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. Also provided herein are nucleic acids encoding chemokine receptors. Additionally described herein are oncolytic viruses comprising the nucleic acids described herein. The oncolytic virus optionally comprises a mutation or deletion of a gene expressing IFN-gamma. Also described are compositions described herein for the treatment of cancer.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 417,484, filed on October 19, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application includes a sequence listing, which has been submitted electronically in ST.26 xml format and is hereby incorporated by reference in its entirety. The xml copy created on September 28, 2023 is named 199249-725601_SL.xml and is 112,890 bytes in size.

[0005] background

[0006] Tumor cells express programmed cell death protein 1 ligand (PD-L1) to drive checkpoint inhibition and evade immune responses. PD-L1 binds to PD-1 on T cells at the checkpoint to inhibit T cell responses. Blocking PD-L1 can enhance T cell function in cancer therapy. Truncated PD-1 lacking a transmembrane domain provides soluble PD-1. Soluble PD-1 can bind to PD-L1 on tumor cells as a decoy receptor, blocking the PD-L1:PD-1 signaling pathway and acting as a checkpoint inhibitor. This article describes improved cancer therapies that integrate checkpoint inhibitor interventions. IL-12 enhances immune responses by activating T cells and NK cells. This article describes the use of soluble PD-1 in combination with IL-12 for treating cancer.

[0007] Brief Description

[0008] Nucleic acids are described herein, wherein the nucleic acids comprise sequences encoding at least two polypeptides, wherein the at least two polypeptides comprise: interleukin-12 (IL-12) or a functional variant thereof; and soluble PD-1 (sPD-1) or a functional variant thereof.

[0009] Nucleic acids are described herein, wherein the nucleic acids comprise: 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.

[0010] Compositions are described herein, wherein the composition comprises: 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.

[0011] Described herein are oncolytic viruses, wherein the oncolytic virus comprises: an insert comprising, in 5' to 3' order, at the TK locus: a first promoter region, wherein the promoter is P7.5; a first region encoding IL-12; a second promoter region, wherein the promoter is P285; and a second region encoding a PD-1 variant.

[0012] Described herein are pharmaceutical compositions, wherein the pharmaceutical composition comprises: a nucleic acid as described herein or a composition as described herein; and a pharmaceutically acceptable excipient.

[0013] Described herein are methods for treating cancer comprising administering to a subject having cancer a pharmaceutical composition as described herein in an amount sufficient to treat the cancer.

[0014] Described herein are methods for activating an anti-tumor immune response comprising administering to a subject having cancer a pharmaceutical composition as described herein.

[0015] Described herein are methods for reducing the incidence of tumor cell growth comprising: administering to tumor cells a pharmaceutical composition as described herein in an amount effective to reduce the incidence of tumor cell growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The novel features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by referring to the following detailed description of illustrative embodiments that utilize the principles of the present disclosure and the accompanying drawings, in which:

[0018] Figure 1A Schematic representation of the PD-1 / PD-L1 interaction between tumor cells and T cells.

[0019] Figure 1B Schematic diagram of sPD-1 binding to PD-L1 on T cells, preventing PD-1 / PD-L1 interaction.

[0020] Figure 2 Figure 2 is a diagram of a transgene inserted at the TK locus with a P7.5 promoter driving expression of a gene encoding an IL-12 polypeptide comprising IL-12 β and α subunits connected by a 22-residue glycine-rich linker and a P28 promoter driving expression of a gene encoding a soluble PD-1 polypeptide.

[0021] Figure 3AFigure 2 is a graph showing treatment groups on the x-axis and the volume of Renca cell tumors induced in mice 45 days after treatment on the y-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 measured at least 1000 mm 3 , and the average size of the treatment group was about 200mm 3 The treatment group that received the modified virus showed a complete response (CR) of 40%.

[0022] Figure 3B Figure 2 is a graph showing treatment groups on the x-axis and the volume of B16 cell tumors induced in mice 38 days after treatment on the y-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 measured at least 1400 mm 3 The group receiving the modified virus showed a 90% CR.

[0023] Figure 4 Figure 2 is a graph showing treatment groups on the x-axis and LLC tumor volume in mice 31 days after treatment on the y-axis; treatment groups received buffer control, TK-control, TK- / B8R-control, or TK- / B8R-virus modified to express murine IL-12 and murine sPD-1. Tumors in the buffer and TK-control groups were measured to be at least 1400 mm 3 The mean size of TK and treated groups was below detectable levels. The treated group receiving the modified virus showed 80% CR.

[0024] Figure 5 is a diagram of a transgene inserted into the A52R locus to drive expression of the CXCR3 chemokine receptor using the A52R promoter.

[0025] Details

[0026] Tumor cells employ various mechanisms to avoid detection and attack by the host immune system. These mechanisms can impact the effectiveness of cancer immunotherapy. Described herein are compositions comprising a combination of immune checkpoint inhibitors and proinflammatory cytokines to enhance the immune response to tumor cells, either alone or in combination with other therapeutic modalities.

[0027] Programmed cell death protein 1 (PD-1) is an immune checkpoint protein expressed on the surface of T cells and B cells. When bound to its ligand PD-L1, found on, for example, macrophages, the T cell response is inhibited. This protein helps regulate autoimmunity by suppressing the inflammatory response of T cells to "self" cells. Some tumor cells take advantage of 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 inhibits anti-tumor activity. Inhibition of the PD-L1 / PD-1 interaction or immune checkpoint inhibition can enhance T cell responses and increase anti-tumor activity. This article describes a modified PD-1 protein lacking the transmembrane and cytoplasmic domains, which produces a soluble variant of PD-1 (sPD-1). sPD-1 can bind to PD-L1, preventing PD-L1 / PD-1 checkpoint inhibition ( Figure 1B ), allowing the T cell response to proceed. In some embodiments, the sPD-1 described herein has sustained binding activity to PD-L1. Also described herein are nucleic acids encoding sPD-1 and vectors comprising such nucleic acids.

[0028] IL-12 activates anti-tumor cytotoxic immune responses, modulating T cell, NK cell, and antigen-presenting cell responses. This cytokine enhances anti-tumor immune responses and inhibits immunosuppression. Provided herein are compositions comprising nucleic acids encoding co-expressed sPD-1 and IL-12.

[0029] Provided herein are compositions for treating cancer and their uses. The compositions described herein may comprise one or more nucleic acids encoding polypeptides as described herein. The nucleic acids provided herein may comprise DNA, RNA, nucleic acid analogs, or any combination thereof. Briefly, described herein are (1) nucleic acids encoding expression of sPD-1 and IL-12, (2) nucleic acids encoding expression of chemokine receptors, (3) vectors for expressing the described nucleic acids, (4) modified oncolytic viruses, (5) conditions to be treated, and (6) dosages, forms, and methods of administering the compositions described herein.

[0030] definition

[0031] The terminology used herein is for the purpose of describing the particular instance only and is not intended to be limiting. Unless the context clearly dictates otherwise, as used herein, the singular forms "a," "an," and "the" include the plural forms. Furthermore, to the extent that the terms "contain," "containing," "including," "include," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0032] The term "about" or "approximately" can mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, for example, limitations of the measurement system. Where particular values ​​are described in the application and claims, unless otherwise stated, the term "about" should be assumed to mean an acceptable error range for the particular value, such as ±10% of the value modified by the term "about."

[0033] As used herein, the terms "heterologous nucleic acid sequence" or "exogenous nucleic acid sequence" or "transgene" in relation to a particular virus may refer to a nucleic acid sequence that is derived from a source other than the specified virus.

[0034] As used herein, the term "mutation" may refer to a deletion, insertion of a heterologous nucleic acid, an inversion or a substitution, including the elimination of a mutated open reading frame as generally understood in the art.

[0035] As used herein, the term "gene" may refer to a segment of nucleic acid that encodes a single protein or RNA (also referred to as a "coding sequence" or "coding region"), optionally together with associated regulatory regions such as promoters, operators, terminators, etc., which may be located upstream or downstream of the coding sequence.

[0036] As used herein, a "promoter" can be a control sequence in a nucleic acid sequence region that controls transcription initiation and transcription rate. In certain embodiments, a promoter can comprise a genetic element to which regulatory proteins and molecules can bind such as RNA polymerase and other transcription factors. The terms "operably positioned," "operably connected," "under control," and "under transcriptional control" can mean that a promoter is in correct functional position and / or orientation relative to a nucleic acid sequence to control transcription initiation and / or expression of the sequence. In certain embodiments, a promoter can be used in conjunction with or without an "enhancer," which refers to a cis-acting regulatory sequence that participates in the transcriptional activation of a nucleic acid sequence.

[0037] As used herein, the term "homology" can be a calculation of "homology" or "homology percentage" between two or more nucleotide or amino acid sequences, which can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced into the sequence of the first sequence). The 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, if a position in the first sequence can be occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent homology between the two sequences can be a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced and the length of each gap for optimal alignment of the two sequences. In some embodiments, the length of the sequences aligned for comparison purposes can be at least about: 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the length of the reference sequence. Search can determine the homology between two sequences. Homology can be between the overall length of two sequences or between parts of the overall length of two sequences. The two sequences can be genes, nucleotide sequences, protein sequences, peptide sequences, amino acid sequences or fragments thereof. The actual comparison of two sequences can be completed by known methods, for example, using a mathematical algorithm. When using BLAST and GappedBLAST programs, any relevant parameters of the corresponding program (for example, NBLAST) can be used. For example, the parameters for sequence comparison can be set to score=100, word length=12, or can vary (for example, W=5 or W=20). Other examples include Myers and Miller, CABIOS (1989) algorithm, ADVANCE, ADAM, BLAT and FASTA.

[0038] The term "subject" can 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 when referring to, for example, a mammalian subject such as a human subject.

[0039] The terms "treat," "treating," and "treatment" may be intended to include alleviating or eliminating a disorder, disease, or condition, or one or more symptoms associated with the disorder, disease, or condition; or alleviating or eradicating the cause of the disorder, disease, or condition itself.

[0040] The term "therapeutically effective amount" may refer to an amount of a compound that, when administered, is sufficient to prevent the development of or alleviate to some extent one or more symptoms of the disorder, disease or condition being treated.

[0041] As used herein, the term "oncolysis" can refer to the killing of cancer cells or tumor cells by an agent, such as an oncolytic poxvirus, such as an oncolytic vaccinia virus, for example, by directly lysing the cells via stimulation of an immune response against the cells, apoptosis, expression of toxic proteins, autophagy and termination of protein synthesis, induction of anti-tumor immunity, or any combination thereof. Direct lysis of cancer cells 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 "oncolysis" can refer to the killing of cancer cells or tumor cells without lysing the cells.

[0042] As used herein, the term "oncolytic virus" may refer to a virus that preferentially infects and kills tumor cells. In some embodiments, oncolytic viruses may include but are not limited to: (i) naturally preferentially replicate in cancer cells and are generally not pathogenic in humans due to increased sensitivity to innate antiviral signal transduction or dependence on oncogenic signal transduction pathways; and (ii) viruses that are genetically manipulated for use. In some embodiments, the oncolytic virus may be measles virus, poliovirus, poxvirus, vaccinia virus, adenovirus, adeno-associated virus, herpes simplex virus, vesicular stomatitis virus, reovirus, Newcastle disease virus, Seneca virus, slow virus, Mengo virus or myxoma virus. In certain embodiments, the oncolytic virus may be a poxvirus. In certain embodiments, the oncolytic virus may be a vaccinia virus.

[0043] As used herein, the term "modified oncolytic virus" may refer to an oncolytic virus comprising modifications to its components, such as, but not limited to, modifications in the natural genome ("skeleton") of the virus, such as mutations or deletions of viral genes, introduction of exogenous nucleic acids, chemical modifications of viral nucleic acids or viral proteins, and introduction of exogenous proteins or modified viral proteins into viral capsids. Typically, oncolytic viruses can be modified (also referred to as "engineering") to obtain improved therapeutic effects for tumor cells. In 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.

[0044] The terms "systemic delivery" and "systemic administration," which are used interchangeably herein, may in some cases refer to a route of administration of a drug, 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, intraarterial administration, or any combination thereof. Soluble programmed cell death protein 1 (sPD-1)

[0045] Immune checkpoint inhibitors block checkpoint proteins from binding to their receptors, allowing the immune response to proceed. The use of checkpoint inhibitors in cancer treatment circumvents the ability of cancer cells to avoid T cell attack. Natural programmed cell death protein 1 (PD-1) contains a transmembrane domain that anchors the protein to the surface of expressing cells. Truncated PD-1 variants before the transmembrane domain provide a soluble form of PD-1 (sPD-1). sPD-1 can act as a dominant negative decoy receptor to bind and sequester PD-L1, blocking the PD-1:PD-L1 signaling pathway. Blocking binding to PD-L1 allows for increased T cell activity and T cell-mediated killing ( Figure 1B ).

[0046] Provided herein are compositions comprising nucleic acids encoding PD-1 immune checkpoint inhibitors. In some embodiments, the PD-1 inhibitor prevents PD-1 on tumor cells from interacting with PD-L1 on T cells. In some embodiments, the PD-1 inhibitor comprises a modified PD-1. In some embodiments, the PD-1 is mouse PD-1. In some embodiments, the PD-1 is human PD-1.

[0047] Native PD-1 is a membrane-bound protein of 288 amino acids. The domains starting from the amino terminus include the extracellular domain, the transmembrane domain, and the 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.

[0048] Table 1. Amino acid sequence of PD-1

[0049]

[0050]

[0051] Provided herein are compositions comprising nucleic acids encoding murine sPD-1. In some embodiments, the nucleic acid sequence encodes the peptide described by SEQ ID NO: 2. In some embodiments, the encoded murine 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.

[0052] Provided herein are compositions comprising nucleic acids encoding human sPD-1. In some embodiments, the nucleic acid sequence encodes the 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.

[0053] In some embodiments, human sPD1 further comprises one or more mutations compared to the wild-type sequence (SEQ ID NO: 3). In some embodiments, the one or more mutations provide an increase in the binding affinity between soluble PD-1 and PD-L1. In some embodiments, the one or more mutations include E61V, M70I, Q75F, K78W, K78L, E84F, S87W, A129H, A132L, K135M, or any combination thereof. The mutation positions are based on the positions in SEQ ID NO: 3.

[0054] IL-12

[0055] Cytokines typically control the growth and activity of immune system cells. IL-12 induces T cells to differentiate into T helper 1 (Th1) cells. Th1 cells help clear pathogens from the system. Provided herein are compositions comprising nucleic acids encoding IL-12 or functional variants thereof. In some embodiments, the nucleic acid encodes a first polypeptide comprising interleukin 12 (IL-12) or a functional variant thereof. In some embodiments, IL-12 comprises a heterodimer comprising subunit β (IL-12b) and subunit α (IL-12a). In some embodiments, the nucleic acid encodes a murine IL-12 (mIL-12) sequence as 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 as depicted 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.

[0056] 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.

[0057] Provided herein are compositions comprising a nucleic acid encoding murine 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.

[0058] Provided herein are compositions comprising nucleic acids 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.

[0059] Provided herein are compositions comprising nucleic acids encoding human IL-12 subunit beta (hIL-12b) (UniProtKB Accession ID P29460). In some embodiments, the nucleic acid sequence encodes the peptide described by SEQ ID NO: 10. In some cases, the encoded hIL-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.

[0060] Exemplary amino acid sequences of IL-12 regions for inclusion in the compositions described herein are listed in Table 2.

[0061] Table 2. IL-12 amino acid sequence.

[0062]

[0063]

[0064] Linker and signaling domains

[0065] Provided herein are compositions comprising nucleic acids encoding linkers. In some embodiments, the nucleic acids encoding linkers are located between the various encoded biological functional units described herein. In some embodiments, the encoded linkers are flexible or rigid. In other embodiments, the encoded linkers are cleavable linkers. In other embodiments, the encoded cleavable linkers comprise a disulfide bond. In other embodiments, the encoded cleavable linkers comprise a protease-sensitive domain. A non-limiting list of exemplary linkers encoded by the nucleic acids contained in the compositions described herein is listed in Table 3. In some embodiments, the compositions described herein comprise nucleic acids encoding linkers having a sequence as described by SEQ ID NO: 11. In some embodiments, the nucleic acids encode linkers having 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.

[0066] Table 3. Connectors.

[0067]

[0068] Subscripts in the sequence indicate repetitions.

[0069] Arrows in the sequences indicate the positions of the cleavage sites.

[0070] Combined nucleic acid sequences

[0071] Provided herein are compositions of nucleic acids encoding a combination of technical features. 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, mouse IL-2 comprises the sequence of SEQ ID NO: 5. In some embodiments, 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, human IL-12 comprises the sequence of SEQ ID NO: 8. In some embodiments, human sPD-1 comprises the sequence as listed in SEQ ID NO: 4.

[0072] In an alternative embodiment, two nucleic acids are provided, wherein the first nucleic acid encodes a first polypeptide comprising IL-12 or a functional variant thereof, and the second nucleic acid encodes a second polypeptide comprising a sPD-1 (sPD-1) protein or a functional variant thereof. In some embodiments, 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.

[0073] Chemokine receptors

[0074] In some embodiments, provided herein are modified oncolytic viruses comprising exogenous nucleic acids, which are also referred to herein as transgenics, encoding chemokine receptors. In some cases, the exogenous nucleic acids are therapeutic transgenics. In some embodiments, provided herein are modified oncolytic viruses comprising exogenous nucleic acids encoding membrane-associated proteins such as hyaluronidase that degrade hyaluronic acid. In some embodiments, provided herein are modified oncolytic viruses comprising exogenous nucleic acids encoding both chemokine receptors and hyaluronidase.

[0075] Chemokines are chemotactic cytokines that regulate the transport and positioning of cells by activating seven transmembrane chemokine receptors. In some cases, based on the position of the first two N-terminal cysteine ​​residues, chemokines are divided into four subfamilies, including CC, CXC, CX3C and XC subfamilies. The differential expression of chemokine receptors on leukocytes optionally leads to the selective recruitment of specific cell types under specific conditions, providing an appropriate and effective immune response suitable for infectious pathogens or external damage. In addition to the key role in the coordinated migration of immune cells to inflammatory sites, in many cases, chemokines also play an important role in the development of lymphoid tissue, the maturation of immune cells and the generation and delivery of adaptive immune responses.

[0076] Tumors are increasingly being recognized as complex microenvironments composed of many different cell types that coexist and communicate with each other within intricate signaling networks. Chemokines are important coordinators of cell migration and intercellular interactions, and therefore have a profound impact on tumor development. Within the tumor microenvironment, tumor-associated host and cancer cells release a diverse array of chemokines, leading to the recruitment and activation of diverse cell types, which mediate the balance between anti-tumor and pro-tumor responses. In addition to their primary role as chemoattractants, chemokines are, in many cases, also involved in other tumor-related processes, including tumor cell growth, angiogenesis, and metastasis.

[0077] Tumor cells have been shown to acquire the ability to produce growth-promoting chemokines. For example, melanoma has been found to express a number of chemokines, including CXCL1, CXCL2, CXCL3, CXCL8, CCL2, and CCL5, which are associated with tumor growth and progression. CCL2 levels can be found to be increased in neuroblastoma cell lines and primary tumor cells isolated from human patients. Immunostaining studies have also shown that CXCL12 expression levels are elevated in a variety of cancers, including breast cancer, carcinoid tumors, cervical cancer, colorectal cancer, endometrial cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, and gastric cancer.

[0078] Chemokine receptors are cytokine receptors found on the surface of certain cells that interact with chemokines. Twenty different chemokine receptors have been discovered in humans. Each has a 7-transmembrane structure and couples to G proteins for intracellular signal transduction, making them members of the large protein family of G protein-coupled receptors. After interacting with its specific chemokine ligand, the chemokine receptor triggers the intracellular calcium (Ca) 2+ ) ions (calcium signaling). This causes cellular responses, including the initiation of a process called chemotaxis, which transports cells to desired locations within the organism. Generally, the term "chemokine receptor" as used herein may refer to a membrane-associated protein that selectively binds a chemokine ligand and induces chemotaxis toward the chemokine ligand.

[0079] It should be understood that the chemokine receptors disclosed herein in some cases refer not only to naturally occurring chemokine receptors identified in humans, but also include chemokine receptors from other sources, such as but not limited to: (1) naturally occurring chemokine receptors identified in animals such as pigs, dogs, cattle, and sheep; and (2) non-naturally occurring chemokine receptors, such as mutant proteins, chimeric receptors, and designer proteins that have binding affinity for a certain type of chemokine. In some instances, if the function of a fragment of a naturally occurring chemokine receptor to bind and respond to the corresponding chemokine and direct the chemotaxis of the cell is retained in the fragment, the fragment is also considered to be a chemokine receptor. As provided herein, in some embodiments, when the virus hijacks the gene expression machinery of the host cell, the virus containing an exogenous nucleic acid encoding the chemokine receptor forces the virus-infected cell to express the chemokine receptor.

[0080] In some cases, the modified oncolytic virus includes exogenous nucleic acids encoding cytokine receptors expressed by its homologous cytokines in the tumor microenvironment (for example, IL15-R has homologous cytokine IL15 expressed in the tumor microenvironment). In some cases, the modified oncolytic virus encodes chemokine receptors (for example, CXCR4 has homologous chemokines CXCL12 expressed on tumors; CCR2 has target CCL2 expressed on tumors) that its homologous chemokines may express on tumors, and is delivered systemically as naked viruses. After the modified oncolytic virus enters the bloodstream, it is delivered systemically, and the virus infects lymphocytes, such as B cells, and the infected B cells are redirected to the tumor, resulting in a significant increase in viral load in the tumor. In certain embodiments, the viral load increased in the tumor is achieved shortly after systemic delivery. The ability to deliver the modified oncolytic virus disclosed herein in a systemic manner provides an advantage over traditional intratumoral delivery methods of oncolytic viruses. While intratumoral delivery is helpful for treating easily accessible tumors, in some cases, it is crucial to treat inaccessible or metastatic cancers, which are said to be the main cause of death from the disease. In this case, oncolytic viruses that rely on intratumoral delivery are ineffective because they would require systemic dissemination after administration to distant sites. However, this dissemination is often short-lived and ineffective, at least in part, due to the development of an immune response to the viral infection.

[0081] Chemokine receptors are divided into different families. Non-limiting examples of chemokine receptors as described herein include CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors, which correspond to 4 different subfamilies of chemokines to which they bind. Among the CXC chemokine receptors, CXCR1 and CXCR2 are closely related, while CXCR1 binds to CXCL8 and CXCL6, and 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 CC chemokine receptors, the ligands of CCR1 include CCL4, CCL5, CCL6, CCL14, CCL15, CCL16, and CCL23; the ligands of CCR2 include CCL2, CCL8, and CCL16; the ligands of CCR3 include CCL11, CCL26, CCL7, CCL13, CCL15, CCL24, CCL5, CCL28, and CCL18; the ligands of CCR4 include CCL3, CCL5, CCL17, and CCL22. CCR5 ligands include CCL3, CCL4, CCL5, CCL8, CCL11, CCL13, CCL14, and CCL16; CCR6 ligands include CCL20; CCR7 ligands include CCL19 and CCL21; CCR8 ligands include CCL1 and CCL16; CCR9 ligands include CCL25; CCR10 ligands include CCL27 and CCL28; and CCR11 ligands include CCL19, CCL21, and CCL25. The CX3C chemokine receptor CX3CR1 has a ligand, CXCL1. The XC chemokine receptor XCR1 binds to both XCL1 and XCL2.

[0082] The 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 a CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, or any combination thereof.

[0083] In certain embodiments, the modified oncolytic virus comprises an exogenously expressed nucleic acid of CXCR4. In certain embodiments, the modified oncolytic virus comprises an exogenously expressed nucleic acid of CCR2. Certain embodiments disclose a modified oncolytic virus, wherein the modified oncolytic poxvirus comprises an exogenous nucleic acid encoding both CXCR4 and CCR2, and both chemokines are expressed from the same virus. In some cases, CXCL12 and / or CCL2, which are usually expressed in the tumor microenvironment, attract lymphocytes expressing CXCR4 and / or CCR2 or other migratory cells infected by the modified oncolytic virus, thereby enhancing the tumor-targeted delivery of the modified oncolytic virus. The nucleic acid and amino acid sequences of the selected chemokine receptors are listed in Table 4.

[0084] Table 4. Chemokine receptor sequences.

[0085]

[0086]

[0087]

[0088] In the compositions provided herein, the oncolytic viral gene can be mutated or replaced by a nucleic acid encoding a chemokine receptor listed in Table 4. In some embodiments, the chemokine receptor is a mouse CXCR3 described by 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 a human CXCR3 described by 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.

[0089] promoter

[0090] 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 that proteins bind 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 a promoter. In some embodiments, a promoter drives transcription of all polypeptides encoded on the nucleic acid. In some embodiments, the nucleic acids described herein comprise a separate promoter for each polypeptide encoded on the nucleic acid. In some embodiments, the nucleic acid comprises two promoters, each of which drives transcription of one of the two polypeptides encoded on the nucleic acid.

[0091] The timing of expression can be modulated by the structure of the promoter 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 drive expression earlier than promoters with fewer or lower affinity binding sites.

[0092] The use of relative time expression of proteins can be used to express specific factors from the modified viruses described herein earlier or later in the infection process. Early promoters have repeated transcription factor binding sites. Late promoters have fewer binding sites than early promoters. In some embodiments, early promoters are used to express receptors. Expression in the early stages of infection allows expression and processing by cells before cellular processes are disrupted. In some embodiments, late promoters are used to express one or more cytokines.

[0093] 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 includes A52R, PB8, mH5, I4L, LEO, PF11, I3L, P7.5, TK promoter, F7L, H5R, short synthetic promoter (SSP), or any variant 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 the selected promoters are listed in Table 5.

[0094] Table 5. Promoter nucleic acid sequences.

[0095]

[0096] 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 at the TK locus is shown in FIG. 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.

[0097] 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.

[0098] Provided herein are compositions comprising exogenous nucleic acids. In some embodiments, the exogenous nucleic acid comprises RNA. In some embodiments, the exogenous nucleic acid comprises DNA. In some embodiments, the DNA comprises an IL-12β subunit, a linker, an IL-12α subunit, and sPD-1 in 5' to 3' order. In some embodiments, the DNA comprises 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 5' to 3' order. In some embodiments, the DNA comprises 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 5' to 3' order. In some embodiments, the DNA comprises, in 5' to 3' order, a 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), a 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, a P7.5 promoter (SEQ ID NO: 44), human IL-12 (SEQ ID NO: 60), a P28 promoter (SEQ ID NO: 41), and human sPD-1 (SEQ ID NO: 60). In some embodiments, the exogenous nucleic acids described herein are integrated into the viral genome.

[0099] carrier

[0100] Provided herein are compositions comprising vectors. Typically, a vector is a vehicle designed to carry nucleic acid into a cell. In some embodiments, the vector comprises a plasmid, a phage, a virus, a cosmid, or an artificial chromosome. For the purposes of this application, the vector may also include a transfection agent and method, such as liposomes, nanoparticles, electroporation, microinjection, a gene gun, piercing effect (impalefection), hydrostatic pressure, continuous infusion, ultrasonic treatment, or any combination thereof. Provided herein are compositions comprising a vector as described herein and one or more nucleic acids. In some embodiments, the vector comprises a virus. In some embodiments, the virus is a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, or a 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 comprises a mutation or deletion of one or more genes. In some embodiments, the virus comprises one or more exogenous nucleic acids as described herein.

[0101] Oncolytic viruses

[0102] Provided herein are compositions comprising oncolytic viruses, wherein the oncolytic viruses include modified nucleic acids described herein. As used herein, oncolytic viruses kill cancer cells or tumor cells by such as directly cracking the cells, by stimulating the immune response to the cells, apoptosis, expressing toxic proteins, autophagy and terminating protein synthesis, inducing anti-tumor immunity or any combination thereof. In some embodiments, oncolytic viruses as described herein replicate in cells. In some embodiments, oncolytic viruses as described herein replicate in tumor cells, immune cells, somatic cells, hematopoietic cells or another type of cells. Exemplary oncolytic viruses for inclusion in compositions described herein include but are not limited to poxvirus, vaccinia virus, adeno-associated virus, adenovirus, reovirus, slow virus, herpes simplex virus, vesicular stomatitis virus, Mengo virus, myxoma virus, Newcastle disease virus, Seneca virus, retrovirus, measles virus, Maraba virus (marabavirus), Coxsackie virus or poliovirus. These oncolytic viruses have a tendency to specifically target cancer cells and cause significant cell death and tumor regression when the virus replicates. In some embodiments, oncolytic viruses are vaccinia viruses. Exemplary vaccinia viruses include, but are not limited to, the following strains for modification by inclusion of the exogenous nucleic acids described herein: Western Reserve Vaccinia Virus (ATCC VR-1354), Ankara Vaccinia Virus (ATCC VR-1508), Ankara Vaccinia Virus (ATCC VR-1566), Wyeth Vaccinia Virus (ATCC VR-1536), or Wyeth Vaccinia Virus (ATCC VR-325). Furthermore, in some embodiments, the recombinant vaccinia virus is a modified form of a wild-type or attenuated vaccinia virus strain. Non-limiting examples of vaccinia virus strains include 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 TashKent strains of vaccinia virus.

[0103] Oncolytic viruses are 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 enables the virus to have selective replication ability. The modified basic oncolytic virus strains listed herein optionally include one or more mutations or one or more deletions relative to their parental strains. In some embodiments, the modification includes mutations or complete or partial deletions 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 selected from the group consisting of: complete or partial deletion of the A1 gene; complete or partial deletion of the A2 gene; complete or partial deletion of the VH1 gene; complete or partial deletion of the A33 gene; complete or partial deletion of the I7 gene; complete or partial deletion of the A52R gene; complete or partial deletion of the TK gene; complete or partial deletion of the B15R gene; complete or partial deletion of the K7R gene; complete or partial deletion of the B14R gene; complete or partial deletion of the N1L gene; complete or partial deletion of the K1L gene; complete or partial deletion of the M2L gene; complete or partial deletion of the A49R gene; complete or partial deletion of the A46R gene; complete or partial deletion of the B8R gene; complete or partial deletion of the C12L gene; complete or partial deletion of the B18R gene; complete or partial deletion of the A52R gene; complete or partial deletion of the F3L gene; complete or partial deletion of the C4 gene; complete or partial deletion of the C16 gene. As used herein, reference to viral genes is made by reference to proteins encoded by genes (for example, A33 genes refer to genes encoding A33 proteins). In some embodiments, viral backbone mutations, including any combination of substitutions, insertions, and deletions, produce sequences with less than 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90% or less sequence homology to the wild-type sequence of viral genes or viral proteins encoded by the genes. In some embodiments, the viral backbone comprises 1, 2, 3, 4, 5 or more mutations in the amino acid sequence of viral proteins (for example, viral antigens). In some embodiments, present disclosure provides a recombinant oncolytic virus comprising one or more mutations in the viral genome (viral backbone) such that the mutation increases the T-cell arm (T-cell arm) of an immune response. Mutations can be additions, deletions, or substitutions of one or more nucleic acids in the viral genome (wild-type or attenuated natural strains of oncolytic viruses). In a non-limiting example, the mutation is a complete or partial deletion of a gene known to inhibit a cytokine involved in a Th1 immune response.In some embodiments, the mutation is a deletion of a nucleic acid encoding B8R (interferon gamma (IFN-g) binding protein). In some embodiments, the mutation is a deletion of a nucleic acid encoding C12L (interleukin-18 (IL-18) binding protein). In some embodiments, the mutation is a complete or partial deletion of a gene in innate immune signaling. In some embodiments, the mutation is a complete or partial deletion of a nucleic acid encoding B18R (type I interferon (IFN) binding protein). In some embodiments, the mutation is a complete or partial deletion of a nucleic acid encoding A52R (nuclear factor kappa B (NF-κB) inhibitor protein). In some embodiments, the mutation is a complete or partial deletion of a nucleic acid encoding E3L (protein kinase (PKR) inhibitor). In some embodiments, the mutation is a complete or partial deletion of a nucleic acid encoding C4 or C16 (STING pathway inhibitor).

[0104] As described herein, oncolytic viruses contain one or more additional insertions or partial insertions of exogenous nucleic acids encoding one or more proteins. In some embodiments, one or more proteins include chemokine receptors or their functional variants, soluble PD1 or their functional variants, or interleukin-12 or their functional variants. In some embodiments, one or more proteins include sPD-1 or its functional variant and interleukin-12 or its functional variant. Exemplary chemokine receptors for inclusion include but are not limited to wild-type and / or mutant CXCR3, CXCR4, CCR2 or CCL2. The oncolytic viruses of the present disclosure also include 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 their functional domains or fragments or variants or any combination thereof. In some cases, the oncolytic viruses provided herein contain a complete or partial deletion of at least one of the following: A52R or TK viral genes, and an insert 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.

[0105] In some embodiments, oncolytic viruses are modified oncolytic viruses with one or more modifications compared to other identical viruses that do not comprise modification, resulting in a greater therapeutic effect for tumor cells. In some non-limiting examples, greater therapeutic effects include each or any combination of the following: enhanced viral immune escape, enhanced viral tumor-targeted systemic delivery, enhanced viral intratumoral and intertumoral spread, and enhanced viral tumor-specific replication, or immunomodulators and antitumor agents are released into the extracellular matrix. In some cases, the modified oncolytic viruses of the present disclosure are used as platform carriers for systemic delivery.

[0106] As described herein, the oncolytic virus comprises an exogenous nucleic acid as described herein. In some embodiments, the oncolytic virus provided herein comprises a complete or partial deletion of the TK gene and an insertion of a region encoding at least one of soluble PD-1 and cytokines such as IL-12. Exemplary sequences for integration are previously described herein.

[0107] In some embodiments, the oncolytic virus provided herein contains 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 the chemokine receptor comprises a sequence selected from Table 4. In some embodiments, the promoter driving the 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 the expression of the chemokine receptor. In some embodiments, the A52R promoter drives the expression of the region encoding CXCR3.

[0108] In some embodiments, provided herein are modified oncolytic viruses comprising modifications that enhance the immune response against tumors. Typically, oncolytic viruses are (a) administered systemically, (b) inoculated locally on tumors, or (c) injected directly into tumors ("intratumoral delivery").

[0109] In some embodiments, provided herein are modified oncolytic viruses comprising modifications that enhance the intratumoral and intertumoral spread of the virus. The enhanced spread of oncolytic viruses within and between tumors is an effective way to enhance 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 acids. In some embodiments, provided herein are modified oncolytic viruses comprising modifications to the genome of the virus. In some embodiments, provided herein are modified oncolytic viruses comprising modifications in the genome of the exogenous nucleic acids and the virus.

[0110] In some embodiments, oncolytic viruses include, but are not limited to: (i) viruses that naturally replicate preferentially in cancer cells and are generally not pathogenic in humans due to increased sensitivity to innate antiviral signaling or dependence on oncogenic signaling pathways; and (ii) viruses that have been genetically manipulated for use.

[0111] In some embodiments, a modified oncolytic virus is used. Generally speaking, such a virus includes modifications to its components, such as, but not limited to, modifications in the natural genome ("skeleton") of the virus, such as mutations or deletions in viral genes, introduction of exogenous nucleic acids, chemical modification of viral nucleic acids or viral proteins, and introduction of exogenous proteins or modified viral proteins into the viral capsid.

[0112] In some embodiments, the modified oncolytic virus comprises a mutation or deletion of 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 of 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.

[0113] 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, wherein the A52R promoter is maintained and further comprises an exogenous nucleic acid encoding a CXCR3 receptor.

[0114] In some cases, the modified oncolytic virus includes the full-length viral backbone gene or viral backbone protein described above, or a truncated form thereof, or a functional domain thereof, or a fragment thereof, or a variant thereof. In various examples, the modified oncolytic virus includes a mutation or deletion of one or more viral backbone genes or viral backbone proteins as described above. Mutations in viral backbone genes and viral backbone proteins include insertions, deletions, substitutions, or modifications of amino acids in nucleotides and protein sequences in nucleic acid sequences. In some instances, deletions include complete or partial deletions of viral backbone genes or proteins.

[0115] In some embodiments, the modification of the oncolytic virus results in an increase in 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, 7.2-fold, 7.5-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 times, 7.8 times, 8 times, 8.2 times, 8.5 times, 8.8 times, 9 times, 9.2 times, 9.5 times, 9.8 times, 10 times, 12 times, 14 times, 15 times, 16 times, 18 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 150 times, 200 times, 250 times, 500 times, 800 times, 1000 times, 2500 times, 5000 times, 10 4 times, 2.5×10 4 times, 5×10 4 times, 7.5×10 4 times, 2.5×10 5 times, 5×10 5 times, 7.5×10 5 times, 10 6 times, 2.5×10 6 times, 5×10 6 times, 7.5×10 6 times, 10 7 times, 2.5×10 7 times, 5×10 7 times, 7.5×10 7 times, 10 8 times, 2.5×10 8 times, 5×10 8 times, 7.5×10 8 times, 10 9 times, 2.5×10 9 times, 5×10 9 times, 7.5×10 9 times, 10 10Times or even higher times. In certain embodiments, the efficacy of the tumor-targeted systemic delivery of the virus is measured by the virus of quantitatively infecting tumor cells, and is optionally compared with the virus of non-tumor cells in the infected body. For example, in some cases, the virus is quantified by staining the viral particles in the tissue section, or in the case of leukemia, lymphoma or myeloma, the blood smear is stained. In some cases, such quantitative is carried out by being engineered to be expressed by viral reporter molecules (for example, luciferase and fluorescent protein). In some cases, such quantitative is carried out by the viral genome in quantitative tumor. It is not limited to, and the tumor-targeted systemic delivery of the virus can also be measured by some downstream effects of viral infection in quantitative tumor cells (such as cytokines accumulated in response to viral infection or lymphocytes). In some embodiments, oncolytic virus includes exogenous nucleic acid encoding CXCR3, CXCR4, CCR2 or any combination thereof. In some embodiments, compared with the otherwise identical oncolytic virus not including exogenous nucleic acid, the presence of exogenous nucleic acid causes the efficacy of the tumor-targeted systemic delivery of the virus to increase by about 5 times to 10 times.

[0116] In some embodiments, provided herein are modified oncolytic viruses comprising exogenous nucleic acids encoding soluble immune checkpoint inhibitors. Expression of soluble immune checkpoint inhibitors by modified oncolytic viruses results in an enhanced immune response against infected tumors. After infecting the tumor, the modified oncolytic virus replicates in tumor cells and results in expression of soluble immune checkpoint inhibitors in the tumor environment. These soluble inhibitors act as decoy receptors, bind to checkpoint ligands, and block immune response suppression in tumors. Therefore, compared to other identical viruses that do not comprise nucleic acids encoding chemokine receptors, the immunosuppressive microenvironment in the tumor is changed, resulting in enhanced immunotherapy activity of the modified oncolytic virus. 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, 8.5 fold, 8.8 fold, .5 times, 8.8 times, 9 times, 9.2 times, 9.5 times, 9.8 times, 10 times, 12 times, 14 times, 15 times, 16 times, 18 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 150 times, 200 times, 250 times, 500 times, 800 times, 1000 times, 2500 times, 5000 times, 10 4 times, 2.5×10 4times, 5×10 4 times, 7.5×10 4 times, 2.5×10 5 times, 5×10 5 times, 10 6 Without limitation, the increased immunotherapeutic activity is reflected by increased B cell accumulation in the tumor, increased T cell response to tumor-associated immunogens, or both. B cell accumulation is measured, for example, by quantifying B cells in the tumor, and T cell immune activity is measured, for example, by interferon-γ (interferon-gamma) secretion in an ELISPOT assay.

[0117] In some embodiments, provided herein are modified oncolytic viruses comprising exogenous nucleic acids encoding chemokine receptors, and the forced expression of modified oncolytic viruses to chemokine receptors results in an enhanced immune response for infected tumors. After infection of the tumor, modified oncolytic viruses replicate in tumor cells and cause the expression of chemokine receptors on the surface of tumor cells. These membrane receptors act as decoy receptors, combining and isolating the immunosuppressive chemokines (e.g., CXCL12 and / or CCL2) in the tumor. Therefore, compared with other identical viruses that do not comprise nucleic acids encoding chemokine receptors, the immunosuppressive microenvironment in the tumor is changed, resulting in enhanced immunotherapy activity of modified oncolytic viruses. 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, 8.5 fold, 8.8 fold, .5 times, 8.8 times, 9 times, 9.2 times, 9.5 times, 9.8 times, 10 times, 12 times, 14 times, 15 times, 16 times, 18 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 150 times, 200 times, 250 times, 500 times, 800 times, 1000 times, 2500 times, 5000 times, 10 4 times, 2.5×10 4 times, 5×10 4 times, 7.5×10 4 times, 2.5×10 5 times, 5×10 5 times, 10 6Without limitation, the increased immunotherapeutic activity is reflected by increased B cell accumulation in the tumor, increased T cell responses to tumor-associated immunogens, or both. B cell accumulation is measured, for example, by quantifying B cells in the tumor, and T cell immune activity is measured, for example, by interferon-γ (interferon-gamma) secretion in an ELISPOT assay.

[0118] In some embodiments, provided herein are modified oncolytic viruses comprising exogenous nucleic acids encoding chemokine receptors, and compared to other identical viruses that do not comprise nucleic acids encoding chemokine receptors, the forced expression of chemokine receptors by the modified oncolytic viruses results in increased replication of the virus in tumor cells. In some embodiments, the modified oncolytic virus comprises exogenously expressed CXCR3 nucleic acids. In some embodiments, the modified oncolytic virus comprises nucleic acids expressing exogenous CCR2, which increases the tumor-specific replication of the virus. In some embodiments, the modified oncolytic virus comprises nucleic acids expressing exogenous CCR5, which increases the 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, 8.5-fold, 8.8-fold, 9-fold, 10-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 .5 times, 8.8 times, 9 times, 9.2 times, 9.5 times, 9.8 times, 10 times, 12 times, 14 times, 15 times, 16 times, 18 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 150 times, 200 times, 250 times, 500 times, 800 times, 1000 times, 2500 times, 5000 times, 10 4 times, 2.5×10 4 times, 5×10 4 times, 7.5×10 4 times, 2.5×10 5 times, 5×10 5 times, 10 6 Exemplary methods for measuring increases in viral delivery and spread in tumors include, but are not limited to, fluorescence- or bioluminescence-based imaging of reporter gene expression, quantitative PCR for detecting tumor concentrations of viral genomes, or plaque determination of plaque-forming units, or immunohistochemistry for viral proteins.

[0119] Conditions being treated

[0120] Provided herein are methods for treating 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 includes a tumor. It is envisioned that the treatment includes delivering 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, renal cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate epithelial cancer, hepatocellular carcinoma, bile duct sarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm or sarcoma.

[0121] In some embodiments, the compositions described herein are administered to cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicles, tongue, or uterus. In addition, the cancer is optionally of the following histological types, but is not limited to these: neoplasm, malignant; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; bile duct carcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and bile duct carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli solid carcinoma; carcinoid tumor, malignant; tracheo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; oncocytic carcinoma; aerophilic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinomas; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; ceroid adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; signet ring cell ovarian carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; follicle tumor, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; adipocyte tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; angiosarcoma; malignant melanoma; amelanotic melanoma melanoma; superficial spreading melanoma; malignant melanoma in giant nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrohistiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; mesenchymal sarcoma; mixed tumor, malignant; mullerian mixed tumor; Wilms' tumor; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant; Brenner's tumor tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; goiter of the ovary, malignant; choriocarcinoma; mesonephroblastoma, malignant; angiosarcoma; angioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;Oligodendroglioblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides Granuloma; other specific non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma; or hairy cell leukemia. In some cases, a modified oncolytic virus of the present disclosure, such as a modified oncolytic vaccinia virus that facilitates systemic delivery, is used to treat metastatic solid cancers. In some cases, a modified oncolytic virus of the present disclosure, such as a modified oncolytic vaccinia virus that facilitates systemic delivery, is used to treat solid cancers that are inaccessible or difficult to access for the purpose of intratumoral delivery of therapeutic agents. In some embodiments, the compositions described herein are used to treat cancers associated with increased free fatty acid expression. ;

[0122] The present disclosure also contemplates methods for suppressing or preventing local invasion or metastasis or both of primary cancers of any type. In exemplary embodiments, the primary cancer is melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, liver cancer, retinoblastoma, astrocytoma, glioblastoma, gum cancer, tongue cancer, leukemia, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, brain cancer, colon cancer or bladder cancer. In certain embodiments, the primary cancer is lung cancer. For example, lung cancer is non-small cell lung cancer. In addition, the present disclosure is optionally used to prevent cancer or treat pre-cancer cells or premalignant cells, including metaplasia, dysplasia and hyperplasia. It can also be used to suppress undesirable but benign cells, such as squamous metaplasia, dysplasia, benign prostatic hyperplasia cells, hyperplastic lesions, etc. In some embodiments, progression to cancer or a more severe form of cancer is stopped, disrupted, or delayed by the methods of the disclosure involving the modified oncolytic viruses discussed herein.

[0123] Provided herein are methods for treating a subject by administering one or more modified oncolytic viruses as disclosed herein. "Individual" or "subject," as used interchangeably herein, refers to a human or non-human subject. Non-limiting examples of non-human subjects include non-human primates, dogs, cats, mice, rats, guinea pigs, rabbits, pigs, poultry, horses, cattle, goats, sheep, cetaceans, and the like. In some embodiments, the subject is human.

[0124] A method of producing a toxic effect in a cancer cell is provided, comprising administering to the cancer cell a therapeutically effective amount of a modified virus, such as an oncolytic vaccinia virus as described above, or a pharmaceutical composition containing the oncolytic vaccinia virus. The present disclosure also provides a method of inhibiting at least one of the growth and proliferation of a second cancer cell, the method comprising administering to a first cancer cell a modified oncolytic virus as described above, such that the first cancer cell is infected with the virus. Therefore, in some embodiments of the methods disclosed herein, it is expected that not every cancer cell or tumor cell is infected after administering a therapeutically effective amount of an oncolytic vaccinia virus as described herein or a pharmaceutical composition comprising the oncolytic vaccinia virus, and that the growth of uninfected cells is inhibited in the absence of direct infection.

[0125] In some examples, in order to use the methods and compositions of the present disclosure to induce oncolysis, kill cells, inhibit growth, inhibit metastasis, reduce tumor size, and otherwise reverse or reduce the malignant phenotype of tumor cells, cancer cells or tumors are contacted with a therapeutically effective dose of an exemplary oncolytic vaccinia virus as described herein or a pharmaceutical composition comprising the oncolytic vaccinia virus. In certain embodiments, an effective amount of a modified oncolytic virus of the present disclosure, such as an oncolytic vaccinia virus or a pharmaceutical composition thereof as described herein, may comprise an amount sufficient to induce oncolysis, destroy or lyse cancer cells, or inhibit or reduce the growth or size of cancer cells. For example, a reduction in the growth of cancer cells is manifested as cell death, or a slowing of the replication rate or a reduction in the growth rate of a tumor comprising the cells, or an extension of the survival of a subject comprising cancer cells.

[0126] Provided herein are methods for treating subjects with cancer or tumors, comprising administering to the subject an effective amount of a modified virus as described above. The effective amount in such a method includes slowing the growth rate or spread of the cancer, or extending the subject's survival. The present disclosure provides a method for slowing tumor growth, comprising administering to the tumor an effective amount of a modified oncolytic virus as described above. In certain embodiments, the effective amount of the modified virus or its pharmaceutical composition includes an amount sufficient to induce a slowdown, inhibition, or reduction in tumor growth or size, and includes eradicating the tumor. For example, the slowing down of tumor growth is manifested as a reduction in growth rate or an extension of the survival of subjects containing tumors. In certain embodiments, an effective amount of the modified virus or its pharmaceutical composition includes an amount sufficient to activate an anti-tumor response. In some embodiments, activating an anti-tumor response includes activating T cells. In certain embodiments, an effective amount of the modified virus or its pharmaceutical composition includes an amount sufficient to reduce the incidence 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.

[0127] Provided herein are methods for determining the infectivity or anti-tumor activity or the amount of tumor-specific viral replication of an oncolytic vaccinia virus as described herein, the method comprising: (i) administering to a subject a therapeutically effective amount of an oncolytic vaccinia virus or a pharmaceutical composition according to the present disclosure that also expresses a luciferase reporter gene, alone or in combination with another therapy; (ii) collecting a first biological sample from the subject immediately after administration of the virus and determining the level of the luciferase reporter gene in the first biological sample; (iii) collecting a second biological sample from the subject after administration in step (ii) and detecting the level of the luciferase reporter gene in the second biological sample, wherein if the level of luciferase in step (iii) is higher than that in step (ii), the oncolytic vaccinia virus is determined to be infectious, exhibit anti-tumor activity, and exhibit tumor-specific viral replication. The second biological sample is collected about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 15 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 1 month to about 2 months after the administration in step (i). In some embodiments, the above method also includes detecting in steps (ii) and (iii) the modified oncolytic virus of the present disclosure administered to the subject in a therapeutically effective amount, such as an oncolytic vaccinia virus as described herein or a pharmaceutical composition comprising the oncolytic vaccinia virus, from a plasma sample collected from the subject one or more of the determination 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) levels. In some embodiments of the present disclosure, compared to the modified virus that is otherwise identical but does not comprise the modified oncolytic virus, the increase in luciferase bioluminescence of the modified oncolytic virus as described herein between steps (ii) and (iii) is higher. Other exemplary techniques for detecting and monitoring viral load after administering a modified oncolytic virus include real-time quantitative PCR.

[0128] Provided herein are methods for monitoring pharmacokinetics after administering a therapeutically effective amount of a modified oncolytic virus according to the present disclosure, such as an oncolytic vaccinia virus or a pharmaceutical composition containing a vaccinia virus, as described herein. An exemplary method for monitoring pharmacokinetics comprises the following steps: (i) administering a therapeutically effective amount of an oncolytic vaccinia virus or a pharmaceutical composition comprising the oncolytic vaccinia virus to a subject, alone or in combination with another therapy; (ii) administering in step (i) a therapeutically effective amount of an oncolytic vaccinia virus or a pharmaceutical composition comprising the oncolytic vaccinia virus to a subject for a period of time selected from the group consisting of 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, or 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, or about 10 hours. , about 12 hours, about 15 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 1 month to about 2 months one or more time points collect biological sample from experimenter, and (iii) detect the amount of viral genome (or the reporter gene inserted in viral genome, such as luciferase) in the biological sample collected at the above-mentioned time points. In some cases, viral genome copies / mL is the highest in the sample collected at 15 minutes time points, and further, the sample collected at 240 minutes time points does not comprise the viral genome of detectable amount. Therefore, in some cases, virus peak is observed at about 15 minutes after administration, and most viruses are removed from experimenter's system after about 240 minutes (or 4 hours). In some cases, a first viral peak is observed after about 15 minutes after administration, and a second viral peak is observed in a biological sample collected at a subsequent time point (e.g., at about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes). In an exemplary embodiment, the biological sample is blood, and the amount of viral genomes / mL is determined by quantitative PCR or other suitable techniques. In some examples, a first viral peak is observed after about 15 minutes after administration of a modified oncolytic virus of the present disclosure, such as an oncolytic vaccinia virus as described herein, and a second viral peak is observed after about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 15 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 1 month to about 2 months after administration.

[0129] In some cases, the tumor selective replication of modified viruses (such as oncolytic vaccinia viruses) is measured by using reporter genes (such as luciferase genes). In some embodiments, the luciferase gene is inserted into the genome of the virus and the tumor cells are infected with the virus. The bioluminescence in the infected tumor cells is measured to monitor tumor selective replication. Some examples show that the luciferase reporter molecule bioluminescence in the modified oncolytic virus of the present disclosure is increased compared to the oncolytic vaccinia virus that is otherwise identical but does not include the modification in the modified oncolytic virus.

[0130] dose

[0131] In some embodiments, the amount of a modified oncolytic virus described herein administered to a subject is about 10 3 and 10 12 Infectious virus particles or plaque forming units (PFU), or between about 10 5 PFU and 10 10 PFU, or about 10 5 PFU and 10 8 PFU, or about 10 8 PFU and 10 10 In some embodiments, the amount of the modified oncolytic virus of the present disclosure administered to a subject is between about 10 3 and 10 12 Virus particles or plaque forming units (PFU), or between about 10 5 PFU and 10 10 PFU, or about 10 5 PFU and 10 8 PFU, or about 10 8 PFU and 10 10 In some embodiments, the modified oncolytic virus of the present disclosure comprises about 10 3 PFU / dose is about 10 4 PFU / dose, about 10 4 PFU / dose is about 10 5 PFU / dose, about 10 5 PFU / dose is about 10 6 PFU / dose, about 10 7 PFU / dose is about 10 8 PFU / dose, about 10 9 PFU / dose is about 10 10 PFU / dose, about 10 10 PFU / dose is about 10 11 PFU / dose, about 10 11 PFU / dose is about 10 12PFU / dose, about 10 12 PFU / dose is about 10 13 PFU / dose, about 10 13 PFU / dose is about 10 14 PFU / dose, or about 10 14 PFU / dose is about 10 15 In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose of about 2×10 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, about 10 4 PFU / dose, about 2×10 4 PFU / dose, about 3×10 4 PFU / dose, about 4×10 4 PFU / dose, about 5×10 4 PFU / dose, about 6×10 4 PFU / dose, about 7×10 4 PFU / dose, about 8×10 4 PFU / dose, about 9×10 4 PFU / dose, about 10 5 PFU / dose, 2×10 5 PFU / dose, 3×10 5 PFU / dose, 4×10 5 PFU / dose, 5×10 5 PFU / dose, 6×10 5 PFU / dose, 7×10 5 PFU / dose, 8×10 5 PFU / dose, 9×10 5 PFU / dose, about 10 6 PFU / dose, about 2×10 6 PFU / dose, about 3×10 6 PFU / dose, about 4×10 6 PFU / dose, about 5×10 6 PFU / dose, about 6×10 6 PFU / dose, about 7×10 6 PFU / dose, about 8×10 6 PFU / dose, about 9×10 6 PFU / dose, about 107 PFU / dose, about 2×10 7 PFU / dose, about 3×10 7 PFU / dose, about 4×10 7 PFU / dose, about 5×10 7 PFU / dose, about 6×10 7 PFU / dose, about 7×10 7 PFU / dose, about 8×10 7 PFU / dose, about 9×10 7 PFU / dose, about 10 8 PFU / dose, about 2×10 8 PFU / dose, about 3×10 8 PFU / dose, about 4×10 8 PFU / dose, about 5×10 8 PFU / dose, about 6×10 8 PFU / dose, about 7×10 8 PFU / dose, about 8×10 8 PFU / dose, about 9×10 8 PFU / dose, about 10 9 PFU / dose, about 2×10 9 PFU / dose, about 3×10 9 PFU / dose, about 4×10 9 PFU / dose, about 5×10 9 PFU / dose, about 6×10 9 PFU / dose, about 7×10 9 PFU / dose, about 8×10 9 PFU / dose, about 9×10 9 PFU / dose, about 10 10 PFU / dose, about 2×10 10 PFU / dose, about 3×10 10 PFU / dose, about 4×10 10 PFU / dose, about 5×10 10 PFU / dose, about 6×10 10 PFU / dose, about 7×10 10 PFU / dose, about 8×10 10 PFU / dose, about 9×10 10 PFU / dose, about 10 10 PFU / dose, about 2×10 10 PFU / dose, about 3×10 10 PFU / dose, about 4×10 10 PFU / dose, about 5×10 10 PFU / dose, about 6×10 10 PFU / dose, about 7×1010 PFU / dose, about 8×10 10 PFU / dose, about 9×10 10 PFU / dose, about 10 11 PFU / dose, about 2×10 11 PFU / dose, about 3×10 11 PFU / dose, about 4×10 11 PFU / dose, about 5×10 11 PFU / dose, about 6×10 11 PFU / dose, about 7×10 11 PFU / dose, about 8×10 11 PFU / dose, about 9×10 11 PFU / dose, or about 10 12 PFU / dose, about 10 12 PFU / dose is about 10 13 PFU / dose, about 10 13 PFU / dose is about 10 14 PFU / dose, or about 10 14 PFU / dose is about 10 15 In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose of 5×10 9 In some embodiments, the modified oncolytic viruses of the present disclosure are administered in a dose comprising up to 5×10 9 PFU / dose was administered.

[0132] In some embodiments, the modified oncolytic viruses of the present disclosure are prepared by comprising about 10 3 Virus particles / dose to about 10 4 Virus particles / dose, about 10 4 Virus particles / dose to about 10 5 Virus particles / dose, about 10 5 Virus particles / dose to about 10 6 Virus particles / dose, about 10 7 Virus particles / dose to about 10 8 Virus particles / dose, about 10 9 Virus particles / dose to about 10 10 Virus particles / dose, about 10 10 Virus particles / dose to about 10 11 Virus particles / dose, about 10 11 Virus particles / dose to about 10 12 Virus particles / dose, about 10 12 Virus particles / dose to about 10 13 Virus particles / dose, about 10 13Virus particles / dose to about 10 14 Virus particles / dose or about 10 14 Virus particles / dose to about 10 15 The dose is administered at a rate of 10 viral particles / dose.

[0133] In some embodiments, the modified oncolytic viruses of the present disclosure are prepared by comprising about 10 3 PFU / kg is about 10 4 PFU / kg, about 10 4 PFU / kg is about 10 5 PFU / kg, about 10 5 PFU / kg is about 10 6 PFU / kg, about 10 7 PFU / kg is about 10 8 PFU / kg, about 10 9 PFU / kg is about 10 10 PFU / kg, about 10 10 PFU / kg is about 10 11 PFU / kg, about 10 11 PFU / kg is about 10 12 PFU / kg, about 10 12 PFU / kg is about 10 13 PFU / kg, about 10 13 PFU / kg is about 10 14 PFU / kg or about 10 14 PFU / kg is about 10 15 In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose of about 2×10 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, about 10 4 PFU / kg, about 2×10 4 PFU / kg, about 3×10 4 PFU / kg, about 4×10 4 PFU / kg, about 5×10 4 PFU / kg, about 6×10 4 PFU / kg, about 7×10 4 PFU / kg, about 8×104 PFU / kg, about 9×10 4 PFU / kg, about 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, about 10 6 PFU / kg, about 2×10 6 PFU / kg, about 3×10 6 PFU / kg, about 4×10 6 PFU / kg, about 5×10 6 PFU / kg, about 6×10 6 PFU / kg, about 7×10 6 PFU / kg, about 8×10 6 PFU / kg, about 9×10 6 PFU / kg, about 10 7 PFU / kg, about 2×10 7 PFU / kg, about 3×10 7 PFU / kg, about 4×10 7 PFU / kg, about 5×10 7 PFU / kg, about 6×10 7 PFU / kg, about 7×10 7 PFU / kg, about 8×10 7 PFU / kg, about 9×10 7 PFU / kg, about 10 8 PFU / kg, about 2×10 8 PFU / kg, about 3×10 8 PFU / kg, about 4×10 8 PFU / kg, about 5×10 8 PFU / kg, about 6×10 8 PFU / kg, about 7×10 8 PFU / kg, about 8×10 8 PFU / kg, about 9×10 8 PFU / kg, about 10 9 PFU / kg, about 2×10 9 PFU / kg, about 3×10 9PFU / kg, about 4×10 9 PFU / kg, about 5×10 9 PFU / kg, about 6×10 9 PFU / kg, about 7×10 9 PFU / kg, about 8×10 9 PFU / kg, about 9×10 9 PFU / kg, about 10 10 PFU / kg, about 2×10 10 PFU / kg, about 3×10 10 PFU / kg, about 4×10 10 PFU / kg, about 5×10 10 PFU / kg, about 6×10 10 PFU / kg, about 7×10 10 PFU / kg, about 8×10 10 PFU / kg, about 9×10 10 PFU / kg, about 10 10 PFU / kg, about 2×10 10 PFU / kg, about 3×10 10 PFU / kg, about 4×10 10 PFU / kg, about 5×10 10 PFU / kg, about 6×10 10 PFU / kg, about 7×10 10 PFU / kg, about 8×10 10 PFU / kg, about 9×10 10 PFU / kg, about 10 11 PFU / kg, about 2×10 11 PFU / kg, about 3×10 11 PFU / kg, about 4×10 11 PFU / kg, about 5×10 11 PFU / kg, about 6×10 11 PFU / kg, about 7×10 11 PFU / kg, about 8×10 11 PFU / kg, about 9×10 11 PFU / kg or about 10 12 PFU / kg, about 10 12 PFU / kg is about 10 13 PFU / kg, about 10 13 PFU / kg is about 10 14 PFU / kg or about 10 14 PFU / kg is about 10 15In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose of 5×10 9 In some embodiments, the modified oncolytic viruses of the present disclosure are administered at a dose of at most 5×10 9 PFU / kg dose was administered.

[0134] In some embodiments, the modified oncolytic viruses of the present disclosure include about 10 3 Virus particles / kg to about 10 4 Virus particles / kg, about 10 4 Virus particles / kg to about 10 5 Virus particles / kg, about 10 5 Virus particles / kg to about 10 6 Virus particles / kg, about 10 7 Virus particles / kg to about 10 8 Virus particles / kg, about 10 9 Virus particles / kg to about 10 10 Virus particles / kg, about 10 10 Virus particles / kg to about 10 11 Virus particles / kg, about 10 11 Virus particles / kg to about 10 12 Virus particles / kg, about 10 12 Virus particles / kg to about 10 13 Virus particles / kg, about 10 13 Virus particles / kg to about 10 14 virus particles / kg or about 10 14 Virus particles / kg to about 10 15 The dose was 100 virus particles / kg.

[0135] In certain embodiments, a liquid dosage form of an oncolytic virus as described herein comprises about 10 3 PFU / mL is about 10 4 PFU / mL, about 10 4 PFU / mL is about 10 5 PFU / mL, about 10 5 PFU / mL is about 10 6 PFU / mL, about 10 7 PFU / mL is about 10 8 PFU / mL, about 10 9 PFU / mL is about 10 10 PFU / mL, about 10 10 PFU / mL is about 10 11PFU / mL, about 10 11 PFU / mL is about 10 12 PFU / mL, about 10 12 PFU / mL is about 10 13 PFU / mL, about 10 13 PFU / mL is about 10 14 PFU / mL or about 10 14 PFU / mL is about 10 15 In some embodiments, the modified oncolytic virus of the present disclosure is prepared to contain about 2×10 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, about 10 4 PFU / mL, about 2×10 4 PFU / mL, about 3×10 4 PFU / mL, about 4×10 4 PFU / mL, about 5×10 4 PFU / mL, about 6×10 4 PFU / mL, about 7×10 4 PFU / mL, about 8×10 4 PFU / mL, about 9×10 4 PFU / mL, about 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, about 10 6 PFU / mL, about 2×10 6 PFU / mL, about 3×10 6 PFU / mL, about 4×10 6 PFU / mL, about 5×10 6 PFU / mL, about 6×10 6PFU / mL, about 7×10 6 PFU / mL, about 8×10 6 PFU / mL, about 9×10 6 PFU / mL, about 10 7 PFU / mL, about 2×10 7 PFU / mL, about 3×10 7 PFU / mL, about 4×10 7 PFU / mL, about 5×10 7 PFU / mL, about 6×10 7 PFU / mL, about 7×10 7 PFU / mL, about 8×10 7 PFU / mL, about 9×10 7 PFU / mL, about 10 8 PFU / mL, about 2×10 8 PFU / mL, about 3×10 8 PFU / mL, about 4×10 8 PFU / mL, about 5×10 8 PFU / mL, about 6×10 8 PFU / mL, about 7×10 8 PFU / mL, about 8×10 8 PFU / mL, about 9×10 8 PFU / mL, about 10 9 PFU / mL, about 2×10 9 PFU / mL, about 3×10 9 PFU / mL, about 4×10 9 PFU / mL, about 5×10 9 PFU / mL, about 6×10 9 PFU / mL, about 7×10 9 PFU / mL, about 8×10 9 PFU / mL, about 9×10 9 PFU / mL, about 10 10 PFU / mL, about 2×10 10 PFU / mL, about 3×10 10 PFU / mL, about 4×10 10 PFU / mL, about 5×10 10 PFU / mL, about 6×10 10 PFU / mL, about 7×10 10 PFU / mL, about 8×10 10 PFU / mL, about 9×10 10 PFU / mL, about 10 10PFU / mL, about 2×10 10 PFU / mL, about 3×10 10 PFU / mL, about 4×10 10 PFU / mL, about 5×10 10 PFU / mL, about 6×10 10 PFU / mL, about 7×10 10 PFU / mL, about 8×10 10 PFU / mL, about 9×10 10 PFU / mL, about 10 11 PFU / mL, about 2×10 11 PFU / mL, about 3×10 11 PFU / mL, about 4×10 11 PFU / mL, about 5×10 11 PFU / mL, about 6×10 11 PFU / mL, about 7×10 11 PFU / mL, about 8×10 11 PFU / mL, about 9×10 11 PFU / mL or about 10 12 PFU / mL, about 10 12 PFU / mL is about 10 13 PFU / mL, about 10 13 PFU / mL is about 10 14 PFU / mL or about 10 14 PFU / mL or about 10 15 In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose of 5×10 9 In some embodiments, the modified oncolytic viruses of the present disclosure are administered at a dose of at most 5×10 9 PFU / mL dose was administered.

[0136] In some cases, when the modified oncolytic virus is administered by injection, the dosage comprises about 10 3 Virus particles, 10 per injection 4 Virus particles, 10 per injection 5 Virus particles, 10 per injection 6 Virus particles, 10 per injection 7 Virus particles, 10 per injection 8 Virus particles, 10 per injection 9 Virus particles, 10 per injection 10 Virus particles, 10 per injection 11 Virus particles, 10 per injection12 Virus particles, 2×10 per injection 12 Virus particles, 10 per injection 13 Virus particles, 10 per injection 14 Virus particles, or 10 per injection 15 In other cases, when the modified oncolytic virus is administered by injection, the dosage comprises about 10 3 Infectious virus particles, 10 per injection 4 Infectious virus particles, 10 per injection 5 Infectious virus particles, 10 per injection 6 Infectious virus particles, 10 per injection 7 Infectious virus particles, 10 per injection 8 Infectious virus particles, 10 per injection 9 Infectious virus particles, 10 per injection 10 Infectious virus particles, 10 per injection 11 Infectious virus particles, 10 per injection 12 Infectious virus particles, 2×10 12 Infectious virus particles, 10 per injection 13 Infectious virus particles, 10 per injection 14 infectious virus particles, or 10 15In some embodiments, the virus is administered in an amount sufficient to induce oncolysis in at least about 20% of the cells in the tumor, at least about 30% of the cells in the tumor, at least about 40% of the cells in the tumor, at least about 50% of the cells in the tumor, at least about 60% of the cells in the tumor, at least about 70% of the cells in the tumor, at least about 80% of the cells in the tumor, or at least about 90% of the cells in the tumor. In certain embodiments, a single dose of virus refers to the amount administered to the subject or tumor within a time period of 1 hour, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, or 24 hours. In certain embodiments, the dose is extended over time or by a separate injection. In certain embodiments, more than one dose (e.g., 2, 3, 4, 5, 6, or more doses) of vaccinia virus is administered to the subject, for example, wherein the second treatment is performed within 1, 2, 3, 4, 5, 6, 7 days or weeks after the first treatment. In certain embodiments, the modified oncolytic virus of more than one dosage is applied to the subject in the time period of 1, 2, 3, 4, 5, 6, 7 or more days or weeks.In certain embodiments, oncolytic virus as described herein or pharmaceutical composition is applied in about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks or about 52 weeks or longer time period.In some cases, the frequency of administration of oncolytic vaccinia virus or pharmaceutical composition as described herein is once a day, twice a day, once a week, once every three weeks, once every four weeks (or once a month), once every 8 weeks (or once every 2 months), once every 12 weeks (or once every 3 months) or once every 24 weeks (once every 6 months). In some embodiments of the methods disclosed herein, the oncolytic vaccinia virus or pharmaceutical composition is independently administered with an initial dose for the first time period, an intermediate dose for the second time period, and a high dose for the third time period. In some embodiments, the initial dose is lower than the intermediate dose, and the intermediate dose is lower than the high dose. In some embodiments, the first time period, the second time period, and the third time period 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.

[0137] The modified oncolytic virus of the present disclosure, such as oncolytic vaccinia virus as described herein, or a pharmaceutical composition comprising the modified oncolytic virus is delivered to cancer cells or tumor cells by intravenous injection, for example, via infusion, parenteral, intravenous, intradermal, intramuscular, transdermal, rectal, intraurethral, ​​intravaginal, intranasal, intrathecal or intraperitoneal administration. However, alternative methods of administration are also used, for example, via intratumoral injection. The route of administration varies with the position and properties of the tumor. In certain embodiments, the route of administration is intradental, transdermal, parenteral, intraperitoneal, intravenous, intramuscular, intranasal, subcutaneous, local (regional) (for example, near the tumor, particularly the blood vessels or adjacent blood vessels of the tumor), transdermal, intrathecal, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, by lavage or oral administration. The oncolytic virus of an injectable dose is administered as a bolus injection or slow infusion. In certain embodiments, the modified oncolytic virus is administered to the patient from a source implanted in the patient. In certain embodiments, the modified oncolytic virus is administered by continuous infusion over a selected time period. In some cases, an oncolytic vaccinia virus as described herein or a pharmaceutical composition comprising the oncolytic vaccinia virus is administered at a therapeutically effective dose by infusion over a period of about 15 min, about 30 min, about 45 min, about 50 min, about 55 min, about 60 min, about 75 min, about 90 min, about 100 min, or about 120 min or longer. The oncolytic viruses or pharmaceutical compositions of the present disclosure are administered as a liquid dose, wherein the total volume administered is about 1 mL to about 5 mL, about 5 mL to 10 mL, about 15 mL to about 20 mL, about 25 mL to about 30 mL, about 30 mL to about 50 mL, about 50 mL to about 100 mL, about 100 mL to 150 mL, about 150 mL to about 200 mL, about 200 mL to about 250 mL, about 250 mL to about 300 mL, about 300 mL to about 350 mL, about 350 mL to about 400 mL, about 400 mL to about 450 mL, about 450 mL to 500 mL, about 500 mL to 750 mL, or about 750 mL to 1000 mL.

[0138] preparation

[0139] Pharmaceutical compositions containing modified viruses (such as oncolytic vaccinia viruses) as described herein are prepared as solutions, dispersions in glycerol, liquid polyethylene glycol, and any combination thereof in oils, solid dosage forms, as inhalable dosage forms, as intranasal dosage forms, as liposomal formulations, dosage forms comprising nanoparticles, dosage forms comprising microparticles, polymeric dosage forms, or any combination thereof. In some embodiments, pharmaceutical compositions as described herein comprise stabilizers and buffers. In some embodiments, pharmaceutical compositions as described herein may comprise solubilizers, such as sterile water, Tris buffer. In some embodiments, pharmaceutical compositions as described herein may comprise excipients. Non-limiting examples of suitable excipients may include buffers, preservatives, stabilizers, binders, compacting agents, lubricants, chelating agents, dispersion enhancers, disintegrants, flavorings, sweeteners, colorants.

[0140] In certain embodiments, the buffer comprises phosphate-buffered saline (PBS), Dulbecco's PBS (DPBS), TRIS-buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), standard saline citrate (SSC), HEPES-buffered saline (HBS), or Gey's balanced salt solution.

[0141] In certain embodiments, the pharmaceutical composition of the present disclosure comprises an effective amount of modified viruses disclosed herein, in combination with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" includes any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and / or is non-toxic to the patient to which it is administered. Non-limiting examples of suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents and sterile solutions. Other non-limiting examples of pharmaceutically compatible carriers include gels, bioabsorbable matrix materials, implant elements comprising modified oncolytic viruses, or any other suitable vehicles, delivery or dispersion tools or materials. Such carriers are formulated by conventional methods and administered to subjects in an effective amount.

[0142] Generation method

[0143] 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 bred in suitable host cells (for example, HeLa cells, 293 cells or Vero cells), separated from the host cells and stored under conditions promoting the stability and integrity of the virus, so that the infectivity loss over time is minimized. In certain exemplary methods, a cell stack (cell stack), a roller bottle or a perfusion bioreactor are used to breed the modified oncolytic virus in the host cell. In some instances, the downstream method for purifying the modified oncolytic virus includes filtration (for example, depth filtration, tangential flow filtration or a combination thereof), ultracentrifugation or chromatographic capture. For example, by freezing or drying, such as by lyophilization, to store the modified oncolytic virus. In certain embodiments, before administration, the stored modified oncolytic virus is reconstructed (if dry storage) and diluted in a pharmaceutically acceptable carrier for administration.

[0144] Some embodiments provide that the modified oncolytic virus as described herein exhibits higher titers in HeLa cells and 293 cells compared to a virus that is otherwise identical but does not comprise the modified oncolytic virus. In some cases, higher titers of the modified oncolytic virus are observed in HeLa cells and 293 cells.

[0145] medicine box

[0146] In embodiments, the present disclosure provides a medicine box for administering a modified oncolytic virus as described herein. In certain embodiments, the medicine box of the present disclosure comprises a modified oncolytic virus as described above or a pharmaceutical composition comprising a modified oncolytic virus. In certain embodiments, the medicine box of the present disclosure further comprises one or more components, such as instructions for use, devices and other reagents, and components for performing the method disclosed above, such as tubes, containers and syringes. In certain embodiments, the medicine box of the present disclosure further comprises one or more agents, for example, at least one of an anticancer agent, an immunomodulator or any combination thereof, which is administered in combination with a modified virus.

[0147] In certain embodiments, the kits of the present disclosure comprise one or more containers containing modified viruses disclosed herein. For example, and not by way of limitation, the kits of the present disclosure comprise one or more containers containing modified oncolytic viruses of the present disclosure.

[0148] In certain embodiments, the kit of the present disclosure includes instructions for use, a device for administering a modified oncolytic virus to a subject, or a device for administering another agent or compound to a subject. For example, and not by way of limitation, the instructions for use include a description of the modified oncolytic virus and optionally other components contained in the kit and a method of administration, including a method for determining the appropriate state of the subject, an appropriate dosage amount, and an appropriate method of administration for administering the modified virus. The instructions for use also optionally include a guide for monitoring the subject during the duration of the treatment time.

[0149] In certain embodiments, the kit of the present disclosure includes a device for administering a modified oncolytic virus to a subject. Any of a variety of devices known in the art for administering drugs and pharmaceutical compositions is included in the kit provided herein. For example, and not by way of limitation, such devices include hypodermic needles, intravenous needles, catheters, needle-free injection devices, inhalers, and liquid dispensers such as eye drops. In certain embodiments, a modified oncolytic virus to be systemically delivered, for example, by intravenous injection, intratumoral injection, or intraperitoneal injection is included in a kit with a hypodermic needle and a syringe.

[0150] Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will now appreciate that many variations, changes, and substitutions without departing from the present disclosure are possible. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the present disclosure. The appended claims are intended to define the scope of the present disclosure and thus cover methods and structures within the scope of these claims and their equivalents.

[0151] Exemplary embodiments

[0152] Provided herein are nucleic acids, wherein the nucleic acids comprise sequences encoding at least two polypeptides, wherein the at least two polypeptides comprise: interleukin-12 (IL-12) or a functional variant thereof; and soluble PD-1 (sPD-1) or a functional variant thereof. Also provided herein are nucleic acids, wherein the nucleic acids comprise DNA or RNA. Also provided herein are nucleic acids, wherein IL-12 is murine IL-12 or human IL-12. Also provided herein are nucleic acids, wherein IL-12 or a functional variant thereof comprises an α subunit and a β subunit. Also provided herein are nucleic acids, wherein the α subunit and the β subunit are connected by a linker. Also provided herein are nucleic acids, wherein the sequence encoding the IL-12 α subunit comprises a sequence having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 55 or SEQ ID NO: 69. Also provided herein are nucleic acids, wherein the sequence encoding the IL-12 α subunit comprises the sequence of SEQ ID NO: 55 or SEQ ID NO: 69. Also provided herein are nucleic acids, wherein the sequence encoding the IL-12β subunit comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 53 or SEQ ID NO: 68. Also provided herein are nucleic acids, wherein the sequence encoding the IL-12β subunit comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 53 or SEQ ID NO: 68. Also provided herein are nucleic acids, wherein the sequence encoding the linker comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 54. Also provided herein are nucleic acids, wherein the sequence encoding the linker comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 54. Also provided herein are nucleic acids, wherein sPD-1 comprises a domain from murine PD-1, a domain from human PD-1, or any combination thereof. Also provided herein are nucleic acids, wherein sPD-1 comprises an extracellular domain of murine PD-1, human PD-1, or any combination thereof. Also provided herein are nucleic acids, wherein sPD-1 comprises a domain that binds to PD-L1. Also provided herein are nucleic acids, 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. Also provided herein are nucleic acids, wherein the sequence encoding sPD-1 comprises the sequence of SEQ ID NO: 58 or SEQ ID NO: 70. Also provided herein are nucleic acids, wherein the nucleic acid further comprises at least one promoter region. Also provided herein are nucleic acids, wherein at least one promoter region drives expression of at least two polypeptides. Also provided herein are nucleic acids, 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.Also provided herein are nucleic acids, wherein the first and second promoters each comprise any one of a 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, 28 kDa promoter, or any variant or combination thereof. Also provided herein are nucleic acids, wherein the first promoter comprises a P7.5 promoter. Also 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. Also provided herein are nucleic acids, wherein the P7.5 promoter comprises the sequence of SEQ ID NO: 32. Also provided herein are nucleic acids, wherein the second promoter comprises a P28 promoter. Also 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. Also provided herein are nucleic acids, wherein the P28 promoter comprises the sequence of SEQ ID NO:29. Also provided herein are nucleic acids, wherein the nucleic acid comprises DNA, wherein the DNA comprises the sequence of SEQ ID NO:53, SEQ ID NO:11, SEQ ID NO:55, and SEQ ID NO:58 in 5' to 3' order. Also provided herein are nucleic acids, wherein the nucleic acid comprises DNA, wherein the DNA comprises the sequence of SEQ ID NO:68, SEQ ID NO:11, SEQ ID NO:69, and SEQ ID NO:70 in 5' to 3' order. Also provided herein are nucleic acids, wherein the nucleic acid comprises DNA, wherein the DNA comprises a sequence encoding SEQ ID NO:52 and SEQ ID NO:58 in 5' to 3' order. Also provided herein are nucleic acids, wherein the nucleic acid comprises DNA, wherein the DNA comprises a sequence encoding SEQ ID NO:67 and SEQ ID NO:70 in 5' to 3' order. Also provided herein are nucleic acids, wherein the nucleic acid comprises DNA, wherein the DNA comprises the sequence of SEQ ID NO: 66. Also provided herein are nucleic acids, wherein the nucleic acid comprises DNA, wherein the DNA comprises the sequence of SEQ ID NO: 71. Also provided herein are nucleic acids further comprising a sequence encoding a chemokine receptor or a functional variant thereof. Also 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.Also 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.

[0153] 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. Also provided herein are nucleic acids wherein the first polypeptide comprises the sequence of SEQ ID NO:5 or SEQ ID NO:8. Also provided herein are nucleic acids wherein the second polypeptide comprises the sequence of SEQ ID NO:2 or SEQ ID NO:4. Also provided herein are nucleic acids wherein the second polypeptide comprising the sequence of SEQ ID NO:4 further comprises one or more substitutions as described by E61V, M70I, Q75F, K78W, K78L, E84F, S87W, A129H, A132L, K135M. Also provided herein are nucleic acids further comprising at least one promoter region. Also provided herein are nucleic acids wherein the at least one promoter region drives expression of the first region and the second region. Also provided herein are nucleic acids, wherein the nucleic acid comprises a first promoter driving expression of the first region and a second promoter driving expression of the second region. Also provided herein are nucleic acids, wherein 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 variant or combination thereof. Also provided herein are nucleic acids, wherein the first promoter comprises a P7.5 promoter. Also provided herein are nucleic acids, wherein the second promoter comprises a P28 promoter. Also provided herein are nucleic acids, 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. Also provided herein are nucleic acids further comprising a sequence encoding a chemokine receptor or a functional variant thereof. Also provided herein are nucleic acids, wherein the chemokine receptor comprises at least one of the following: a CXC receptor, a CC receptor, a CX3C receptor, an XC receptor, a functional fragment thereof, a functional variant thereof, or any combination thereof.Also 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.

[0154] Also provided herein is nucleic acid as described herein, wherein the nucleic acid is present in an oncolytic virus. Also provided herein is nucleic acid, wherein the oncolytic virus is poxvirus, adeno-associated virus, adenovirus, reovirus, slow virus, herpes simplex virus, vesicular stomatitis virus, Mengo virus, myxoma virus, Newcastle disease virus, measles virus or poliovirus. Also provided herein is nucleic acid, wherein poxvirus is vaccinia virus. Also provided herein is nucleic acid, wherein vaccinia virus is Western Reserve vaccinia virus (ATCC VR-1354), Ankara vaccinia virus (ATCC VR-1508), Ankara vaccinia virus (ATCC VR-1566), Wyeth vaccinia virus strain (ATCC VR-1536) or Wyeth vaccinia virus (ATCC VR-325) modified strain. Also provided herein is nucleic acid, wherein the nucleic acid is inserted into viral genome. Also provided herein are nucleic acids that further comprise 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. Also provided herein are nucleic acids that further comprise a mutation or deletion of the TK gene. Also provided herein are nucleic acids that further comprise a mutation or deletion of the B8R gene.

[0155] Provided herein are compositions, wherein the compositions comprise: 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. Also provided herein are compositions, wherein the cytokine comprises IL-12 or a functional variant thereof. Also provided herein are compositions, wherein 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. Also provided herein are compositions, wherein IL-12 comprises a sequence of SEQ ID NO: 5 or SEQ ID NO: 8. Also provided herein are compositions, 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. Also provided herein are compositions, wherein the protein that binds to PD-L1 is an anti-PD-L1 antibody. Also provided herein are compositions, wherein the protein that binds to PD-L1 is a soluble variant of PD-1 (sPD-1). Also 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. Also provided herein are compositions, wherein sPD-1 comprises the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Also provided herein are compositions, wherein sPD-1 comprising the sequence of SEQ ID NO: 4 further comprises one or more substitutions as described by E61V, M70I, Q75F, K78W, K78L, E84F, S87W, A129H, A132L, K135M. Also provided herein are compositions further comprising at least one promoter region. Also provided herein are compositions, wherein at least one promoter region drives the expression of cytokines and PD-L1 receptors. Also provided herein are compositions, wherein at least one promoter region comprises a first promoter that drives cytokine expression and a second promoter that drives PD-L1 receptor expression. Also provided herein are compositions wherein the at least one promoter comprises any 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, 28 kDa promoter, or any variant or combination thereof. Also provided herein are compositions wherein the first promoter comprises a P7.5 promoter. Also provided herein are compositions wherein the second promoter comprises a P28 promoter. Also provided herein are compositions wherein the exogenous nucleic acid comprises a combined sequence of SEQ ID NO: 66. Also provided herein are compositions wherein the exogenous nucleic acid comprises a combined sequence of SEQ ID NO: 71. Also provided herein are compositions further comprising an exogenous nucleic acid comprising a sequence encoding a chemokine receptor or a functional variant thereof.Also provided herein is a composition, wherein the chemokine receptor comprises at least one of the following: CXC receptor, CC receptor, CX3C receptor, XC receptor, its functional fragment, its functional variant or any combination thereof. Also provided herein is a composition, wherein the chemokine receptor comprises at least one of the following: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, its functional fragment or its functional variant or any combination thereof. Also provided herein is a composition, wherein the carrier comprises a plasmid, a phage, a virus, a clay, an artificial chromosome, a liposome, a nanoparticle or any combination thereof. Also provided herein is a composition, wherein the carrier is an oncolytic virus. Compositions are also provided herein, wherein the oncolytic virus is poxvirus, adeno-associated virus, adenovirus, reovirus, slow virus, herpes simplex virus, vesicular stomatitis virus, Mengo virus, myxoma virus, Newcastle disease virus, measles virus or poliovirus. Compositions are also provided herein, wherein the poxvirus is vaccinia virus. Compositions are also provided herein, wherein the vaccinia virus is a modified strain of Western Reserve vaccinia virus (ATCC VR-1354), Ankara vaccinia virus (ATCC VR-1508), Ankara vaccinia virus (ATCC VR-1566), Wyeth vaccinia virus strain (ATCC VR-1536) or Wyeth vaccinia virus (ATCC VR-325). Compositions are also provided herein, wherein exogenous nucleic acid is inserted into the viral genome. Compositions are also provided herein, wherein the oncolytic virus includes at least one genome modification. Also provided herein are compositions wherein the at least one modification comprises a mutation or deletion in 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, functional fragments thereof, or any combination thereof. Also provided herein are compositions comprising a mutation or deletion in the TK gene. Also provided herein are compositions further comprising a mutation or deletion in the B8R gene.

[0156] Provided herein are oncolytic viruses, wherein the oncolytic virus comprises: an insert comprising, in 5' to 3' order, at the TK locus: 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.

[0157] Pharmaceutical compositions are provided herein, wherein the pharmaceutical compositions comprise: a nucleic acid as described herein or a composition as described herein; and a pharmaceutically acceptable excipient. Pharmaceutical compositions are also provided herein, wherein the composition is in liquid dosage form. Pharmaceutical compositions are also provided herein, wherein the pharmaceutically acceptable excipient is buffered saline. Pharmaceutical compositions are also provided herein, wherein the buffered saline is phosphate buffered saline (PBS), Dulbecco's PBS (DPBS), TRIS buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), standard citrate saline (SSC), HEPES buffered saline (HBS), or Gey's balanced salt solution. Pharmaceutical compositions are also provided herein, wherein the composition further comprises liposomes or nanoparticles. Pharmaceutical compositions are also provided herein, wherein the nucleic acid or vector is associated with the liposomes or nanoparticles.

[0158] Provided herein are methods for treating cancer, including administering a pharmaceutical composition as described herein to a subject with cancer in an amount sufficient to treat the cancer. Also provided herein are methods for treating cancer, wherein the cancer is a solid tumor, leukemia, or lymphoma. Also provided herein are methods 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 epithelial cancer, hepatocellular carcinoma, bile duct sarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma. Also provided herein are methods for treating cancer, wherein administration includes intratumoral administration. Also provided herein are methods for treating cancer, wherein administration includes systemic administration. Also provided herein are methods for treating cancer, wherein systemic administration comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof.

[0159] Provided herein are methods for activating an anti-tumor immune response, comprising administering a pharmaceutical composition described herein to a subject suffering from cancer. Also provided herein are methods for activating an anti-tumor immune response, wherein the cancer is a solid tumor, leukemia, or lymphoma. Also provided herein are methods for activating an anti-tumor immune response, wherein the cancer includes melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, renal cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate epithelial cancer, hepatocellular carcinoma, bile duct sarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma. Also provided herein are methods for activating an anti-tumor immune response, wherein the administering step is intratumoral administration. Also provided herein are methods for activating an anti-tumor immune response, wherein the administering step is systemic administration. Also provided herein are methods for activating an anti-tumor immune response, wherein systemic administration comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof.

[0160] Provided herein are methods for reducing the incidence of tumor cell growth, comprising administering a pharmaceutical composition as described herein to the tumor cells in an effective amount sufficient to reduce the incidence of tumor cell growth. Also provided herein are methods for reducing the incidence of tumor cell growth, wherein the tumor cells are from solid tumors, leukemias, or lymphomas. Also provided herein are methods for reducing the incidence of tumor cell growth, wherein the tumor cells are from melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemias, lymphomas, renal cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate epithelial cancer, hepatocellular carcinoma, bile duct sarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasms, or sarcoma. Also provided herein are methods for reducing the incidence of tumor cell growth, wherein the administering step is intratumoral administration. Example

[0161] The following examples further illustrate the described embodiments but do not limit the scope of the disclosure.

[0162] Example 1: Construction of mouse IL-12 and mouse sPD-1 expression system

[0163] The vaccinia virus Western Reserve strain was modified by replacing the gene encoding thymidine kinase (VACV094, J2R) with nucleic acids encoding murine IL-12 (mIL-12), soluble murine PD-1 (murine sPD-1), and a fluorescent reporter gene. A plasmid transfer vector containing the following, in order, without a gap, was generated: a 5' recombination guide sequence (SEQ ID NO:47), an SbfI cloning site (CCTGCAGG), a P7.5 promoter (SEQ ID NO:44), a KpnI cloning site (GGTACC), an open reading frame encoding murine IL-12 (SEQ ID NO:48), a SalI cloning site followed by a short spacer (SEQ ID NO:52), a P28 promoter (SEQ ID NO:41), a SpeI cloning site (ACTAGT), soluble murine PD-1 (SEQ ID NO:53), a sacI cloning site (GAGCTC), a loxP sequence (SEQ ID NO:54), a spacer sequence A (SEQ ID NO:55), a viral 454 promoter (SEQ ID NO:42), 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 a 3' recombination guide sequence (SEQ ID NO:48). NO:58). After recombination and treatment with cre recombinase, the viral genome contains the integrated sequence of SEQ ID NO:59. The selected sequences are shown in Table 6. The schematic diagram of the promoter and transgene inserted at the TK locus is shown in Figure 2 Shown in.

[0164] Table 6. Murine IL-12 and sPD-1 recombinant sequences.

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172] Example 2: Construction of human IL-12 and sPD-1 expression system

[0173] The vaccinia virus Western Reserve 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 gene. A plasmid transfer vector containing the following, in order, without a gap, was generated: a 5' recombination guide sequence (SEQ ID NO: 47), an SbfI cloning site (CCTGCAGG), a 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 short spacer (SEQ ID NO: 52), promoter P28 (SEQ ID NO: 41), a SpeI cloning site (ACTAGT), an open reading frame encoding human sPD-1 (SEQ ID NO: 63), a Sad 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 Pad cloning site (TTAATTA), spacer sequence B (SEQ ID NO: 57), a loxP sequence (SEQ ID NO: 54), and a 3' recombination guide sequence (SEQ ID NO: 5 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.

[0174] Table 7. Human IL-12 and human sPD-1 recombinant sequences.

[0175]

[0176]

[0177]

[0178]

[0179]

[0180] Example 3: Measurement of tumor size after treatment with viruses expressing sPD-1 and IL-12

[0181] Renca cells were implanted into the flanks of Balb / c mice. Twelve days later, the mice were divided into groups so that each treatment group had 50-100 mm 3 Mice were treated with 1E7 PFU of virus via intratumoral injection. Tumor size was measured 45 days later.

[0182] B16F10 cells were mixed with Matrigel 1:1 and implanted into the flanks of C57 / Black 6 mice. After 5 days, the mice were divided into groups so that each treatment group had 50-100 mm 3 Mice were treated with 1E7 PFU of virus via intratumoral injection. Tumor size was measured 38 days later.

[0183] Mice with induced tumors were divided into the following treatment groups as described in Example 1: buffer control, TK-vaccinia virus, and TK-vaccinia virus expressing IL-12 and murine sPD-1. The mean tumor volume in each treatment group was measured.

[0184] The tumor volume of the Renca tumor group was Figure 3A Mice sacrificed due to tumor burden before the final measurement were Figure 3A The display is 1000mm 3 Mice with induced Renca tumors treated with a virus expressing a combination of IL-12 and murine sPD-1 showed approximately 200 mm 3 In contrast, the control groups treated with buffer control or TK-virus had an average tumor volume of at least 1000 mm and a complete response (CR) of 40%. 3 The average tumor volume.

[0185] The tumor volume of the B16 tumor group was Figure 3B Mice sacrificed due to tumor burden before the final measurement were Figure 3B The display shows 1400mm 3 Mice bearing induced B16 tumors treated with a virus expressing IL-12 and murine sPD-1 had mean tumor volumes below detection and a 90% CR 38 days after treatment. In contrast, controls treated with buffer control or TK-virus had tumors of at least 1400 mm 3 The average tumor volume.

[0186] Example 4: Deletion of the B8R gene (IFNg binding protein)

[0187] Interferon gamma (IFNg) plays a role in anti-cancer immunity by promoting the activity of various immune cells. It is expected that IL-12 and sPD-1 require IFNg production for their activity. The vaccinia IFNg binding protein expressed by the B8R gene acts as a secreted decoy receptor, removing extracellular IFNg. The virus described in Example 1 was further modified to remove the B8R gene.

[0188] A transfer vector was generated that contained, in order: a reporter vector 5' recombinant guide sequence (SEQ ID NO: 65), a spacer sequence C (SEQ ID NO: 66), a 454 promoter (SEQ ID NO: 42), a fluorescent reporter protein (SEQ ID NO: 67), and a reporter vector 3' recombinant guide sequence (SEQ ID NO: 68). The components of the vector were replaced with nucleic acid expressing a fluorescent reporter gene for the B8R open reading frame for plaque selection purposes. Selected plaques were then treated with a second transfer vector containing a reporter vector 5' recombinant guide sequence (SEQ ID NO: 65) and a reporter vector 3' recombinant guide sequence (SEQ ID NO: 68) to remove the promoter and fluorescent reporter gene. The final sequence of the modified B8R locus is shown 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.

[0189] Table 8. B8R removal vector sequences

[0190]

[0191]

[0192]

[0193] Example 5: Measurement of tumor size after treatment with B8R-virus expressing sPD-1 and IL-12

[0194] Lewis lung carcinoma (LLC) cells were implanted into the flanks of C57 / Black mice. Five days later, the mice were divided into groups so that each treatment group had 50-100 mm 3 Mice were treated with 1E7 PFU of virus via intratumoral injection. Tumor size was measured 31 days later.

[0195] As described in Example 4, mice with induced tumors 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. The mean tumor volume in each treatment group was measured. The results of the measurements on day 31 were given in Table 1. Figure 4 Mice sacrificed due to tumor burden before the final measurement were Figure 4 The display shows 1400mm 3 .

[0196] Mice treated with buffer control or TK-vaccinia showed at least 1400 mm 3 The mean tumor volume of mice treated with TK- / B8R-vaccinia virus was approximately 900 mm3 Mice treated with TK- / B8R-vaccinia virus expressing IL-12 and murine sPD-1 had an average tumor volume below detectable levels and an 80% CR after 31 days.

[0197] Example 6: Addition of mouse CXCR3 expression system

[0198] The vaccinia virus of any of the foregoing examples is modified by replacing the gene encoding A52 with nucleic acid encoding murine CXCR3 and a fluorescent reporter gene. A plasmid transfer vector is generated that contains, in order, the following, unnicked: upstream recombination guide sequence B (SEQ ID NO: 70), an open reading frame encoding murine CXCR3 (SEQ ID NO: 71), a stop codon, a Sad cloning site and a short spacer (SEQ ID NO: 72), a loxP site (SEQ ID NO: 54), a spacer followed by a viral promoter driving expression of a GFP-pac reporter gene (SEQ ID NO: 73), a Pad cloning site (TTAATTAA), a short spacer B (SEQ ID NO: 57), and downstream recombination guide sequence B (SEQ ID NO: 74). After recombination and treatment with cre recombinase, the viral genome contains the integration sequence of SEQ ID NO: 75. The sequence is shown in Table 9. A schematic diagram of the promoter and transgene inserted at the A52R gene is shown in Figure 5 Shown in.

[0199] Table 9. Murine CXCR3 recombinant sequences.

[0200]

[0201]

[0202]

[0203]

[0204] Example 7: Addition of chemokine receptor sequences

[0205] The modified vaccinia virus described in Example 2 was further modified by replacing the gene encoding A52 (VACV094, J2R) with nucleic acids encoding human CXCR3 and a fluorescent reporter gene. This generated a non-nicked plasmid transfer vector containing, in order: an upstream recombination guide sequence B (SEQ ID NO: 70), an open reading frame encoding human CXCR3 isoform 1 (SEQ ID NO: 76), a stop codon, a Sad cloning site and a short spacer (SEQ ID NO: 72), a loxP site (SEQ ID NO: 54), a spacer followed by a viral promoter driving expression of a GFP-pac reporter gene (SEQ ID NO: 73), a Pad cloning site (TTAATTAA), a short spacer B (SEQ ID NO: 57), a loxP site (SEQ ID NO: 54), and a downstream recombination guide sequence B (SEQ ID NO: 74). Reporter gene positive viruses were isolated and then treated with a reporter gene-free transfer vector containing an upstream recombination guide sequence B (SEQ ID NO: 70), an open reading frame encoding human CXCR3 isoform 1 (SEQ ID NO: 76), and a second downstream recombination guide sequence B (SEQ ID NO: 77). After recombination, the viral genome contained the integration sequence of SEQ ID NO: 78. The selected sequences are shown in Table 10.

[0206] Table 10. Human CXCR3 recombinant sequences.

[0207]

[0208]

[0209]

[0210] The foregoing description and accompanying drawings illustrate many representative embodiments of the present invention. Various modifications, additions, and alternative designs will, of course, become apparent to those skilled in the art based on the foregoing teachings without departing from the scope of the foregoing teachings, which is indicated by the appended claims rather than the foregoing description. All changes and variations that come within the meaning and range of equivalency of the claims are intended to be embraced within their scope.

Claims

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

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

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

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

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

4.

10. The composition of claim 9, wherein the sPD-1 comprises the sequence of SEQ ID NO: 4, further comprising 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. The composition of claim 11, wherein the at least one promoter region comprises a first promoter driving expression of the cytokine and a second promoter driving expression of the PD-L1 receptor.

14. The composition of claim 12, wherein the at least one promoter comprises any 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 variant or combination thereof.

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

59.

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

64.

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

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. 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, a functional variant thereof, or any combination thereof.

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

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

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

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

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

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

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

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

30. The composition of claim 29, wherein the at least one modification 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. The composition according to claim 30 , comprising a mutation or deletion of the TK gene.

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

33. A nucleic acid, wherein the nucleic acid comprises a sequence encoding at least two polypeptides, wherein the at least two polypeptides comprise: Interleukin-12 (IL-12) or a functional variant thereof; and Soluble PD-1 (sPD-1) or its functional variants.

34. The nucleic acid of claim 33, wherein the nucleic acid comprises DNA or RNA.

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

36. The nucleic acid according to claim 33, wherein the IL-12 or a functional variant thereof comprises an α subunit and a β subunit.

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

38. The nucleic acid of claim 36, wherein the sequence encoding the IL-12α 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. The nucleic acid of claim 38, wherein the sequence encoding the IL-12α subunit comprises the sequence of SEQ ID NO: 51 or SEQ ID NO:

62.

40. The nucleic acid of claim 36, wherein the sequence encoding the IL-12β 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. The nucleic acid of claim 40, wherein the sequence encoding the IL-12β subunit comprises the sequence of SEQ ID NO: 49 or SEQ ID NO:

61.

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

50.

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

45. The nucleic acid of claim 44, wherein the sPD-1 comprises the extracellular domain of the murine 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. The nucleic acid of claim 33, wherein the sequence encoding the 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. The nucleic acid of claim 47, wherein the sequence encoding the sPD-1 comprises the sequence of SEQ ID NO: 53 or SEQ ID NO:

63.

49. The nucleic acid of claim 33, wherein the nucleic acid further comprises at least one promoter region.

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

51. The nucleic acid of claim 49, wherein the nucleic acid comprises a first promoter upstream of the sequence encoding the IL-12 and a second promoter upstream of the 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. The nucleic acid of claim 51 , wherein the first promoter and the second promoter each comprise any one of a 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 variant or combination thereof.

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

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

44.

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

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

41.

59. The nucleic acid of claim 34, wherein the nucleic acid comprises DNA, wherein the DNA comprises the sequences of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:53 in 5' to 3' order.

60. The nucleic acid of claim 34, wherein the nucleic acid comprises DNA, wherein the DNA comprises the sequence of SEQ ID NO: 61, SEQ ID NO: 50, SEQ ID NO: 62, and SEQ ID NO: 63 in 5' to 3' order.

61. The nucleic acid of claim 34, wherein the nucleic acid comprises DNA, wherein the DNA comprises a sequence encoding SEQ ID NO: 49 and SEQ ID NO: 63 in 5' to 3' order.

62. The nucleic acid of claim 34, wherein the nucleic acid comprises DNA, wherein the DNA comprises a sequence encoding SEQ ID NO: 60 and SEQ ID NO: 63 in 5' to 3' order.

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

59.

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

64.

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

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. 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 nucleic acid, wherein the nucleic acid comprises: 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 The second region encodes 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.

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

4.

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

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. The nucleic acid of claim 73, wherein the nucleic acid comprises a first promoter driving expression of the first region and a second promoter driving expression of the second region.

75. The nucleic acid of claim 74, wherein the first promoter and the second promoter each comprise any one of a 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 variant or combination thereof.

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

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

78. The nucleic acid of claim 68, 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.

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

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. 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 33-81, wherein the nucleic acid is present in an oncolytic virus.

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

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

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

86. The nucleic acid of claim 82, wherein the nucleic acid is inserted into a viral genome.

87. The nucleic acid of claim 86, further comprising 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.

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

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

90. An oncolytic virus, wherein the oncolytic virus comprises: The insert contains the following in 5' to 3' order at the TK locus: a first promoter region, wherein the promoter is P7.5; The first region encoding IL-12; a second promoter region, wherein the promoter is P28; and The second region encoding the PD-1 variant.

91. A pharmaceutical composition, wherein the pharmaceutical composition comprises: The composition of any one of claims 1 to 32 or the nucleic acid of any one of claims 33 to 89; and Pharmaceutically acceptable excipients.

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

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

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), Earle's balanced salt solution (EBSS), standard saline citrate (SSC), HEPES buffered saline (HBS), or Gey's balanced salt solution.

95. The pharmaceutical composition of claim 91, wherein the composition further comprises liposomes or nanoparticles.

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

97. A method for treating cancer comprising 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. The method of claim 97, wherein the cancer is a solid tumor, leukemia, or lymphoma.

99. 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, renal cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate epithelial cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal-type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma.

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

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

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

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

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

106. 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, renal cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate epithelial cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal-type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma.

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

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

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

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. A method for reducing the incidence of tumor cell growth, comprising: The pharmaceutical composition of any one of claims 91 to 96 is administered to tumor cells in an amount effective to reduce the incidence of tumor cell growth.

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

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

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

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

Images