protein.

[0086] In some embodiments, the modification of the oncolytic virus increases the efficacy of tumor-targeted systemic delivery of the virus by at least about 1.1 fold, 1.2 fold, 1.5 fold, 1.8 fold, 2 fold, 2.2 fold, 2.5 fold, 2.8 fold, 3 fold, 3.2 fold, 3.5 fold, 3.8 fold, 4 fold, 4.2 fold, 4.5 fold, 4.8 fold, 5 fold, 5.2 fold, 5.5 fold, 5.8 fold, 6 fold, 6.2 fold, 6.5 fold, 6.8 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, 24 fold, 25 fold, 26 fold, 27 fold, 28 fold, 29 fold, 30 fold, 31 fold, 32 fold, 33 fold, 34 fold, 35 fold, 36 fold, 37 fold, 38 fold, 39 fold, 40 fold, 41 fold, 42 fold, 43 fold, 44 fold, 45 fold, 46 fold, 47 fold, 48 fold, 49 fold, 50 fold, 51 fold, 52 fold, 53 fold, 54 fold, 55 fold, 56 fold, 57 fold, 58 fold, 59 fold, 60 fold, 6 x, 7.2x, 7.5x, 7.8x, 8x, 8.2x, 8.5x, 8.8x, 9x, 9.2x, 9.5x, 9.8x, 10x, 12x, 14x, 15x, 16x, 18x, 20x, 25x, 30x, 35x, 40x 100x 4 times, 2.5 x 10 4 Double, 5 x 10 4 times, 7.5 x 10 4 times, 2.5 x 10 5 Double, 5 x 10 5 times, 7.5 x 10 5 double, 10 6 times, 2.5 x 10 6 Double, 5 x 10 6 times, 7.5 x 10 6 double, 10 7 times, 2.5 x 10 7 Double, 5 x 10 7 times, 7.5 x 10 7 double, 10 8 times, 2.5 x 10 8 Double, 5 x 10 8 times, 7.5 x 10 8 double, 10 9 times, 2.5 x 10 9 Double, 5 x 10 9 times, 7.5 x 10 9 double, 10 10In certain embodiments, the efficacy of tumor-targeted systemic delivery of a virus is measured by quantifying the virus that infects tumor cells, optionally compared with the virus that infects non-tumor cells in the body. For example, in some cases, virus quantification is performed by staining viral particles in tissue sections or blood smears in the case of leukemia, lymphoma, or myeloma. In some cases, such quantification is performed by using reporter molecules, such as luciferase and fluorescent proteins, engineered to be expressed by the virus. In some cases, such quantification is performed by quantifying viral genomes in tumors. It is also possible to measure tumor-targeted systemic delivery of a virus by quantifying certain downstream effects of viral infection in tumor cells, such as, but not limited to, cytokines in response to viral infection or lymphocyte accumulation. In some embodiments, the oncolytic virus comprises an exogenous nucleic acid encoding CXCR3, CXCR4, CCR2, or any combination thereof. In some embodiments, the presence of exogenous nucleic acid results in an approximately 5-10 fold increase in the efficacy of tumor-targeted systemic delivery of the virus compared to an otherwise identical oncolytic virus lacking the exogenous nucleic acid.

[0087] In some embodiments, provided herein are modified oncolytic viruses comprising an exogenous nucleic acid encoding a soluble immune checkpoint inhibitor. Expression of the soluble immune checkpoint inhibitor by the modified oncolytic virus results in a boosted immune response against the infected tumor. After infecting a tumor, the modified oncolytic virus replicates in tumor cells, resulting in the expression of soluble immune checkpoint inhibitors in the tumor environment. These soluble inhibitors act as decoy receptors, binding to checkpoint ligands and blocking the inhibition of immune responses in tumors. As a result, the immunosuppressive microenvironment in the tumor is altered, resulting in enhanced immunotherapeutic activity of the modified oncolytic virus compared to an otherwise identical virus lacking the nucleic acid encoding a chemokine receptor. In some embodiments, the increase in immunotherapeutic activity is at least about 1.1 fold, 1.1 fold, 1.2 fold, 1.5 fold, 1.8 fold, 2 fold, 2.2 fold, 2.5 fold, 2.8 fold, 3 fold, 3.2 fold, 3.5 fold, 3.8 fold, 4 fold, 4.2 fold, 4.5 fold, 4.8 fold, 5 fold, 5.2 fold, 5.5 fold, 5.8 fold, 6 fold, 6.2 fold, 6.5 fold, 6.8 fold, 7 fold, 7.2 fold, 7.5 fold, 7.8 fold, 8 fold, 8.2 fold , 8.5x, 8.8x, 9x, 9.2x, 9.5x, 9.8x, 10x, 12x, 14x, 15x, 16x, 18x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55 100x 4 times, 2.5 x 10 4 Double, 5 x 10 4 times, 7.5 x 10 4 times, 2.5 x 10 5 Double, 5 x 10 5 double, 10 62-fold or even higher. Increased immunotherapeutic activity is reflected, but not limited to, by increased B cell accumulation in tumors, increased T cell responses to tumor-associated immunogens, or both. B cell accumulation is measured, for example, by quantifying B cells in tumors, and T cell immune activity is measured, for example, by interferon-γ (interferon-gamma) secretion in an ELISPOT assay.

[0088] In some embodiments, the present invention provides a modified oncolytic virus comprising an exogenous nucleic acid encoding a chemokine receptor, and the forced expression of the chemokine receptor by the modified oncolytic virus results in a boosted immune response against the infected tumor. After infecting a tumor, the modified oncolytic virus replicates in tumor cells, resulting in the expression of chemokine receptors on the surface of the tumor cells. These membrane receptors act as decoy receptors, binding to and sequestering immunosuppressive chemokines (e.g., CXCL12 and / or CCL2) within the tumor. As a result, the immunosuppressive microenvironment in the tumor is altered, resulting in enhanced immunotherapeutic activity of the modified oncolytic virus compared to an identical virus that does not contain a nucleic acid encoding a chemokine receptor. In some embodiments, the increase in immunotherapeutic activity is at least about 1.1 fold, 1.1 fold, 1.2 fold, 1.5 fold, 1.8 fold, 2 fold, 2.2 fold, 2.5 fold, 2.8 fold, 3 fold, 3.2 fold, 3.5 fold, 3.8 fold, 4 fold, 4.2 fold, 4.5 fold, 4.8 fold, 5 fold, 5.2 fold, 5.5 fold, 5.8 fold, 6 fold, 6.2 fold, 6.5 fold, 6.8 fold, 7 fold, 7.2 fold, 7.5 fold, 7.8 fold, 8 fold, 8.2 fold , 8.5x, 8.8x, 9x, 9.2x, 9.5x, 9.8x, 10x, 12x, 14x, 15x, 16x, 18x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55 100x 4 times, 2.5 x 10 4 Double, 5 x 10 4 times, 7.5 x 10 4times, 2.5 x 10 5 Double, 5 x 10 5 double, 10 6 2-fold or even higher. Increased immunotherapeutic activity is reflected, but not limited to, by increased B cell accumulation in tumors, increased T cell responses to tumor-associated immunogens, or both. B cell accumulation is measured, for example, by quantifying B cells in tumors, and T cell immune activity is measured, for example, by interferon-γ (interferon-gamma) secretion in an ELISPOT assay.

[0089] In some embodiments, provided herein are modified oncolytic viruses comprising an exogenous nucleic acid encoding a chemokine receptor, wherein forced expression of the chemokine receptor by the modified oncolytic virus results in increased replication of the virus in tumor cells compared to an identical virus that does not comprise a nucleic acid encoding a chemokine receptor. In some embodiments, the modified oncolytic virus comprises an exogenous CXCR3-expressing nucleic acid. In some embodiments, the modified oncolytic virus comprises an exogenous CCR2-expressing nucleic acid, which increases tumor-specific replication of the virus. In some embodiments, the modified oncolytic virus comprises an exogenous CCR5-expressing nucleic acid, which increases tumor-specific replication of the virus. In some embodiments, the increase in tumor-specific replication is at least about 1.1 fold, 1.1 fold, 1.2 fold, 1.5 fold, 1.8 fold, 2 fold, 2.2 fold, 2.5 fold, 2.8 fold, 3 fold, 3.2 fold, 3.5 fold, 3.8 fold, 4 fold, 4.2 fold, 4.5 fold, 4.8 fold, 5 fold, 5.2 fold, 5.5 fold, 5.8 fold, 6 fold, 6.2 fold, 6.5 fold, 6.8 fold, 7 fold, 7.2 fold, 7.5 fold, 7.8 fold, 8 fold, 8.2 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, 24 fold, 25 fold, 26 fold, 27 fold, 28 fold, 29 fold, 30 fold, 31 fold, 32 fold, 33 fold, 34 fold, 35 fold, 36 fold, 37 fold, 38 fold, 39 fold, 40 fold, 41 fold, 42 fold, 43 fold, 44 fold, 45 fold, 46 fold, 47 fold, 48 fold, 49 fold, 50 fold, 51 fold, 52 fold, 53 fold, 54 fold, 55 fold, 56 fold, 57 fold, x, 8.5x, 8.8x, 9x, 9.2x, 9.5x, 9.8x, 10x, 12x, 14x, 15x, 16x, 18x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x 100x 4 times, 2.5 x 10 4 Double, 5 x 104 times, 7.5 x 10 4 times, 2.5 x 10 5 Double, 5 x 10 5 double, 10 6 Exemplary methods for measuring increased viral delivery and spread in tumors include, but are not limited to, fluorescence- or bioluminescence-based imaging of reporter gene expression, quantitative PCR for detection of tumor concentrations of viral genomes, or plaque determination of plaque-forming units, or immunohistochemistry of viral proteins.

[0090] Treatment conditions Provided herein are methods for the treatment of cancer, comprising administering a composition described herein. In some embodiments, the method of treatment is for a hyperproliferative disease. In some embodiments, the hyperproliferative disease is cancer. In some embodiments, the hyperproliferative disease comprises a tumor. Treatments are contemplated that include delivery of a modified oncolytic virus, such as an oncolytic vaccinia virus, described herein. In some embodiments, the cancer is melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial carcinoma, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal-type gastric adenocarcinoma, cervical squamous-cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma.

[0091] In some embodiments, the compositions described herein are administered to cancer cells originating from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testicle, tongue, or uterus. In addition, the cancer may optionally be of the following histological types, including, but not limited to, malignant neoplasm, carcinoma, undifferentiated carcinoma, giant cell and spindle cell carcinoma, small cell carcinoma, papillary carcinoma, squamous cell carcinoma, lymphoepithelial carcinoma, basal cell carcinoma, pilomatrix carcinoma, transitional cell carcinoma, papillary transitional cell carcinoma, adenocarcinoma, malignant gastrinoma, cholangiocarcinoma, hepatocellular carcinoma, combined hepatocellular carcinoma and cholangiocarcinoma, trabecular adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma in adenomatous polyps, familial polyposis coli adenocarcinoma, solid tumors, malignant carcinoid tumor, bronchiolo-alveolar adenocarcinoma, papillary adenocarcinoma, chromophobe carcinoma, eosinophilic carcinoma, oxyphilic adenocarcinoma adenocarcinoma), basophilic carcinoma, clear cell adenocarcinoma, granular cell carcinoma, follicular adenocarcinoma, papillary and follicular adenocarcinoma, nonencapsulating sclerosing carcinoma, adrenocortical carcinoma, endometroid carcinoma, skin appendage carcinoma, apocrine adenocarcinoma, sebaceous carcinoma, auditory canal adenocarcinoma (ceruminous)adenocarcinoma, mucoepidermoid carcinoma, cystadenocarcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, invasive ductal carcinoma of the breast, medullary carcinoma, lobular carcinoma, inflammatory carcinoma, Paget's disease of the breast, pancreatic 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 angiosarcoma, malignant melanoma, amelanotic melanoma, superficial spreading melanoma, malignant melanoma in giant pigmented 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, mixed Müllerian tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, malignant mesenchymoma, malignant Brenner tumor, malignant phyllodes tumor, synovial sarcoma, malignant mesothelioma, dysgerminoma, embryonal carcinoma, malignant teratoma, malignant ovarian stromal tumor, choriocarcinoma, malignant mesonephroma, angiosarcoma, malignant hemangioendothelioma, Kaposi's sarcoma, malignant hemangiopericytoma, lymphangiosarcoma, osteosarcoma, parosteal osteosarcoma, chondrosarcoma, malignant chondroblastoma, mesenchymal chondrosarcoma, giant cell tumor of bone, Ewing's sarcoma, malignant odontogenic tumor, ameloblastic odontogenic sarcoma odontosarcoma, malignant ameloblastoma, ameloblastic fibrosarcoma, malignant pinealoma, chordoma, malignant glioma, ependymoma, astrocytoma, protoplasmic astrocytoma, fibrous astrocytoma, astroblastoma, glioblastoma, oligodendroglioma, oligodendroblastoma, primitive neuroectodermal tumorneuroectodermal, cerebellar sarcoma, ganglioneuroblastoma, neuroblastoma, retinoblastoma, olfactory neurogenic tumor, malignant meningioma, neurofibrosarcoma, malignant neurilemmoma, malignant granular cell tumor, malignant lymphoma, Hodgkin's disease, Hodgkin's lymphoma, paragranuloma, malignant small lymphocytic lymphoma, malignant large cell diffuse lymphoma, malignant follicular lymphoma, mycosis fungoides, other specified non-Hodgkin's lymphoma, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small intestinal disease, leukemia, lymphoid leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia leukemia), myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, or hairy cell leukemia. In some cases, metastatic solid cancers are treated using the modified oncolytic viruses of the present disclosure, e.g., modified oncolytic vaccinia viruses that are advantageous for systemic delivery. In some cases, solid cancers that are inaccessible or difficult to access, such as for intratumoral delivery of therapeutic agents, are treated using the modified oncolytic viruses of the present disclosure, e.g., modified oncolytic vaccinia viruses that are advantageous for systemic delivery. In some embodiments, the compositions described herein are used to treat cancers associated with increased expression of free fatty acids.

[0092] The present disclosure also contemplates a method for inhibiting or preventing the local invasiveness or metastasis, or both, of any type of primary cancer. In exemplary embodiments, the primary cancer is melanoma, non-small cell lung, small cell lung, lung, hepatocellular carcinoma, retinoblastoma, astrocytoma, glioblastoma, gum, tongue, leukemia, neuroblastoma, head, neck, breast, pancreas, prostate, kidney, bone, testis, ovary, mesothelioma, cervix, gastrointestinal tract, lymphoma, brain, colon, or bladder. In certain embodiments, the primary cancer is lung cancer. For example, the lung cancer is non-small cell lung cancer. Furthermore, the present disclosure can be used to prevent cancer or treat pre-cancerous or pre-malignant cells, including metaplasia, dysplasia, and hyperplasia, as needed. It can also be used to inhibit unwanted but benign cells, such as squamous cell metaplasia, dysplasia, benign prostatic hyperplasia, hyperplastic lesions, etc. In some embodiments, cancer or the progression to more severe forms of cancer is stopped, destroyed, or slowed by the methods of the present disclosure, including the modified oncolytic viruses discussed herein.

[0093] Provided herein is a method for treating a subject by administering one or more modified oncolytic viruses disclosed herein.In this specification, "individual" or "subject" are used interchangeably to refer to human or non-human subjects.Non-limiting examples of non-human subjects include non-human primates, dogs, cats, mice, rats, guinea pigs, rabbits, pigs, poultry, horses, cows, goats, sheep, cetaceans, etc.In some embodiments, the subject is human.

[0094] Provided is a method for producing toxic effects in cancer cells, comprising administering a therapeutically effective amount of the modified virus described above, for example, an oncolytic vaccinia virus, or a pharmaceutical composition comprising the same to cancer cells.The present disclosure further provides a method for inhibiting at least one of the growth and proliferation of a second cancer cell, comprising administering the modified oncolytic virus described above to a first cancer cell so that the first cancer cell is infected with the virus.Therefore, in some embodiments of the method disclosed herein, it is contemplated that when a therapeutically effective amount of the oncolytic vaccinia virus described herein or a pharmaceutical composition comprising the same is administered, not all cancer or tumor cells are infected, and the growth of uninfected cells is inhibited without direct infection.

[0095] In some examples, the methods and compositions of the present disclosure are used to contact cancer cells or tumors with a therapeutically effective dose of the exemplary oncolytic vaccinia virus described herein or a pharmaceutical composition comprising the same, to induce tumor lysis, kill cells, inhibit growth, inhibit metastasis, reduce tumor size, and otherwise reverse or reduce the malignant phenotype of tumor cells.In certain embodiments, the effective amount of the modified oncolytic virus of the present disclosure, such as the oncolytic vaccinia virus described herein, or a pharmaceutical composition thereof, can comprise an amount sufficient to induce tumor lysis, destruction or lysis of cancer cells, or inhibit or reduce the growth or size of cancer cells.The reduction of cancer cell growth is indicated, for example, by cell death, or a slower replication rate or reduced growth rate of tumors comprising the cells, or the prolonged survival of subjects comprising cancer cells.

[0096] Provided herein is a method for treating a subject with cancer or a tumor, comprising administering to the subject an effective amount of the modified virus described above. In such methods, an effective amount includes an amount that reduces the growth rate or spread of the cancer or prolongs survival in the subject. The present disclosure provides a method for reducing tumor growth, comprising administering to a tumor an effective amount of the modified oncolytic virus described above. In certain embodiments, the effective amount of the modified virus or pharmaceutical composition thereof comprises an amount sufficient to induce slowing, inhibition, or reduction in tumor growth or size, including eradication of the tumor. Reduction of tumor growth is indicated, for example, by a reduced growth rate or prolonged survival of the tumor-bearing subject. In certain embodiments, the effective amount of the modified virus or pharmaceutical composition thereof comprises an amount sufficient to activate an anti-tumor response. In some embodiments, activating an anti-tumor response comprises activating T cells. In certain embodiments, the effective amount of the modified virus or pharmaceutical composition thereof comprises an amount sufficient to reduce the occurrence of tumor growth. In some embodiments, reducing the incidence of tumor growth includes inhibiting metastasis, preventing primary tumor growth, inhibiting existing tumor growth, or any combination thereof.

[0097] Provided herein is a method for determining the infectivity or anti-tumor activity of an oncolytic vaccinia virus described herein, or the amount of tumor-specific viral replication thereof, comprising the steps of: (i) administering to a subject a therapeutically effective amount of an oncolytic vaccinia virus or pharmaceutical composition according to the present disclosure that further expresses a luciferase reporter gene, alone or in combination with an additional therapy; (ii) collecting a first biological sample from the subject immediately after administering the virus and determining the level of the luciferase reporter in the first biological sample; (iii) collecting a second biological sample from the subject after administration in step (ii); and (iii) detecting the level of the luciferase reporter in the second biological sample; wherein if the level of luciferase is higher in step (iii) than in step (ii), the oncolytic vaccinia virus is determined to be infectious, exhibit anti-tumor activity, and exhibit tumor-specific viral replication. The second biological sample is collected at 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, or about 2 months after administration in step (i). In some embodiments, the above-described method in steps (i) and (iii) further comprises detecting the levels of one or more assayed 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 to the subject a therapeutically effective amount of a modified oncolytic virus of the present disclosure, e.g., an oncolytic vaccinia virus described herein, or a pharmaceutical composition comprising the same.In some embodiments of the present disclosure, the increase in luciferase bioluminescence between steps (ii) and (iv) above is greater for the modified oncolytic viruses described herein compared to the same virus except that the modified oncolytic virus does not contain the modification. Other exemplary techniques for detecting and monitoring viral load after administration of the modified oncolytic virus include real-time quantitative PCR.

[0098] Provided herein is a method for monitoring pharmacokinetics after administration of a therapeutically effective amount of a modified oncolytic virus according to the present disclosure, such as an oncolytic vaccinia virus described herein or a pharmaceutical composition comprising a vaccinia virus. An exemplary method for monitoring pharmacokinetics includes the following steps: (i) administering to a subject a therapeutically effective amount of an oncolytic vaccinia virus or a pharmaceutical composition comprising the same, alone or in combination with an additional therapy; and (ii) measuring the pharmacokinetics at about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 120 minutes, about 180 minutes, and about 240 minutes, 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 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 120 minutes, about 180 minutes, and about 240 minutes after administration in step (i). The method includes (iii) collecting a biological sample from the subject at one or more time points selected from about 15 minutes, 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, and about 2 months later; and (iii) detecting the amount of viral genome (or a reporter gene inserted into the viral genome, e.g., luciferase) in the biological sample collected at the aforementioned time points. In some cases, the viral genome copies / mL are highest in the sample collected at 15 minutes, and further, the sample collected at 240 minutes does not contain a detectable amount of viral genome. Thus, in some cases, the viral peak is observed at about 15 minutes after administration, and the majority of the virus is cleared from the subject's system after about 240 minutes (or 4 hours). In some cases, a first viral peak is observed about 15 minutes after administration, and a second viral peak is observed in a biological sample taken at a subsequent time point, for example, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes. In an exemplary embodiment, the biological sample is blood, and the amount of viral genomes per mL is determined by quantitative PCR or other suitable technique.In some examples, a first viral peak is observed about 15 minutes after administration, and a second viral peak is observed about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 15 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 1 month, or up to about 2 months after administration of a modified oncolytic virus of the present disclosure, such as an oncolytic vaccinia virus described herein.

[0099] In some cases, tumor-selective replication of the modified virus, e.g., oncolytic vaccinia virus, is measured through the use of a reporter gene, e.g., a luciferase gene. In some embodiments, the luciferase gene is inserted into the genome of the virus, and tumor cells are infected with the virus. Bioluminescence in the infected tumor cells is measured to monitor tumor-selective replication. Some examples show an increase in luciferase reporter bioluminescence in the modified oncolytic virus of the present disclosure compared to the same oncolytic vaccinia virus that does not contain the modifications in the modified oncolytic virus.

[0100] Dosage In some embodiments, the amount of a modified oncolytic virus described herein administered to a subject is about 10 3 ~10 12 infectious viral particles or plaque-forming units (PFU), or approximately 10 5 ~10 10 PFU, or approximately 10 5 ~10 8 PFU, or approximately 10 8 ~10 10 In some embodiments, the amount of modified oncolytic virus of the present disclosure administered to a subject is about 10 PFU. 3 ~10 12 viral particles or plaque-forming units (PFU), or approximately 105 ~10 10 PFU, or approximately 10 5 ~10 8 PFU, or approximately 10 8 ~10 10 In some embodiments, the modified oncolytic viruses of the present disclosure are at about 10 PFU. 3 PFU / dose~about 10 4 PFU / dose, approximately 10 4 PFU / dose~about 10 5 PFU / dose, approximately 10 5 PFU / dose~about 10 6 PFU / dose, approximately 10 7 PFU / dose~about 10 8 PFU / dose, approximately 10 9 PFU / dose~about 10 10 PFU / dose, approximately 10 10 PFU / dose~about 10 11 PFU / dose, approximately 10 11 PFU / dose~about 10 12 PFU / dose, approximately 10 12 PFU / dose~about 10 13 PFU / dose, approximately 10 13 PFU / dose~about 10 14 PFU / dose, or approximately 10 14 PFU / dose~about 10 15 In some embodiments, the modified oncolytic viruses of the present disclosure are administered at a dose comprising about 2 x 10 PFU / dose. 3 PFU / dose, 3×10 3 PFU / dose, 4×10 3 PFU / dose, 5×10 3 PFU / dose, 6×10 3 PFU / dose, 7×10 3 PFU / dose, 8×10 3 PFU / dose, 9×10 3 PFU / dose, approximately 10 4 PFU / dose, approximately 2 x 10 4 PFU / dose, approximately 3 x 10 4 PFU / dose, approximately 4 x 10 4 PFU / dose, approximately 5 x 10 4 PFU / dose, approximately 6 x 10 4 PFU / dose, approximately 7 x 104 PFU / dosage, approximately 8 x 10 4 PFU / dosage, approximately 9 x 10 4 PFU / dosage, approximately 10 5 PFU / dosage, 2×10 5 PFU / dosage, 3×10 5 PFU / dosage, 4×10 5 PFU / dosage, 5×10 5 PFU / dosage, 6×10 5 PFU / dosage, 7×10 5 PFU / dosage, 8×10 5 PFU / dosage, 9×10 5 PFU / dosage, approximately 10 6 PFU / dosage, approximately 2 × 10 6 PFU / dosage, approximately 3 x 10 6 PFU / dosage, approximately 4 x 10 6 PFU / dosage, approximately 5 x 10 6 PFU / dosage, approximately 6 x 10 6 PFU / dosage, approximately 7 x 10 6 PFU / dosage, approximately 8 x 10 6 PFU / dosage, approximately 9 x 10 6 PFU / dosage, approximately 10 7 PFU / dosage, approximately 2 × 10 7 PFU / dosage, approximately 3 x 10 7 PFU / dosage, approximately 4 x 10 7 PFU / dosage, approximately 5 x 10 7 PFU / dosage, approximately 6 x 10 7 PFU / dosage, approximately 7 x 10 7 PFU / dosage, approximately 8 x 10 7 PFU / dosage, approximately 9 x 10 7 PFU / dosage, approximately 10 8 PFU / dosage, approximately 2 × 10 8 PFU / dosage, approximately 3 x 10 8 PFU / dosage, approximately 4 x 10 8 PFU / dosage, approximately 5 x 10 8 PFU / dosage, approximately 6 x 10 8 PFU / dosage, approximately 7 x 10 8 PFU / dosage, approximately 8 x 10 8 PFU / dosage, approximately 9 x 10 8 PFU / dosage, approximately 10 9 PFU / dosage, approximately 2 × 10 9PFU / dose, approximately 3 x 10 9 PFU / dose, approximately 4 x 10 9 PFU / dose, approximately 5 x 10 9 PFU / dose, approximately 6 x 10 9 PFU / dose, approximately 7 x 10 9 PFU / dose, approximately 8 x 10 9 PFU / dose, approximately 9 x 10 9 PFU / dose, approximately 10 10 PFU / dose, approximately 2 x 10 10 PFU / dose, approximately 3 x 10 10 PFU / dose, approximately 4 x 10 10 PFU / dose, approximately 5 x 10 10 PFU / dose, approximately 6 x 10 10 PFU / dose, approximately 7 x 10 10 PFU / dose, approximately 8 x 10 10 PFU / dose, approximately 9 x 10 10 PFU / dose, approximately 10 10 PFU / dose, approximately 2 x 10 10 PFU / dose, approximately 3 x 10 10 PFU / dose, approximately 4 x 10 10 PFU / dose, approximately 5 x 10 10 PFU / dose, approximately 6 x 10 10 PFU / dose, approximately 7 x 10 10 PFU / dose, approximately 8 x 10 10 PFU / dose, approximately 9 x 10 10 PFU / dose, approximately 10 11 PFU / dose, approximately 2 x 10 11 PFU / dose, approximately 3 x 10 11 PFU / dose, approximately 4 x 10 11 PFU / dose, approximately 5 x 10 11 PFU / dose, approximately 6 x 10 11 PFU / dose, approximately 7 x 10 11 PFU / dose, approximately 8 x 10 11 PFU / dose, approximately 9 x 10 11 PFU / dose, or approximately 10 12 PFU / dose, approximately 10 12 PFU / dose~about 10 13 PFU / dose, approximately 10 13 PFU / dose~about 10 14 PFU / dose, or approximately 10 14 PFU / dose~about 1015 In some embodiments, the modified oncolytic viruses of the present disclosure are administered at a dose comprising 5×10 PFU / dose. 9 In some embodiments, the modified oncolytic viruses of the present disclosure are administered in doses comprising up to 5×10 PFU / dose. 9 The dose is administered in a volume containing PFU / dose.

[0101] In some embodiments, the modified oncolytic viruses of the present disclosure are administered in an amount of about 10 3 Virus particles / dose ~ approx. 10 4 Viral particles / dose, approximately 10 4 Virus particles / dose ~ approx. 10 5 Viral particles / dose, approximately 10 5 Virus particles / dose ~ approx. 10 6 Viral particles / dose, approximately 10 7 Virus particles / dose ~ approx. 10 8 Viral particles / dose, approximately 10 9 Virus particles / dose ~ approx. 10 10 Viral particles / dose, approximately 10 10 Virus particles / dose ~ approx. 10 11 Viral particles / dose, approximately 10 11 Virus particles / dose ~ approx. 10 12 Viral particles / dose, approximately 10 12 Virus particles / dose ~ approx. 10 13 Viral particles / dose, approximately 10 13 Virus particles / dose ~ approx. 10 14 viral particles / dose, or approximately 10 14 Virus particles / dose ~ approx. 10 15 The dose containing the viral particles / dose is administered.

[0102] In some embodiments, the modified oncolytic viruses of the present disclosure are administered in an amount of about 10 3 PFU / kg ~ approx. 10 4 PFU / kg, approximately 10 4 PFU / kg ~ approx. 10 5 PFU / kg, approximately 10 5 PFU / kg ~ approx. 10 6 PFU / kg, approximately 10 7 PFU / kg ~ approx. 108 PFU / kg, approximately 10 9 PFU / kg ~ approx. 10 10 PFU / kg, approximately 10 10 PFU / kg ~ approx. 10 11 PFU / kg, approximately 10 11 PFU / kg ~ approx. 10 12 PFU / kg, approximately 10 12 PFU / kg ~ approx. 10 13 PFU / kg, approximately 10 13 PFU / kg ~ approx. 10 14 PFU / kg, or approximately 10 14 PFU / kg ~ approx. 10 15 In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose comprising about 2 x 10 PFU / kg. 3 PFU / kg, 3 × 10 3 PFU / kg, 4 × 10 3 PFU / kg, 5 × 10 3 PFU / kg, 6 × 10 3 PFU / kg, 7 × 10 3 PFU / kg, 8 × 10 3 PFU / kg, 9 × 10 3 PFU / kg, approximately 10 4 PFU / kg, approximately 2×10 4 PFU / kg, approximately 3×10 4 PFU / kg, approximately 4×10 4 PFU / kg, approximately 5×10 4 PFU / kg, approximately 6×10 4 PFU / kg, approximately 7×10 4 PFU / kg, approximately 8×10 4 PFU / kg, approximately 9×10 4 PFU / kg, approximately 10 5 PFU / kg, 2 × 10 5 PFU / kg, 3 × 10 5 PFU / kg, 4 × 10 5 PFU / kg, 5 × 10 5 PFU / kg, 6 × 10 5 PFU / kg, 7 × 10 5 PFU / kg, 8 × 10 5 PFU / kg, 9 × 10 5 PFU / kg, approximately 10 6 PFU / kg, approximately 2×106 PFU / kg, approximately 3×10 6 PFU / kg, approximately 4×10 6 PFU / kg, approximately 5 × 10 6 PFU / kg, approximately 6×10 6 PFU / kg, approximately 7×10 6 PFU / kg, approximately 8×10 6 PFU / kg, approximately 9×10 6 PFU / kg, approximately 10 7 PFU / kg, approximately 2×10 7 PFU / kg, approximately 3×10 7 PFU / kg, approximately 4×10 7 PFU / kg, approximately 5 × 10 7 PFU / kg, approximately 6×10 7 PFU / kg, approximately 7×10 7 PFU / kg, approximately 8×10 7 PFU / kg, approximately 9×10 7 PFU / kg, approximately 10 8 PFU / kg, approximately 2×10 8 PFU / kg, approximately 3×10 8 PFU / kg, approximately 4×10 8 PFU / kg, approximately 5 × 10 8 PFU / kg, approximately 6×10 8 PFU / kg, approximately 7×10 8 PFU / kg, approximately 8×10 8 PFU / kg, approximately 9×10 8 PFU / kg, approximately 10 9 PFU / kg, approximately 2×10 9 PFU / kg, approximately 3×10 9 PFU / kg, approximately 4×10 9 PFU / kg, approximately 5 × 10 9 PFU / kg, approximately 6×10 9 PFU / kg, approximately 7×10 9 PFU / kg, approximately 8×10 9 PFU / kg, approximately 9×10 9 PFU / kg, approximately 10 10 PFU / kg, approximately 2×10 10 PFU / kg, approximately 3×10 10 PFU / kg, approximately 4×10 10 PFU / kg, approximately 5 × 10 10 PFU / kg, approximately 6×10 10PFU / kg, approximately 7×10 10 PFU / kg, approximately 8×10 10 PFU / kg, approximately 9×10 10 PFU / kg, approximately 10 10 PFU / kg, approximately 2×10 10 PFU / kg, approximately 3×10 10 PFU / kg, approximately 4×10 10 PFU / kg, approximately 5×10 10 PFU / kg, approximately 6×10 10 PFU / kg, approximately 7×10 10 PFU / kg, approximately 8×10 10 PFU / kg, approximately 9×10 10 PFU / kg, approximately 10 11 PFU / kg, approximately 2×10 11 PFU / kg, approximately 3×10 11 PFU / kg, approximately 4×10 11 PFU / kg, approximately 5×10 11 PFU / kg, approximately 6×10 11 PFU / kg, approximately 7×10 11 PFU / kg, approximately 8×10 11 PFU / kg, approximately 9×10 11 PFU / kg, or approximately 10 12 PFU / kg, approximately 10 12 PFU / kg ~ approx. 10 13 PFU / kg, approximately 10 13 PFU / kg ~ approx. 10 14 PFU / kg, or approximately 10 14 PFU / kg ~ approx. 10 15 In some embodiments, the modified oncolytic viruses of the present disclosure are administered at a dose comprising 5×10 PFU / kg. 9 In some embodiments, the modified oncolytic viruses of the present disclosure are administered at a dose comprising up to 5×10 PFU / kg. 9 The dose is administered in the amount of PFU / kg.

[0103] In some embodiments, the modified oncolytic viruses of the present disclosure are administered in an amount of about 10 3 Virus particles / kg ~ approx. 10 4 Virus particles / kg, approximately 10 4 Virus particles / kg ~ approx. 10 5Virus particles / kg, approximately 10 5 Virus particles / kg ~ approx. 10 6 Virus particles / kg, approximately 10 7 Virus particles / kg ~ approx. 10 8 Virus particles / kg, approximately 10 9 Virus particles / kg ~ approx. 10 10 Virus particles / kg, approximately 10 10 Virus particles / kg ~ approx. 10 11 Virus particles / kg, approximately 10 11 Virus particles / kg ~ approx. 10 12 Virus particles / kg, approximately 10 12 Virus particles / kg ~ approx. 10 13 Virus particles / kg, approximately 10 13 Virus particles / kg ~ approx. 10 14 virus particles / kg, or approximately 10 14 Virus particles / kg ~ approx. 10 15 It is administered in a dose containing viral particles / kg.

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

[0105] In some cases, when the modified oncolytic virus is administered by injection, the dosage is about 10 per injection. 3 10 viral particles per injection 4 10 viral particles per injection 5 10 viral particles per injection 6 10 viral particles per injection 7 10 viral particles per injection 8 10 viral particles per injection 9 10 viral particles per injection 10 10 viral particles per injection 11 10 viral particles per injection 12 2 x 10 viral particles per injection 12 10 viral particles per injection13 10 viral particles per injection 14 virus particles, or 10 per injection 15 In a further example, when the modified oncolytic virus is administered by injection, the dosage is about 10 viral particles per injection. 3 10 infectious viral particles per injection 4 10 infectious viral particles per injection 5 10 infectious viral particles per injection 6 10 infectious viral particles per injection 7 10 infectious viral particles per injection 8 10 infectious viral particles per injection 9 10 infectious viral particles per injection 10 10 infectious viral particles per injection 11 10 infectious viral particles per injection 12 2 x 10 infectious viral particles per injection 12 10 infectious viral particles per injection 13 10 infectious viral particles per injection 14 infectious viral particles, or 10 per injection 15In certain embodiments, the virus is administered in an amount sufficient to induce tumor lysis 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 refers to the amount administered to a subject or tumor over a period of 1 hour, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, or 24 hours. In certain embodiments, the dose is spread over time or by separate injections. In certain embodiments, multiple doses (e.g., 2, 3, 4, 5, 6, or more doses) of vaccinia virus are administered to a subject, e.g., the second treatment occurs within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or 7 weeks, of the first treatment. In certain embodiments, multiple doses of the modified oncolytic virus are administered to a subject over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more, or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or more. In certain embodiments, the oncolytic viruses or pharmaceutical compositions described herein are administered for about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks, or about 52 weeks, or longer.The administration frequency of the oncolytic vaccinia virus or pharmaceutical composition described herein is, in certain cases, once a day, twice a day, once a week, once every three weeks, once every four weeks (or once a month), once every eight weeks (or once every two months), once every 12 weeks (or once every three months), or once every 24 weeks (once every six months). In some embodiments of the methods disclosed herein, the oncolytic vaccinia virus or pharmaceutical composition is independently administered at an initial dose for a first period, at an intermediate dose for a second period, and at a high dose for a third period. In some embodiments, the initial dose is lower than the intermediate dose, and the intermediate dose is lower than the high dose. In some embodiments, the first, second, and third periods are independently about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks, or about 52 weeks, or longer.

[0106] An exemplary method for delivering the modified oncolytic virus of the present disclosure, such as the oncolytic vaccinia virus described herein, or a pharmaceutical composition comprising the same to cancer or tumor cells is by intravenous administration, for example, by injection, parenteral, intravenous, intradermal, intramuscular, transdermal, rectal, intraurethral, ​​intravaginal, intranasal, intrathecal, or intraperitoneal administration. However, alternative administration methods, such as intratumoral injection, are also used. The route of administration varies depending on the location and nature of the tumor. In certain embodiments, the route of administration is intraocular, transdermal, parenteral, intraperitoneal, intravenous, intramuscular, intranasal, subcutaneous, regional (e.g., near the tumor, particularly using the tumor vasculature or adjacent vasculature), percutaneous, intrathecal, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, or oral. The injection dose of the oncolytic virus is administered as a bolus injection or slow infusion. In certain embodiments, modified oncolytic virus is administered to patient from a source implanted in patient.In certain embodiments, modified oncolytic virus is administered by continuous infusion over a selected period of time.In some cases, the oncolytic vaccinia virus described herein or the pharmaceutical composition comprising it is administered at a therapeutically effective dose by infusion over 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 oncolytic viruses or pharmaceutical compositions of the present disclosure are administered as a liquid dosage, 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.

[0107] formulation Pharmaceutical compositions comprising the modified viruses described herein, such as oncolytic vaccinia viruses, can be prepared as solutions, dispersions in glycerol, liquid polyethylene glycol, and any combination thereof in oil, solid dosage forms, inhalable dosage forms, intranasal dosage forms, liposomal formulations, nanoparticle dosage forms, microparticle dosage forms, polymeric dosage forms, or any combination thereof. In some embodiments, the pharmaceutical compositions described herein include stabilizers and buffers. In some embodiments, the pharmaceutical compositions described herein can include solubilizers, such as sterile water and Tris buffer. In some embodiments, the pharmaceutical compositions described herein can include additives. Non-limiting examples of suitable additives include buffering agents, preservatives, stabilizers, binders, compaction agents, lubricants, chelating agents, dispersion enhancers, disintegrants, flavoring agents, sweeteners, and coloring agents.

[0108] In certain embodiments, the buffering agent includes phosphate buffered saline (PBS), Dulbecco's PBS (DPBS), TRIS buffered saline (TBS), Hank's balanced salt solution (HBSS), Earl's balanced salt solution (EBSS), standard citrate saline (SSC), HEPES buffered saline (HBS), or Gey's balanced salt solution.

[0109] In certain embodiments, the pharmaceutical composition of the present disclosure comprises an effective amount of the modified virus 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 not toxic to the patient to whom it is administered. Non-limiting examples of suitable pharmaceutical carriers include phosphate-buffered saline solution, water, emulsions, such as oil / water emulsions, various types of wetting agents, and sterile solutions. Additional non-limiting examples of pharmaceutically compatible carriers include gels, bioabsorbable matrix materials, implanted elements containing the modified oncolytic virus, or any other suitable vehicle, delivery, or dispensing means or material. Such carriers are formulated by conventional methods and administered to a subject in an effective amount.

[0110] Method of production The modified oncolytic virus of the present disclosure is produced by methods known to those skilled in the art. In certain embodiments, the modified oncolytic virus is propagated in suitable host cells, such as HeLa cells, 293 cells, or Vero cells, isolated from the host cells, and stored under conditions that promote the stability and integrity of the virus so that the decrease in infectivity over time is minimized. In certain exemplary methods, the modified oncolytic virus is propagated in host cells using cell stacks, roller bottles, or perfusion bioreactors. In some examples, downstream methods for purifying the modified oncolytic virus include filtration (e.g., depth filtration, tangential flow filtration, or a combination thereof), ultracentrifugation, or chromatographic capture. The modified oncolytic virus is stored, for example, by freezing or drying, for example, by lyophilization. In certain embodiments, prior to administration, the stored modified oncolytic virus is reconstituted (if dried for storage) and diluted in a pharmaceutically acceptable carrier for administration.

[0111] Some embodiments provide that the modified oncolytic viruses described herein exhibit higher titers in HeLa cells and 293 cells compared to the same virus but without the modifications in the modified oncolytic virus. In certain cases, higher titers are found in HeLa cells and 293 cells for the modified oncolytic viruses.

[0112] kit In some embodiments, the present disclosure provides a kit for administering the modified oncolytic virus described herein. In certain embodiments, the kit of the present disclosure includes the above-mentioned modified oncolytic virus or a pharmaceutical composition containing the modified oncolytic virus. In certain embodiments, the kit of the present disclosure further includes one or more components, such as instructions for use, devices, and additional reagents, and components for carrying out the methods disclosed above, such as tubes, containers, and syringes. In certain embodiments, the kit of the present disclosure further includes one or more drugs, such as at least one of an anticancer agent, an immunomodulatory agent, or any combination thereof, to be administered in combination with the modified virus.

[0113] In certain embodiments, the kits of the present disclosure include one or more containers containing the modified viruses disclosed herein. For example, and without limitation, the kits of the present disclosure include one or more containers containing the modified oncolytic viruses of the present disclosure.

[0114] In certain embodiments, the kits of the present disclosure include instructions for use, a device for administering the modified oncolytic virus to a subject, or a device for administering an additional agent or compound to a subject. For example, and without limitation, the instructions include instructions for the modified oncolytic virus and other components optionally included in the kit, as well as instructions for administration, including methods for determining the appropriate condition of the subject for administering the modified virus, the appropriate dosage, and the appropriate administration method. The instructions also optionally include guidance for monitoring the subject over the treatment period.

[0115] In certain embodiments, the kit of the present disclosure includes a device for administering modified oncolytic viruses to a subject.Any of the various devices known in the art for administering medicaments and pharmaceutical compositions can be included in the kit provided herein.For example, but not limited to, such devices include hypodermic needles, intravenous needles, catheters, needleless injection devices, inhalers, and liquid dispensers, such as eye droppers.In certain embodiments, the modified oncolytic viruses to be delivered systemically, for example, by intravenous injection, intratumoral injection, or intraperitoneal injection, are included in the kit together with hypodermic needles and syringes.

[0116] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will 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 utilized in practicing the disclosure. It is intended that the following claims define the scope of the disclosure, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0117] Illustrative Embodiments Provided herein are nucleic acids comprising sequences encoding at least two polypeptides, including interleukin-12 (IL-12) or a functional variant thereof and soluble PD-1 (sPD-1) or a functional variant thereof. Further provided herein are nucleic acids comprising DNA or RNA. Further provided herein are nucleic acids in which the IL-12 is mouse IL-12 or human IL-12. Further provided herein are nucleic acids in which the IL-12 or a functional variant thereof comprises an alpha subunit and a beta subunit. Further provided herein are nucleic acids in which the alpha subunit and the beta subunit are connected by a linker. Further provided herein are nucleic acids in which the sequence encoding the IL-12 alpha subunit comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:55 or SEQ ID NO:69. Further provided herein are nucleic acids in which the sequence encoding the IL-12 alpha subunit comprises the sequence of SEQ ID NO:55 or SEQ ID NO:69. Further provided herein are nucleic acids wherein the sequence encoding the IL-12 beta subunit comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:53 or SEQ ID NO:68. Further provided herein are nucleic acids wherein the sequence encoding the IL-12 beta subunit comprises the sequence of SEQ ID NO:53 or SEQ ID NO:68. Further provided herein are nucleic acids wherein the sequence encoding a linker comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:54. Further provided herein are nucleic acids wherein the sequence encoding a linker comprises the sequence of SEQ ID NO:54. Further provided herein are nucleic acids wherein the sPD-1 comprises a domain derived from mouse PD-1, a domain derived from human PD-1, or any combination thereof. Further provided herein are nucleic acids wherein the sPD-1 comprises the extracellular domain of mouse PD-1, human PD-1, or any combination thereof. Further provided herein are nucleic acids wherein the sPD-1 comprises a domain that binds to PD-L1.Further provided herein is a nucleic acid wherein the sequence encoding sPD-1 comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:58 or SEQ ID NO:70. Further provided herein is a nucleic acid wherein the sequence encoding sPD-1 comprises the sequence of SEQ ID NO:58 or SEQ ID NO:70. Further provided herein is a nucleic acid further comprising at least one promoter region. Further provided herein is a nucleic acid wherein the at least one promoter region drives expression of at least two polypeptides. Further provided herein is a nucleic acid wherein the nucleic acid comprises a first promoter upstream of the sequence encoding IL-12 and a second promoter upstream of the sequence encoding sPD-1, wherein the first promoter region drives expression of IL-12 and the second promoter region drives expression of sPD-1. Further provided herein are nucleic acids wherein the first promoter and the second promoter each comprise any one of the PD-1 promoter, SSP, P7.5, P28, P135, 454, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28kDa promoter, or any variation or combination thereof. Further provided herein are nucleic acids wherein the first promoter comprises the P7.5 promoter. Further provided herein are nucleic acids wherein the P7.5 promoter comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 32. Further provided herein are nucleic acids wherein the P7.5 promoter comprises the sequence of SEQ ID NO: 32. Further provided herein are nucleic acids wherein the second promoter comprises the P28 promoter. Further provided herein are nucleic acids wherein the P28 promoter comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 29. Further provided herein are nucleic acids wherein the P28 promoter comprises the sequence of SEQ ID NO: 29.Further provided herein is a nucleic acid wherein the nucleic acid comprises DNA, wherein the DNA comprises, in 5' to 3' order, the sequences of SEQ ID NO:53, SEQ ID NO:11, SEQ ID NO:55, and SEQ ID NO:58. Further provided herein is a nucleic acid wherein the nucleic acid comprises DNA, wherein the DNA comprises, in 5' to 3' order, the sequences of SEQ ID NO:68, SEQ ID NO:11, SEQ ID NO:69, and SEQ ID NO:70. Further provided herein is a nucleic acid wherein the nucleic acid comprises DNA, wherein the DNA comprises, in 5' to 3' order, the sequences encoding SEQ ID NO:52 and SEQ ID NO:58. Further provided herein is a nucleic acid wherein the nucleic acid comprises DNA, wherein the DNA comprises, in 5' to 3' order, the sequences encoding SEQ ID NO:67 and SEQ ID NO:70. Further provided herein is a nucleic acid wherein the nucleic acid comprises DNA, wherein the DNA comprises the sequence of SEQ ID NO:66. Further provided herein is a nucleic acid wherein the nucleic acid comprises DNA, wherein the DNA comprises the sequence of SEQ ID NO:71. Further provided herein is a nucleic acid further comprising a sequence encoding a chemokine receptor or a functional variant thereof. Further provided herein are nucleic acids wherein the chemokine receptor comprises at least one of a CXC receptor, a CC receptor, a CX3C receptor, an XC receptor, a functional fragment thereof, a functional variant thereof, or any combination thereof. Further provided herein are nucleic acids wherein the chemokine receptor comprises at least one of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, a functional fragment thereof, or a functional variant thereof, or any combination thereof.

[0118] Provided herein are nucleic acids comprising a first region encoding a first polypeptide comprising a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:5 or SEQ ID NO:8, and a second region encoding a second polypeptide comprising a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:2 or SEQ ID NO:4. Further provided herein are nucleic acids wherein the first polypeptide comprises the sequence of SEQ ID NO:5 or SEQ ID NO:8. Further provided herein are nucleic acids wherein the second polypeptide comprises the sequence of SEQ ID NO:2 or SEQ ID NO:4. Further provided herein are nucleic acids wherein the second polypeptide comprising the sequence of SEQ ID NO:4 further comprises one or more substitutions described by E61V, M70I, Q75F, K78W, K78L, E84F, S87W, A129H, A132L, K135M. Further provided herein are nucleic acids further comprising at least one promoter region. Further provided herein is a nucleic acid in which at least one promoter region drives the expression of a first region and a second region. Further provided herein is a nucleic acid comprising a first promoter driving the expression of the first region and a second promoter driving the expression of the second region. Further provided herein is a nucleic acid in which the first promoter and the second promoter each comprise any one of SSP, P7.5, P28, P135, 454, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28kDa promoter, or any variation or combination thereof. Further provided herein is a nucleic acid in which the first promoter comprises a P7.5 promoter. Further provided herein is a nucleic acid in which the second promoter comprises a P28 promoter. Further provided herein is a nucleic acid wherein the first region encodes a polypeptide comprising the sequence of SEQ ID NO: 5 or SEQ ID NO: 8, and the second region encodes a polypeptide comprising the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Further provided herein is a nucleic acid further comprising a sequence encoding a chemokine receptor or a functional variant thereof.Further provided herein are nucleic acids wherein the chemokine receptor comprises at least one of a CXC receptor, a CC receptor, a CX3C receptor, an XC receptor, a functional fragment thereof, a functional variant thereof, or any combination thereof. Further provided herein are nucleic acids wherein the chemokine receptor comprises at least one of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, a functional fragment thereof, or a functional variant thereof, or any combination thereof.

[0119] The present invention provides a nucleic acid described herein that is present in an oncolytic virus. Furthermore, the present invention provides a nucleic acid in which the oncolytic virus is a poxvirus, adeno-associated virus, adenovirus, reovirus, lentivirus, herpes simplex virus, vesicular stomatitis virus, mengovirus, myxoma virus, Newcastle disease virus, measles virus, or poliovirus. Furthermore, the present invention provides a nucleic acid in which the poxvirus is a vaccinia virus. Furthermore, the present invention provides a nucleic acid in which the vaccinia virus is a Western Reserve vaccinia virus (ATCC VR-1354), an Ankara vaccinia virus (ATCC VR-1508), an Ankara vaccinia virus (ATCC VR-1566), a Wyeth vaccinia virus (ATCC VR-1536), or a modified strain of the Wyeth vaccinia virus (ATCC VR-325). Furthermore, the present invention provides a nucleic acid that is inserted into a viral genome. Further provided herein is a nucleic acid that further comprises a mutation or deletion of at least one viral gene selected from the group consisting of thymidine kinase (TK), F13L, A36R, A34R, A33R, A52R, B5R, B8R, B18R, SPI-1, SPI-2, B15R, VGF, E3L, K3L, A41L, K7R, or N1L, a functional fragment thereof, or any combination thereof. Further provided herein is a nucleic acid that further comprises a mutation or deletion of the TK gene. Further provided herein is a nucleic acid that further comprises a mutation or deletion of the B8R gene.

[0120] Provided herein is a composition comprising a vector, an exogenous nucleic acid comprising a sequence encoding a cytokine or a functional variant thereof, and an exogenous nucleic acid comprising a sequence encoding a PD-L1 receptor. Further provided herein is a composition wherein the cytokine comprises IL-12 or a functional variant thereof. Further provided herein is a composition wherein the IL-12 comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:5 or SEQ ID NO:8. Further provided herein is a composition wherein the IL-12 comprises the sequence of SEQ ID NO:5 or SEQ ID NO:8. Further provided herein is a composition wherein the PD-L1 receptor comprises a PD-1 dominant negative, a soluble PD-1 dominant negative, or a protein that binds to PD-L1. Further provided herein is a composition wherein the PD-L1-binding protein is an anti-PD-L1 antibody. Further provided herein is a composition wherein the PD-L1-binding protein is a soluble variant of PD-1 (sPD-1). Further provided herein are compositions wherein sPD-1 comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:2 or SEQ ID NO:4. Further provided herein are compositions wherein sPD-1 comprises the sequence of either SEQ ID NO:2 or SEQ ID NO:4. Further provided herein are compositions wherein sPD-1 comprising the sequence of SEQ ID NO:4 further comprises one or more substitutions described by E61V, M70I, Q75F, K78W, K78L, E84F, S87W, A129H, A132L, K135M. Further provided herein are compositions further comprising at least one promoter region. Further provided herein are compositions wherein at least one promoter region drives expression of a cytokine and the PD-L1 receptor. Further provided herein are compositions wherein at least one promoter region comprises a first promoter driving expression of a cytokine and a second promoter driving expression of the PD-L1 receptor.Further provided herein are compositions wherein at least one promoter comprises any one of SSP, P7.5, P28, P135, 454, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28kDa promoter, or any variation or combination thereof. Further provided herein are compositions wherein a first promoter comprises a P7.5 promoter. Further provided herein are compositions wherein a second promoter comprises a P28 promoter. Further provided herein are compositions wherein an exogenous nucleic acid comprises a combined sequence of SEQ ID NO: 66. Further provided herein are compositions wherein an exogenous nucleic acid comprises a combined sequence of SEQ ID NO: 71. Further provided herein are compositions wherein an exogenous nucleic acid further comprises an exogenous nucleic acid comprising a sequence encoding a chemokine receptor or a functional variant thereof. Further provided herein is a composition in which the chemokine receptor comprises at least one of a CXC receptor, a CC receptor, a CX3C receptor, an XC receptor, a functional fragment thereof, a functional variant thereof, or any combination thereof. Further provided herein is a composition in which the chemokine receptor comprises at least one of a CXCR1, a CXCR2, a CXCR3, a CXCR4, a CXCR5, a CXCR6, a CXCR7, a CCR1, a CCR2, a CCR3, a CCR4, a CCR5, a CCR6, a CCR7, a CCR8, a CCR9, a CCR10, a CCR11, a CX3CR1, an XCR1, a functional fragment thereof, a functional variant thereof, or any combination thereof. Further provided herein is a composition in which the vector comprises a plasmid, a phage, a virus, a cosmid, an artificial chromosome, a liposome, a nanoparticle, or any combination thereof. Further provided herein is a composition in which the vector is an oncolytic virus. Further provided herein are compositions wherein the oncolytic virus is a poxvirus, adeno-associated virus, adenovirus, reovirus, lentivirus, herpes simplex virus, vesicular stomatitis virus, mengovirus, myxoma virus, Newcastle disease virus, measles virus, or poliovirus.Further provided herein is a composition wherein the poxvirus is a vaccinia virus. Further provided herein is a composition wherein the vaccinia virus is Western Reserve vaccinia virus (ATCC VR-1354), vaccinia virus Ankara (ATCC VR-1508), vaccinia virus Ankara (ATCC VR-1566), vaccinia virus strain Wyeth (ATCC VR-1536), or a modified strain of vaccinia virus Wyeth (ATCC VR-325). Further provided herein is a composition wherein an exogenous nucleic acid is inserted into the viral genome. Further provided herein is a composition wherein the oncolytic virus comprises at least one genome modification. Further provided herein are compositions wherein the at least one modification comprises a mutation or deletion of at least one gene selected from the group consisting of thymidine kinase, F13L, A36R, A34R, A33R, A52R, B5R, B8R, B18R, SPI-1, SPI-2, B15R, VGF, E3L, K3L, A41L, K7R, or N1L, a functional fragment thereof, or any combination thereof. Further provided herein are compositions comprising a mutation or deletion of the TK gene. Further provided herein are compositions further comprising a mutation or deletion of the B8R gene.

[0121] Provided herein is an oncolytic virus comprising an insertion at the TK locus, the insertion comprising, in 5' to 3' order, a first promoter region, wherein the promoter is P7.5, a first region encoding IL-12, a second promoter region, wherein the promoter is P728, and a second region encoding a PD-1 variant.

[0122] Provided herein is a pharmaceutical composition comprising a nucleic acid described herein or a composition described herein and a pharmaceutically acceptable excipient. Also provided herein is a pharmaceutical composition in a liquid dosage form. Also provided herein is a pharmaceutical composition wherein the pharmaceutically acceptable excipient is buffered saline. Also provided herein is a pharmaceutical composition wherein the buffered saline is phosphate-buffered saline (PBS), Dulbecco's PBS (DPBS), TRIS-buffered saline (TBS), Hank's balanced salt solution (HBSS), Earl's balanced salt solution (EBSS), normal citrate saline (SSC), HEPES-buffered saline (HBS), or Gey's balanced salt solution. Also provided herein is a pharmaceutical composition wherein the composition further comprises liposomes or nanoparticles. Also provided herein is a pharmaceutical composition wherein the nucleic acid or vector is associated with liposomes or nanoparticles.

[0123] Provided herein is a method for treating cancer, comprising administering to a subject with cancer a pharmaceutical composition described herein in an amount sufficient to treat the cancer. Also provided herein is a method for treating cancer, wherein the cancer is a solid tumor, leukemia, or lymphoma. Also provided herein is a method for treating cancer, wherein the cancer includes melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal-type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma. Further provided herein are methods for treating cancer, wherein the administering step comprises intratumoral administration. Further provided herein are methods for treating cancer, wherein the administering step comprises systemic administration. Further provided herein are methods for treating cancer, wherein the systemic administration comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof.

[0124] Provided herein is a method for activating an anti-tumor immune response, comprising administering a pharmaceutical composition described herein to a subject with cancer. Also provided herein is a method for activating an anti-tumor immune response, wherein the cancer is a solid tumor, leukemia, or lymphoma. Also provided herein is a method for activating an anti-tumor immune response, wherein the cancer is melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal-type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma. Further provided herein are methods for activating an anti-tumor immune response, wherein the administering step is intratumoral administration. Further provided herein are methods for activating an anti-tumor immune response, wherein the administering step is systemic administration. Further provided herein are methods for activating an anti-tumor immune response, wherein the systemic administration comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof.

[0125] Provided herein is a method for reducing the incidence of tumor cell growth, comprising administering to tumor cells a pharmaceutical composition described herein in an effective amount sufficient to reduce the incidence of tumor cell growth. Further provided herein is a method for reducing the incidence of tumor cell growth, wherein the tumor cells are derived from a solid tumor, leukemia, or lymphoma. Further provided herein are methods for reducing the incidence of tumor cell growth, wherein the tumor cells are derived from melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial carcinoma, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal-type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma. Further provided herein are methods for reducing the incidence of tumor cell growth, wherein the administering step is intratumoral administration. [Example]

[0126] The following examples further illustrate the described embodiments without limiting the scope of the disclosure.

[0127] Example 1 Construction of mouse IL-12 and mouse sPD-1 expression systems The Western Reserve strain of vaccinia virus was modified by replacing the gene encoding thymidine kinase (VACV094, J2R) with nucleic acids encoding mouse IL-12 (mIL-12), soluble mouse PD-1 (mouse sPD-1), and a fluorescent reporter. A plasmid transfer vector was generated containing, in order without gaps, the 5' recombination induction sequence (SEQ ID NO:47), an SbfI cloning site (CCTGCAGG), the P7.5 promoter (SEQ ID NO:44), a KpnI cloning site (GGTACC), an open reading frame encoding mouse IL-12 (SEQ ID NO:48), a SalI cloning site followed by a spacer (SEQ ID NO:52), the P28 promoter (SEQ ID NO:41), an SpeI cloning site (ACTAGT), soluble mouse PD-1 (SEQ ID NO:53), a sacI cloning site (GAGCTC), a loxP sequence (SEQ ID NO:54), spacer sequence A (SEQ ID NO:55), the viral 454 promoter (SEQ ID NO:34), a fluorescent reporter protein (SEQ ID NO:56), a PacI cloning site (TTAATTAA), a short spacer B (SEQ ID NO:57), a loxP sequence (SEQ ID NO:54), and the 3' recombination induction sequence (SEQ ID NO:58). After recombination and treatment with cre recombinase, the viral genome contained the integrated sequence of SEQ ID NO: 59. Selected sequences are shown in Table 6. A schematic diagram of the promoter and transgene inserted into the TK locus is shown in Figure 2. Table 6. Mouse IL-12 and sPD-1 recombinant sequences. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7]

[0128] Example 2 Construction of human IL-12 and sPD-1 expression system The Western Reserve vaccinia virus strain was modified by replacing the gene encoding thymidine kinase (VACV094, J2R) with nucleic acids encoding human IL-12 (hIL-12), soluble human PD-1 (human sPD-1), and a fluorescent reporter. A plasmid transfer vector is generated containing, in order without gaps, the 5' recombination induction sequence (SEQ ID NO:47), an SbfI cloning site (CCTGCAGG), the P7.5 promoter (SEQ ID NO:44), a KpnI cloning site (GGTACC), an open reading frame encoding human IL-12 (SEQ ID NO:60), a SalI cloning site followed by a spacer (SEQ ID NO:52), promoter P28 (SEQ ID NO:41), an SpeI cloning site (ACTAGT), an open reading frame encoding human sPD-1 (SEQ ID NO:63), a SacI cloning site (GAGCTC), a loxP sequence (SEQ ID NO:54), spacer sequence A (SEQ ID NO:55), promoter 454 (SEQ ID NO:42), a fluorescent reporter protein (SEQ ID NO:56), a PacI cloning site (TTAATTAA), spacer sequence B (SEQ ID NO:57), a loxP sequence (SEQ ID NO:54), and the 3' recombination induction sequence (SEQ ID NO:58). After recombination and treatment with cre recombinase, the viral genome contains the integrated sequence of SEQ ID NO: 64. The sequence is shown in Table 7. Table 7. Human IL-12 and human sPD-1 recombinant sequences. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6]

[0129] Example 3 Measurement of tumor size after treatment with viruses expressing sPD-1 and IL-12 Renca cells were implanted into the flanks of Balb / c mice. After 12 days, the mice were divided into 50-100 mm 3 Mice were classified as having tumors of 1E7 PFU. Mice were treated with 1E7 PFU of virus by intratumoral injection. Tumor size was measured 45 days later.

[0130] B16F10 cells were mixed with Matrigel at a 1:1 ratio and implanted into the flanks of C57 / Black 6 mice. Five days later, mice were divided into 50-100 mm 3 Mice were classified as having tumors of 1E7 PFU. Mice were treated with 1E7 PFU of virus by intratumoral injection. Tumor size was measured 38 days later.

[0131] Tumor-induced mice were divided into the following treatment groups: buffer control, TK-vaccinia virus, and TK-vaccinia virus expressing IL-12 and murine sPD-1 as described in Example 1. The mean tumor volume was measured in each treatment group.

[0132] The tumor volumes of the Renca tumor group are shown in Figure 3A. Mice sacrificed due to tumor burden before final measurement were 1000 mm in Figure 3A. 3 Renca tumor-inducing mice treated with viruses expressing a combination of IL-12 and murine sPD-1 grew to approximately 200 mm 45 days after treatment.3 and a 40% complete response (CR). In comparison, buffer control or TK-virus treated control groups had a mean tumor volume of at least about 1000 mm 3 The mean tumor volume was 1.01.

[0133] The tumor volumes of the B16 tumor group are shown in Figure 3B. Mice sacrificed due to tumor burden before final measurement had a tumor volume of 1400 mm in Figure 3B. 3 The data are expressed as mean tumor volume (mTc) and mean tumor volume (mTc) of the B16 tumor-inducing mice treated with a virus expressing IL-12 and murine sPD-1 had a mean tumor volume below the level of detection and a 90% CR 38 days after treatment. In comparison, control groups treated with buffer control or TK-virus had tumor volumes of at least 1400 mm 3 The mean tumor volume was 1.01.

[0134] Example 4 Deletion of the B8R gene (IFNg-binding protein) Interferon gamma (IFNg) plays a role in anti-cancer immunity by promoting the activity of various immune cells. IL-12 and sPD-1 are expected to require the production of IFNg for their activity. The vaccinia IFNg-binding protein expressed by the B8R gene acts as a secreted decoy receptor and removes extracellular IFNg. The virus described in Example 1 was further modified to remove the B8R gene.

[0135] A transfer vector was generated containing, in order, the reporter vector 5' recombination-inducing sequence (SEQ ID NO: 65), spacer sequence C (SEQ ID NO: 66), 454 promoter (SEQ ID NO: 42), fluorescent reporter protein (SEQ ID NO: 67), and reporter vector 3' recombination-inducing sequence (SEQ ID NO: 68). The vector components replaced the B8R open reading frame with a nucleic acid expressing a fluorescent reporter gene for plaque selection purposes. Selected plaques were then treated with a second transfer vector containing the reporter vector 5' recombination-inducing sequence (SEQ ID NO: 65) and the reporter vector 3' recombination-inducing sequence (SEQ ID NO: 68) to remove the promoter and fluorescent reporter gene. The final sequence of the modified B8R locus is set forth in SEQ ID NO: 69. The original position of the B8R open reading frame is indicated by paired brackets [ ]. The sequence is shown in Table 8. Table 8. B8R removal vector sequence [Table 8-1] [Table 8-2] [Table 8-3]

[0136] Example 5 Measurement of tumor size after treatment with B8R-virus expressing sPD-1 and IL-12 Lewis lung carcinoma (LLC) cells were implanted into the flanks of C57 / Black mice. Five days later, mice were divided into 50-100 mm 3 Mice were classified as having tumors of 1E7 PFU. Mice were treated with 1E7 PFU of virus by intratumoral injection. Tumor size was measured 31 days later.

[0137] Tumor-induced mice were divided into the following treatment groups: buffer control, TK-vaccinia virus, TK- / B8R-vaccinia virus, and TK- / B8R-vaccinia virus expressing IL-12 and murine sPD-1 as described in Example 4. The mean tumor volume was measured in each treatment group. Measurements at day 31 are shown in Figure 4. Mice sacrificed due to tumor burden before the final measurement are shown as 1400 mm in the figure. 3 It is expressed as:

[0138] Mice treated with buffer control or TK-vaccinia had a mean age of at least 1400 mm after 31 days. 3 Mice treated with TK- / B8R-vaccinia virus showed a mean tumor volume of approximately 900 mm 3 Mice treated with TK- / B8R-vaccinia virus expressing IL-12 and murine sPD-1 had below-detectable mean tumor volumes and an 80% CR after 31 days.

[0139] Example 5 Addition of mouse CXCR3 expression system The vaccinia virus of any of the preceding examples is modified by replacing the gene encoding A52 with nucleic acids encoding mouse CXCR3 and a fluorescent reporter. A plasmid transfer vector is generated containing, without a gap, the upstream recombination sequence B (SEQ ID NO: 70), an open reading frame encoding mouse CXCR3 (SEQ ID NO: 71), a stop codon, a SacI cloning site, and a short spacer (SEQ ID NO: 72), a loxP site (SEQ ID NO: 54), a viral promoter driving expression of the GFP-pac reporter (SEQ ID NO: 73), a PacI cloning site (TTAATTAA), a short spacer B (SEQ ID NO: 57), and a downstream recombination sequence B (SEQ ID NO: 74). After recombination and treatment with cre recombinase, the viral genome contains the integrated sequence of SEQ ID NO: 75. The sequence is shown in Table 9. A schematic diagram of the promoter and transgene inserted into the A52R gene is shown in Figure 5. Table 9. Mouse CXCR3 recombinant sequence. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5]

[0140] Example 8 Addition of chemokine receptor sequences The modified vaccinia virus described in Example 2 is further modified by replacing the gene encoding A52 (VACV094, J2R) with nucleic acids encoding human CXCR3 and a fluorescent reporter. A plasmid transfer vector is generated containing, in order without a gap, the upstream recombination sequence B (SEQ ID NO: 70), an open reading frame encoding human CXCR3 isoform 1 (SEQ ID NO: 76), a stop codon, a SacI cloning site, and a short spacer (SEQ ID NO: 72), a loxP site (SEQ ID NO: 54), the spacer followed by a viral promoter driving expression of the GFP-pac reporter (SEQ ID NO: 73), a PacI cloning site (TTAATTAA), a short spacer B (SEQ ID NO: 57), a loxP site (SEQ ID NO: 54), and a downstream recombination sequence B (SEQ ID NO: 74). Reporter-positive viruses are isolated and then treated with a reporter-free transfer vector containing an upstream recombinogenic sequence B (SEQ ID NO: 70), an open reading frame encoding human CXCR3 isoform 1 (SEQ ID NO: 76), and a second downstream recombinogenic sequence B (SEQ ID NO: 77). After recombination, the viral genome contains the integrated sequence of SEQ ID NO: 78. Selected sequences are shown in Table 10. Table 10. Human CXCR3 recombinant sequences. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4]

[0141] The foregoing description and accompanying drawings describe certain presently representative embodiments. Various modifications, additions, and alternative designs will, of course, be apparent to those skilled in the art in light of the foregoing teachings without departing from the scope thereof, which is indicated not by the foregoing description but by the following claims. All changes and variations that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

1. Vector and a first exogenous nucleic acid comprising a sequence encoding a cytokine or a functional variant thereof; a second exogenous nucleic acid comprising a sequence encoding the PD-L1 receptor; and A composition comprising:

2. The composition of claim 1, wherein the encoded cytokine comprises IL-12 or a functional variant thereof.

3. 3. The composition of claim 2, wherein the IL-12 comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:5 or SEQ ID NO:

8.

4. The composition of claim 2, wherein the IL-12 comprises the sequence of SEQ ID NO:5 or SEQ ID NO:

8.

5. The composition of claim 1, wherein the encoded PD-L1 receptor comprises a PD-1 dominant negative or a protein that binds to PD-L1.

6. The composition according to claim 5, wherein the protein that binds to PD-L1 is an anti-PD-L1 antibody.

7. The composition of claim 5, wherein the protein that binds to PD-L1 is a soluble variant of PD-1 (sPD-1).

8. 8. The composition of claim 7, wherein the sPD-1 comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:2 or SEQ ID NO:

4.

9. 8. The composition of claim 7, wherein the sPD-1 comprises the sequence of SEQ ID NO:2 or SEQ ID NO:

4.

10. 10. The composition of claim 9, wherein the sPD-1 comprises the sequence of SEQ ID NO:4 and further comprises one or more substitutions described by E61V, M70I, Q75F, K78W, K78L, E84F, S87W, A129H, A132L, K135M.

11. The composition of claim 1 , further comprising at least one promoter region.

12. The composition of claim 11, wherein the at least one promoter region drives expression of the cytokine and the PD-L1 receptor.

13. 12. The composition of claim 11, wherein the at least one promoter region comprises a first promoter that drives expression of the cytokine and a second promoter that drives expression of the PD-L1 receptor.

14. 13. The composition of claim 12, wherein the at least one promoter comprises any one of P7.5, P28, P135, SSP, 454, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28kDa promoter, or any variation or combination thereof.

15. 14. The composition of claim 13, wherein the first promoter comprises a P7.5 promoter.

16. The composition of claim 13 , wherein the second promoter comprises a P28 promoter.

17. 2. The composition of claim 1, wherein the first exogenous nucleic acid and the second exogenous nucleic acid comprise the combined sequence of SEQ ID NO:

59.

18. 2. The composition of claim 1, wherein the first exogenous nucleic acid and the second exogenous nucleic acid comprise the combined sequence of SEQ ID NO:

64.

19. The composition of claim 1 , further comprising a third exogenous nucleic acid comprising a sequence encoding a chemokine receptor or a functional variant thereof.

20. 20. The composition of claim 19, wherein the chemokine receptor comprises at least one of a CXC receptor, a CC receptor, a CX3C receptor, an XC receptor, a functional fragment thereof, a functional variant thereof, or any combination thereof.

21. 20. The composition of claim 19, wherein the chemokine receptor comprises at least one of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, a functional fragment thereof, or a functional variant thereof, or any combination thereof.

22. 20. The composition of claim 19, wherein the chemokine receptor comprises the amino acid sequence of any one of SEQ ID NOs: 27-40.

23. 23. The composition of any one of claims 1 to 22, wherein the vector comprises a plasmid, a phage, a virus, a cosmid, an artificial chromosome, a liposome, a nanoparticle, or any combination thereof.

24. 24. The composition of any one of claims 23, wherein the vector is an oncolytic virus.

25. 25. The composition of claim 24, wherein the oncolytic virus is a poxvirus, adeno-associated virus, adenovirus, reovirus, lentivirus, herpes simplex virus, vesicular stomatitis virus, mengovirus, myxoma virus, Newcastle disease virus, measles virus, or poliovirus.

26. 26. The composition of claim 25, wherein the poxvirus is a vaccinia virus.

27. 27. The composition of claim 26, wherein the vaccinia virus is Western Reserve vaccinia virus (ATCC VR-1354), vaccinia virus Ankara (ATCC VR-1508), vaccinia virus Ankara (ATCC VR-1566), vaccinia virus strain Wyeth (ATCC VR-1536), or a modified strain of vaccinia virus Wyeth (ATCC VR-325).

28. 20. The composition of claim 19, wherein the first exogenous nucleic acid, the second exogenous nucleic acid, and the third exogenous nucleic acid are inserted into a viral genome.

29. 25. The composition of claim 24, wherein the oncolytic virus comprises at least one genome modification.

30. 30. The composition of claim 29, wherein the at least one alteration comprises a mutation or deletion of at least one gene selected from the group consisting of thymidine kinase (TK), F13L, A36R, A34R, A33R, A52R, B5R, B8R, B18R, SPI-1, SPI-2, B15R, VGF, E3L, K3L, A41L, K7R, or N1L, a functional fragment thereof, or any combination thereof.

31. 31. The composition of claim 30, comprising a mutation or deletion in the TK gene.

32. 32. The composition of claim 31, further comprising a mutation or deletion in the B8R gene.

33. A nucleic acid comprising a sequence encoding at least two polypeptides, said at least two polypeptides comprising: Interleukin-12 (IL-12) or a functional variant thereof, and Soluble PD-1 (sPD-1) or functional variants thereof A nucleic acid comprising:

34. 34. The nucleic acid of claim 33, comprising DNA or RNA.

35. The nucleic acid of claim 33, wherein the IL-12 is mouse IL-12 or human IL-12.

36. 34. The nucleic acid of claim 33, wherein the IL-12 or functional variant thereof comprises an alpha subunit and a beta subunit.

37. 37. The nucleic acid of claim 36, wherein the alpha subunit and the beta subunit are connected by a linker.

38. 37. The nucleic acid of claim 36, wherein the sequence encoding the IL-12 alpha subunit comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:51 or SEQ ID NO:

62.

39. 39. The nucleic acid of claim 38, wherein the sequence encoding the IL-12 alpha subunit comprises the sequence of SEQ ID NO: 51 or SEQ ID NO:

62.

40. 37. The nucleic acid of claim 36, wherein the sequence encoding the IL-12 beta subunit comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:49 or SEQ ID NO:

61.

41. 41. The nucleic acid of claim 40, wherein the sequence encoding the IL-12 beta subunit comprises the sequence of SEQ ID NO: 49 or SEQ ID NO:

61.

42. 38. The nucleic acid of claim 37, wherein the sequence encoding the linker comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

50.

43. 43. The nucleic acid of claim 42, wherein the sequence encoding the linker comprises the sequence of SEQ ID NO:

50.

44. 34. The nucleic acid of claim 33, wherein the sPD-1 comprises a domain derived from mouse PD-1, a domain derived from human PD-1, or any combination thereof.

45. 45. The nucleic acid of claim 44, wherein the sPD-1 comprises the extracellular domain of the mouse PD-1, the human PD-1, or any combination thereof.

46. The nucleic acid of claim 33, wherein the sPD-1 comprises a domain that binds to PD-L1.

47. 34. The nucleic acid of claim 33, wherein the sequence encoding sPD-1 comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:53 or SEQ ID NO:

63.

48. 48. The nucleic acid of claim 47, wherein the sequence encoding sPD-1 comprises the sequence of SEQ ID NO: 53 or SEQ ID NO:

63.

49. 34. The nucleic acid of claim 33, further comprising at least one promoter region.

50. 50. The nucleic acid of claim 49, wherein said at least one promoter region drives expression of said at least two polypeptides.

51. 50. The nucleic acid of claim 49, wherein the nucleic acid comprises a first promoter upstream of a sequence encoding the IL-12 and a second promoter upstream of a sequence encoding the sPD-1, wherein the first promoter region drives expression of the IL-12 and the second promoter region drives expression of the sPD-1.

52. 52. The nucleic acid of claim 51 , wherein the first promoter and the second promoter each comprise any one of PD-1 promoter, P7.5, P28, P135, SSP, 454, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28 kDa promoter, or any variation or combination thereof.

53. 53. The nucleic acid of claim 52, wherein the first promoter comprises a P7.5 promoter.

54. 54. The nucleic acid of claim 53, wherein the P7.5 promoter comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

44.

55. 55. The nucleic acid of claim 54, wherein the P7.5 promoter comprises the sequence of SEQ ID NO:

44.

56. 53. The nucleic acid of claim 52, wherein the second promoter comprises a P28 promoter.

57. 57. The nucleic acid of claim 56, wherein the P28 promoter comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

41.

58. 58. The nucleic acid of claim 57, wherein the P28 promoter comprises the sequence of SEQ ID NO:

41.

59. 35. The nucleic acid of claim 34, wherein the nucleic acid comprises the DNA, and the DNA comprises, in 5' to 3' order, the sequence of SEQ ID NO: 49, the sequence of SEQ ID NO: 50, the sequence of SEQ ID NO: 51, and the sequence of SEQ ID NO:

53.

60. The nucleic acid of claim 34, wherein the nucleic acid comprises the DNA, and the DNA comprises, in 5' to 3' order, the sequence of SEQ ID NO: 61, the sequence of SEQ ID NO: 50, the sequence of SEQ ID NO: 62, and the sequence of SEQ ID NO:

63.

61. 35. The nucleic acid of claim 34, wherein the nucleic acid comprises the DNA, and the DNA comprises, in 5' to 3' order, a sequence encoding SEQ ID NO:49 and a sequence encoding SEQ ID NO:

63.

62. 35. The nucleic acid of claim 34, wherein the nucleic acid comprises the DNA, and the DNA comprises, in 5' to 3' order, a sequence encoding SEQ ID NO: 60 and a sequence encoding SEQ ID NO:

63.

63. 35. The nucleic acid of claim 34, wherein the nucleic acid comprises the DNA, and the DNA comprises the sequence of SEQ ID NO:

59.

64. 35. The nucleic acid of claim 34, wherein the nucleic acid comprises the DNA, and the DNA comprises the sequence of SEQ ID NO:

64.

65. 34. The nucleic acid of claim 33, further comprising a sequence encoding a chemokine receptor or a functional variant thereof.

66. 66. The nucleic acid of claim 65, wherein the chemokine receptor comprises at least one of a CXC receptor, a CC receptor, a CX3C receptor, an XC receptor, a functional fragment thereof, a functional variant thereof, or any combination thereof.

67. 66. The nucleic acid of claim 65, wherein the chemokine receptor comprises at least one of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, a functional fragment thereof, or a functional variant thereof, or any combination thereof.

68. a first region encoding a first polypeptide comprising a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:5 or SEQ ID NO:8; a second region encoding a second polypeptide comprising a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:2 or SEQ ID NO:4; A nucleic acid comprising:

69. 69. The nucleic acid of claim 68, wherein the first polypeptide comprises the sequence of SEQ ID NO:5 or SEQ ID NO:

8.

70. 69. The nucleic acid of claim 68, wherein the second polypeptide comprises the sequence of SEQ ID NO:2 or SEQ ID NO:

4.

71. 71. The nucleic acid of claim 70, wherein the second polypeptide comprising the sequence of SEQ ID NO: 4 further comprises one or more substitutions described by E61V, M70I, Q75F, K78W, K78L, E84F, S87W, A129H, A132L, K135M.

72. 69. The nucleic acid of claim 68, further comprising at least one promoter region.

73. 73. The nucleic acid of claim 72, wherein the at least one promoter region drives expression of the first region and the second region.

74. 74. The nucleic acid of claim 73, comprising a first promoter that drives expression of the first region and a second promoter that drives expression of the second region.

75. 75. The nucleic acid of claim 74, wherein the first promoter and the second promoter each comprise any one of P7.5, P28, P135, SSP, 454, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28 kDa promoter, or any variation or combination thereof.

76. 76. The nucleic acid of claim 75, wherein the first promoter comprises a P7.5 promoter.

77. 76. The nucleic acid of claim 75, wherein the second promoter comprises a P28 promoter.

78. the first region encodes a polypeptide comprising the sequence of SEQ ID NO:5 or SEQ ID NO:8; the second region encodes a polypeptide comprising the sequence of SEQ ID NO: 2 or SEQ ID NO: 4; 69. The nucleic acid of claim 68.

79. 69. The nucleic acid of claim 68, further comprising a sequence encoding a chemokine receptor or a functional variant thereof.

80. 80. The nucleic acid of claim 79, wherein the chemokine receptor comprises at least one of a CXC receptor, a CC receptor, a CX3C receptor, an XC receptor, a functional fragment thereof, a functional variant thereof, or any combination thereof.

81. 80. The nucleic acid of claim 79, wherein the chemokine receptor comprises at least one of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, a functional fragment thereof, or a functional variant thereof, or any combination thereof.

82. The nucleic acid of any one of claims 33 to 81, which is present in an oncolytic virus.

83. 83. The nucleic acid of claim 82, wherein the oncolytic virus is a poxvirus, adeno-associated virus, adenovirus, reovirus, lentivirus, herpes simplex virus, vesicular stomatitis virus, mengovirus, myxoma virus, Newcastle disease virus, measles virus, or poliovirus.

84. 84. The nucleic acid of claim 83, wherein the poxvirus is a vaccinia virus.

85. 85. The nucleic acid of claim 84, wherein the vaccinia virus is Western Reserve vaccinia virus (ATCC VR-1354), vaccinia virus Ankara (ATCC VR-1508), vaccinia virus Ankara (ATCC VR-1566), vaccinia virus strain Wyeth (ATCC VR-1536), or a modified strain of vaccinia virus Wyeth (ATCC VR-325).

86. 83. The nucleic acid of claim 82, which is inserted into a viral genome.

87. 87. The nucleic acid of claim 86, further comprising a mutation or deletion in at least one viral gene selected from the group consisting of thymidine kinase (TK), F13L, A36R, A34R, A33R, A52R, B5R, B8R, B18R, SPI-1, SPI-2, B15R, VGF, E3L, K3L, A41L, K7R, or N1L, a functional fragment thereof, or any combination thereof.

88. 88. The nucleic acid of claim 87, comprising a mutation or deletion in the TK gene.

89. 89. The nucleic acid of claim 88, further comprising a mutation or deletion in the B8R gene.

90. 1. An oncolytic virus comprising an insertion at the TK locus, said insertion comprising, in 5' to 3' order: a first promoter region, wherein the promoter is P7.5; a first region encoding IL-12, a second promoter region, wherein the promoter is P28; and A second region encoding a PD-1 variant Oncolytic viruses, including

91. A composition according to any one of claims 1 to 32 or a nucleic acid according to any one of claims 33 to 89, a pharmaceutically acceptable excipient; A pharmaceutical composition comprising:

92. 92. The pharmaceutical composition of claim 91, in liquid dosage form.

93. 92. The pharmaceutical composition of claim 91, wherein the pharmaceutically acceptable excipient is buffered saline.

94. 94. The pharmaceutical composition of claim 93, wherein the buffered saline is phosphate buffered saline (PBS), Dulbecco's PBS (DPBS), TRIS buffered saline (TBS), Hank's balanced salt solution (HBSS), Earl's balanced salt solution (EBSS), standard citrate saline (SSC), HEPES buffered saline (HBS), or Gey's balanced salt solution.

95. 92. The pharmaceutical composition of claim 91, wherein the composition further comprises a liposome or a nanoparticle.

96. 96. The pharmaceutical composition of claim 95, wherein the nucleic acid or vector is associated with the liposome or nanoparticle.

97. 100. A method for the treatment of cancer, comprising the step of administering to a subject having cancer the pharmaceutical composition of any one of claims 91 to 96 in an amount sufficient to treat the cancer.

98. 98. The method of claim 97, wherein the cancer is a solid tumor, leukemia, or lymphoma.

99. 98. The method of claim 97, wherein the cancer comprises melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial carcinoma, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal-type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma.

100. 98. The method of claim 97, wherein the administering step comprises intravenous administration.

101. 98. The method of claim 97, wherein the administering step comprises intratumoral administration.

102. 98. The method of claim 97, wherein the administering step comprises systemic administration.

103. 103. The method of claim 102, wherein the systemic administration comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof.

104. 97. A method for activating an anti-tumor immune response, comprising administering to a subject with cancer the pharmaceutical composition of any one of claims 91 to 96.

105. 105. The method of claim 104, wherein the cancer is a solid tumor, leukemia, or lymphoma.

106. 105. The method of claim 104, wherein the cancer comprises melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial carcinoma, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal-type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma.

107. 105. The method of claim 104, wherein the administering step is intravenous administration.

108. 105. The method of claim 104, wherein the administering step is intratumoral administration.

109. 105. The method of claim 104, wherein the administering step is systemic administration.

110. 110. The method of claim 109, wherein the systemic administration comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof.

111. 1. A method for reducing the incidence of tumor cell growth, comprising: Administering to the tumor cells a pharmaceutical composition according to any one of claims 91 to 96 in an effective amount sufficient to reduce the occurrence of tumor cell growth. A method comprising:

112. 112. The method of claim 111, wherein the tumor cells are derived from a solid tumor, leukemia, or lymphoma.

113. 112. The method of claim 111, wherein the tumor cells are derived from melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial carcinoma, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, bile duct sarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal-type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma.

114. 112. The method of claim 111, wherein the administering step is intratumoral administration.

115. 112. The method of claim 111, wherein the administering step is intratumoral administration.

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