Enhanced system for the treatment of cell-mediated oncolytic viruses

Abstract

To provide enhanced modified viruses for enhancing cell-mediated oncolytic viral therapy, and compositions including such modified viruses.SOLUTION: The present invention provides a composition or a combination, comprising (a) an oncolytic virus and (b)supra adventitialadipose stromal cells (SA-ASC;CD235a- / CD45- / CD34+ / CD146- / CD31-) or pericytes (CD235a- / CD45- / CD34- / CD146+ / CD31-), where the composition is a mixture of cells and viruses, and the combination is two separate compositions, where one composition includes viruses, and the other composition includes cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related applications The benefit of priority is claimed in U.S. Provisional Application No. 62 / 756,550, filed November 6, 2018, by inventor Antonio Fernandez Santidrian and applicant Calidi Biotherapeutics, Inc., entitled “ENHANCED SYSTEMS FOR CELL-MEDIATED ONCOLYTIC VIRAL THERAPY,” and in U.S. Provisional Application No. 62 / 789,458, filed January 7, 2019, entitled “ENHANCED SYSTEMS FOR CELL-MEDIATED ONCOLYTIC VIRAL THERAPY.” This application also relates to U.S. Patent Application No. 16 / 676,416, filed on the same day, entitled "ENHANCED SYSTEMS FOR CELL-MEDIATED ONCOLYTIC VIRAL THERAPY" by inventors Antonio Fernandez Santidrian, Duong Nguyen, Dobrin Draganov, and applicant Calidi Biotherapeutics, Inc. The subject matter and disclosures of each of these applications are incorporated in whole by reference.

[0002] Integration by referencing electronically provided sequence listings An electronic version of the sequence listing is submitted with this specification, and its contents are incorporated in their entirety by reference. The electronic file was created on November 6, 2019, is 793 kilobytes in size, and is titled 2601SEQ001.txt.

[0003] Field of Invention A cell-assisted viral expression system for improving oncolytic virus therapy, the use of the system, and a method for treating cancer by administering the system to subjects requiring such treatment are provided. [Background technology]

[0004] background The ability of delivered or administered viruses to infect tumors and to colonize and / or replicate within them can be inhibited by circulating neutralizing antibodies, innate and adaptive immune mechanisms, and other elimination mechanisms against viruses and / or carrier cells. Cells have been used as carriers for the delivery of oncolytic viruses for cancer treatment. Improved systems are needed to enhance the therapeutic efficacy of oncolytic viruses. [Overview of the Initiative]

[0005] overview The use of cells, such as stem cells, for the delivery of oncolytic viruses can be improved by incubating the cells and the virus for a sufficient time for the virus to express and / or replicate the gene it encodes. After incubation, the cells can be stored at low temperatures for subsequent use. Longer incubation periods enhance the efficacy of oncolytic virus therapy compared to naked viruses and compared to previous use of cells that have not been incubated with the virus for a sufficient time. The resulting cells can be stored for subsequent use by cryopreservation or other means. The resulting cells, which provide a more effective oncolytic virus delivery medium, can be administered via systemic administration as well as other routes, including intratumoral, intraperitoneal, and local administration.

[0006] A cell-assisted viral expression system (CAVES) for enhancing oncolytic virus therapy is provided herein. This system comprises (1) a cell, such as a carrier cell, that allows viral infection and replication; (2) an oncolytic virus; and (3) at least one expressible immunomodulatory or therapeutic gene encoded by the virus expressed in the cell. CAVES are produced by incubating the cell and virus under conditions that allow the virus to infect the cell and express its gene. Exemplary cells enhance oncolytic virus therapy by having one or more characteristics for this purpose, e.g., the ability to: (a) amplify the expression of the virus and its encoded protein; (b) protect the virus from inactivation by the humoral immune system or other serum components; and / or (c) promote colonization and / or spread of viral infection within a tumor. The systems provided herein can be generated using any oncolytic virus, as well as any cell that enables viral amplification and expression of its encoded protein. Generally, the cells / carrier cells are not limited to tumor cells or inactivated tumor cells, but include, however, stem cells and fibroblasts.

[0007] The systems provided herein can generate cells that are permissible to viral infection and replication, as well as viruses such as oncolytic viruses, by incubating them together ex vivo for a predetermined period sufficient for viral infection and expression of at least one virally encoding immunomodulatory protein and / or at least one virally encoding and recombinant expression therapeutic protein, as well as optionally one or more cell / recombinant cell proteins.

[0008] To construct a CAVES that does not use cells as a standard delivery medium, but rather uses cells as part of a system to enhance delivered oncolytic virus therapy, the typical time required is generally 2-4 hours or more, generally about 5 or 6 hours to 72 hours or more, for example, generally at least about 5 or 6 hours, or about 5 or 6 hours, or more than about 5 or 6 hours to at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2 An incubation period of 3, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 hours or longer is required, for example, about 6 to 18 hours, or about 12 to 48 hours. However, this period depends on the specific oncolytic virus and / or carrier cells used in the system, and their combinations. This period is sufficient time for the virus to infect the cells and for the expression of virus-encoded immunomodulatory proteins or encoded therapeutic proteins. For example, in the vesicular stomatitis virus (VSV) replication cycle, the time for expressing recombinant proteins such as virus-encoding immunomodulatory proteins and / or therapeutic proteins is generally relatively short, about 2-3 hours, while in the case of vaccinia virus, the time for expressing virus-encoding immunomodulatory proteins and / or recombinant therapeutic proteins is generally longer, about 6-12 hours or more. To prepare vaccinia virus caves, it is generally necessary to incubate the virus with cells for at least about 6 hours.

[0009] When administered, the Cell-Assisted Viral Expression System (CAVES) provides an immediate supply of immunomodulatory and / or therapeutic proteins, regardless of tumor tolerance to viral infection and / or amplification. Promoting the expression of virally encoded immunomodulatory and / or therapeutic genes offers several advantages. These include improved adaptation to allogeneic situations, as the virally encoded immunomodulators are expressed before exposure to cancer / tumor so that they act immediately upon administration to prevent viral and / or cellular rejection by the patient's immune system. The Cell-Assisted Viral Expression System (CAVES) can promote the expression of proteins that block complement inhibition.

[0010] While cell-assisted viral expression systems (CAVES) can be produced using autologous cells, CAVES provide a method using allogeneic cells and can be manufactured and stored for administration using standard protocols. Since viral amplification and expression of one or more virally encoded immunomodulatory proteins and / or recombinantly expressed therapeutic genes are initiated ex vivo over a predetermined time before administration or before storage for future administration, the cell-assisted viral expression systems (CAVES) provided herein can be standardized to treat patient populations. Since the virus that infects the cells expresses the encoded proteins upon administration, the therapeutic effect of the systems provided herein is not affected, or is less affected, by variability caused by differences in the tumor microenvironment of patients in a population. The cell-assisted viral expression systems (CAVES) provided herein overcome or mitigate difficulties associated with intolerant cancer / tumors, unfavorable microenvironments, and / or malnourished environments within tumors, which can interfere with viral amplification and virally encoded gene expression. Viral release and spread in tumors can occur immediately after administration due to the initiation of viral amplification prior to ex vivo administration, as well as the expression of virally encoded immunomodulation and / or therapeutic genes.

[0011] In any of the compositions and methods provided herein, the cells and / or viruses used to generate CAVES and used in the associated methods are modified as provided herein. The modifications provided herein can result in improved therapeutic benefits, for example by incorporating an encoded therapeutic product, or can help overcome immune and other barriers, such as tumor vasoblockage, to improve therapeutic efficacy. In some embodiments, cells are modified for conditional immortalization, i.e., they can be stably proliferated to produce a large population by activating immortalization and then inactivated before administration to a subject so that uncontrolled cell division does not continue in the subject. For example, cells ("carrier cells") can be modified to express one or more wild-type or modified (mutated, or, for example, as fusion proteins) c-myc, v-myc, E6 / E7, hTERT, SV40 large tumor antigen, loxP, and / or tetR in order to adapt the cellular components of CAVES to conditional immortalization. The proliferation of the thus modified carrier cell population can be activated at a first time point (or more) before the preparation of CAVES and / or before administration of CAVES to the subject, and the proliferation of the carrier cell population can be inactivated at a second time point (after the expansion of CAVES and before administration to the subject).

[0012] The systems provided herein can be stored stably and indefinitely under cryogenic storage conditions, such as -80°C, and can be thawed as needed or desired before administration. For example, the systems provided herein can be stored at a storage temperature such as -20°C or -80°C, for at least or about several hours or several hours, 1, 2, 3, 4 or 5 hours, or for a period of days including at least about several years, for example, but not limited to at least about 1, 2, 3 or more years, for example, from at least about 1, 2, 3, 4 or 5 hours to at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 hours before thawing for administration. It can be stored for 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 68, 69, 60, 70, 71 or 72 hours, or 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 days, or 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12 months, or for 1, 2, 3, 4 or 5 years or more. The systems provided herein can also be stored stably under refrigerated conditions such as 4°C and / or transported on ice to the administration site for treatment. For example, the systems provided herein can be stored at 4°C or on ice for at least or about several hours, for example, 1, 2, 3, 4 or 5 hours before administration for treatment, and at least or about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 hours or more.

[0013] The cell-assisted viral expression systems (CAVES) provided herein include pre-expressed viral-coding proteins, such as immunomodulators and / or recombinant therapeutic proteins, before administration for the treatment of the target. This allows the tumor microenvironment to respond more rapidly to the treatment. The amount of virus used to construct the systems provided herein can be less than when the virus is administered directly without ex vivo amplification. Since viral amplification and expression of viral-coding immunomodulators and / or recombinant therapeutic proteins occur ex vivo over a predetermined time before administration to the tumor site, viral infection and release at the tumor site can occur before host immune cells initiate a response to the cells and / or virus, thus enabling the generation of the systems provided herein even using allogeneic cells. Since viral amplification and expression of viral-coding immunomodulators and / or recombinant therapeutic proteins occur ex vivo over a predetermined time before administration to the tumor site, extracellular enveloped virus particles (eeVs) that can survive longer in circulation can be produced in situ immediately after administration of the system to the host.

[0014] Methods of treatment are also provided herein, which include administering the system provided herein to subjects requiring such treatment. The system can be administered alone or in combination with other immuno-oncology therapies, including, but not limited to, checkpoint inhibitors, CAR-T cells, costimulatory molecules, therapeutic antibodies, bi-antibodies, and antibody-drug conjugates. Furthermore, the following items are provided herein: [Section 1] A cell-assisted viral expression system (CAVES) that includes carrier cells, The carrier cells contain an oncolytic virus, The aforementioned oncolytic virus is not an adenovirus, The carrier cell is a cell in which the virus can replicate, The carrier cells are not tumor cells or immune cells, and A cell-assisted viral expression system in which the carrier cells express at least one immunomodulatory protein or recombinant therapeutic protein encoded by the virus and expressed by association between the virus and the carrier cells. [Section 2] A cell-assisted viral expression system (CAVES) that includes carrier cells, The carrier cells contain an oncolytic virus, The aforementioned oncolytic virus is not the measles virus. The carrier cell is a stem cell in which the virus can replicate, and A cell-assisted viral expression system in which the carrier cell expresses at least one immunomodulatory protein or recombinant therapeutic protein encoded by the virus and expressed upon association between the virus and the carrier cell. [Section 3] A cell-assisted viral expression system (CAVES) that includes carrier cells, The carrier cells contain an oncolytic virus, The carrier cell is a cell in which the virus can replicate, The carrier cell expresses at least one immunomodulatory protein or recombinant therapeutic protein encoded by the virus and expressed upon association between the virus and the carrier cell. A cell-assisted viral expression system wherein the carrier cells are treated or modified, or both, to enhance the immunosuppressive and / or immune privileged properties of the cells for administration to a human subject, and optionally, the cells are treated or modified to enhance viral amplification in the cells. [Section 4] A cell-assisted viral expression system (CAVES) that includes carrier cells, The carrier cells contain an oncolytic virus, The aforementioned oncolytic virus is not an adenovirus, The aforementioned carrier cells are stem cells, The carrier cell expresses at least one immunomodulatory protein or recombinant therapeutic protein encoded by the virus and expressed upon association between the virus and the carrier cell, and A cell-assisted viral expression system in which the carrier cells are infected with the oncolytic virus, and the oncolytic virus is incubated for 6 hours or more. [Section 5] The aforementioned virus is a vaccinia virus, as described in item 4 above, and is a cell-assisted viral expression system (CAVES). [Section 6] A cell-assisted viral expression system (CAVES) according to item 4 or 5 above, produced by infecting a carrier cell containing a virus with an infection multiplicity (MOI) of 0.001 to 10. [Section 7] A cell-assisted viral expression system (CAVES) described in any of items 1 to 6 above, which is cryopreserved. [Section 8] A cell-assisted viral expression system (CAVES) as described in any of items 1 to 6 above, refrigerated for at least 24 hours. [Section 9] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 6 above, wherein the temperature of the composition is -20°C to -80°C or about -20°C to about -80°C. [Section 10] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 7 above, wherein the carrier cells are stored at a temperature of 5°C to -200°C for at least 24 hours, and the carrier cells contain approximately 100, 50, or fewer than 10 viral particles, the number of viral particles being evaluated by sonicating the cells and measuring the plaque-forming units. [Section 11] A cell-assisted viral expression system (CAVES) as described in any of items 7, 9, and 10 above, which has been stored at a temperature of approximately -80°C to -200°C or in between for at least 24 hours. [Section 12] A cell-assisted viral expression system (CAVES) as described in any of items 1 to 11 above, wherein the virus is a TK+ vaccinia virus. [Section 13] A cell-assisted viral expression system (CAVES) as described in any of items 1 to 11 above, in which the virus has been attenuated. [Section 14] A cell-assisted viral expression system (CAVES) that includes carrier cells, The carrier cells contain an oncolytic virus, The carrier cell is a cell in which the virus can replicate, The carrier cell expresses at least one immunomodulatory protein or recombinant therapeutic protein encoded by the virus and expressed by the association of the virus with the carrier cell, and A cell-assisted viral expression system in which the CAVES is cryopreserved or present in a composition containing a cryoprotectant. [Section 15] The aforementioned virus is not an adenovirus, and is a cell-assisted viral expression system (CAVES) as described in item 14 above. [Section 16] A cell-assisted viral expression system (CAVES) according to item 14 or item 5, wherein the temperature of the composition is exactly or about -200°C to -20°C. [Section 17] A cell-assisted viral expression system (CAVES) according to item 7 or 14, wherein the CAVES is in a composition comprising either or both DMSO and glycerol for cryopreservation. [Section 18] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 14 above, produced by infecting a carrier cell having an oncolytic virus with the virus at an infection multiplicity (MOI) of only up to 10 virus particles / cell. [Section 19] A cell-assisted viral expression system (CAVES) as described in item 18 above, wherein the MOI is 0.001 to 10. [Section 20] A cell-assisted viral expression system (CAVES) according to item 19, wherein the MOI is at least 0.1, at least 0.3, or at least 0.5. [Section 21] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 19 above, wherein the carrier cells contain the oncolytic viral genome ranging from about 900 or about 1,000 copies to about 10,000 or about 11,000 copies. [Section 22] A cell-assisted viral expression system (CAVES) as described in any of items 1 to 19 above, containing fewer than 100 viral genomes / cells. [Section 23] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 22 above, wherein the carrier cells are stored at a temperature of 5°C to -200°C for at least 24 hours, and the carrier cells contain the oncolytic viral genome in quantities ranging from about 900 or about 1,000 copies to about 10,000 or about 11,000 copies. [Section 24] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 23 above, wherein the carrier cells contain about 100, 50, or fewer than 10 viral particles, and the number of viral particles can be evaluated by sonicating the cells and measuring the plaque-forming units. [Section 25] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 24 above, wherein the carrier cells contain 1 to less than 200 viral particles / cells, and the number of viral particles can be evaluated by sonicating the cells and measuring the plaque-forming units. [Section 26] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 24 above, wherein the carrier cell contains 1 to less than 200 viral particles / cell. [Section 27] The cells are treated or modified, or both treated and modified, to enhance their immunosuppressive or immunoprivileged properties for administration to human subjects. and / or the cells are treated or modified to enhance the amplification of the virus within the cells, A cell-assisted viral expression system (CAVES) as described in any of the above sections 1, 2, and 7-26. [Section 28] The cells are treated or modified, or both treated and modified, to enhance the immunosuppressive or immunoprivileged properties of the cells for administration to human subjects, and The cells are treated or modified to enhance the amplification of the virus within the cells. A cell-assisted viral expression system (CAVES) as described in any of items 1 to 27 above. [Section 29] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 28 above, wherein the carrier cells are tolerant to oncolytic virus amplification, accumulate within the tumor, and / or are not recognized by the target immune system for a sufficient time to deliver the virus to the target tumor. [Section 30] A cell-assisted viral expression system (CAVES) according to any one of items 3 to 29 above, wherein the carrier cells are selected from treated or modified stem cells, immune cells, and tumor cells. [Section 31] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 29 above, wherein the carrier cell is an embryonic epithelial cell or a fibroblast. [Section 32] A cell-assisted viral expression system (CAVES) according to any one of items 3 to 29 above, wherein the carrier cell is an immune cell. [Section 33] The cell-assisted viral expression system (CAVES) described in item 32 above, wherein the immune cells are selected from granulocytes, mast cells, monocytes, dendritic cells, natural killer cells, lymphocytes, T cell receptor (TCR) transgenic cells that target tumor-specific antigens, and CAR-T cells that target tumor-specific antigens. [Section 34] A cell-assisted viral expression system (CAVES) according to any one of items 3 to 29 above, wherein the carrier cells are modified or treated cells from a hematological malignancy cell line. [Section 35] The cell line is selected from among human leukemia, T-cell leukemia, myelomonocytic leukemia, lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), erythroleukemia, myelomonoblastic leukemia, malignant non-Hodgkin NK lymphoma, myeloma / plasmacytoma, multiple myeloma, and macrophage cell lines, as described in item 34 above. [Section 36] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 32 above, wherein the carrier cell is a stem cell. [Section 37] The cell-assisted viral expression system (CAVES) described in item 36 above, wherein the stem cells are selected from adult stem cells, embryonic stem cells, fetal stem cells, neural stem cells, mesenchymal stem cells, totipotent stem cells, pluripotent stem cells, induced pluripotent stem cells, compound pluripotent stem cells, oligopluripotent stem cells, unipotent stem cells, adipocyte stem cells, endothelial stem cells (e.g., endothelial progenitor cells, placental endothelial progenitor cells, angiogenic endothelial cells, pericytes), adult peripheral blood stem cells, myoblasts, small immature stem cells, cutaneous fibroblast stem cells, tissue / tumor-associated fibroblasts, epithelial stem cells, and embryonic epithelial stem cells. [Section 38] The cell-assisted viral expression system (CAVES) described in item 36 above, wherein the stem cells are selected from mesenchymal cells. [Section 39] The cell-assisted virus expression system (CAVES) described in paragraph 38 above, wherein the mesenchymal cells are isolated from / derived from adult bone marrow, adipose tissue, blood, dental pulp, neonatal umbilical cord, umbilical cord blood, placental mesenchymal cells, placental-derived adherent stromal cells, placental-derived decidual stromal cells, endometrial regenerative cells, placental dipotent endothelial / mesenchymal progenitor cells, amniotic membrane or amniotic fluid mesenchymal stem cells, amniotic fluid-derived progenitor cells, Howartan jelly mesenchymal stem cells, pelvic girdle stem cells, chorionic villous mesenchymal stromal cells, subcutaneous white adipose mesenchymal stem cells, pericytes, perisinus reticular cells, hair follicle-derived stem cells, hematopoietic stem cells, periosteum-derived mesenchymal stem cells, lateral plate mesenchymal stem cells, deciduous tooth stem cells, periodontal ligament stem cells, dental follicle progenitor cells, dental papilla-derived stem cells, muscle satellite cells, and other such cells. [Section 40] The cell-assisted viral expression system (CAVES) described in any of items 1 to 36 above, wherein the cells are selected from endothelial progenitor cells, neural stem cells, adult bone marrow cells, and mesenchymal stem cells. [Section 41] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 36 above, wherein the cells are mesenchymal stem cells isolated from or derived from adult bone marrow, adipose tissue, blood, dental pulp, neonatal umbilical cord, umbilical cord blood, placental mesenchymal cells, placental-derived adherent stromal cells, placental-derived decidual stromal cells, endometrial regenerative cells, placental dipotent endothelial / mesenchymal progenitor cells, amniotic membrane or amniotic fluid mesenchymal stem cells, amniotic fluid-derived progenitor cells, Howartan jelly mesenchymal stem cells, pelvic girdle stem cells, chorionic villous mesenchymal stromal cells, subcutaneous white adipose mesenchymal stem cells, pericytes, perisinus reticular cells, hair follicle-derived stem cells, hematopoietic stem cells, periosteum-derived mesenchymal stem cells, lateral plate mesenchymal stem cells, deciduous tooth stem cells, periodontal ligament stem cells, dental follicle progenitor cells, dental papilla-derived stem cells, and muscle satellite cells. [Section 42] The cell-assisted viral expression system (CAVES) described in item 41 above, wherein the mesenchymal stem cells are isolated from adipocytes. [Section 43] A cell-assisted viral expression system (CAVES) as described in any of items 1 to 36 above, wherein the carrier cells include adipocytes. [Section 44] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 36 above, wherein the cells are MSCs isolated from umbilical cord blood, peripheral blood, muscle, cartilage, or amniotic fluid, or a mixture thereof. [Section 45] The aforementioned carrier cells are derived from stromal vascular cells (SVF) of adipocytes, and the cell-assisted viral expression system (CAVES) is one of the above items 1 to 44. [Section 46] The cell-assisted viral expression system (CAVES) according to any one of items 1 to 45 above, wherein the cells are stem cells derived from the superepithelial adipose-derived stromal cells (CD34+SA-ASC) produced by culturing superepithelial adipose-derived stromal cells. [Section 47] The cell-assisted viral expression system (CAVES) described in item 38 above, wherein the mesenchymal cells are derived from adipocytes. [Section 48] A cell-assisted viral expression system (CAVES) according to any of items 1 to 47 above, wherein the carrier cells are adipocytes selected from superembryonic adipocytes (SA-ASCs; CD235a- / CD45- / CD34+ / CD146- / CD31-) and pericytes (CD235a- / CD45- / CD34- / CD146+ / CD31-). [Section 49] The cell-assisted viral expression system (CAVES) according to any one of items 1 to 48 above, wherein the carrier cells are adipose-cultured adipose-mesenchymal stem cells (AD-MSCs) derived from superembryonic adipose-derived adipose-stromal cells (CD34+SA-ASCs). [Section 50] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 49 above, wherein the carrier cells and oncolytic virus are co-cultured in vitro for a sufficient time before administration to a subject or storage for a sufficient time for the immunomodulatory or therapeutic protein encoded by the virus to be expressed and / or for a sufficient time for the virus to undergo at least one replication cycle. [Section 51] The cell-assisted viral expression system (CAVES) according to item 50, wherein the carrier cells and the virus are co-cultured for a sufficient time to express an oncolytic viral immunomodulatory protein and for the virus to undergo at least one replication cycle. [Section 52] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 51 above, wherein the cells are autologous to the subject being treated. [Section 53] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 51 above, wherein the cells are allogeneic with respect to the subject being treated. [Section 54] A cell-assisted viral expression system (CAVES) as described in any of items 1 to 53 above, wherein the virus is selected from poxvirus, herpes simplex virus, adeno-associated virus, reovirus, vesicular stomatitis virus (VSV), coxsackievirus, Semryki forest virus, Seneca Valley virus, Newcastle disease virus, Sendai virus, dengue virus, picornavirus, poliovirus, parvovirus, retrovirus, alphavirus, flavivirus, rhabdovirus, papillomavirus, influenza virus, mumps virus, gibbon leukemia virus, maraba virus, and Sindbisvirus. [Section 55] A cell-assisted viral expression system (CAVES) as described in any of items 1 and 3 to 54 above, wherein the aforementioned virus is the measles virus. [Section 56] The aforementioned virus is a retrovirus, as described in item 54 above, a cell-assisted viral expression system (CAVES). [Section 57] The cell-assisted viral expression system (CAVES) described in item 54 above, wherein the aforementioned virus is a vaccinia virus poxvirus. [Section 58] The cell-assisted viral expression system (CAVES) described in item 57 above, wherein the vaccinia virus is TK+. [Section 59] The aforementioned virus has been attenuated, and is a cell-assisted viral expression system (CAVES) as described in item 54 above. [Section 60] The cell-assisted viral expression system (CAVES) described in item 54 above, wherein the virus is a poxvirus selected from the strains of Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, Modified Vaccinia Ankara (MVA), New York City Board of Health, Dairen, Ikeda, LC16M8, Tashkent, Wyeth, IHD-J, IHD-W, Brighton, Dairen I, and Connaught. [Section 61] The cell-assisted viral expression system (CAVES) described in item 60 above, wherein the virus is ACAM1000 or ACAM2000. [Section 62] The expression of immunomodulatory proteins and / or therapeutic proteins encoded by the virus occurs under conditions that, after the virus infects the carrier cells, at least one immunomodulatory protein or therapeutic protein encoded by the virus is expressed, or the carrier cells can proliferate, or the virus can replicate, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 A cell-assisted viral expression system (CAVES) as described in any of items 1 to 57 above, achieved by incubation for 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 hours or more. [Section 63] A cell-assisted viral expression system (CAVES) according to item 62, wherein the expression of immunomodulatory proteins and / or therapeutic proteins encoded by the virus is achieved by incubating the carrier cells with the virus for 6 hours or more after infection. [Section 64] The cell-assisted viral expression system (CAVES) described in item 63 above, wherein the aforementioned virus is a vaccinia virus. [Section 65] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 62 above, wherein the carrier cells having the virus are incubated after infection for a time sufficient for the expression of the protein and replication of the virus. [Section 66] A cell-assisted viral expression system (CAVES) according to item 62 or item 60, wherein the carrier cells containing the virus are stored at -5°C to -200°C for at least 24 hours after incubation. [Section 67] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 66 above, obtained by storing the CAVES or a composition containing the CAVES at -5°C to -200°C for at least 24 hours. [Section 68] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 66 above, obtained by storing the CAVES or a composition containing the CAVES at -80°C to -200°C for at least 24 hours. [Section 69] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 68 above, wherein the virus expresses an immunomodulatory protein presented on the surface of the carrier cell. [Section 70] The cell-assisted viral expression system (CAVES) described in item 61 above, wherein the immunomodulatory viral protein is selected from VCP(C3L), B5R, HA(A56R), B18R / B19R, and B8R. [Section 71] The carrier cells are stem cells, which are mesenchymal cells derived from adipocytes. The aforementioned virus is a vaccinia virus, and The carrier cells containing the virus are produced by incubating the cells with the virus for 6 hours or more under conditions in which at least one immunomodulatory protein or therapeutic protein encoded by the virus is expressed, or under conditions in which the carrier cells can proliferate or the virus can replicate. A cell-assisted viral expression system (CAVES) as described in any of items 1 to 69 above. [Section 72] A cell-assisted viral expression system (CAVES) as described in item 71 above, wherein the incubation is performed for any duration between 6 hours and the next day, up to 24 hours, up to 26 hours, up to 3 hours, up to 3 days, up to 4 days, up to 5 days, or between 6 hours and 1 week. [Section 73] A cell-assisted viral expression system (CAVES) according to any one of items 62 to 71 above, wherein the above conditions include incubation in a cell culture medium at a temperature of approximately 25°C to 40°C. [Section 74] A cell-assisted viral expression system (CAVES) according to any of items 1 to 73 above, wherein the aforementioned virus is a vaccinia virus selected from those described in any of items 280 to 310 above. [Section 75] The aforementioned virus is a vaccinia virus selected from among vaccinia viruses. a) A nucleic acid sequence shown in Sequence ID No. 71 or a sequence having at least 95% sequence identity therewith, b) At least one therapeutic gene or detectable marker gene inserted into the intergenetic region between the corresponding vaccinia virus open reading frame 157 (ORF_157) and open reading frame 158 (ORF_158), c) A locus of the corresponding unmodified vaccinia virus that is partially or completely deleted and in which the F1L gene is not expressed, d) A B8R locus in which the corresponding unmodified vaccinia virus is partially or completely deleted, where the B8R gene is not expressed, e) One or more of the following modifications: (i) At least one therapeutic gene or marker gene inserted into the intergenetic region between open reading frame 157 (ORF_157) and open reading frame 158 (ORF_158) of the corresponding unmodified vaccinia virus, (ii) A locus of the corresponding unmodified vaccinia virus that is partially or completely deleted, in which the F1L gene is not expressed, and / or (ii) A B8R locus in which the corresponding unmodified vaccinia virus is partially or completely deleted, and a locus in which the B8R gene is not expressed. A cell-assisted viral expression system (CAVES) as described in any of items 1 to 73 above, including the above. [Section 76] A cell-assisted viral expression system (CAVES) according to any of items 75a) to e) above, comprising the corresponding unmodified vaccinia virus shown in SEQ ID NO: 70 or SEQ ID NO: 71. [Section 77] A cell-assisted viral expression system (CAVES) according to any one of items 75b), e) and 76, wherein the modification includes at least one therapeutic gene inserted into the intergenetic region between open reading frame 157 (ORF_157) and open reading frame 158 (ORF_158) of the corresponding unmodified vaccinia virus. [Section 78] The cell-assisted viral expression system (CAVES) described in item 77 above, wherein the therapeutic gene(s) are selected from among immune checkpoint inhibitors, costimulators, cytokines, growth factors, photosensitizers, radionuclides, toxins, antimetabolites, signaling modulators, anticancer antibodies, and angiogenesis inhibitors. [Section 79] A cell-assisted viral expression system (CAVES) as described in item 78 above, wherein the therapeutic gene is a costimulatory factor that is OX40L or CD40L. [Section 80] A cell-assisted viral expression system (CAVES) according to any of the above items 75b) and e), and 76-78, wherein the modification includes at least one marker gene inserted into the intergenetic region between open reading frame 157 (ORF_157) and open reading frame 158 (ORF_158) of the corresponding unmodified vaccinia virus. [Section 81] The cell-assisted viral expression system (CAVES) described in item 80 above, wherein the marker gene(s)(or more) are selected from green fluorescent protein (GFP), high-sensitivity green fluorescent protein (eGFP), blue fluorescent protein (BFP), TurboFP635, and phosphoribosyltransferase (gpt). [Section 82] Because the aforementioned modification is a B8R locus in the corresponding unmodified virus that is partially or completely deleted, the B8R gene is not expressed. The modified virus comprises at least one therapeutic gene and / or marker gene inserted into a partially or completely deleted region of the B8R locus. A cell-assisted viral expression system (CAVES) as described in any of the above sections 75d) to f). [Section 83] Because the aforementioned modification is a partially or completely deleted F1L locus in the corresponding unmodified virus, the F1L gene is not expressed. The modified virus comprises at least one therapeutic gene and / or marker gene inserted into a partially or completely deleted region of the F1L locus. A cell-assisted viral expression system (CAVES) as described in any of items 75c), e), and f) above. [Section 84] A cell-assisted viral expression system (CAVES) as described in item 82 or 83 above, encoding at least one therapeutic gene. [Section 85] A cell-assisted viral expression system (CAVES) as described in item 84 above, wherein the therapeutic gene(s) are selected from among immune checkpoint inhibitors, costimulators, cytokines, growth factors, photosensitizers, radionuclides, toxins, antimetabolites, signaling modulators, anticancer antibodies, and angiogenesis inhibitors. [Section 86] A cell-assisted viral expression system (CAVES) as described in any of sections 82 to 85 above, comprising at least one marker gene. [Section 87] The cell-assisted viral expression system (CAVES) described in item 86 above, wherein the marker gene(s)(or more) are selected from green fluorescent protein (GFP), high-sensitivity green fluorescent protein (eGFP), blue fluorescent protein (BFP), TurboFP635, and phosphoribosyltransferase (gpt). [Section 88] A cell-assisted viral expression system (CAVES) according to any of items 75b) and f) and 76-87 above, wherein at least one therapeutic gene is an anti-cancer antibody. [Section 89] The cell-assisted viral expression system (CAVES) described in item 88 above, wherein the anti-cancer antibody is an anti-VEGF antibody or an anti-CTLA-4 antibody. [Section 90] A cell-assisted viral expression system (CAVES) as described in item 88 or 89 above, wherein the anti-cancer antibody is a single-chain antibody. [Section 91] A cell-assisted viral expression system (CAVES) according to any one of items 88 to 90 above, further comprising a nucleic acid encoding an IgK signal peptide for promoting the secretion of the aforementioned antibody. [Section 92] A cell-assisted viral expression system (CAVES) according to any one of items 88 to 91 above, further comprising a nucleic acid encoding a FLAG tag for facilitating the detection of the aforementioned antibody. [Section 93] A cell-assisted viral expression system (CAVES) according to any of items 75b) and e) and 76-87, wherein the virus comprises a gene encoding a therapeutic product and / or a detectable marker gene selected from sodium-iodine cotransporter (NIS), OX40L, and 4-IBBL. [Section 94] A cell-assisted viral expression system (CAVES) according to any one of items 74 and 76-93 above, wherein the unmodified vaccinia virus is ACAM2000 or ACAM1000. [Section 95] The cell-assisted viral expression system (CAVES) described in item 94 above, wherein the unmodified vaccinia virus is ACAM2000. [Section 96] A cell-assisted viral expression system (CAVES) according to any one of items 3 to 75 above, wherein the cells are derived from a tumor to which the composition is administered. [Section 97] A cell-assisted viral expression system (CAVES) as described in item 96 above, encoding a therapeutic product. [Section 98] The cell-assisted viral expression system (CAVES) described in item 97 above, wherein the therapeutic product is one or more of cytokines, chemokines, costimulators, prodrug activators, and therapeutic antibodies. [Section 99] The cell-assisted viral expression system (CAVES) described in item 98 above, wherein the therapeutic product is one or more single-chain antibodies against GM-CSF, IL2, IL10, IL12, IL-15, IL-17, IL-18, IL-21, TNF, MIP1a, FLt3L, IFN-b, IFN-g, CCl5, CCl2, CCl19, CXCl11, OX40L, 41BBL, CD40L, B7.1 / CD80, GITRL, LIGHT, CD70, BITE, as well as VEGFA, VEGFB, PGF, VEGFR2, PDGFR, Ang-1, Ang-2, ANGPT1, ANGPT2, HGF, lacZ, cytosine deaminase enzyme, and human sodium-iodine cotransporter. [Section 100] The cell-assisted viral expression system (CAVES) described in item 98 above, wherein the therapeutic product is an immune checkpoint inhibitor. [Section 101] The cell-assisted viral expression system (CAVES) according to item 100 above, wherein the immune checkpoint inhibitor is an antibody against PD-1, PD-L1, CTLA4, TIM-3, BTLA, VISTA, PD-L1, or LAG-3. [Section 102] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 96 above, wherein the oncolytic virus encodes a therapeutic product that inhibits or reduces angiogenesis. [Section 103] The cell-assisted viral expression system (CAVES) described in item 97 above, wherein the encoded therapeutic product is selected from among an anti-VEGF or anti-VEGFR single-chain antibody, VEGFR or its extracellular domain, which is optionally directly or indirectly linked to the Fc portion of human IgG1, an anti-angiopoietin-2 (ANGPT) single-chain antibody, and combinations thereof. [Section 104] A pharmaceutical composition comprising a cell-assisted viral expression system (CAVES) described in any of items 1 to 103 above in a pharmaceutically acceptable medium. [Section 105] A pharmaceutical composition as described in item 104 above, which is stored by freezing. [Section 106] A pharmaceutical composition according to item 104 or 105 above, which is formulated for direct administration without dilution. [Section 107] A pharmaceutical composition as described in any of items 104 to 106 above, which is formulated as a single dose. [Section 108] A pharmaceutical composition according to any one of items 104 to 107, wherein the pharmaceutically acceptable medium is suitable for parenteral administration. [Section 109] A pharmaceutical composition according to any one of items 104 to 108 above, formulated for intratumor, intraperitoneal, intravenous, subcutaneous, oral, mucosal, or rectal administration. [Section 110] A pharmaceutical composition according to any one of items 104 to 109 above, which is formulated for systemic administration. [Section 111] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 103 above, wherein the virus expresses an immunomodulatory protein presented on the surface of the carrier cell. [Section 112] The cell-assisted viral expression system (CAVES) described in item 111 above, wherein the aforementioned virus is a vaccinia virus. [Section 113] A cell-assisted viral expression system (CAVES) as described in item 111 or 112 above, wherein the immunomodulatory viral protein is selected from VCP(C3L), B5R, HA(A56R), B18R / B19R, and B8R. [Section 114] A cell-assisted viral expression system (CAVES) as described in any of items 1 to 110 above, wherein the oncolytic virus is selected from oncolytic vaccinia virus, herpesvirus, adeno-associated virus, reovirus, varicella stomatitis virus (VSV), coxsackievirus, Semryki forest virus, Seneca Valley virus, Newcastle disease virus, Sendai virus, dengue virus, picornavirus, poliovirus, parvovirus, lentivirus, alphavirus, flavivirus, rhabdovirus, papillomavirus, influenza virus, mumps virus, gibbon leukemia virus, maraba virus, and Sindbis virus. [Section 115] A cell-assisted viral expression system (CAVES) according to any of items 1 to 114 above, wherein the aforementioned virus encodes a therapeutic product. [Section 116] The cell-assisted viral expression system (CAVES) according to item 115, wherein the therapeutic product is a polypeptide or nucleic acid. [Section 117] The cell-assisted viral expression system (CAVES) described in item 116 above, wherein the therapeutic product is a polypeptide. [Section 118] The cell-assisted viral expression system (CAVES) described in item 116 above, wherein the therapeutic product is a nucleic acid that is double-stranded RNA. [Section 119] The cell-assisted viral expression system (CAVES) described in item 116 above, wherein the cells are T cells. [Section 120] The cell-assisted viral expression system (CAVES) described in item 116 above, wherein the therapeutic product is an anticancer drug. [Section 121] A cell-assisted viral expression system (CAVES) according to any one of items 115 to 120 above, wherein the therapeutic product is an antibody or an antigen-binding fragment thereof. [Section 122] The cell-assisted viral expression system (CAVES) described in item 121 above, wherein the therapeutic product is an anti-CTLA4, anti-PD-1, or anti-PD-L1 antibody or its antigen-binding fragment. [Section 123] A cell-assisted viral expression system (CAVES) according to any one of items 115 to 121 above, wherein the therapeutic product is an immune checkpoint inhibitor or a product that modulates an immune pathway. [Section 124] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 123 above, contained in a cryopreserved composition. [Section 125] The cell-assisted viral expression system (CAVES) according to item 124, wherein the cryopreservation composition comprises one or more glycerols, DMSOs, or other cryopreserving agents. [Section 126] A pharmaceutical composition comprising a cell-assisted viral expression system (CAVES) or a combination thereof, as described in any of items 1 to 103 and 111 to 125 above. [Section 127] A pharmaceutical composition as described in item 126 above, formulated for parenteral administration. [Section 128] A pharmaceutical composition as described in item 126 above, formulated for local or systemic administration. [Section 129] A pharmaceutical composition according to any one of items 126 to 128 above, formulated for oral, intravenous, intratumor, rectal, subcutaneous, or mucosal administration. [Section 130] A method for treating cancer, including solid tumors or hematological malignancies, in a subject, comprising administering a cell-assisted viral expression system (CAVES) as described in any of items 1 to 125 above, or administering a pharmaceutical composition as described in any of items 126 to 129 above. [Section 131] A method for treating cancer, including solid tumors or hematological malignancies, in a subject, a) Infecting cells with oncolytic viruses with an infection multiple (MOI) of 10 or less, b) Incubating or co-culturing the cells with the oncolytic virus for a sufficient amount of time for the virus to produce a cell-assisted viral expression system (CAVES) by expressing therapeutic or immunomodulatory proteins encoded in the viral genome, c) A method comprising administering the cell-assisted viral expression system (CAVES) to a subject in order to treat the solid tumor or hematological malignancy. [Section 132] The method according to item 131, wherein the MOI is 0.001 to 10, or 0.1 to 10, or 0.1, or less than 0.3, or less than 0.1, or less than 0.5. [Section 133] The method according to any one of items 130 to 132, wherein the carrier cells and virus are incubated or co-cultured for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 40 hours or longer. [Section 134] The method according to any one of items 130 to 133, wherein the cells and virus are incubated or co-cultured for at least 6 hours. [Section 135] The method according to any one of items 130 to 134, wherein the cells and viruses are incubated or co-cultured for at least or up to 1, 2, 3, 4, 5, 6, or 7 days. [Section 136] The method according to any one of items 130 to 135 above, wherein the cells and virus are incubated or co-cultured, and the resulting cell-assisted viral expression system (CAVES) is stored. [Section 137] The method according to item 136, wherein storage is carried out at a temperature of -5°C to -200°C, or in a refrigerator, or in liquid nitrogen or CO2. [Section 138] The method according to item 136 or 137, wherein the cell-assisted viral expression system (CAVES) is cryopreserved. [Section 139] The method according to any one of items 136 to 138 above, wherein the cell-assisted viral expression system (CAVES) is stored for at least 24 hours. [Section 140] The method according to any one of items 136 to 139 above, wherein the cell-assisted viral expression system (CAVES) is stored for a maximum of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or for a maximum of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years or longer. [Section 141] The method according to any one of items 131 to 140, wherein the cell-assisted viral expression system (CAVES) is stored after incubation or co-culture (step b) and before administration (step c). [Section 142] The method according to any one of items 130 to 141, wherein the cell-assisted viral expression system (CAVES) is refrigerated, frozen, or stored at a temperature of approximately or just -5°C to -200°C. [Section 143] The method according to item 142, wherein the cell-assisted viral expression system (CAVES) is stored at a temperature of approximately -80°C to -200°C, or in liquid nitrogen or CO2. [Section 144] The method according to any one of items 130 to 143 above, wherein the carrier cells are not immune cells and / or tumor cells. [Section 145] The method according to any one of items 130 to 143 above, wherein the carrier cell is an immune cell, or a tumor cell, or a tumor cell line, or a stem cell. [Section 146] The method according to any one of items 130 to 145 above, wherein the carrier cell is a stem cell. [Section 147] The carrier cells are treated or modified, or both treated and modified, to enhance the immunosuppressive or immunoprivileged properties of the cells for administration to human subjects, and / or The method according to any one of items 130 to 146, wherein the carrier cells are treated and / or modified to enhance the amplification of the virus in the cells. [Section 148] The method according to any one of items 130 to 147 above, wherein the carrier cells are selected from treated or modified carrier cells selected from stem cells, immune cells, and tumor cells. [Section 149] The method according to any one of items 130 to 147 above, wherein the carrier cell is an embryonic epithelial cell or a fibroblast. [Section 150] The method according to any one of the above items 130 to 147, wherein the carrier cell is an immune cell. [Section 151] The method according to any one of items 130 to 147 above, wherein the carrier cell is selected from granulocytes, mast cells, monocytes, dendritic cells, natural killer cells, lymphocytes, T cell receptor (TCR) transgenic cells that target tumor-specific antigens, and CAR-T cells that target tumor-specific antigens. [Section 152] The method according to any one of items 130 to 147, wherein the carrier cells are modified or treated cells from a hematological malignancy cell line, and the cells are treated or modified, or both treated and modified, to enhance the immunosuppressive or immunoprivileged properties of the cells for administration to a human subject and / or to enhance the amplification of the virus in the cells. [Section 153] The method according to any one of items 130 to 152 above, wherein the carrier cell is a cell line selected from human leukemia, T-cell leukemia, myelomonocytic leukemia, lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), erythroleukemia, myelomonoblastic leukemia, malignant non-Hodgkin NK lymphoma, myeloma / plasmacytoma, multiple myeloma, and macrophage cell lines. [Section 154] The method according to any one of items 130 to 152 above, wherein the carrier cell is a stem cell. [Section 155] The method according to items 130 to 154 above, wherein the stem cells are selected from adult stem cells, embryonic stem cells, fetal stem cells, neural stem cells, mesenchymal stem cells, totipotent stem cells, pluripotent stem cells, induced pluripotent stem cells, compound pluripotent stem cells, oligopluripotent stem cells, unipotent stem cells, adipocyte stem cells, endothelial stem cells (e.g., endothelial progenitor cells, placental endothelial progenitor cells, angiogenic endothelial cells, pericytes), adult peripheral blood stem cells, myoblasts, small immature stem cells, cutaneous fibroblast stem cells, tissue / tumor-associated fibroblasts, epithelial stem cells, and embryonic epithelial stem cells. [Section 156] The method according to item 155 above, wherein stem cells are selected from mesenchymal cells. [Section 157] The method according to item 156, wherein the mesenchymal cells are isolated from / derived from adult bone marrow, adipose tissue, blood, dental pulp, neonatal umbilical cord, umbilical cord blood, placental mesenchymal cells, placental-derived adherent stromal cells, placental-derived decidual stromal cells, endometrial regenerative cells, placental dipotent endothelial / mesenchymal progenitor cells, amniotic membrane or amniotic fluid mesenchymal stem cells, amniotic fluid-derived progenitor cells, Howartan jelly mesenchymal stem cells, pelvic girdle stem cells, chorionic villous mesenchymal stromal cells, subcutaneous white adipose mesenchymal stem cells, pericytes, perisinus reticular cells, hair follicle-derived stem cells, hematopoietic stem cells, periosteum-derived mesenchymal stem cells, lateral plate mesenchymal stem cells, deciduous tooth stem cells, periodontal ligament stem cells, dental follicle progenitor cells, dental papilla-derived stem cells, muscle satellite cells, and other such cells. [Section 158] The method according to any one of items 130 to 157 above, wherein the carrier cell is selected from endothelial progenitor cells, neural stem cells, adult bone marrow cells, and mesenchymal stem cells. [Section 159] The method according to any one of items 130 to 158 above, wherein the cells are mesenchymal stem cells isolated from or derived from adult bone marrow, adipose tissue, blood, dental pulp, neonatal umbilical cord, umbilical cord blood, placental mesenchymal cells, placental-derived adherent stromal cells, placental-derived decidual stromal cells, endometrial regenerative cells, placental dipotent endothelial / mesenchymal progenitor cells, amniotic membrane or amniotic fluid mesenchymal stem cells, amniotic fluid-derived progenitor cells, Howartan jelly mesenchymal stem cells, pelvic girdle stem cells, chorionic villous mesenchymal stromal cells, subcutaneous white adipose mesenchymal stem cells, pericytes, perisinus reticular cells, hair follicle-derived stem cells, hematopoietic stem cells, periosteum-derived mesenchymal stem cells, lateral plate mesenchymal stem cells, deciduous tooth stem cells, periodontal ligament stem cells, dental follicle progenitor cells, dental papilla-derived stem cells, and muscle satellite cells. [Section 160] The method according to item 159 above, wherein mesenchymal stem cells are isolated from adipocytes. [Section 161] The method according to any one of items 130 to 160 above, wherein the carrier cells include adipocytes. [Section 162] The method according to any one of items 130 to 161 above, wherein the cells are MSCs isolated from umbilical cord blood, peripheral blood, muscle, cartilage, or amniotic fluid, or a mixture thereof. [Section 163] The method according to any one of items 130 to 162 above, wherein the carrier cells are derived from stromal vascular cells (SVF) of adipocytes. [Section 164] The method according to any one of items 130 to 163 above, wherein the cells are stem cells derived from the superepithelial adipose-derived stromal cells (CD34+SA-ASC) produced by culturing superepithelial adipose-derived stromal cells. [Section 165] The method according to item 164, wherein the mesenchymal cells are derived from adipocytes. [Section 166] The method according to any one of the above items 130 to 164, wherein the carrier cells are adipocytes selected from superembryonic adipocytes (SA-ASCs; CD235a- / CD45- / CD34+ / CD146- / CD31-) and pericytes (CD235a- / CD45- / CD34- / CD146+ / CD31-). [Section 167] The method according to any one of items 130 to 166 above, wherein the carrier cells are adipose-cultured adipose-mesenchymal stem cells (AD-MSCs) derived from superembryonic adipose-derived stromal cells (CD34+SA-ASCs). [Section 168] The method according to any one of items 130 to 167 above, wherein the cells are allogeneic to the subject being treated. [Section 169] The method according to any one of items 130 to 167 above, wherein the cells are autologous to the subject being treated. [Section 170] The method according to any one of items 130 to 169 above, wherein the virus is selected from poxvirus, herpes simplex virus, adeno-associated virus, reovirus, vesicular stomatitis virus (VSV), coxsackievirus, Semryki forest virus, Seneca Valley virus, Newcastle disease virus, Sendai virus, dengue virus, picornavirus, poliovirus, parvovirus, retrovirus, alphavirus, marabavirus, flavivirus, rhabdovirus, papillomavirus, influenza virus, mumps virus, gibbon leukemia virus, and Sindbisvirus. [Section 171] The method according to item 170 above, wherein the virus is the measles virus. [Section 172] The method according to item 170 above, wherein the virus is a retrovirus that is a lentivirus. [Section 173] The method according to item 170 above, wherein the virus is a poxvirus that is a vaccinia virus. [Section 174] The method according to item 170, wherein the virus is a poxvirus selected from strains of Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, Modified Vaccinia Ankara (MVA), New York City Board of Health, Dairen, Ikeda, LC16M8, Tashkent, Wyeth, IHD-J, IHD-W, Brighton, Dairen I, and Connaught. [Section 175] The method according to item 174 above, wherein the virus is ACAM1000 or ACAM2000. [Section 176] The aforementioned virus is a vaccinia virus. The carrier cells are stem cells derived from adipocytes. The method described in any of the above items 130 to 175. [Section 177] The method according to any one of items 130 to 176, wherein the CAVES expresses an immunomodulatory protein encoded by the virus. [Section 178] The method according to any one of items 130 to 177 above, wherein the cancer includes a solid tumor. [Section 179] The method according to any one of items 130 to 177 above, wherein the cancer is a hematological malignancy. [Section 180] The method according to any one of paragraphs 130 to 179 above, wherein the subject has cancer including solid tumors or hematological malignancies selected from lung cancer, pancreatic cancer, breast cancer, colon cancer, head and neck cancer, liver cancer, melanoma, leukemia, lymphoma, and kidney cancer. [Section 181] The solid tumors or hematological malignancies include bladder tumors, breast tumors, prostate tumors, carcinomas, basal cell carcinomas, biliary tract cancers, bladder cancers, bone cancers, brain cancers, central nervous system (CNS) cancers, gliomas, cervical cancers, choroidal cancers, colon and rectal cancers, connective tissue cancers, digestive system cancers, endometrial cancers, esophageal cancers, eye cancers, head and neck cancers, stomach cancers, carcinomas in situ, kidney cancers, laryngeal cancers, leukemias, liver cancers, lung cancers, lymphomas, and Hodgkin's cancers. Parkinson's cancer, non-Hodgkin's lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, kidney cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, urinary tract cancer, lymphosarcoma, osteosarcoma, mammary gland tumor, mast cell tumor, brain tumor, adenosquamous carcinoma, carcinoid lung tumor, bronchial gland tumor, bronchial gland cancer, small cell lung cancer, non-small cell lung cancer, fibroma, mucosal carcinoma Hypochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma, Wilms' tumor, Burkitt lymphoma, microglia, osteoclastoma, oral neoplasm, fibrosarcoma, genital squamous cell carcinoma, sexually transmitted disease tumors, testicular tumors, seminoma, Sertoli cell tumor, hemangiocytoma, histiocytoma, chloroplastoma, granulocytic sarcoma, corneal papilloma, corneal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, gastric tumor, adrenal cancer, oral The method according to item 180 above, selected from papillomatosis, hemangioendothelioma, cystadenoma, follicular lymphoma, enteric lymphosarcoma, pulmonary squamous cell carcinoma, leukemia, hemangioepoidoma, ocular neoplasm, prepistagosomal fibrosarcoma, ulcerative squamous cell carcinoma, prepistagosomal carcinoma, connective tissue neoplasm, hepatocellular carcinoma, pulmonary adenomatosis, pulmonary sarcoma, Rous sarcoma, reticuloendotheliopathy, nephroblastoma, B-cell lymphoma, lymphocytic leukemia, retinoblastoma, hepatic neoplasm, lymphosarcoma, plasmacytoid leukemia, swim bladder sarcoma (fish), caseous lymphadenitis, lung cancer, insulinoma, neuroma, pancreatic islet cell tumor, gastric MALT lymphoma, and gastric adenocarcinoma. [Section 182] The method according to any one of items 130 to 181 above, wherein the oncolytic virus encodes a heterogeneous gene product. [Section 183] The method according to item 182 above, wherein the heterogeneous gene product is an anticancer drug. [Section 184] The method according to item 183, wherein the product is an antibody or its antigen-binding portion. [Section 185] The method according to any of items 130 to 184 above, wherein the subject is a human. [Section 186] The method according to any one of items 130 to 184 above, wherein the subject is a non-human animal. [Section 187] The method according to any of items 130 to 184 above, wherein the subject is an animal in a farm or zoo. [Section 188] The method according to any one of items 130 to 184 above, wherein the subject is a dog or a cat. [Section 189] The method according to item 187 above, wherein the subject is a cattle, horse, goat, mule, zebra, giraffe, gorilla, bonobo, donkey, pig, llama, chimpanzee, or alpaca. [Section 190] The method described in any of items 130 to 185 above, wherein the subject is a pediatric human. [Section 191] The method according to any one of items 130 to 190 above, wherein the cell-assisted viral expression system (CAVES) is administered by intratumor, intravenous, intraperitoneal, intrathecal, intraventricular, intraarticular, or intraocular injection. [Section 192] The method according to any one of items 130 to 190 above, wherein the cell-assisted viral expression system (CAVES) is administered by intramuscular or subcutaneous administration. [Section 193] The method described in any of paragraphs 130 to 192 above, including administering another anti-cancer treatment or therapy. [Section 194] The method according to paragraph 193, wherein the other cancer treatment or therapy is immunotherapy. [Section 195] The method according to item 194, wherein the immunotherapy comprises administering a checkpoint inhibitor or a procedure that inhibits a checkpoint or immunosuppressive pathway. [Section 196] The method according to any one of items 130 to 195, comprising administering a treatment to activate the T cell response within the subject, wherein the treatment comprises administering a blocking antibody against a negative costimulatory molecule or an agonist antibody against an activating costimulatory molecule. [Section 197] The method according to item 196, wherein the procedure comprises administering a blocking antibody against a negative costimulatory molecule. [Section 198] The method according to item 196 or 197, wherein the blocking antibody is against a negative costimulatory molecule selected from CTLA-1, CTLA-4, PD-1, TIM-3, BTLA, VISTA, PD-L1, and LAG-3. [Section 199] The method according to any one of the above paragraphs 196 to 198, wherein the treatment includes the administration of an inhibitor of the PD-1 pathway. [Section 200] The method according to item 199, wherein the inhibitor of the PD-1 pathway is selected from antibodies against PD-1 and soluble PD-1 ligands. [Section 201] The method according to item 199, wherein the inhibitor of the PD-1 pathway is an antibody selected from AMP-244, MEDI-4736, MPDL328 OA, and MIH1. [Section 202] The method according to any one of items 196 to 198, wherein the treatment comprises administering an anti-CTLA-4 antibody, an anti-PD-L1 antibody, or an anti-PD-1 antibody. [Section 203] The method according to any one of items 196 to 198, wherein the treatment comprises administering a blocking antibody against a negative costimulatory molecule selected from PD-L1 and CTLA-4. [Section 204] The method according to any one of items 130 to 203 above, comprising a procedure including administering an agonist antibody against a costimulatory molecule. [Section 205] The method according to item 204, wherein the co-stimulatory molecule is selected from CD28, OX40, CD40, GITR, CD137, CD27, and HVEM. [Section 206] A cell-assisted viral expression system (CAVES) according to any of items 1 to 125 above, or a pharmaceutical composition according to any of items 126 to 129 above, for use in treating cancer including solid tumors or hematological malignancies. [Section 207] Use of a cell-assisted viral expression system (CAVES) as described in any of items 1 to 125 above, or a pharmaceutical composition as described in any of items 126 to 129 above, for use in treating cancer, including solid tumors or hematological malignancies. [Section 208] The use of a cell-assisted viral expression system (CAVES) for the use described in item 206 or item 207 above, wherein the cell-assisted viral expression system is a) Infecting cells with oncolytic viruses with an infection multiple (MOI) of 10 or less; and b) Use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) produced by incubating or co-culturing the oncolytic virus with the oncolytic virus under conditions that allow the cells to grow and / or replicate for a sufficient amount of time for the oncolytic virus to express therapeutic or immunomodulatory proteins encoded in the genome of the virus and produce the CAVES. [Section 209] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in item 208 above, wherein the MOI is 0.001 to 10, 0.1 to 10, 0.1 to 1, 0.1 to less than 1, 0.1 to 0.5, or 0.1. [Section 210] Use of a cell-assisted viral expression system (CAVES) or cell-assisted viral expression system (CAVES) for use as described in any of items 206 to 209 above, which is produced by incubating or co-culturing the carrier cells and virus for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 40 hours or longer. [Section 211] Use of a cell-assisted viral expression system (CAVES) or cell-assisted viral expression system (CAVES) for use according to any one of items 206 to 210 above, wherein the cells and virus are incubated or co-cultured for at least 6 hours. [Section 212] Use of a cell-assisted viral expression system (CAVES) or cell-assisted viral expression system (CAVES) for use according to any of items 206 to 211 above, wherein the cells and viruses are incubated or co-cultured for at least or up to 1, 2, 3, 4, 5, 6, or 7 days. [Section 213] The cell-assisted viral expression system (CAVES) is stored for use as described in any of paragraphs 206 to 212 above, or the use of the cell-assisted viral expression system (CAVES). [Section 214] The use of a cell-assisted viral expression system (CAVES) or cell-assisted viral expression system (CAVES) for use as described in item 213 above, wherein the storage is carried out at a temperature of -5°C to -200°C, or in a refrigerator, or in liquid nitrogen or CO2. [Section 215] The cell-assisted viral expression system (CAVES) is cryopreserved, or the use of the cell-assisted viral expression system (CAVES) as described in paragraph 213 or 214 above. [Section 216] The use of the cell-assisted viral expression system (CAVES) or the cell-assisted viral expression system (CAVES) as described in any of items 213 to 215 above, wherein the cell-assisted viral expression system (CAVES) is stored for at least 24 hours. [Section 217] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in any of paragraphs 213 to 216 above, wherein the cell-assisted viral expression system (CAVES) is stored for a maximum of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or for a maximum of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years or longer. [Section 218] The cell-assisted viral expression system (CAVES) obtained is stored after incubation of the carrier cells with the oncolytic virus, or the use of a cell-assisted viral expression system (CAVES) as described in any of items 213 to 217 above. [Section 219] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of sections 206 to 218 above, wherein the cells are stored, refrigerated, or frozen at a temperature of approximately or just -5°C to -200°C. [Section 220] The use of a cell-assisted viral expression system (CAVES) or cell-assisted viral expression system (CAVES) for the use described in item 219 above, wherein the cells are stored at a temperature of approximately -80°C to -200°C, or in liquid nitrogen or CO2. [Section 221] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in any of paragraphs 206 to 220 above, wherein the carrier cells are not immune cells and / or tumor cells. [Section 222] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use according to any one of the above paragraphs 206 to 220, wherein the carrier cell is an immune cell, or a tumor cell, or a tumor cell line, or a stem cell. [Section 223] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use according to any one of the above paragraphs 206 to 222, wherein the carrier cell is a stem cell. [Section 224] The carrier cells are treated or modified, or both treated and modified, to enhance the immunosuppressive or immunoprivileged properties of the cells for administration to human subjects, and / or The carrier cells are treated and / or modified to enhance the amplification of the virus within the cells. Use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in any of the above sections 206 to 223. [Section 225] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use according to any of the above paragraphs 206 to 224, wherein the carrier cells are selected from treated or modified cells selected from stem cells, immune cells, and tumor cells. [Section 226] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use according to any one of the above items 206 to 224, wherein the carrier cell is an embryonic epithelial cell or a fibroblast. [Section 227] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use according to any of the above paragraphs 206 to 224, wherein the carrier cell is an immune cell. [Section 228] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of items 206 to 224 above, wherein the carrier cells are selected from granulocytes, mast cells, monocytes, dendritic cells, natural killer cells, lymphocytes, T cell receptor (TCR) transgenic cells that target tumor-specific antigens, and CAR-T cells that target tumor-specific antigens. [Section 229] Use of a cell-assisted viral expression system (CAVES) or a modified or treated cell derived from a hematological malignancy cell line for use according to any of paragraphs 206 to 224, wherein the carrier cells are treated or modified, or both treated and modified, to enhance the immunosuppressive or immunoprivileged properties of the cells for administration to a human subject and / or to enhance the amplification of the virus in the cells. [Section 230] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of items 206 to 229 above, wherein the carrier cell is a cell line selected from human leukemia, T-cell leukemia, myelomonocytic leukemia, lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), erythroleukemia, myelomonoblastic leukemia, malignant non-Hodgkin NK lymphoma, myeloma / plasmacytoma, multiple myeloma, and macrophage cell lines. [Section 231] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of items 206 to 229 above, wherein the carrier cell is a stem cell. [Section 232] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of paragraphs 206 to 231 above, wherein the carrier cell is a stem cell selected from adult stem cells, embryonic stem cells, fetal stem cells, neural stem cells, mesenchymal stem cells, totipotent stem cells, pluripotent stem cells, induced pluripotent stem cells, compound pluripotent stem cells, minimal pluripotent stem cells, unipotent stem cells, adipocytes, endothelial stem cells (e.g., endothelial progenitor cells, placental endothelial progenitor cells, angiogenic endothelial cells, pericytes), adult peripheral blood stem cells, myoblasts, small immature stem cells, dermal fibroblast stem cells, tissue / tumor-associated fibroblasts, epithelial stem cells, and embryonic epithelial stem cells. [Section 233] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in item 232 above, wherein the stem cells are selected from mesenchymal cells. [Section 234] The mesenchymal cells are isolated from / derived from adult bone marrow, adipose tissue, blood, dental pulp, neonatal umbilical cord, umbilical cord blood, placental mesenchymal cells, placental-derived adherent stromal cells, placental-derived decidual stromal cells, endometrial regenerative cells, placental dipotent endothelial / mesenchymal progenitor cells, amniotic or amniotic fluid mesenchymal stem cells, amniotic fluid-derived progenitor cells, Howarton's jelly mesenchymal stem cells, pelvic girdle stem cells, chorionic villous mesenchymal stromal cells, subcutaneous white adipose mesenchymal stem cells, pericytes, perisinus reticular cells, hair follicle-derived stem cells, hematopoietic stem cells, periosteum-derived mesenchymal stem cells, lateral plate mesenchymal stem cells, deciduous tooth stem cells, periodontal ligament stem cells, dental follicle progenitor cells, dental papilla-derived stem cells, muscle satellite cells, and other such cells, for use as described in Section 233 above. [Section 235] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of items 206 to 234 above, wherein the carrier cells are selected from endothelial progenitor cells, neural stem cells, adult bone marrow cells, and mesenchymal stem cells. [Section 236] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use according to any of the above sections 206 to 235, wherein the cells are adult bone marrow, adipose tissue, blood, dental pulp, neonatal umbilical cord, umbilical cord blood, placental mesenchymal cells, placental-derived adherent stromal cells, placental-derived decidual stromal cells, endometrial regenerative cells, placental dipotent endothelial / mesenchymal progenitor cells, amniotic membrane or amniotic fluid mesenchymal stem cells, amniotic fluid-derived progenitor cells, Howarton's jelly mesenchymal stem cells, pelvic girdle stem cells, chorionic villous mesenchymal stromal cells, subcutaneous white adipose mesenchymal stem cells, pericytes, perisinus reticular cells, hair follicle-derived stem cells, hematopoietic stem cells, periosteum-derived mesenchymal stem cells, lateral plate mesenchymal stem cells, deciduous tooth stem cells, periodontal ligament stem cells, dental follicle progenitor cells, dental papilla-derived stem cells, muscle satellite cells, and other mesenchymal stem cells isolated from or derived from such cells. [Section 237] The use of a cell-assisted viral expression system (CAVES) for the use described in item 236 above, wherein the mesenchymal stem cells are isolated from adipocytes. [Section 238] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use according to any one of the above items 206 to 237, wherein the carrier cells include adipocytes. [Section 239] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in any of sections 206 to 238 above, wherein the cells are MSCs isolated from umbilical cord blood, peripheral blood, muscle, cartilage, or amniotic fluid, or a mixture thereof. [Section 240] The use of a cell-assisted viral expression system (CAVES) for use according to any of the above items 206 to 239, wherein the carrier cells are derived from stromal vascular cells (SVF) of adipocytes. [Section 241] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use according to any of the above items 206 to 240, wherein the cells are stem cells derived from the above-mentioned [Section 242] The use of a cell-assisted viral expression system (CAVES) for the use described in item 241 above, wherein the mesenchymal stem cells are derived from adipocytes. [Section 243] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of items 206 to 240 above, wherein the carrier cells are adipocytes selected from superembryonic adipocytes (SA-ASCs; CD235a- / CD45- / CD34+ / CD146- / CD31-) and pericytes (CD235a- / CD45- / CD34- / CD146+ / CD31-). [Section 244] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of items 206 to 243 above, wherein the carrier cells are adipose-cultured adipose-mesenchymal stem cells (AD-MSCs) derived from superembryonic adipose-derived adipose-mediated stromal cells (CD34+SA-ASCs). [Section 245] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of paragraphs 206 to 244 above, wherein the cells are allogeneic with respect to the subject being treated. [Section 246] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of paragraphs 206 to 244 above, wherein the cells are autologous to the subject being treated. [Section 247] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in any of sections 206 to 246 above, wherein the virus is selected from poxvirus, herpes simplex virus, adeno-associated virus, reovirus, varicella stomatitis virus (VSV), coxsackievirus, Semryki forest virus, Seneca Valley virus, Newcastle disease virus, Sendai virus, dengue virus, picornavirus, poliovirus, parvovirus, retrovirus, alphavirus, flavivirus, rhabdovirus, papillomavirus, influenza virus, mumps virus, gibbon leukemia virus, and Sindbisvirus. [Section 248] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in item 247 above, wherein the aforementioned virus is the measles virus. [Section 249] The use of a cell-assisted viral expression system (CAVES) or cell-assisted viral expression system (CAVES) for the use described in item 247 above, wherein the virus is a lentivirus retrovirus. [Section 250] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in item 247 above, wherein the virus is a vaccinia virus poxvirus. [Section 251] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in item 247 above, wherein the virus is a poxvirus selected from the strains of Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Western Reserve, Modified Vaccinia Ankara (MVA), New York City Board of Health, Dairen, Ikeda, LC16M8, Copenhagen, Tashkent, Wyeth, IHD-J, IHD-W, Brighton, Dairen I, and Connaught. [Section 252] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in item 251 above, wherein the virus is ACAM1000 or ACAM2000. [Section 253] The cell-assisted viral expression system (CAVES) according to item 252 above, wherein the genome of the aforementioned virus comprises the nucleotide sequence shown in SEQ ID NO: 70 or 71. [Section 254] The aforementioned virus is a vaccinia virus, and The carrier cells are stem cells derived from adipocytes. Use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in any of the above sections 206 to 252. [Section 255] The cell-assisted viral expression system (CAVES) according to item 254, wherein the carrier cells and virus are incubated for at least 6 hours, and the incubation is performed under conditions in which the virus infects the cells and expresses the encoded protein. [Section 256] A cell-assisted viral expression system (CAVES) as described in item 255 above, wherein the incubation is 10, 12, 14, 16, or 20 hours or less. [Section 257] The use of a cell-assisted viral expression system (CAVES) or cell-assisted viral expression system (CAVES) for the use described in any of the above sections 206 to 254, wherein the CAVES expresses an immunomodulatory protein encoded by the virus. [Section 258] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use according to any of the above paragraphs 206 to 257, wherein the cancer includes a solid tumor. [Section 259] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of paragraphs 206 to 258 above, wherein the cancer is a hematological malignancy. [Section 260] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of paragraphs 206 to 259 above, wherein the subject being treated has cancer including solid tumors or hematological malignancies selected from lung cancer, pancreatic cancer, breast cancer, colon cancer, head and neck cancer, liver cancer, melanoma, leukemia, lymphoma, and kidney cancer. [Section 261] The solid tumors or hematological malignancies include bladder tumors, breast tumors, prostate tumors, carcinomas, basal cell carcinomas, biliary tract cancers, bladder cancers, bone cancers, brain cancers, central nervous system (CNS) cancers, gliomas, cervical cancers, choroidal cancers, colon and rectal cancers, connective tissue cancers, digestive system cancers, endometrial cancers, esophageal cancers, eye cancers, head and neck cancers, stomach cancers, carcinomas in situ, kidney cancers, laryngeal cancers, leukemias, liver cancers, lung cancers, lymphomas, and Hodgkin's cancers. Parkinson's cancer, non-Hodgkin's lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, kidney cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, urinary tract cancer, lymphosarcoma, osteosarcoma, mammary gland tumor, mast cell tumor, brain tumor, adenosquamous carcinoma, carcinoid lung tumor, bronchial gland tumor, bronchial gland cancer, small cell lung cancer, non-small cell lung cancer, fibroma, mucosal carcinoma Hypochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma, Wilms' tumor, Burkitt lymphoma, microglia, osteoclastoma, oral neoplasm, fibrosarcoma, genital squamous cell carcinoma, sexually transmitted disease tumors, testicular tumors, seminoma, Sertoli cell tumor, hemangiocytoma, histiocytoma, chloroplastoma, granulocytic sarcoma, corneal papilloma, corneal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, gastric tumor, adrenal cancer, oral Use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in item 259 above, selected from among papillomatosis, hemangioendothelioma, cystadenoma, follicular lymphoma, enteric lymphosarcoma, pulmonary squamous cell carcinoma, leukemia, hemangioepidermoma, ocular neoplasm, prepistagosomal fibrosarcoma, ulcerative squamous cell carcinoma, prepistagosomal carcinoma, connective tissue neoplasm, hepatocellular carcinoma, pulmonary adenomatosis, pulmonary sarcoma, Rous sarcoma, reticuloendotheliopathy, nephroblastoma, B-cell lymphoma, lymphocytic leukemia, retinoblastoma, hepatic neoplasm, lymphosarcoma, plasmacytoid leukemia, swim bladder sarcoma (fish), caseous lymphadenitis, lung cancer, insulinoma, neuroma, islet cell tumor, gastric MALT lymphoma, and gastric adenocarcinoma. [Section 262] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in any of items 206 to 261 above, wherein the oncolytic virus encodes a heterologous gene product. [Section 263] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in item 262 above, wherein the heterogeneous gene product is an anticancer drug. [Section 264] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in item 262 or item 187 above, wherein the product is an antibody or its antigen-binding portion. [Section 265] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of paragraphs 193 to 264 above, wherein the subject being treated is a human. [Section 266] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of paragraphs 206 to 264 above, wherein the subject being treated is a non-human animal. [Section 267] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of paragraphs 206 to 264 above, wherein the subject being treated is an animal in a farm or zoo. [Section 268] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of paragraphs 206 to 264 above, wherein the subject being treated is a dog or a cat. [Section 269] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in paragraph 187 above, wherein the subject to be treated is a cattle, horse, goat, mule, zebra, giraffe, gorilla, bonobo, donkey, pig, llama, chimpanzee, or alpaca. [Section 270] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of paragraphs 206 to 265 above, wherein the subject being treated is a pediatric human. [Item 271] The use of the cell-assisted viral expression system (CAVES) or the cell-assisted viral expression system (CAVES) for use according to any one of Items 206 to 270 above, wherein the CAVES is for administration by intratumoral, intravenous, intraperitoneal, intramedullary, intraventricular, intra-articular, or intraocular injection. [Item 272] The use of the cell-assisted viral expression system (CAVES) or the cell-assisted viral expression system (CAVES) for use according to any one of Items 206 to 270 above, wherein the cell-assisted viral expression system (CAVES) is for intramuscular or subcutaneous administration. [Item 273] The use of the cell-assisted viral expression system (CAVES) or the cell-assisted viral expression system (CAVES) for use according to any one of Items 206 to 272 above, in combination with another different anti-cancer treatment or therapy. [Item 274] The use of the cell-assisted viral expression system (CAVES) or the cell-assisted viral expression system (CAVES) for use according to Item 209 above, wherein the other cancer treatment or therapy is immunotherapy. [Item 275] The use of the cell-assisted viral expression system (CAVES) or the cell-assisted viral expression system (CAVES) for use according to Item 274 above, wherein the immunotherapy includes a checkpoint inhibitor or a treatment that inhibits a checkpoint or immunosuppressive pathway. [Item 276] The use of the cell-assisted viral expression system (CAVES) or the cell-assisted viral expression system (CAVES) for use according to any one of Items 206 to 275 above, including a treatment that activates a T cell response within the subject, wherein the treatment includes a blocking antibody against a negative costimulatory molecule or an agonist antibody against an activating costimulatory molecule. [Item 277] The use of the cell-assisted viral expression system (CAVES) or the cell-assisted viral expression system (CAVES) for use according to Item 276 above, wherein the treatment includes a blocking antibody against a negative costimulatory molecule. [Item 278] The use of a cell-assisted viral expression system (CAVES) or cell-assisted viral expression system (CAVES) for the use described in item 276 or 213 above, wherein the blocking antibody is against a negative costimulatory molecule selected from CTLA-1, CTLA-4, PD-1, TIM-3, BTLA, VISTA, PD-L1, and LAG-3. [Section 279] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of items 276 to 278 above, wherein the treatment includes an inhibitor of the PD-1 pathway. [Section 280] The use of a cell-assisted viral expression system (CAVES) or cell-assisted viral expression system (CAVES) for the use described in item 279 above, wherein the inhibitor of the PD-1 pathway is selected from antibodies against PD-1 and soluble PD-1 ligands. [Section 281] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in item 279 above, wherein the inhibitor of the PD-1 pathway is an antibody selected from AMP-244, MEDI-4736, MPDL328 OA, and MIH1. [Section 282] The treatment comprises an anti-CTLA-4 antibody, an anti-PD-L1 antibody, or an anti-PD-1 antibody, and the use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) as described in any of sections 276 to 278 above. [Section 283] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use according to any one of the above sections 276 to 278, wherein the treatment comprises blocking an antibody against a negative costimulatory molecule selected from PD-L1 and CTLA-4. [Section 284] Use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for use as described in any of sections 206 to 283 above, including a treatment comprising a costimulatory molecule, an agonist antibody against CAR-T and / or TIL. [Section 285] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in item 284 above, wherein the aforementioned co-stimulatory molecule is selected from CD28, OX40, GITR, CD40, CD137, CD27, and HVEM. [Section 286] A vaccinia virus containing a genome having the nucleotide sequence shown in SEQ ID NO: 71. [Section 287] A modified ACAM2000 virus comprising at least one therapeutic gene or marker gene inserted into the intergenetic region between the corresponding unmodified ACAM2000 virus open reading frame 157 (ORF_157) and open reading frame 158 (ORF_158). [Section 288] A modified ACAM2000 virus containing a partially or completely deleted or interrupted F1L locus of the corresponding unmodified ACAM2000 virus, wherein the F1L gene is not expressed. [Section 289] A modified ACAM2000 virus containing a partially or completely deleted or interrupted B8R locus of the corresponding unmodified ACAM2000 virus, wherein the B8R gene is not expressed. [Section 290] Modified ACAM2000 virus including one or more of the following modifications: (a) At least one therapeutic gene or marker gene inserted into the intergenetic region between open reading frame 157 (ORF_157) and open reading frame 158 (ORF_158) of the corresponding unmodified ACAM2000 virus; (b) an F1L locus in which the corresponding unmodified ACAM2000 virus is partially or completely deleted, and in which the F1L gene is not expressed; and / or (c) A B8R locus in which the corresponding unmodified ACAM2000 virus is partially or completely deleted, and in which the B8R gene is not expressed. [Section 291] A modified ACAM2000 virus that includes one or more of the following modifications in the corresponding unmodified ACAM2000 virus: At least one therapeutic gene or marker gene inserted into the intergenetic region between open reading frame 174 (ORF_174) and open reading frame 175 (ORF_175); and / or At least one truncated open reading frame (ORF) at one or more of the following loci: ORF72, ORF73, ORF156, ORF157, ORF158, ORF159, ORF160, ORF174, and ORF175. [Section 292] The modified ACAM2000 virus according to paragraph 291, wherein the modification includes at least one therapeutic gene or marker gene inserted into the intergenetic region between the open reading frame 174 (ORF_174) and the open reading frame 175 (ORF_175) of the corresponding unmodified ACAM2000 virus. [Section 293] The therapeutic gene(s) mentioned above are selected from among immune checkpoint inhibitors, cytokines, growth factors, photosensitizers, radionuclides, toxins, antimetabolites, signaling modulators, anticancer antibodies, and angiogenesis inhibitors; The aforementioned marker gene(s) may be selected from among green fluorescent protein (GFP), high-sensitivity green fluorescent protein (eGFP), blue fluorescent protein (BFP), TurboFP635, and phosphoribosyltransferase (GPT). The modified ACAM2000 virus described in item 292 above. [Section 294] A modified ACAM2000 virus as described in item 291 above, comprising at least one cleaved open reading frame (ORF) at one or more of the following loci: ORF72, ORF73, ORF156, ORF157, ORF158, ORF159, ORF160, ORF174, and ORF175, and further comprising at least one therapeutic gene and / or marker gene inserted into the cleaved region of the one or more loci: ORF72, ORF73, ORF156, ORF157, ORF158, ORF159, ORF160, ORF174, and ORF175. [Section 295] The therapeutic gene(s) mentioned above are selected from among immune checkpoint inhibitors, cytokines, growth factors, photosensitizers, radionuclides, toxins, antimetabolites, signaling modulators, anticancer antibodies, and angiogenesis inhibitors; The aforementioned marker gene(s) may be selected from among green fluorescent protein (GFP), high-sensitivity green fluorescent protein (eGFP), blue fluorescent protein (BFP), TurboFP635, and phosphoribosyltransferase (GPT). The modified ACAM2000 virus described in item 294 above. [Section 296] The modified ACAM2000 virus according to any one of items 287 to 295 above, wherein the corresponding unmodified ACAM2000 includes the nucleic acid sequence shown in SEQ ID NO: 70 or SEQ ID NO: 71. [Section 297] The modified ACAM2000 virus according to paragraphs 287, 290, or 296, wherein the modification includes at least one therapeutic gene inserted into the intergenetic region between open reading frame 157 (ORF_157) and open reading frame 158 (ORF_158) of the corresponding unmodified ACAM2000 virus. [Section 298] The modified ACAM2000 virus as described in item 297 above, wherein the therapeutic gene(s) are selected from among immune checkpoint inhibitors, cytokines, growth factors, photosensitizers, radionuclides, toxins, antimetabolites, signaling modulators, anticancer antibodies, and angiogenesis inhibitors. [Item 299] The modified ACAM2000 virus according to any one of Items 287, 290, 291, 296 or 297, wherein the modification comprises at least one marker gene inserted into the intergenic region between open reading frame 157 (ORF_157) and open reading frame 158 (ORF_158) of the corresponding unmodified ACAM2000 virus. [Item 300] The modified ACAM2000 virus according to Item 299, wherein the marker gene(s) is selected from green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), blue fluorescent protein (BFP), TurboFP635 and phosphoribosyltransferase (gpt). [Item 301] Since the modification is a partially or completely deleted B8R locus of the corresponding unmodified ACAM2000 virus, the B8R gene is not expressed. The modified virus according to any one of Items 289, 290 or 296, comprising at least one therapeutic gene and / or marker gene inserted into the region of the partially or completely deleted B8R locus. The modified ACAM2000 virus according to any one of Items 289, 290 or 296. [Item 302] Since the modification is a partially or completely deleted F1L locus of the corresponding unmodified ACAM2000 virus, the F1L gene is not expressed. The modified virus according to any one of Items 288, 290 or 296, comprising at least one therapeutic gene and / or marker gene inserted into the region of the partially or completely deleted F1L locus. The modified ACAM2000 virus according to any one of Items 288, 290 or 296. [Item 303] The modified ACAM2000 virus according to Item 301 or 302, comprising at least one therapeutic gene. [Item 304] The modified ACAM2000 virus as described in item 303 above, wherein the therapeutic gene(s) are selected from among immune checkpoint inhibitors, cytokines, growth factors, photosensitizers, radionuclides, toxins, antimetabolites, signaling modulators, anticancer antibodies, and angiogenesis inhibitors. [Section 305] A modified ACAM2000 virus as described in any of sections 301 to 304 above, comprising at least one marker gene. [Section 306] The modified ACAM2000 virus described in item 305 above, wherein the marker gene(s)(or more)(s [Section 307] A modified ACAM2000 virus as described in any of sections 287 and 290-306 above, wherein at least one therapeutic gene is an anti-cancer antibody. [Section 308] The modified ACAM2000 virus as described in item 307 above, wherein the anti-cancer antibody is an anti-VEGF antibody or an anti-CTLA-4 antibody. [Section 309] The modified ACAM2000 virus as described in item 307 or 308 above, wherein the anti-cancer antibody is a single-chain antibody. [Section 310] A modified ACAM2000 virus according to any one of items 307 to 309, further comprising a nucleic acid encoding an IgK signal peptide for promoting the secretion of the aforementioned antibody. [Section 311] A modified ACAM2000 virus according to any one of items 307 to 309 above, further comprising a nucleic acid encoding a FLAG tag for facilitating the detection of the aforementioned antibody. [Section 312] A modified ACAM2000 virus as described in any of sections 287 and 290-311 above, wherein at least one gene encoding a detectable marker gene and / or therapeutic product is sodium-iodine cotransporter (NIS), OX40L, or 4-IBBL. [Section 313] ACAM2000 virus as described in item 1 above or a modified ACAM2000 virus as described in any of items 387 to 312 above, further comprising at least one inactivating gene selected from thymidine kinase (TK), hemagglutinin (HA), interferon alpha / beta blocker receptor, or immunomodulator. [Section 314] ACAM2000 virus as described in item 1 above, or a modified ACAM2000 virus as described in any of items 287 to 313 above, further modified to enhance EEV (extracellular enveloped virus) production. [Section 315] A further modification for enhancing EEV production is a mutation in the gene encoding the A34R protein, as described in item 314 above, for the ACAM2000 virus or modified ACAM2000 virus. [Section 316] The ACAM2000 virus or modified ACAM2000 virus described in item 315 above, wherein the mutation encodes the A34R protein containing the mutation K151E. [Section 317] A method for treating a solid tumor or hematological malignancy in a subject, comprising administering to the subject a composition comprising an oncolytic virus and adipocytes, wherein the adipocytes are selected from superembryonic adipocytes (SA-ASC; CD235a- / CD45- / CD34+ / CD146- / CD31-) and pericytes (CD235a- / CD45- / CD34- / CD146+ / CD31-). [Section 318] A method for treating a solid tumor or hematological malignancy in a subject, comprising administering to the subject a composition comprising an oncolytic virus and cultured adipocyte-mesenchymal stem cells (AD-MSCs) derived from superembryonic adipocytes (CD34+SA-ASCs). [Section 319] The method according to item 317 or 318, wherein the cells and oncolytic virus are co-cultured in vitro before administration to the subject. [Section 320] The method according to item 319, wherein the cells are co-cultured for a time sufficient to allow the oncolytic viral immunomodulatory protein to be expressed, or for a time sufficient to allow the virus to undergo at least one replication cycle. [Section 321] The method according to item 318, wherein the adipose mesenchymal stem cells (AD-MSCs) are derived from epiperitoneal adipose stromal cells (CD34+SA-ASCs) by culturing epiperitoneal adipose stromal cells to produce AD-MSCs. [Section 322] The method according to item 319 or 320, wherein the cells and viruses are co-cultured for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 40 hours or longer. [Section 323] The method according to any one of items 317 to 322 above, wherein the oncolytic virus is selected from poxvirus, adenovirus, herpes simplex virus, Newcastle disease virus, varicella stomatitis virus, measles virus, reovirus, cytomegalovirus (CMV), and lentivirus. [Section 324] The method according to item 323 above, wherein the oncolytic virus is vaccinia virus. [Section 325] The method according to item 324, wherein the vaccinia virus is a poxvirus selected from strains of Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, Modified Vaccinia Ankara (MVA), New York City Board of Health, Dairen, Ikeda, LC16M8, Copenhagen, Tashkent, Wyeth, IHD-J, IHD-W, Brighton, Dairen I, and Connaught. [Section 326] The method according to any one of the above items 317 to 325, wherein the tumor is a solid tumor. [Section 327] The method according to any one of items 317 to 326 above, wherein the subject has a cancer selected from lung cancer, pancreatic cancer, breast cancer, colon cancer, head and neck cancer, liver cancer, melanoma, and kidney cancer. [Section 328] The solid tumors or hematological malignancies include bladder tumors, breast tumors, prostate tumors, carcinomas, basal cell carcinomas, biliary tract cancers, bladder cancers, bone cancers, brain cancers, central nervous system (CNS) cancers, gliomas, cervical cancers, choroidal cancers, colon and rectal cancers, connective tissue cancers, digestive system cancers, endometrial cancers, esophageal cancers, eye cancers, head and neck cancers, stomach cancers, carcinomas in situ, kidney cancers, laryngeal cancers, leukemias, liver cancers, lung cancers, lymphomas, and Hodgkin's cancers. Parkinson's cancer, non-Hodgkin's lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, kidney cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, urinary tract cancer, lymphosarcoma, osteosarcoma, mammary gland tumor, mast cell tumor, brain tumor, adenosquamous carcinoma, carcinoid lung tumor, bronchial gland tumor, bronchial gland cancer, small cell lung cancer, non-small cell lung cancer, fibroma, mucosal carcinoma Hypochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma, Wilms' tumor, Burkitt lymphoma, microglia, osteoclastoma, oral neoplasm, fibrosarcoma, genital squamous cell carcinoma, sexually transmitted disease tumors, testicular tumors, seminoma, Sertoli cell tumor, hemangiocytoma, histiocytoma, chloroplastoma, granulocytic sarcoma, corneal papilloma, corneal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, gastric tumor, adrenal cancer, oral The method according to item 326 above, selected from papillomatosis, hemangioendothelioma, cystadenoma, follicular lymphoma, enteric lymphosarcoma, pulmonary squamous cell carcinoma, leukemia, hemangioepoidoma, ocular neoplasm, prepistagosomal fibrosarcoma, ulcerative squamous cell carcinoma, prepistagosomal carcinoma, connective tissue neoplasm, hepatocellular carcinoma, pulmonary adenomatosis, pulmonary sarcoma, Rous sarcoma, reticuloendotheliopathy, nephroblastoma, B-cell lymphoma, lymphocytic leukemia, retinoblastoma, hepatic neoplasm, lymphosarcoma, plasmacytoid leukemia, swim bladder sarcoma (fish), caseous lymphadenitis, lung cancer, insulinoma, neuroma, pancreatic islet cell tumor, gastric MALT lymphoma, and gastric adenocarcinoma. [Section 329] The method according to any one of items 317 to 328 above, wherein the oncolytic virus encodes a heterogeneous gene product. [Section 330] The method according to item 329 above, wherein the heterogeneous gene product is an anticancer drug. [Section 331] The method according to item 330 above, wherein the product is an antibody. [Section 332] The method according to any of items 317 to 331 above, wherein the subject is a human. [Section 333] The method according to item 332 above, wherein the subject is a pediatric patient. [Section 334] The method according to any one of items 317 to 333 above, wherein the cells are autologous to the subject. [Section 335] The method according to any one of the above items 317 to 333, wherein the cells are allogeneic with respect to the subject. [Section 336] The method according to any one of items 317 to 335, wherein the virus is administered by intratumoral injection, intravenous injection, intraperitoneal injection, intrathecal injection, intraventricular injection, intraarticular injection, or intraocular injection. [Section 337] The method according to any one of items 317 to 336, wherein the cells are administered by intratumoral injection, intravenous injection, intraperitoneal injection, intrathecal injection, intraventricular injection, intraarticular injection, or intraocular injection. [Section 338] The method described in any of sections 317 to 337 above, including administering another anti-cancer treatment or therapy. [Section 339] The method according to item 338, wherein the other cancer treatment or therapy is immunotherapy. [Section 340] The method according to paragraph 339, wherein the immunotherapy comprises administering a checkpoint inhibitor or a treatment that inhibits a checkpoint or immunosuppressive pathway. [Section 341] The method according to any one of items 317 to 338, comprising administering a treatment to activate the T cell response within the subject, wherein the treatment comprises administering a blocking antibody against a negative costimulatory molecule or an agonist antibody against an activating costimulatory molecule. [Section 342] The method according to item 341, wherein the procedure comprises administering a blocking antibody against a negative costimulatory molecule. [Section 343] The method according to item 341 or 342, wherein the blocking antibody is against a negative costimulatory molecule selected from CTLA-1, CTLA-4, PD-1, TIM-3, BTLA, VISTA, PD-L1, and LAG-3. [Section 344] The method according to any one of items 341 to 343 above, wherein the treatment includes the administration of an inhibitor of the PD-1 pathway. [Section 345] The method according to item 344, wherein the inhibitor of the PD-1 pathway is selected from antibodies against PD-1 and soluble PD-1 ligands. [Section 346] The method according to item 344, wherein the inhibitor of the PD-1 pathway is an antibody selected from AMP-244, MEDI-4736, MPDL328 OA, and MIH1. [Section 347] The method according to any one of items 341 to 343, wherein the treatment comprises administering an anti-CTLA-4 antibody, an anti-PD-L1 antibody, or an anti-PD-1 antibody. [Section 348] The method according to any one of items 341 to 343, wherein the treatment comprises administering a blocking antibody against a negative costimulatory molecule selected from PD-L1 and CTLA-4. [Section 349] The method according to any one of items 317 to 348 above, comprising a procedure including administering an agonist antibody against a costimulatory molecule. [Section 350] The method according to item 349, wherein the co-stimulatory molecule is selected from CD28, OX40, GITR, CD137, CD27, and HVEM. [Section 351] A composition or combination, (a) Oncolytic viruses and, (b) comprising superembryonic adipocytes (SA-ASC; CD235a- / CD45- / CD34+ / CD146- / CD31-) or pericytes (CD235a- / CD45- / CD34- / CD146+ / CD31-), A composition or combination in which the composition is a mixture of cells and viruses, and the combination is two separate compositions, one of which contains viruses and the other composition contains cells. [Section 352] The composition or combination described in item 351 above, wherein the oncolytic virus is selected from poxvirus, adenovirus, herpes simplex virus, Newcastle disease virus, varicella stomatitis virus, measles virus, reovirus, cytomegalovirus (CMV), and lentivirus. [Section 353] The composition or combination according to item 352, wherein the oncolytic virus is vaccinia virus. [Section 354] The composition or combination according to item 353, wherein the vaccinia virus is a poxvirus selected from strains of Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, Modified Vaccinia Ankara (MVA), New York City Board of Health, Dairen, Ikeda, LC16M8, Copenhagen, Tashkent, Wyeth, IHD-J, IHD-W, Brighton, Dairen I, and Connaught. [Section 355] A composition or combination according to any one of sections 351 to 354 above, for use in treating solid tumors or hematological malignancies. [Section 356] The solid tumors or hematological malignancies include bladder tumors, breast tumors, prostate tumors, carcinomas, basal cell carcinomas, biliary tract cancers, bladder cancers, bone cancers, brain cancers, central nervous system (CNS) cancers, gliomas, cervical cancers, choroidal cancers, colon and rectal cancers, connective tissue cancers, digestive system cancers, endometrial cancers, esophageal cancers, eye cancers, head and neck cancers, stomach cancers, carcinomas in situ, kidney cancers, laryngeal cancers, leukemias, liver cancers, lung cancers, lymphomas, and Hodgkin's lymphomas. Tumors, non-Hodgkin lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, kidney cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, urinary tract cancer, lymphosarcoma, osteosarcoma, mammary gland tumors, mast cell tumors, brain tumors, adenosquamous cell carcinoma, carcinoid lung tumors, bronchial gland tumors, bronchial gland cancers, small cell lung cancer, non-small cell lung cancer, fibroma, myxoid cartilage Tumors, pulmonary sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma, Wilms' tumor, Burkitt lymphoma, microglia, osteoclastoma, oral neoplasms, fibrosarcoma, genital squamous cell carcinoma, sexually transmitted disease tumors, testicular tumors, seminoma, Sertoli cell tumor, hemangioepoid tumor, histiocytoma, chloroplastoma, granulocytic sarcoma, corneal papilloma, corneal squamous cell carcinoma, angiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, gastric tumor, adrenal cancer, oral papillomatosis The compositions or combinations described in item 355 above, selected from hemangioendothelioma, cystadenoma, follicular lymphoma, intestinal lymphosarcoma, pulmonary squamous cell carcinoma, leukemia, hemangioepoidoma, ocular neoplasm, prepistagosomal fibrosarcoma, ulcerative squamous cell carcinoma, prepistagosomal carcinoma, connective tissue neoplasm, mast cell tumor, hepatocellular carcinoma, pulmonary adenomatosis, pulmonary sarcoma, Rous sarcoma, reticuloendotheliopathy, nephroblastoma, B-cell lymphoma, lymphocytic leukemia, retinoblastoma, hepatic neoplasm, lymphosarcoma, plasmacytoid leukemia, swim bladder sarcoma (fish), caseous lymphadenitis, lung cancer, insulinoma, neuroma, pancreatic islet cell tumor, gastric MALT lymphoma, and gastric adenocarcinoma. [Section 357] A combination therapy for use in the treatment of solid tumors or hematological malignancies in the subject, (a) Stem cells, The aforementioned stem cells contain an oncolytic virus, The stem cells are adipocytes selected from superembryonic adipocytes (SA-ASC; CD235a- / CD45- / CD34+ / CD146- / CD31-) and pericytes (CD235a- / CD45- / CD34- / CD146+ / CD31-), (b) A combination therapy comprising a treatment for activating the T cell response in the subject, which includes a blocking antibody against a negative costimulatory molecule or an agonist antibody against an activating costimulatory molecule. [Section 358] (b) The combination described in paragraph 357 above, wherein the treatment includes a blocking antibody against a negative costimulatory molecule. [Section 359] The combination for use described in item 358 above, wherein the blocking antibody is against a negative costimulatory molecule selected from CTLA-1, CTLA-4, PD-1, TIM-3, BTLA, VISTA, and LAG-3. [Section 360] (b) The combination of any of the items 357 to 359 above, wherein the treatment includes an inhibitor of the PD-1 pathway. [Section 361] The combination for use described in item 360 above, wherein the inhibitor of the PD-1 pathway is selected from antibodies against PD-1 and soluble PD-1 ligands. [Section 362] The combination for use described in item 360 above, wherein the inhibitor of the PD-1 pathway is an antibody selected from AMP-244, MEDI-4736, MPDL328 OA, and MIH1. [Section 363] (b) The treatment is a combination for use as described in any of sections 357 to 359 above, comprising an anti-CTLA-4 antibody, an anti-PD-L1 antibody, or an anti-PD-1 antibody. [Section 364] (b) The combination according to paragraph 357 or 358, wherein the treatment comprises blocking an antibody against a negative costimulatory molecule selected from PD-L1 and CTLA-4. [Section 365] (b) A combination therapy for use according to any one of the above paragraphs 357 to 364, wherein the treatment comprises an agonist antibody against a costimulatory molecule. [Section 366] The combination described in item 365 above, wherein the co-stimulatory molecule is selected from CD28, OX40, GITR, CD137, CD27, and HVEM. [Section 367] The combination therapy described in any of paragraphs 357 to 366 above, wherein (a) is to be used after (b). [Section 368] The combination therapy described in any of paragraphs 357 to 366 above, wherein (a) is to be used before (b). [Section 369] A combination therapy for use according to any one of paragraphs 357 to 368, further comprising a procedure to induce apoptosis of cells within the tumor, wherein the procedure is selected from radiotherapy, chemotherapy, immunotherapy, phototherapy, or a combination thereof. [Section 370] The combination therapy described in paragraph 369 above, wherein the treatment for inducing apoptosis includes radiotherapy. [Section 371] A combination therapy for use according to any one of the above paragraphs 357 to 370, further comprising a procedure for modifying the tumor microenvironment, wherein the procedure includes a cytokine blocker. [Section 372] The combination therapy for use as described in any of paragraphs 357 to 371 above, wherein the lytic virus is selected from poxvirus, adenovirus, herpes simplex virus, Newcastle disease virus, varicella stomatitis virus, mumps virus, influenza virus, measles virus, reovirus, human immunodeficiency virus (HIV), hantavirus, myxoma virus, cytomegalovirus (CMV), and lentivirus. [Section 373] The combination therapy for use according to any one of paragraphs 357 to 371 above, wherein the oncolytic virus is vaccinia virus. [Section 374] The aforementioned stem cells are administered in combination with agents selected from TLR agonist intravenous immunoglobulin (IVIG); monocyte-conditioned medium; supernatant from peripheral blood mononuclear cells exposed to neutrophil extracellular traps; peptidoglycan isolated from Gram-positive bacteria; lipoarabinomannan isolated from mycobacteria; zymosan isolated from yeast cell walls; polyadenylic acid; poly(IC); lipopolysaccharide; monophosphoryl lipid A; flagellin; galdiquemod; imiquimod; R848; oligonucleosides containing CpG motifs; and 23S ribosomal RNA; or The combination therapy for use according to any one of the above sections 357 to 373, wherein the stem cells are co-cultured with IVIG-pretreated monocytes, T cells, T cells exposed to T cell stimulation, or natural killer cells. [Section 375] The aforementioned use is for bladder tumors, breast tumors, prostate tumors, carcinomas, basal cell carcinomas, biliary tract cancers, bladder cancers, bone cancers, brain cancers, central nervous system (CNS) cancers, gliomas, cervical cancers, choroidal cancers, colon and rectal cancers, connective tissue cancers, digestive system cancers, endometrial cancers, esophageal cancers, eye cancers, head and neck cancers, stomach cancers, carcinomas in situ, kidney cancers, laryngeal cancers, leukemias, liver cancers, lung cancers, lymphomas, Hodgkin lymphomas, and non-Hodgkin lymphomas. Parkinson's disease, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, kidney cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, urinary tract cancer, lymphosarcoma, osteosarcoma, mammary gland tumor, mast cell tumor, brain tumor, adenosquamous cell carcinoma, carcinoid lung tumor, bronchial gland tumor, bronchial gland cancer, small cell lung cancer, non-small cell lung cancer, fibroma, myxochondroma, pulmonary sarcoma, Neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma, Wilms' tumor, Burkitt lymphoma, microglioma, osteoclastoma, oral neoplasm, fibrosarcoma, genital squamous cell carcinoma, sexually transmitted disease tumors, testicular tumors, seminoma, Sertoli cell tumor, hemangioepoid tumor, histiocytoma, chloroplastoma, granulocytic sarcoma, corneal papilloma, corneal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, gastric tumor, adrenal cancer, oral papillomatosis, blood vessels Combination therapy for use as described in any of sections 357-374 above, for the treatment of endothelioma, cystadenoma, follicular lymphoma, enteric lymphosarcoma, pulmonary squamous cell carcinoma, leukemia, hemangioepoidoma, ocular neoplasm, prepistagocheal fibrosarcoma, ulcerative squamous cell carcinoma, prepistagocheal carcinoma, connective tissue neoplasm, mast cell tumor, hepatocellular carcinoma, pulmonary adenomatosis, pulmonary sarcoma, Rous sarcoma, reticuloendotheliopathy, nephroblastoma, B-cell lymphoma, lymphocytic leukemia, retinoblastoma, hepatic neoplasm, lymphosarcoma, plasmacytoid leukemia, swim bladder sarcoma (fish), caseous lymphadenitis, lung cancer, insulinoma, neuroma, pancreatic islet cell tumor, gastric MALT lymphoma, and gastric adenocarcinoma. [Section 376] The combination therapy for the use described in any of paragraphs 357 to 375 above, wherein the use is for the treatment of glioblastoma, breast cancer, lung cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, central nervous system tumors, pancreatic adenocarcinoma, or melanoma. [Section 377] The combination for use described in any of paragraphs 357 to 376 above, wherein the stem cells are autologous. [Section 378] The combination for use described in any of paragraphs 357 to 376 above, wherein the stem cells are allogeneic. [Section 379] A cell-assisted viral expression system (CAVES) according to any one of items 1 to 103 and 111 to 125 above, wherein the carrier cells are treated or modified for conditional immortalization. [Section 380] The cell-assisted viral expression system (CAVES) described in paragraph 379 above, wherein the carrier cells are modified to express one or more of c-myc, v-myc, E6 / E7, hTERT, wild-type or modified SV40 large tumor antigen, loxP, and / or tetR. [Section 381] A pharmaceutical composition comprising a cell-assisted viral expression system (CAVES) as described in item 379 or 380 above in a pharmaceutically acceptable medium. [Section 382] A pharmaceutical composition as described in item 381 above, which is stored by freezing. [Section 383] A pharmaceutical composition according to item 381 or 382 above, which is formulated for direct administration without dilution. [Section 384] A pharmaceutical composition as described in any of items 381 to 383 above, which is formulated as a single dose. [Section 385] A pharmaceutical composition according to any one of items 381 to 384 above, wherein the pharmaceutically acceptable medium is suitable for parenteral administration. [Section 386] A cell-assisted viral expression system (CAVES) as described in item 379 or 380 above, for use in treating cancer, including solid tumors or hematological malignancies. [Section 387] Use of a cell-assisted viral expression system (CAVES) as described in item 379 or 380 above, or any of the pharmaceutical compositions described in items 381 to 385 above, for use in treating cancer, including solid tumors or hematological malignancies. [Section 388] The use of a cell-assisted viral expression system (CAVES) for the use described in item 386 or 387 above, wherein the cell-assisted viral expression system is a) Infecting cells with oncolytic viruses with an infection multiple (MOI) of 10 or less; and b) Use of a cell-assisted viral expression system or the cell-assisted viral expression system (CAVES) produced by incubating or co-culturing the cells with the oncolytic virus under conditions that allow the cells to grow and / or replicate for a sufficient amount of time for the oncolytic virus to express therapeutic or immunomodulatory proteins encoded in the viral genome and produce the CAVES. [Section 389] The use of a cell-assisted viral expression system (CAVES) or a cell-assisted viral expression system (CAVES) for the use described in item 388 above, wherein the MOI is 0.001 to 10, 0.1 to 10, 0.1 to 1, 0.1 to less than 1, 0.1 to 0.5, or 0.1. [Section 390] Use of a cell-assisted viral expression system (CAVES) or cell-assisted viral expression system (CAVES) for use according to any one of items 386 to 389 above, wherein the cells and virus are incubated or co-cultured for at least 6 hours. [Section 391] The carrier cells are treated or modified for conditional immortalization. The proliferation of the modified carrier cell population is activated at a first time point (or more) before the preparation of CAVES and / or before the administration of CAVES to the subject. The method according to any one of items 130 to 205, wherein the proliferation of the carrier cell population is inactivated at a second time point after a first time point (or more) and before the administration of the CAVES to the subject. [Section 392] A method for constructing a cell-assisted viral expression system (CAVES), A cell-assisted viral expression system (CAVES) is produced by incubating carrier cells and oncolytic viruses for viral infection at an MOI of 0.001 to 10 for more than 6 hours and less than 48 hours under conditions that express virally encoded proteins, thereby causing the carrier cells to express at least one virally encoded immunomodulatory protein or recombinant therapeutic protein by association with the carrier cells; To recover the aforementioned cell-supported virus expression system cells; and A method comprising storing them in a cryopreservation medium at low temperatures to produce a preserved cell-assisted viral expression system (CAVES). [Section 393] The method according to paragraph 392, wherein the MOI is 0.001 to 10, 0.1 to 10, 0.1 to 1, 0.1 to less than 1, 0.1 to 0.5, or 0.1. [Section 394] The method according to item 392 or 393, wherein the incubation time is at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 40 hours or more. [Section 395] The method according to item 394, wherein the incubation time is at least 6 hours. [Section 396] The method according to any one of items 392 to 395, wherein the carrier cells are treated or modified for conditional immortalization. [Section 397] The method according to item 396, wherein the carrier cells are modified to express one or more of c-myc, v-myc, E6 / E7, hTERT, wild-type or modified SV40 large tumor antigen, loxP and / or tetR. [Section 398] The method according to any one of items 392 to 397, wherein the virus is treated or modified to encode or include one or more of the following: immune checkpoint inhibitors, costimulators, cytokines, growth factors, photosensitizers, radionuclides, toxins, antimetabolites, signaling modulators, anticancer antibodies, and angiogenesis inhibitors. [Modes for carrying out the invention]

[0015] overview A.Definition B. Selection of components for cell-assisted viral expression systems (CAVES) 1.Cells (i) Cell media sensitized / protected for improved viral amplification and / or immunomodulation (ii) Sensitized for resistance to virus-borne killing (for extended survival time and improved local immunosuppression) (iii) Cell medium manipulated to improve viral amplification and / or immunomodulation (iv) Cell medium engineered to express angiogenesis inhibitors for vascular normalization / tumor angiogenesis reprogramming (v) Cell medium engineered to express transgenes for conditional cell immortalization 2. Virus Production, formulation, storage, and transport of C. caves D. Pharmaceutical compositions, combinations, and kits 1. Pharmaceutical composition 2. Combinations 3. Kit Combination (additional) treatments administered with E.CAVES F. Dosage of CAVES for treatment a. Administration of irradiated or unirradiated CAVES. b. Route of administration c.Device d. Dosage of administration regimen G. Treatment methods and monitoring in conjunction with treatment H. Exemplary types of cancer treated I. Examples

[0016] A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. All patents, patent applications, published applications and publications, GenBank sequences, databases, websites, and other publicly available materials referenced throughout this disclosure are incorporated by reference in their entirety unless otherwise specified. If there are multiple definitions for any term herein, the definition in this section shall prevail. Where URLs or other such identifiers or addresses are referenced, it is understood that such identifiers can change and certain information on the Internet can shift, but equivalent information can be found by searching the Internet. References to them demonstrate the availability and general dissemination of such information.

[0017] As used herein, "virus" refers to any group of infectious entities that cannot reproduce or replicate without a host cell. Viruses typically contain a protein coat and RNA or DNA as genetic material, but lack a semipermeable membrane and can only reproduce and multiply in living cells. Examples include influenza virus, mumps virus, poliovirus, Seneca Valley virus, and Semryqui Forest virus.

[0018] As used herein, “oncolytic virus” refers to a virus that selectively replicates in tumor cells of a tumor target. These include viruses that naturally preferentially replicate and accumulate in tumor cells, such as poxviruses, and viruses that have been engineered to do so. Some oncolytic viruses can kill tumor cells after infection. For example, oncolytic viruses can cause tumor cell death by lysing tumor cells or by inducing tumor cell death. Exemplary oncolytic viruses include, but are not limited to, poxviruses, herpesviruses, adenoviruses, adeno-associated viruses, lentiviruses, retroviruses, rhabdoviruses, papillomaviruses, varicella stomatitis viruses, measles viruses, Newcastle disease viruses, picornaviruses, Sindbisviruses, parvoviruses, reoviruses, and coxsackieviruses.

[0019] As used herein, the term “therapeutic virus” refers to a virus administered for the treatment, diagnosis, and / or both of the following: neoplasms such as cancer, tumors and / or metastases, or diseases or disorders such as inflammation or wounds. Generally, the therapeutic viruses described herein exhibit antitumor activity and minimal toxicity.

[0020] As used herein, the terms "vaccinia virus," "VACV," or "VV" refer to a large, complex enveloped virus belonging to the poxvirus family. It has a linear, double-stranded DNA genome approximately 190 kbp long, encoding approximately 200 proteins. Vaccinia virus strains include, but are not limited to, Western Reserve (WR), Copenhagen (Cop), Bern, Paris, Tashkent, Tian Tan, Lister, Wyeth, IHD-J, IHD-W, Brighton, Ankara, modified vaccinia Ankara (MVA), CVA382, Dairen I, LIPV, LC16M8, LC16M0, ACAM, WR 65-16, Connaught, JX-594 (pexastimogene devacirepvec), GL-ONC1, vvDD TK mutants, New York City Department of Health (NYCBH), EM-63, and NYVAC vaccinia virus strains, strains derived from these, or modified versions thereof.

[0021] As used herein, with respect to an engineered virus, “marker” or “selection marker” refers to a compound such as a protein whose expression and / or presence within and / or on the viral surface enables the selection of viruses having desired engineered characteristics, such as viruses expressing recombinant therapeutic genes or other proteins, including marker proteins.

[0022] As used herein, the Lister strain (LIVP) or LIVP virus strain refers to the attenuated Lister virus strain (ATCC catalog number VR-1549) produced at the Institute of Viral Preparations, Moscow, Russia, through adaptation to calf hide (Al'tshtein et al. (1985) Dokl. Akad. Nauk USSR 285:696-699). The LIVP strain can be obtained, for example, from the Institute of Viral Preparations, Moscow, Russia (see, e.g., Kutinova et al. (1995) Vaccine 13:487-493); from the FSRI SRC VB Vector microbial collection (Kozlova et al. (2010) Environ.Sci.Technol. 44:5121-5126); or from the Moscow Ivanovsky Institute of Virology (C0355 K0602; Agranovski et al. (2006) Atmospheric Environment 40:3924-3929). It is also well known to those skilled in the art, as it was a vaccine strain used for vaccination in the USSR and throughout Asia and India. This strain is currently used by researchers and is well known (see, for example, Altshteyn et al. (1985) Dokl. Akad. Nauk USSR 285:696-699; Kutinova et al. (1994) Arch. Virol. 134:1-9; Kutinova et al. (1995) Vaccine 13:487-493; Shchelkunov et al. (1993) Virus Research 28:273-283; Sroller et al. (1998) Archives Virology 143:1311-1320; Zinoviev et al. (1994) Gene 147:209-214; and Chkheidze et al. (1993) FEBS 336:340-342). LIVP virus strains include any virus strain or viral preparation obtained by the proliferation of LIVP through repeated passage in a cell line.

[0023] As used herein, “modified virus” refers to a virus that has been altered compared to the parental strain of the virus. Typically, a modified virus has one or more cuts, mutations, insertions, or deletions in the viral genome. A modified virus may have one or more modified endogenous viral genes and / or one or more modified intergenetic regions. An exemplary modified virus may have one or more heterologous nucleic acid sequences inserted into the viral genome. A modified virus may include one or more heterologous nucleic acid sequences in the form of a gene expression cassette for the expression of heterologous genes.

[0024] Typically, the genome of a virus is modified by nucleotide substitution, insertion, or deletion. Modifications can be carried out using any method known to those skilled in the art, including those provided herein, such as genetic engineering and recombinant DNA methods. Thus, a modified virus is a virus whose genome is altered compared to the genome of the parent virus. An exemplary modified virus has one or more heterologous nucleic acid sequences inserted into the viral genome. Generally, heterologous nucleic acids include an open reading frame encoding a heterologous protein that can be inserted under the control of a viral promoter or a heterologous non-viral promoter. For example, a modified virus according to this specification may include one or more heterologous nucleic acid sequences in the form of a gene expression cassette for the expression of heterologous genes.

[0025] As used herein, the terms “cell,” “cell vehicle,” “carrier vehicle,” “cell-based delivery vehicle,” and “carrier cell,” as used interchangeably with “cell-based vehicle,” refer to any cell that a virus can infect or be infected with, or that can associate with a virus, for example, through chemical or physical interactions between the virus and surface proteins, or by viral infection of the cell’s cytoplasm or nucleus. As used herein, a carrier cell refers to a cell that can be infected with a virus, such as an oncolytic virus, and that a virus / oncolytic virus can replicate in. The resulting carrier cell contains or associates with an oncolytic virus.

[0026] As used herein, the terms “cell-assisted viral expression system” or “cell-assisted viral enhancement system” (CAVES) refer to carrier cells that associate with a virus, generally an oncolytic virus, and at least one viral-encoding protein, such as an immunomodulatory gene product or therapeutic gene product, which is expressed by association. The term “CAVES” is used herein interchangeably with the term “SNV.” Carrier cells associate with a virus by incubating the virus and cells under conditions in which the virus infects the cells, and viral proteins are expressed by the cells. By the virus, the carrier cells contain or present at least one immunomodulatory protein or therapeutic gene product encoded by the virus on their surface. In exemplary embodiments, the CAVES provided herein are generated by ex vivo or in vitro incubation of carrier cells with a virus over a period of time to achieve the expression of an immunomodulatory or therapeutic protein encoded by the virus. The duration is a function of the specific virus and cell, ranging from over 2 hours to over 72 hours, generally between 3 hours and 72 hours, for example, about or at least 3, 4, 5 or 6 hours to about or at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 3 The time is 8, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 hours or longer, and is generally a temperature that allows expression of at least one virus-encoded immunomodulatory or therapeutic gene, generally about or just 37°C. The specific time and temperature for promoting such expression depends on the type of oncolytic virus used in the system. CAVES are brought about by the expression of virus-encoded products and viral replication in cells. In most embodiments herein, the cells of CAVES are not tumor cells and / or immune cells.

[0027] As used herein, “cryopreservation” refers to the process of cooling and preserving cells, tissues, or organs at very low temperatures to maintain their viability. Typically, cryopreservation is carried out at temperatures of approximately -80°C to -200°C. Cryopreservation media are known to those skilled in the art. For example, such media generally contain the same formulations used to grow cells, and in addition, if the cells are grown in serum-free medium, it is recommended to include up to 20% fetal bovine serum, as well as cryopreservatives such as DMSO (7% to 10%) and / or glycerol (about 10%). The resulting composition is said to be cryopreserved.

[0028] As used herein, “freezing-preserved composition” is a composition that has been stored at a freezing temperature for at least 24 hours.

[0029] As used herein, "multiplicity of infection (MOI)" refers to the number of viral particles added to a cell during infection (i.e., 1 million viral particles added to 1 million cells has an MOI of 1).

[0030] As used herein, “sensitized” or “sensitizing a cell” generally refers to treating a cell before use with a drug that modifies its properties, for example, by inducing gene expression.

[0031] As used herein, viral amplification in carrier cells means that the virus replicates within the cell to maintain its presence or increases the amount of virus within the cell.

[0032] As used herein, "host cell" and "target cell" are used interchangeably to mean a cell that a virus can infect.

[0033] As used herein, the term "tissue" generally refers to a group, collection, or aggregate of similar cells that act to perform a particular function within an organism.

[0034] As used herein, the terms “immunomodulatory protein” or “immunomodulator” refer to proteins expressed by viruses that can protect the virus from attack by the innate and / or adaptive immune systems of target cells, such as tumor cells. Viral immunomodulatory products have evolved to withstand the selective evolutionary pressures imposed by the host immune system. These products can modulate innate and adaptive host immune responses. Exemplary immunomodulatory products encoded by vaccinia include, but are not limited to, VCP(C3L), B5R, HA(A56R), B18R / B19R, B8R, CmrC, and CmrE.

[0035] As used herein, the terms “therapeutic gene product” or “therapeutic polypeptide” refer to any heterologous protein expressed by a therapeutic gene encoded by a virus, such as an oncolytic virus, that improves the symptoms of a disease or disorder, or improves a disease or disorder. A therapeutic gene product may include, but is not limited to, a portion that inhibits cell growth or promotes cell death, a portion that can be activated to inhibit cell growth or promote cell death, or a portion that activates another agent that inhibits cell growth or promotes cell death. Optionally, a therapeutic agent may exhibit or reveal additional properties, such as properties that enable its use as a contrast agent, as described elsewhere herein. Exemplary therapeutic gene products include, for example, cytokines, growth factors, photosensitizers, radionuclides, toxins, antimetabolites, signaling modulators, anticancer antibodies, angiogenesis inhibitors, or combinations thereof.

[0036] As used herein, “fit” between a particular cell carrier (also referred herein as a cell medium) and a subject having cancer to be treated with the carrier cells and virus means that the cell carrier is sufficiently compatible with the host’s immune system to deliver the virus to the target tumor, evading the target’s immune system. Carrier cells can also be matched with a virus, and the matched virus can replicate within the cell. A carrier cell matched with a virus is suitable for administration to the target if the virus amplifies / replicates within the cell and the cell delivers the virus to the target tumor. Assays for identifying carrier cells suitable for a target to be treated and for identifying matching carrier cell / virus combinations are provided in U.S. Provisional Patent Application No. 62 / 680,570 and U.S. Patent Application No. 16 / 536,073, which are incorporated herein by reference in their entirety.

[0037] For the purposes of this specification, the enumeration of “antibody” (e.g., an antibody directed at an antigen expressed on an immune cell population such as T cells, γδ(gd)T cells, NK cells, and NKT cells that are depleted or inhibited to suppress the immune response) includes portions that include full-length antibodies and antibody fragments. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-bonded Fv(dsFv), Fd fragments, Fd' fragments, single-stranded Fv(scFv), single-stranded Fab(scFab), diabodies, anti-idiotype (anti-Id) antibodies, or any of the antigen-binding fragments described above. Antibodies also include synthetic antibodies, recombinant-produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, and intrabodies. The antibodies provided herein include any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a member of a subclass (e.g., IgG2a and IgG2b).

[0038] Antibodies, such as monoclonal antibodies, can be prepared using standard methods known to those skilled in the art (see, for example, Kohler et al., Nature 256:495-497 (1975); Kohler et al., Eur.J.Immunol. 6:511-519 (1976); and International Publication No. 02 / 46455). For example, animals are immunized by standard methods for producing somatic cells that secrete antibodies. These cells are then isolated from the immunized animals for fusion with myeloma cells. Somatic cells capable of producing antibodies, particularly B cells, can be used for fusion with myeloma cell lines. These somatic cells can be derived from the lymph nodes, spleen, and peripheral blood of the primed animals. Specialized myeloma cell lines have been developed from lymphocytic tumors for use in hybridoma-producing fusion procedures (Kohler and Milstein, Eur. J. Immunol. 6:511-519 (1976); Shulman et al., Nature, 276:269-282 (1978); Volk et al., J. Virol., 42:220-227 (1982)). These cell lines possess three useful properties. First, they facilitate the selection of fused hybridomas from non-fused and similarly indefinitely self-proliferating myeloma cells by possessing enzyme deficiencies that prevent them from growing in selective media supporting hybridoma proliferation. Second, they possess the ability to produce antibodies and are unable to produce endogenous light-chain or heavy-chain immunoglobulin chains. Third, they efficiently fuse with other cells. Other methods for producing hybridomas and monoclonal antibodies are well known to those skilled in the art. It is common practice to produce antibodies against any polypeptide, such as antigen markers on immune cell populations or immune checkpoints.

[0039] As used herein, a therapeutic agent is an agent that improves the symptoms of a disease or disorder, or that improves a disease or disorder. Therapeutic agents, therapeutic compounds, or therapeutic regimens include conventional drugs and pharmacotherapies, including vaccines for treatment or prevention (i.e., reducing the risk of developing a particular disease or disorder), which are known to those skilled in the art and are described elsewhere herein. Therapeutic agents for treating neoplastic diseases include, but are not limited to, parts that inhibit cell growth or promote cell death, parts that can be activated to inhibit cell growth or promote cell death, or parts that activate another agent that inhibits cell growth or promotes cell death. Therapeutic agents for use in the manner provided herein may, for example, be anticancer agents. Exemplary therapeutic agents include, for example, therapeutic microorganisms such as therapeutic viruses and bacteria, cytokines, growth factors, photosensitizers, radionuclides, toxins, antimetabolites, signaling modulators, anticancer antibiotics, anticancer antibodies, angiogenesis inhibitors, radiotherapy, chemotherapeutic compounds, or combinations thereof.

[0040] As used herein, tumor cells or cancer cells refer to cells that divide and replicate abnormally because their growth and division are not regulated or controlled, i.e., cells that are susceptible to uncontrolled proliferation. Tumor cells can be benign or malignant. Typically, tumor cells are malignant cells that can spread to other parts of the body in a process known as metastasis.

[0041] As used herein, a viral preparation or viral composition refers to a viral composition obtained by the growth of a viral strain, such as a vaccinia virus strain, a vaccinia virus clone, or a modified or recombinant viral strain, in vivo or in vitro in a culture system. For example, a vaccinia virus preparation refers to a viral composition obtained by the growth of a viral strain in host cells, typically when purified from a culture system using standard methods known in the art. Viral preparations generally consist of several viral particles or virions. If desired, the number of viral particles in a sample or preparation can be determined using a plaque assay, which calculates the number of plaque-forming units (pfu / mL) per unit volume of sample, assuming that each formed plaque represents one infectious viral particle. Each viral particle or virion in a preparation may have the same genomic sequence as other viral particles (i.e., the preparation is sequence-homogeneous) or may have different genomic sequences (i.e., the preparation is sequence-heterogeneous). Those skilled in the art will understand that, in the absence of clonal isolation, heterogeneity or diversity in the viral genome can arise during replication due to homologous recombination events that occur during the natural selection process of viral strains (Plotkin & Orenstein (eds.), "Recombinant Vaccinia Virus Vaccines," in Vaccines, 3rd edition (1999)).

[0042] As used herein, plaque-forming units (pfu) or infectious units (IU) refer to the number of infectious viruses or live viruses. Therefore, this reflects the amount of active virus in the preparation. Pfu can be determined using a viral plaque assay (plaque formation assay) or an endpoint dilution assay, which are standard assays known to those skilled in the art.

[0043] As used herein, “targeting molecule” or “targeting ligand” refers to any molecular signal that directs localization to a specific cell, tissue, or organ. Examples of targeting ligands include, but are not limited to, proteins, carbohydrates, lipids, or other such moieties, and but are not limited to, proteins, polypeptides, or moieties that bind to cell surface molecules. For example, a targeting ligand may include a protein or moiety that binds to a cell surface receptor, or an antibody directed to an antigen selectively expressed on a target cell. Targeting ligands may also include, but are not limited to, growth factors, cytokines, adhesion molecules, neuropeptides, protein hormones, and single-chain antibodies (scFv).

[0044] As used herein, the delivery medium for administration refers to a lipid-based or other polymer-based composition, such as a liposome, micelle or reverse micelle, that associates with a drug, such as a virus, provided herein, for delivery to a host target.

[0045] As used herein, viral accumulation in a particular tissue refers to the distribution or colonization of the virus in a particular tissue of a host organism after a period of time following administration of the virus to the host that is long enough for the virus to infect the host's organs or tissues. Those skilled in the art will recognize that the duration of viral infection varies depending on the virus, the organ(s) or tissue(s)(s) to be infected, the host's immune capacity, and the dose of the virus. Generally, accumulation can be determined at less than about 1 day after viral infection, about 1 day to about 2, 3, 4, 5, 6 or 7 days, about 1 week to about 2, 3 or 4 weeks, and about 1 month to about 2, 3, 4, 5 or 6 months or longer. For the purposes of this specification, the virus preferentially accumulates in immune-privileged tissues such as inflammatory or tumor tissue, but is eliminated from other tissues and organs, such as non-tumor tissues of the host, to the extent that the viral toxicity is mild or tolerable and at most not lethal.

[0046] As used herein, “preferential accumulation” refers to the accumulation of the virus at a first site at a higher level than the accumulation at a second site (i.e., the concentration or titer of viral particles at the first site is higher than the concentration of viral particles at the second site). Therefore, viruses that preferentially accumulate in immune-privileged tissues (tissues protected from the immune system), such as inflammatory tissue and tumor tissue, compared to normal tissue or organs, refer to viruses that accumulate in immune-privileged tissues such as tumors at a higher level (i.e., concentration or viral titer) than the virus accumulates in normal tissue or organs.

[0047] As used herein, activity refers to the in vitro or in vivo activity of the compounds or viruses provided herein. For example, in vivo activity refers to the physiological response that occurs after in vivo administration of the compounds or viruses (or their compositions or other mixtures) provided herein. Thus, activity encompasses the therapeutic and pharmacokinetic effects resulting from such compounds, compositions, and mixtures. Activity can be observed in in vitro and / or in vivo systems designed to test or use such activity.

[0048] As used herein, “anti-tumor activity” or “anti-tumorigenic” refers to a viral strain that prevents or inhibits tumor formation or growth in a subject, either in vitro or in vivo. Antitumor activity can be determined by evaluating one or more parameters that indicate antitumor activity.

[0049] As used herein, “greater” or “improved” activity with respect to antitumor activity or antitumor-forming ability means that the viral strain can prevent or inhibit tumor formation or growth in a subject in vitro or in vivo to a greater extent than a reference virus or control virus, or to a greater extent than in the absence of viral treatment. Whether antitumor activity is “greater” or “improved” can be determined by evaluating the effect of the virus on parameters indicating antitumor activity, and, if necessary, on a control or reference virus. When comparing the activity of two or more different viruses, it is understood that the amount of virus used in the in vitro assay or administered in vivo (e.g., pfu) is the same or similar, and the conditions of the in vitro assay or in vivo evaluation (e.g., in vivo administration regimen) are the same or similar.

[0050] As used herein, "toxicity" (also referred to herein as virulence or pathogenicity) of a virus refers to the adverse or toxic effect on a host upon administration of the virus. In the case of oncolytic viruses such as vaccinia virus, viral toxicity is related to accumulation in non-tumor organs or tissues that can affect the survival of the host or produce adverse or toxic effects. Toxicity can be measured by evaluating one or more parameters that indicate toxicity. These include effects on the survival or health of the administered subject, such as accumulation in non-tumor tissues and effects on body weight.

[0051] As used herein, “reduced toxicity” means that the toxic or adverse effects of administering a virus to a host are attenuated or reduced compared to a host not treated with the virus, or compared to a host administered with another reference or control virus. Whether toxicity is reduced or reduced can be determined by evaluating the effects of the virus on toxicity parameters, and, if necessary, the control or reference virus. When comparing the activity of two or more different viruses, it is understood that the amount of virus used in the in vitro assay or administered in vivo (e.g., pfu) is the same or similar, and the conditions of the in vitro assay or in vivo evaluation (e.g., in vivo administration regimen) are the same or similar. For example, when comparing the effects of a virus and a control or reference virus administered in vivo, the subjects are of the same species, size, and sex, and the viruses are administered in the same or similar amounts under the same or similar administration regimen. In particular, a virus with reduced toxicity may mean that, when administered to a host for purposes such as treating a disease, the virus does not accumulate in the host's non-tumor organs and tissues to the extent that it causes damage or harm to the host, or that it affects the host's survival to a greater extent than the disease being treated or than a control or reference virus. For example, a virus with reduced toxicity includes viruses that do not cause death of the subject during treatment.

[0052] As used herein, “control” or “standard” refers to a sample that is substantially identical to the test sample except that it has not been treated with the test parameters, or, if it is a plasma sample, it may be from a normal volunteer that is not suffering from the condition of interest. A control may also be an internal control. For example, a control may be a sample such as a virus with known properties or activity.

[0053] As used herein, a drug regimen refers to the amount of drugs administered, such as carrier cells or viruses or other drugs, and the frequency of administration over the course of a drug cycle. A drug regimen is a function of the disease or condition being treated and is therefore subject to change.

[0054] As used herein, the frequency of administration refers to the number of times the drug is administered during an administration cycle. For example, the frequency can be several days, several weeks, or several months. For example, the frequency can be one, two, three, four, five, six, or seven doses during an administration cycle. The frequency can refer to consecutive days during an administration cycle. A particular frequency is a function of the specific disease or condition being treated.

[0055] As used herein, “cycle of administration” refers to a repeated schedule of a drug regimen for administering the virus over a series of doses. For example, an exemplary cycle of administration is a 28-day cycle.

[0056] As used herein, immune-privileged cells and immune-privileged tissues refer to cells and tissues, such as solid tumors, that are isolated from the immune system. Immune-privileged cells or tissues tolerate the introduction of an antigen without inducing an inflammatory immune response. For example, administration of a virus to a subject triggers an immune response that eliminates the virus from the subject. However, immune-privileged sites are protected or isolated from the immune response, allowing the virus to survive and generally replicate. Immune-privileged tissues include proliferative tissues such as tumor tissue and other tissues, as well as cells involved in other proliferative disorders, wounds, and other tissues involved in inflammatory responses.

[0057] As used herein, a tumor, also known as a neoplasm, is an abnormal mass of tissue that arises when cells proliferate abnormally rapidly. Tumors include hematopoietic malignancies and solid tumors. Tumors may exhibit a partial or complete lack of structural organization and functional coordination with normal tissue. Tumors can be benign (not malignant) or malignant (malignant).

[0058] As used herein, malignancy, when applied to tumors, refers to a primary tumor having metastatic potential accompanied by loss of growth control and location control.

[0059] As used herein, metastasis primarily refers to the proliferation of abnormal or neoplastic cells that are located away from the site involved in the pathological process.

[0060] As used herein, malignant tumors can be broadly classified into three main types. Carcinomas are malignant tumors that arise from epithelial structures such as the breast, prostate, lungs, colon, and pancreas, but are not limited to these. Sarcomas are malignant tumors that originate from connective tissue or mesenchymal cells such as muscle, cartilage, fat, or bone. Leukemia and lymphoma are malignant tumors that develop in hematopoietic structures (structures involved in the formation of blood cells) that include components of the immune system. Other malignant tumors include, but are not limited to, tumors of the nervous system (e.g., neurofibromatomas), germ cell tumors, and blastocyte tumors.

[0061] As used herein, an excised tumor refers to a tumor from which a substantial portion has been removed. Excision can be performed by surgical procedure (i.e., a tumor surgically removed). Resection can be partial or complete.

[0062] As used herein, disease or disorder refers to a pathological condition of an organism characterized by identifiable symptoms, resulting, for example, from an infection or genetic defect. Illustrative diseases described herein include neoplasms such as cancer.

[0063] As used herein, neoplastic disease refers to any disorder, including cancer, that involves the development, growth, metastasis, and progression of a tumor.

[0064] As used herein, cancer is a term for any disease caused by or characterized by any type of malignant or hematological malignancy, including metastatic cancer, lymphoid neoplasms, and hematological cancers. Exemplary cancers include, but are not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoma, adrenal carcinoma, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brainstem glioma, brain cancer, carcinoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primordial neuroectodermal tumor, visual tract or hypothalamic glioma, Breast cancer, bronchial adenoma / carcinoid tumor, Burkitt lymphoma, carcinoid tumor, carcinoma, central nervous system lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, fibrinogenic small round cell tumor, endometrial cancer, ependymoma, epidermal carcinoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer / intraocular melanoma, eye cancer / retinoblastoma, gallbladder cancer, gallstone tumor, stomach / stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, giant cell tumor Glioblastoma multiforme, glioma, hairy cell tumor, head and neck cancer, cardiac cancer, hepatocellular carcinoma, Hodgkin lymphoma, hyperplasia, hyperplastic corneal neuroma, carcinoma in situ, hypopharyngeal cancer, enteric ganglia neuroma, islet cell tumor, Kaposi's sarcoma, renal cell carcinoma, laryngeal cancer, leiomyoma, lip and oral cancer, liposarcoma, liver cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, macroglobulinemia, malignant carcinoid, malignant fibrous histiocytoma of bone, malignant hypercalcemia, malignant melanoma, Marfan-type tumor, medullary carcinoma, melanoma, Lukell cell carcinoma, mesothelioma, metastatic skin cancer, metastatic cervical squamous cell carcinoma, oral cancer, mucosal neuroma, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, cervical cancer, nerve tissue cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial tumor, ovarian germ cell tumor, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pineoblastoma, pituitary adenoma, pleuropulmonary blastoma, polycythemia vera, primary brain tumor,This includes prostate cancer, rectal cancer, renal cell tumor, reticulum sarcoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, seminoma, Sézary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, cervical squamous cell carcinoma, gastric cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, pharyngeal cancer, thymoma, thyroid cancer, focal skin lesions, gestational trophoblastic tumor, urethral cancer, uterine / endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, or Wilms tumor. Exemplary cancers commonly diagnosed in humans include, but are not limited to, cancers of the bladder, brain, breast, bone marrow, cervix, colon / rectum, kidney, liver, lung / bronchus, ovary, pancreas, prostate, skin, stomach, thyroid, or uterus. Exemplary cancers commonly diagnosed in dogs, cats, and other pets include, but are not limited to, lymphosarcoma, osteosarcoma, mammary gland tumors, mast cell tumors, brain tumors, melanoma, adenosquamous cell carcinoma, carcinoid lung tumors, bronchial gland tumors, bronchial gland carcinoma, fibroma, myxochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma, Wilms' tumor, Burkitt lymphoma, microglia, and neuroblastoma. These include tumors, osteoclastomas, oral neoplasms, fibrosarcomas, osteosarcomas and rhabdomyosarcomas, genital squamous cell carcinomas, Sertoli cell tumors, hemangioepoidomas, histiocytomas, chloroplastomas (e.g., granulocytic sarcomas), corneal papillomas, corneal squamous cell carcinomas, hemangiosarcomas, pleural mesotheliomas, basal cell tumors, thymomas, gastric tumors, adrenal adenocarcinomas, oral papillomatosis, hemangioendotheliomas and cystadenomas, follicular lymphomas, intestinal lymphosarcomas, fibrosarcomas and pulmonary squamous cell carcinomas. Exemplary cancers diagnosed in rodents such as ferrets include, but are not limited to, insulinomas, lymphomas, sarcomas, neuromas, islet cell tumors, gastric MALT lymphomas, and gastric adenocarcinomas. Exemplary neoplasms affecting agricultural livestock include, but are not limited to, leukemia, hemangioepoidoma and bovine ocular neoplasms (cattle); prepistagocheal fibrosarcoma, ulcerative squamous cell carcinoma, prepistagocheal carcinoma, connective tissue neoplasms and mast cell tumors (horses); hepatocellular carcinoma (pig); lymphoma and pulmonary adenomatosis (sheep); pulmonary sarcoma, lymphoma, Rous sarcoma, reticuloendotheliopathy, fibrosarcoma, nephroblastoma, B-cell lymphoma and lymphocytic leukemia (avian species); retinoblastoma, hepatic neoplasms, lymphosarcoma (lymphoblastic lymphoma), plasmacytoid leukemia and swim bladder sarcoma (fish).Caseous lymphadenitis (CLA): This includes a chronic, infectious, and contact-transmitted disease of sheep and goats caused by the bacterium Corynebacterium pseudotuberculosis, as well as contact-transmitted lung tumors in sheep caused by Corynebacterium jaagzikte.

[0065] As used herein, cells involved in a disease or disease process refer to cells whose presence contributes to, exacerbates, causes, or is involved in the pathogenesis of a disease or disease process. Inhibition or killing of such cells can improve the symptoms of a disease or improve the disease. An example of such cells is tumor cells. Killing tumor cells or inhibiting their growth or proliferation results in treatment of the tumor. Another example is immune effector cells involved in inflammatory responses that contribute to the pathogenesis of various diseases. Diseases with inflammatory components can be treated by inhibiting or killing immune effector cells.

[0066] As used herein, “killing or inhibiting growth or proliferation of cells” means that cells are killed or eliminated. “Inhibiting growth or proliferation” means that the number of such cells does not increase but can decrease.

[0067] As used herein, “tumor cell” refers to any cell that is part of a tumor. Typically, the carrier cells provided herein preferentially homing to tumor cells, and the viruses provided herein preferentially infect the tumor cells in question compared to normal cells.

[0068] As used herein, "metastatic cell" refers to a cell that has the ability to metastasize. Metastatic cells have the ability to metastasize from a primary tumor of the target, and can colonize tissue at a different site of the target, thereby forming a secondary tumor at that site.

[0069] As used herein, "tumorigenic cell" refers to a cell that, when introduced to an appropriate site of interest, is capable of forming a tumor. The cell may be non-metastatic or metastatic.

[0070] As used herein, "normal cells" refers to cells that do not originate from a tumor but are derived from healthy, non-disease tissue.

[0071] As used herein, "metastasis" refers to the spread of cancer from one part of the body to another. For example, during metastasis, malignant cells can spread from the site of the primary tumor where they originated, traveling through lymphatic vessels and blood vessels, which transport the cells to normal tissue elsewhere in the organism, where they continue to proliferate. A tumor formed by cells that have spread through metastasis is called a "metastatic tumor," "secondary tumor," or "metastasis."

[0072] As used herein, an anti-cancer agent or anti-cancer compound (used interchangeably with "anti-tumor or anti-neoplastic agent") refers to any agent or compound used in anti-cancer treatment. These include any agent that, when used alone or in combination with other compounds, can alleviate, reduce, improve, prevent, or induce or maintain remission of clinical symptoms or diagnostic markers associated with neoplasms, tumors, and cancer, and which can be used in the methods, combinations, and compositions provided herein. Anti-cancer agents include anti-metastatic agents. Exemplary anticancer agents include, but are not limited to, chemotherapeutic compounds (e.g., toxins, alkylating agents, nitrosoureas, anticancer antibiotics, antimetabolites, mitotic inhibitors, and topoisomerase inhibitors), cytokines, growth factors, hormones, photosensitizers, radionuclides, signaling modulators, immunotherapeutic agents, CAR-T cells, checkpoint inhibitors, CRISPR therapy, anticancer antibodies, anticancer oligopeptides, anticancer oligonucleotides (e.g., antisense RNA and RNAi such as siRNA and shRNA), angiogenesis inhibitors, radiotherapy, or combinations thereof. Exemplary chemotherapeutic compounds include, but are not limited to, Ara-C, cisplatin, carboplatin, paclitaxel, doxorubicin, gemcitabine, camptothecin, irinotecan, cyclophosphamide, 6-mercaptopurine, vincristine, 5-fluorouracil, and methotrexate.

[0073] In this specification, references to anticancer agents or chemotherapeutic agents include, unless otherwise indicated, a combination of anticancer agents or chemotherapeutic agents, or multiple anticancer agents or chemotherapeutic agents.

[0074] As used herein, "subject" includes any living organism, including animals, to which diagnosis, screening, monitoring, or treatment is intended. Animals include mammals, such as primates and domesticated animals. An exemplary primate is humans. "Patient" refers to a subject, such as a mammal, primate, human, or livestock, that is suffering from, or is determined to be suffering from, or is at risk of suffering from, a disease condition.

[0075] As used herein, "patient" refers to a human subject exhibiting symptoms of a disease or disorder.

[0076] As used herein, treatment of a subject having symptoms, disorders or diseases means any method of treatment that improves or beneficially alters the symptoms of the condition, disorder or disease. Treatment includes any medicinal use of cell-assisted viral expression systems described and provided herein.

[0077] As used herein, treatment of a subject with a neoplasm, including a tumor or metastasis, means any form of treatment that improves or beneficially alters the symptoms of the neoplasm. Typically, treatment of a tumor or metastasis in a subject includes any form of treatment that results in delaying tumor growth, lysing tumor cells, reducing tumor size, preventing new tumor growth, or preventing metastasis of a primary tumor, including inhibition of tumor angiogenesis, tumor cell division, tumor cell migration, or degradation of the basement membrane or extracellular matrix.

[0078] As used herein, therapeutic effect means the effect resulting from the treatment of a subject, which alters, typically improves or improves, or cures the symptoms of a disease or condition. The therapeutically effective dose refers to the amount of a composition, molecule, or compound that produces a therapeutic effect after administration to the subject.

[0079] As used herein, improvement or relief of symptoms of a particular disorder, such as by the administration of a particular pharmaceutical composition, means any reduction, whether permanent or temporary, sustained or transient, that may be caused by or related to the administration of the composition.

[0080] As used herein, efficacy means that upon administration of a virus or viral composition, the virus colonizes and replicates in proliferating cells or immune privileged cells, such as tumor cells. Colonization and replication in tumor cells indicate that the treatment is or will become an effective treatment.

[0081] As used herein, effective treatment with cell carriers / viruses is one that can increase survival compared to no treatment. For example, viruses are effective treatments when they stabilize disease, induce tumor regression, reduce disease severity, or delay or reduce tumor metastasis.

[0082] As used herein, an effective dose or therapeutic dose of a virus or compound for treating a particular disease is the amount that improves or reduces in some way the symptoms associated with the disease. The amount varies from person to person and is not limited, but depends on several factors, including age, weight, the patient's overall health, and the severity of the disease. The therapeutic dose may be administered as a single dose or in multiple doses, according to the regimen in which it is effective. This dose may cure the disease, but is typically administered to improve the symptoms of the disease. Repeated doses may be required to achieve the desired improvement of symptoms.

[0083] As used herein, an effective or therapeutically effective amount of a virus or compound for treating a neoplasm, including tumors or metastases, is an amount that improves or in any way reduces symptoms associated with the neoplasm, including, but not limited to, delaying tumor growth, lysing tumor cells, reducing tumor size, preventing new tumor growth, or preventing metastasis of a primary tumor.

[0084] As used herein, preventing a disease or condition means reducing the probability or increase in the likelihood of developing a disease or condition.

[0085] As used herein, “composition” means any mixture of two or more products or compounds. This can be a solution, suspension, liquid, powder, paste, aqueous system, non-aqueous system, or any combination thereof.

[0086] As used herein, a formulation refers to a composition comprising at least one active pharmaceutical or therapeutic agent and one or more excipients.

[0087] As used herein, co-formulation refers to a composition containing two or more activators or pharmaceuticals or therapeutic agents and one or more excipients.

[0088] As used herein, a combination refers to any association between or within two or more items. A combination can be two or more separate items, such as two compositions or two sets of items, a mixture thereof, such as a single mixture of two or more items, or any variation thereof. The elements of a combination are generally functionally associated or related. Exemplary combinations include, but are not limited to, two or more pharmaceutical compositions, two or more active ingredients, such as two viruses, or a virus and an anticancer agent, such as a chemotherapeutic compound, two or more viruses, a virus and a therapeutic agent, a virus and a contrast agent, a virus and multiple therapeutic agents and / or contrast agents, or any association thereof. Such combinations can be packaged as kits.

[0089] As used herein, a composition refers to a mixture of two or more components, such as a therapeutic agent, in or mixed with a pharmaceutically acceptable medium.

[0090] As used herein, direct administration refers to administering the composition without dilution.

[0091] As used herein, a kit is a packaged combination that optionally includes instructions for use of the combination and / or other reactions and components for such use.

[0092] As used herein, “article of manufacture” refers to a product that is manufactured and sold. As used throughout this application, the term is intended to encompass articles containing carrier cells and vaccinia virus alone or in combination with a second therapeutic or therapeutic energy source contained in the same or separate packaging.

[0093] As used herein, "apparatus" refers to an apparatus manufactured or adapted for a particular problem. Illustrative apparatuses herein are those that can cover, coat, or come into contact with the surface of the epidermis or skin. Examples of such apparatuses, but not limited to, include wraps, bandages, binds, dresses, sutures, patches, gauze, or dressings.

[0094] As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise.

[0095] As used herein, ranges and quantities may be expressed as specific values ​​or ranges of "about" or "approximately." "About" or "approximately" also include exact quantities. Therefore, "about 5 milliliters" also means "about 5 milliliters" and "5 milliliters." Generally, "about" includes quantities that are expected to be within the range of experimental error.

[0096] As used herein, “about the same” means within a range of quantities that would be considered the same or within an acceptable margin of error by those skilled in the art. For example, typically, in the case of pharmaceutical compositions, quantities of at least 1%, 2%, 3%, 4%, 5%, or 10% are considered about the same. Such quantities may vary depending on the tolerance for particular compositional variations in the subject matter.

[0097] As used herein, “optional” or “optionally” means whether or not the events or circumstances described later occur, and the descriptions include both cases in which such events or circumstances occur and cases in which they do not occur.

[0098] As used herein, "allogeneic cell" generally refers to a cell that is genetically distinct with respect to a particular subject because it originates from a genetically different individual of the same species. For example, allogeneic stem cells are stem cells derived from a donor other than the patient (or identical twin).

[0099] As used herein, "autologous cell" refers to a cell obtained from the individual being treated with the cell. For example, an autologous cell is obtained from the subject being treated (i.e., the patient). For example, an autologous stem cell is a stem cell derived from the patient.

[0100] As used herein, the term "engineered" with respect to a cell medium or carrier cell means a genetic modification of a cell, such as expressing a protein that can improve or enhance the cell's performance. For example, a cell may be engineered for improved viral amplification and / or improved immunomodulation.

[0101] As used herein, "immunomodulation" refers to any process by which an immune response is modified to a desired level, for example, by inducing, enhancing, or suppressing the immune response.

[0102] As used herein, "immune suppression" or "immunosuppression" refers to the suppression or reduction of the immune response.

[0103] As used herein, “immune privileged” or “immunoprivileged” refers to cells or tissues that do not trigger an immune response and can evade the immune system. Immune-privileged cells and tissues refer to cells and tissues such as solid tumors and the tumor microenvironment that are isolated from the immune system by the immunosuppressive properties of the tumor. As a result, oncolytic viruses preferentially accumulate in tumors in the tumor microenvironment because they are protected from the immune system. However, immune-privileged tissues and cells are protected or isolated from the immune response, allowing viruses to survive and generally replicate.

[0104] As used herein, “disease or disorder” refers to a pathological condition of an organism that results from, for example, an infection or a genetic defect and is characterized by identifiable symptoms.

[0105] As used herein, "resistant" in relation to viral infection refers to cells that do not become infected with the virus, or become infected to a very low degree, upon exposure to the virus.

[0106] As used herein, "permissive" in relation to viral infection refers to cells that readily become infected upon exposure to the virus.

[0107] As used herein, immunological compatibility refers to a cell or virus that is sufficiently compatible with the target / host immune system to evade the target immune system for a sufficient amount of time to deliver the virus to the target tumor or cancerous cells.

[0108] As used herein, "co-culture" refers to a cell culture in which two or more different cell populations are cultured.

[0109] As used herein, the term “loading” in relation to cells may refer to the association of a cell with a drug, either on the cell surface or inside the cell, through chemical or physical interactions between the cell and the drug, such as viruses, small molecules, therapeutic agents, and antibodies or their antigen-binding fragment antibodies.

[0110] As used herein, adipose-derived stem cells or ADSCs are mesenchymal stem cells obtained from donor adipose tissue.

[0111] As used herein, peripheral blood mononuclear cells or PBMCs are any peripheral blood cells having a round nucleus, such as lymphocytes, monocytes, or macrophages.

[0112] As used herein, "L14 VV" or "CAL14 VV" refers to a TK-inserted Turbo-FP635 manipulated LIVP strain of vaccinia virus.

[0113] As used herein, "ACAM2000" (ACAM1000 and ACAM2000, which have the same genome sequence and are deposited as ATCC deposit number PTA-3321; see U.S. Patents 6,723,325, 7,115,270 and 7,645,456) is the wild-type thymidine kinase (TK)-positive Wyeth strain of vaccinia virus. This is the smallpox vaccine strain available from the CDC. ACAM1000 is the name of the virus when grown in MRC5 cells, and ACAM2000 is the name of the virus when grown in Vero cells. In embodiments, the ACAM2000 virus has the sequence shown in Sequence ID No. 70.

[0114] As used herein, "CAL-01," "CAL1," or "WT1," as used interchangeably herein, refers to a virus amplified or cultured from ACAM2000 or ACAM1000. In an exemplary embodiment, the CAL1 virus has the sequence shown in SEQ ID NO: 71.

[0115] As used herein, "CAL-02" or "CAL2" as used interchangeably refers to recombinant forms of the ACAM2000 or CAL1 virus that encode exogenous genes, such as OX40L, 4-IBBL, or single-chain antibodies against checkpoint inhibitors, such as CTLA-4.

[0116] As used herein, “CAL-03” or “CAL3” as used interchangeably refers to a recombinant form of the ACAM2000, CAL1, or CAL2 virus that expresses a single-chain antibody against an anti-angiogenic gene, such as VEGF, in combination with one or more other exogenous genes, such as OX40L, 4-IBBL, or a single-chain antibody against a checkpoint inhibitor, such as CTLA-4.

[0117] As used herein, the terms "SNV-1" or "SNV1", which are used interchangeably herein, refer to CAVES (or SNV) formed by incubating CAL-01 virus with a cell carrier such as a stem cell. When incubating 1×10 7 pfu of the virus with a cell carrier, the resulting SNV is called SNV-1a (or SNV1a). When incubating 1×10 6 pfu of the virus with a cell carrier, the resulting SNV is called SNV-1b (or SNV1b). When incubating 1×10 5 pfu of the virus with a cell carrier, the resulting SNV is called SNV-1c (or SNV1c).

[0118] As used herein, the terms "SNV-2" or "SNV2", which are used interchangeably herein, refer to CAVES (or SNV) formed by incubating CAL-02 virus with a cell carrier such as a stem cell. When incubating 1×10 7 pfu of the virus with a cell carrier, the resulting SNV is called SNV-2a (or SNV2a). When incubating 1×10 6 pfu of the virus with a cell carrier, the resulting SNV is called SNV-2b (or SNV2b). When incubating 1×10 5 pfu of the virus with a cell carrier, the resulting SNV is called SNV-2c (or SNV2c).

[0119] As used herein, the terms "SNV-3" or "SNV3", which are used interchangeably herein, refer to CAVES or SNV formed by incubating CAL-03 virus with a cell carrier such as a stem cell. When incubating 1×10 7 pfu of the virus with a cell carrier, the resulting SNV is called SNV-3a (or SNV3a). When incubating 1×10 6 pfu of the virus with a cell carrier, the resulting SNV is called SNV-3b (or SNV3b). When incubating 1×10 5When a pfu virus is incubated with a cell carrier, the resulting SNVs are called SNV-3c (or SNV3c).

[0120] As used herein, a viral plaque assay (VPA) is an assay used to determine the amount of infectious virus or viral titer, given as plaque-forming units (pfu) per ml or per sample.

[0121] As used herein, a “primed” or “protected” cell medium or carrier cell is one that has been pretreated with and / or carried with a cytokine, such as an interferon (IFN), or an alloinactivating / rejection-determining factor antagonist, in order to protect the cell from an immune response.

[0122] As used herein, treatment refers to the improvement of the symptoms of a disease or disorder.

[0123] As used herein, prevention refers to preventive measures taken to reduce the risk of developing a disease or condition, or to reduce its severity.

[0124] As used herein, "subject" refers to any mammal that can be treated by the methods and uses described herein. Mammals include humans, other primates such as chimpanzees, bonobos and gorillas, dogs, cats, cattle, pigs, goats, and other livestock and pets. "Patient" refers to a human subject.

[0125] As used herein, “inactivation” of a gene or locus means that the expression of one or more products encoded by the gene or locus is partially or completely inhibited, for example by 10% or more, generally by 50% or more, for example by about or just 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. Inactivation can be carried out, for example, by partial or complete cleavage of a locus and / or by insertion of an exogenous gene, such as a therapeutic gene.

[0126] In this specification, abbreviations for protecting groups, amino acids, and other compounds shall conform to their common usage, recognized abbreviations, or those of the IUPAC-IUB Committee on Biochemical Nomenclature (see (1972) Biochem.11:1726), unless otherwise specified.

[0127] To clarify the disclosure, rather than limiting it, we will divide the detailed explanation into the following subsections.

[0128] B. Selection of components for cell-assisted viral expression systems (CAVES) Cells have been used as carriers for therapeutic oncolytic viruses. It was understood that viruses should be loaded onto cells ex vivo with the aim of loading as many viruses as possible per cell (see, e.g., Kim et al. (2015) Viruses 7:6200-6217). Generally, to achieve this, a multiplicity of infection (MOI) of at least 200 or more viruses / cells is used. It was also understood that viruses should be loaded as quickly as possible to avoid premature initiation of viral replication, which reduces the viability of the cell carrier and increases premature presentation of viral antigens on the cell surface, leading to elimination by the host immune system (see, e.g., Kim et al. (2015) Viruses 7:6200-6217). This specification shows that this prior understanding is incorrect. In this specification, cell infection should be performed at a low MOI, generally less than 10, e.g., less than or equal to 0.1, and a maximum of about 1 MOI / cell. The incubation of the virus with the cells should proceed long enough for viral replication to begin and for the expression of viral genes, e.g., immunomodulatory gene products and therapeutic products, to occur. Generally, the virus-containing cells provided herein should contain less than about 100 virus particles / cell at the time of administration or during storage by freezing or refrigeration for future use, and should express proteins encoded by the virus. The specific amount of virus depends on the selected cells and virus. For example, vaccinia virus and stem cells, e.g., MSCs or adipose-derived SVF cells, must be incubated for at least 6 hours, a maximum of about 35, about 40, or about 50 hours. The initial MOI should be less than 10 pfu / cell, e.g., 0.1–10, or 0.01–10, or 0.1–1 virus particles / cell. Generally, the cells are stem cells or primary cells, such as fibroblasts, and not cancer cells, including inactivated cancer cells and / or immune cells.

[0129] 1.Cells Oncolytic viruses (OVs) have the ability to preferentially infect, accumulate in, and kill tumor cells compared to normal cells. This ability may be an inherent feature of the virus (e.g., poxviruses, reoviruses, Newcastle disease viruses, and mumps viruses), or viruses may be modified or selected for this property. Viruses can be genetically attenuated or modified to evade antiviral immunity and other defenses in a target (e.g., vesicular stomatitis virus, herpes simplex virus, adenovirus), and as a result, they preferentially accumulate in tumor cells or the tumor microenvironment, and / or this preference for tumor cells can be selectively applied to or manipulated against viruses using, for example, tumor-specific cell surface molecules, transcription factors, and tissue-specific microRNAs (see, e.g., Cattaneo et al., Nat. Rev. Microbiol., 6(7):529-540 (2008); Dorer et al., Adv. Drug Deliv. Rev., 61(7-8):554-571 (2009); Kelly et al., Mol. Ther., 17(3):409-416 (2009); and Naik et al., Expert Opin. Biol. Ther., 9(9):1163-1176 (2009)).

[0130] Oncolytic viruses can be delivered via direct intratumoral injection. Direct intratumoral delivery minimizes exposure of normal cells to the virus, but it often has limitations, for example, due to inaccessibility to the tumor site (e.g., brain tumors) or for tumors or metastatic diseases that are in the form of several small nodules spreading over a large area. The virus can be delivered systemically or locally, such as by intravenous or intraperitoneal administration, and through other such routes. Systemic delivery can deliver the virus not only to the primary tumor site but also to disseminated metastases.

[0131] However, regardless of the mode of delivery, the success of treatment with oncolytic viruses can induce an immune response that can be impaired by the host's immune system neutralizing the virus (Kerrigan et al. (2017) Cytotherapy 19(4):445-457; Roy and Bell (2013) Oncolytic Virotherapy 2:47-56). For example, intravenously delivered viruses are exposed to complement and various immune cells, as well as antibodies, and are isolated and subsequently eliminated in organs such as the lungs, spleen, and liver (Roy and Bell (2013) Oncolytic Virotherapy 2:47-56). The host's immune system has evolved to eliminate viruses. For example, neutralizing antibodies (NAbs) bind to viruses, block their attachment to cell surface receptors, inhibit viral infection, and thus limit the therapeutic potential of the administered therapeutic virus (Jennings et al. (2014) Int.J.Cancer 134:1091-1101). In addition to the innate immune response, prior exposure that leads to adaptive immunity is more specific and potent and can also limit the therapeutic potential of OV. Physical barriers such as the extracellular matrix of tumors and high interstitial fluid pressure can hinder the efficient delivery of viral particles to tumor cells (Roy and Bell (2013) Oncolytic Virotherapy 2:47-56).

[0132] The vast majority of human individuals have been exposed to several viruses, including measles virus, adenovirus, vaccinia virus, and reovirus, and consequently exhibit pre-existing antiviral immunity that can reduce the therapeutic efficacy of oncolytic virus therapy. For example, most subjects born before the mid-1970s have received smallpox vaccination, which provides pre-existing antiviral immunity to orthopoxviruses, including vaccinia virus. Even if a subject does not yet have pre-existing immunity to a particular OV, the initial dose of the virus elicits an antiviral immune response, limiting the effectiveness of repeated doses that may be required to achieve a potent antitumor response. Strategies to circumvent this include the use of immunosuppressants such as cyclophosphamide and the use of carrier cells (cell media) to deliver OV to the tumor site by bypassing the immune system.

[0133] Transient immunosuppression using immunosuppressants such as cyclophosphamide, tacrolimus, mycophenolate mofetil, and methylprednisolone succinate sodium has been used in organ transplantation, but has had limited success in enhancing tumor delivery of systemically administered OV (Guo et al. (2010) Gene Ther. 17(12):1465-1475). The use of immunosuppressants can also increase the potential virulence of the virus and, furthermore, reduce the antitumor response initiated by the immune system that aids in tumor lysis (Thorne et al. (2010) Molecular Therapy 18(9):1698-1705).

[0134] Carrier cells were used for OV delivery. Carrier cells can mimic the way viruses evolve and spread within a host. For example, human immunodeficiency virus binds to circulating dendritic cells (DCs) and macrophages, which then migrate to lymph nodes, where the virus targets: CD4 +This allows the virus to reach T cells. Furthermore, viruses that replicate by spreading from cell to cell can evade neutralizing antibodies. Clinical trials have shown that oncolytic reoviruses, when administered intravenously, bind to circulating cells, retain their infectivity even in the presence of neutralizing antibodies, and reach tumor cells (Roy and Bell (2013) Oncolytic Virotherapy 2:47-56). Advantages of using cell-based media include the specific delivery of OV to tumor cells, increased therapeutic potential and prevention of extratarget toxicity, as well as the ability to protect OV from existing antiviral immunity.

[0135] The effectiveness of carrier cells for delivering OV is not limited but depends on several factors, including: (1) ex vivo loading of the virus; (2) in vivo accumulation of the virus at the tumor site; and (3) viral amplification / production at the tumor site (Guo et al. (2010) Gene Ther. 17(12):1465-1475). Ideal carrier cells not only protect OV from neutralization by the immune system but also deliver OV specifically to the tumor and possess their own antitumor activity. Carrier cells should be safe to administer, easy to isolate and / or manufacture, susceptible to viral infection, allow for viral replication, and release the virus at the tumor site before being destroyed.

[0136] In the case of CAVES provided herein, the carrier cells must be able to promote ex vivo amplification (replication) of the virus and the expression of at least one virally encoded immunomodulatory protein and / or recombinantly expressed therapeutic protein, in addition to being effective for OV delivery. In the system provided herein, cells such as carrier cells are incubated with the virus for a predetermined time to enable ex vivo replication (replication) of the virus and the expression of at least one virally encoded immunomodulatory protein and / or recombinantly expressed therapeutic protein. The length of time can vary depending on the viral replication cycle, for example, from more than 2 hours, more than 3 hours, more than 4 hours, for example, from 6 to 48 hours to for example, more than 72 hours. For example, VSV is a rapidly replicating virus, and delivery via carrier cells can be achieved by injecting cells 1 to 2 hours after infection. Slowly replicating viruses such as vaccinia virus offer greater flexibility in optimizing the timing of infection and carrier cell delivery.

[0137] The cells used in the systems provided herein may be autologous or allogeneic. The use of autologous carrier cells (cells obtained from the subject being treated) can minimize the innate and immune responses directed towards the carrier cells, but they can be cumbersome for the subject, expensive, and their availability may be limited. Allogeneic carrier cells, which may include a variety of readily isolateable and / or commercially available cells / cell lines, offer ease of availability and non-invasiveness to the subject, but extraordinarily large innate and / or adaptive immune responses can impair therapeutic efficacy and clinical applicability.

[0138] The systems provided herein overcome or mitigate the host immune response to the therapeutic effect of a virus, i.e., by providing a head start through pre-administration ex vivoviral replication and immunomodulation and / or recombinant expression of therapeutic protein(s), thereby enabling the use of allogeneic cells to create the systems. The cells used in the systems provided herein can also be fitted or optimized to be immunologically compatible, or otherwise optimal, in terms of therapeutic efficacy for the specific target being treated. Methods for matching cells with viruses and, furthermore, with targets are described in U.S. Provisional Patent Application No. 62 / 680,570 and U.S. Patent Application No. 16 / 536,073. The cells used in the systems provided herein can also be modified to overcome or mitigate the immune host response, as described in U.S. Provisional Patent Application No. 62 / 680,570 and U.S. Patent Application No. 16 / 536,073, and below. The modified cells can also be fitted with viruses and / or targets being treated.

[0139] In some embodiments, the carrier cells used to construct the CAVES system provided herein are not tumor cells. In other embodiments, the carrier cells are not cancer cells. In yet another embodiment, the carrier cells are not cancer cell lines such as immortalized cancer cell lines. In embodiments, the carrier cells are selected from stromal cells, stem cells, and fibroblasts.

[0140] Exemplary cells, such as carrier cells that can be used to construct the systems provided herein, are listed below.

[0141] Stem cells, immune cells, cancer cell lines Stem cells, immune cells, and tumor / cancerous cells can be used as delivery vehicles for oncolytic viruses (OVs), including HSV-1, parvovirus, measles virus, varicella stomatitis virus (VSV), vaccinia virus, reovirus, Newcastle disease virus, and adenovirus. These cells exhibit tumor-homing properties that enhance the therapeutic effect of OVs. This tumor selectivity is due to the attraction of these cells to the tumor microenvironment, which is characterized by hypoxia, inflammation, and numerous chemoattractant molecules such as cytokines and chemokines.

[0142] stem cells Stem cells possess an intrinsic tumor-homing ability, which makes them attractive as carrier cells for oncolytic virus therapy. This is due to the tumor microenvironment (TME), which is rich in various growth factors, angiogenic factors, cytokines, and chemokines that support unregulated tumor growth. The hypoxic nature of the TME also facilitates the migration of stem cells to tumors. Stem cells are also used as carrier cells because they are highly immunosuppressive and express molecules necessary for antigen processing and presentation at low levels, delaying the immune system's recognition of the virus they carry (Kim et al. (2015) Viruses 7:6200-6217). Examples of stem cells that can be used as carrier cells for OV include endothelial progenitor cells, neural stem cells, and mesenchymal stem cells.

[0143] Endothelial progenitor cells have been shown to return to sites of neovascularization in tumors and have been used to deliver oncolytic measles virus in a mouse model of human glioma (Guo et al. (2008) Biochim Biophys Acta 1785(2):217-231). These cells divide rapidly in vivo but are not immortal, and new cells must be repeatedly isolated from clinical samples (Kim et al. (2015) Viruses 7:6200-6217).

[0144] Neural stem cells (NSCs), which differentiate into various different cells of the nervous system, including neurons and glial cells, were the first stem cells investigated as carrier cells for delivering therapeutic drugs to brain tumors (Kerrigan et al. (2017) Cytotherapy 19(4):445-457). NSCs show strong tropism to glioblastoma tumors due to the expression of hypoxia-inducible factor (HIF)-mediated stromal cell-derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and urokinase plasminogen activator (uPA) in glioma cells (Kim et al. (2015) Viruses 7:6200-6217). NSCs have been used to deliver OVs to gliomas, such as IL-4, IL-12, IL-23, cytosine deaminase, the anti-angiogenic protein thrombospondin, and adenoviruses. NSCs must be isolated from fetal brain tissue or the periventricular zone of adult brains during surgery, which is disadvantageous for their usefulness as carrier cells.

[0145] Adult human bone marrow has been used as an alternative source of stem cells because bone marrow stem cells can be easily obtained and supplied from the patient themselves for autologous transplantation, eliminating immune rejection (Kerrigan et al. (2017) Cytotherapy 19(4):445-457). Among the various bone marrow stem cells available, mesenchymal stem cells (MSCs) are attractive as carrier cells because they can be easily isolated from patients, proliferated in vitro, support OV replication and protection from immediate neutralization by the immune system, are easily manipulated, and essentially homing to tumors in vivo due to tumor-associated expression of inflammatory cytokines. MSCs can even be used as standalone anticancer agents. For example, studies have demonstrated the tumor-homing ability and oncolytic effect of IFN-β expressing MSCs (Nakashima et al. (2010) Cytokine Growth Factor Rev.21(2-3):119-126).

[0146] MSCs express low levels of MHC class I molecules and do not express MHC class II molecules on their cell surface, making them suitable for allogeneic transplantation. MSCs can inhibit T cell proliferation and monocyte differentiation into dendritic cells (DCs), and can suppress the expression of interferon-γ and tumor necrosis factor produced by CD4+ T helper cells (Kim et al. (2015) Viruses 7:6200-6217). MSCs can also degrade the extracellular matrix through the secretion of proteases, which can help overcome physical barriers to oncolytic virus delivery (Ramirez et al. (2015) Oncolytic Virotherapy 4:149-155). Another advantage of using MSCs is that they can be frozen after viral infection and retain active viral replication and antitumor activity upon thawing, allowing for storage (Roy and Bell (2013) Oncolytic Virotherapy 2:47-56). In addition to bone marrow, MSCs can be isolated from adipose tissue, umbilical cord blood, peripheral blood, muscle, cartilage, and amniotic fluid. Adipose tissue is the most attractive source due to its easy accessibility and abundance (Kerrigan et al. (2017) Cytotherapy 19(4):445-457; Nakashima et al. (2010) Cytokine Growth Factor Rev.21(2-3):119-126).

[0147] MSCs have served as carriers of oncolytic adenoviruses for treating pancreatic cancer, brain cancer, renal cell carcinoma, glioblastoma, and ovarian cancer, as well as for treating measles virus for treating ovarian cancer and hepatocellular carcinoma (Kim et al. (2015) Viruses 7:6200-6217). For example, MSCs have been used as carriers of the oncolytic adenovirus ICOVIR-5 for treating children with progressive metastatic neuroblastoma (Kerrigan et al. (2017) Cytotherapy 19(4):445-457; Ramirez et al. (2015) Oncolytic Virotherapy 4:149-155).

[0148] However, one drawback of using MSCs is their ability to promote tumor growth, which has been demonstrated in models of breast cancer, endometrial tumors, and gliomas. To overcome the cause of this potential failure, MSCs can be engineered to reliably destroy themselves upon delivery of OV, for example, by possessing suicide genes (Kerrigan et al. (2017) Cytotherapy 19(4):445-457).

[0149] Examples of stem cells (autologous or allogeneic) that can be used as carrier cells include, for example, adult stem cells, embryonic stem cells, fetal stem cells, neural stem cells, mesenchymal stem cells, totipotent stem cells, pluripotent stem cells, induced pluripotent stem cells, compound pluripotent stem cells, oligopluripotent stem cells, unipotent stem cells, adipocyte stem cells, endothelial stem cells (e.g., endothelial progenitor cells, placental endothelial progenitor cells, angiogenic endothelial cells, pericytes), adult peripheral blood stem cells, myoblasts, small immature stem cells, cutaneous fibroblast stem cells, tissue / tumor-associated fibroblasts, epithelial stem cells, and embryonic epithelial stem cells.

[0150] Mesenchymal cells include, but are not limited to, those from adult bone marrow, adipose tissue, blood, dental pulp, neonatal umbilical cord, umbilical cord blood, placental mesenchymal cells, placental-derived adhesive stromal cells, placental-derived decidual stromal cells, endometrial regenerative cells, placental dipotent endothelial / mesenchymal progenitor cells, amniotic or amniotic fluid mesenchymal stem cells, amniotic fluid-derived progenitor cells, Howarton's jelly mesenchymal stem cells, pelvic girdle stem cells, chorionic villous mesenchymal stromal cells, subcutaneous white adipose mesenchymal stem cells, pericytes, perisinus reticular cells, hair follicle-derived stem cells, hematopoietic stem cells, periosteum-derived mesenchymal stem cells, lateral plate mesenchymal stem cells, deciduous tooth stem cells, periodontal ligament stem cells, dental follicle progenitor cells, dental papilla-derived stem cells, muscle satellite cells, and other mesenchymal stem cells isolated from / derived from such cells.

[0151] A population of cells derived from adipose-derived interstitial vascular cells. In some embodiments, the carrier cells used in the systems and methods provided herein are freshly isolated from adipose tissue stromal vascular cells (SVF) and / or cultured adipose-derived mesenchymal stromal / stem cells (AD-MSC) derived from SVF. Any of the oncolytic viruses known to those skilled in the art and provided herein can be combined with such cells to generate the CAVES systems provided herein and / or used in the manner provided herein. In some embodiments, the oncolytic virus is vaccinia virus (VACV). In embodiments, the VACV is ACAM2000 having the sequence shown in SEQ ID NO: 70 or CAL1 virus having the sequence shown in SEQ ID NO: 71.

[0152] The ability of these carrier cells to protect, deliver, and amplify viruses, as well as to overcome innate and adaptive immune barriers, was analyzed by flow cytometry, microscopy, and viral plaque assays of ex vivo co-cultures of these cells infected with VACV in the presence of human serum or peripheral blood mononuclear cells derived from healthy donors. Comparative analyses were performed to establish statistically significant correlations and to assess the effect of stem cells on the activity of key immune cell populations. SVF cells were found to be able to protect VACV from serum inactivation. Cell sorting demonstrated that epiadipocytes (SA-ASCs; CD235a- / CD45- / CD34+ / CD146- / CD31-) and pericytes (CD235a- / CD45- / CD34- / CD146+ / CD31-) were the primary populations of SVF cells most efficient for the delivery of oncolytic viruses to tumor cells. This demonstrates the validity of their clinical use as a tool for enhancing oncolytic virus therapy in both the self-environment and the allogeneic environment.

[0153] Cultured AD-MSCs (derived from CD34+SA-ASCs) as a delivery medium have been demonstrated herein to protect against serum inactivation and to amplify viruses in the presence of human PBMCs in autologous and allogeneic settings. This may be related to their inherent immunosuppressive properties and avoidance of allogeneic rejection. Hereinafter, these cells are shown to provide transient immunosuppression by inhibiting antiviral responses derived from both innate (NK) and adaptive (T) immune cells, thereby enhancing viral oncolysis and the generation of antitumor immunity.

[0154] SA-ASCs and pericytes for use as carrier cells with oncolytic viruses known to those skilled in the art, including any of those described or provided herein, are provided herein. These include, but are not limited to, poxviruses, adenoviruses, herpes simplex virus, Newcastle disease virus, varicella stomatitis virus, mumps virus, influenza virus, measles virus, reovirus, human immunodeficiency virus (HIV), hantavirus, myxoma virus, cytomegalovirus (CMV), and lentivirus. Examples of viruses include vaccinia viruses, e.g., ACAM1000 and ACAM2000, the example of which is the sequence shown in SEQ ID NO: 70, or CAL1, the example of which is the sequence shown in SEQ ID NO: 71, or minor variations (sequence identity of 85%, 90%, 95%, 96%, 97%, 98%, 99% or more in the genome excluding the ITR, and lower sequence identity in the case of recombination of the ITR during replication). SA-ASCs (CD235a- / CD45- / CD34+ / CD146- / CD31-) and pericytes (CD235a- / CD45- / CD34- / CD146+ / CD31-), SA-ASCs and / or pericytes and / or AD-MSCs produced by culturing such cells can be incubated together for a period sufficient to produce the CAVES system provided herein, with at least one immunomodulatory or recombinant therapeutic protein expressed by an oncolytic virus on the surface or inside the SA-ASC, pericyte, or AD MSC carrier cells.

[0155] Selection of cell populations from adipose-derived SVF that promote viral infection and / or replication can be performed, for example, by incubating the SVF with an oncolytic virus, such as ACAM1000, ACAM2000, or CAL1, at a temperature suitable for such infection, such as room temperature (about 20°C) or higher, such as 32–42°C, for example 35–40°C, typically 37°C. Loading of the virus onto cells at an appropriate MOI, generally a low MOI, for example about 0.001–10, for example 0.01–1.0, or less than 1.0, can be performed, for example, at 20 RPM with continuous rotation for about 20 minutes to about 5 hours, generally about 30 minutes to about 2 hours. In several embodiments, incubation is about 1 hour. After loading SVF cells or their subpopulations, or MSCs produced therefrom, with oncolytic viruses, the cells can be labeled with a panel of antibodies against cell surface markers for different cell populations, such as CD235a, CD45, CD34, CD31, and CD146, and stained for viability with an appropriate stain such as propidium iodide (PI). Subsequently, SVF cells can be sorted by flow cytometry based on the expression of the cell surface markers. In adipose-derived SVF, seven distinct cell populations were identified and selected: erythrocytes (CD235a+); superembryonic adipose-astrophic stromal cells (SA-ASC; CD235a- / CD45- / CD34+ / CD146- / CD31-), which are the major MSC precursors in culture; pericytes (CD235a- / CD45- / CD34- / CD146+ / CD31-), which are also MSC precursors in culture; granulocytes (CD235a- / CD45 medium / high, high lateral scattering (SSC)); lymphocytes (CD235a- / CD45 high, low SSC); monocytes (CD235a- / CD45 high SSC medium); and endothelial progenitor cells (CD235a- / CD45- / CD34+ / CD146+ / CD31+). The composition (%) of the main cell populations is described in Example 4.

[0156] To measure viral infection, individual cell populations selected from SVF are then seeded onto a suitable recipient cell monolayer, e.g., A549 tumor cell monolayer, and incubated for a period allowing plaque formation, e.g., 1–5 days, e.g., 1–3 days, 3 days, or approximately 3 days. The number of plaques formed on the cell monolayer can be measured by fixing and staining with a suitable stain such as crystal violet to determine the number of cells from each selected population harboring the oncolytic virus. In adipose SVF, five different cell populations—erythrocytes, SA-ASCs, pericytes, granulocytes, and lymphocytes—were found to harbor oncolytic viruses such as vaccinia viruses, e.g., ACAM2000 or CAL1. The main cell populations from three SVF fractions harboring vaccinia viruses (e.g., ACAM2000 or CAL1) were identified as SA-ASCs (MSC precursors) and pericytes. These cell populations can also promote viral amplification.

[0157] Accordingly, the Specified Specified Use

[0158] immune cells Immune cells that respond to “danger signals” released from tumors upon transport to cancer sites have been investigated as carrier cells for OV. Immune cells include, but are not limited to, CAR-T cells that target tumor-specific antigens, TCR transgenic cells that target tumor-specific antigens; NKT cells, lymphocytes, monocytes, macrophages, mast cells, granulocytes, dendritic cells (DCs), natural killer (NK) cells, bone marrow-derived suppressor cells, lymphokine-activated killer (LAK) cells, and cytokine-induced killer (CIK) cells. Immune cells are attractive as carrier cells because they circulate throughout the body and can recognize tumors (Roy and Bell (2013) Oncolytic Virotherapy 2:47-56). Furthermore, using immune cells as carriers for OV provides additional antitumor activity, either in the form of direct cytotoxicity or by priming an adaptive antitumor immune response (Jennings et al. (2014) Int. J. Cancer 134:1091-1101).

[0159] For example, tumor antigen-specific T cells have shown direct anti-cancer effector function, and activated T cells have been widely investigated in the delivery of OV to tumors. Because the pro-inflammatory nature of viral infection can prevent T cell silencing and inactivation, it has been shown that loading OV onto adoptive T cells can help combat the immunosuppressive nature of the tumor microenvironment (Roy and Bell (2013) Oncolytic Virotherapy 2:47-56). Intratumoral expression of chemokines such as CCL3, CCL21, and CXCL10 (IP-10) enhances tumor-specific transport of adoptive T cells. T cells can also be genetically engineered to express chemokine receptors such as CXCR2 and directed towards tumors (Guo et al. (2008) Biochim Biophys Acta 1785(2):217-231). Studies have demonstrated that vesicular stomatitis virus, reovirus, herpes simplex virus, Newcastle disease virus, and retroviral particles can adhere to the surface of T cells and be delivered to tumor cells passively or via cellular synapses between the carrier and the tumor cell (Roy and Bell (2013) Oncolytic Virotherapy 2:47-56). Despite the advantages of using T cells as carriers for OV, generating T cell populations against highly tumor-specific antigens from patients remains extremely expensive and difficult, limiting their use (Willmon et al. (2009) Molecular Therapy 17(10):1667-1676).

[0160] Lymphokine-activated killer cells (LAK cells) are used in combination with IL-2 in the treatment of ovarian cancer. Immature dendritic cells (iDCs), LAK cells, and co-cultures thereof (LAKDCs) have been tested as reovirus carriers in the treatment of ovarian cancer, and it has been shown that reovirus-carrying LAKDCs can protect reoviruses from neutralizing antibodies, induce a pro-inflammatory cytokine environment, and generate innate and adaptive antitumor immune responses (Jennings et al. (2014) Int. J. Cancer 134:1091-1101). DC cells have also been used as reovirus carriers for the treatment of melanoma (Jennings et al. (2014) Int. J. Cancer 134:1091-1101).

[0161] CIK cells are another type of immune cell that can be used as carriers for OV. While tumor antigen-specific T cells recognize one antigen, CIK cells recognize NKG2D ligands that are upregulated in various tumor cells, making them more versatile. CIK cells are also easy to isolate from patients and grow ex vivo, can produce high-titer viruses, and have been used to deliver measles and vaccinia viruses to tumors (Roy and Bell (2013) Oncolytic Virotherapy 2:47-56; Willmon et al. (2009) Molecular Therapy 17(10):1667-1676; Power and Bell (2007) Mol.Ther. 15(4):660-665). However, one drawback of using CIK cells is that their production requires the proliferation of primary leukocytes in vivo using cytokines (Kim et al. (2015) Viruses 7:6200-6217).

[0162] Macrophages represent yet another potential class of carrier cells for oncolytic adenoviruses (OV). Tumors typically secrete monocyte chemotactic protein-1, macrophage colony-stimulating factor, and VEGF, so that monocytes spontaneously migrate to tumor sites, localize to hypoxic areas, and differentiate into tumor-associated macrophages, which can enhance tumor growth inhibition (Roy and Bell (2013) Oncolytic Virotherapy 2:47-56). Consequently, macrophages have been preclinically investigated for the delivery of oncolytic adenoviruses and measles viruses. In addition to other types of immune cells discussed, myeloid-derived suppressor cells have also been investigated as carrier cells for the delivery of oncolytic VSVs.

[0163] cancer cells For safety reasons, cancer cells that are typically inactivated by gamma irradiation before administration have also been used as carrier cells for OV. Gamma irradiation can prevent tumorigenicity but maintains virus production. Another safety measure involves manipulating OV to express suicide genes such as thymidine kinase (i.e., to kill them and make them no longer immortal) to ensure that cancer cells do not remain indefinitely within the target. Alternatively, allogeneic cancer cells that are typically eliminated by the recipient's immune system can be used (Power and Bell (2007) Mol.Ther. 15(4):660-665).

[0164] Cancer cells are readily available in large quantities and can exhibit higher levels of viral infectivity and amplification than normal cells (Guo et al. (2010) Gene Ther. 17(12):1465-1475; Roy and Bell (2013) Oncolytic Virotherapy 2:47-56). Furthermore, as seen in metastatic diseases, some tumor cells migrate specifically to certain organs. For example, myeloma cells express high levels of the chemokine receptor CXCR4, leading to myelometra and have been used for the delivery of oncolytic measles virus (Roy and Bell (2013) Oncolytic Virotherapy 2:47-56). Various transformed cell lines have been shown to deliver oncolytic parvovirus, measles virus, and varicella-stomatitis virus in immunonormal and immunodeficient animals. For example, cancer cells infected with VSV or adenovirus have been used to effectively deliver the virus to lung metastases in mice (Willmon et al. (2009) Molecular Therapy 17(10):1667-1676; Power and Bell (2007) Mol.Ther. 15(4):660-665). However, cells derived from solid tumors have been shown to accumulate in the lungs of mice after IV administration due to their larger diameter. Consequently, hematopoietic / blood-derived cancer cells may be a better alternative because they are more widely distributed in the body and can deliver OV to anatomical locations outside the lungs (Power and Bell (2007) Mol.Ther. 15(4):660-665).

[0165] Examples of allogeneic human hematological malignancy cell lines that can be used as carrier cells include leukemia cells (e.g., KASUMI-1, HL-60, THP-1, K-562, RS4;11, MOLT-4, CCRF-CEM, JVM-13, 31E9, ARH-77, MoB, JM1, NALM-1, ProPak-X.36, etc.); T-cell leukemia cells (e.g., HM-2, CEM-CM3, Jurkat / Jurkat clone E6-1, J.CaM1.6, BCL2 Jurkat, BCL2 S87A Jurkat, BCL2 S70A Jurkat, Neo Jurkat, BCL2 AAA) Jurkat, J.RT3-T3.5, J45.01, J.γ1, J.γ1.WT, JK28, P116, P116.cl39, A3, JX17, D1.1, I 9.2, I 2.1 etc); myelomonocytic leukemia cells (e.g., MV-4-11); lymphoma cells (e.g., HT, BC-3, CA46, Raji, Daudi, GA-10-Clone-4, HH, H9); non-Hodgkin lymphoma cells (e.g., SU-DHL-1, SU-DHL-2, SU-DHL-4, SU-DHL-5, SU-DHL-6, SU-DHL-8, SU-DHL-10, SU-DHL-16, NU-DUL-1, NCEB-1, EJ-1, BCP-1, TUR, U-937 etc.); Burkitt lymphoma cells (e.g., Ramos / RA 1, Ramos.2G6.4C10, P3HR-1, Daudi, ST486, Raji, CA46, human gamma herpesvirus 4 / HHV-4 cheek tumors from Burkitt lymphoma patients, DG-75, GA-10, NAMALWA, HS-Sultan, Jiyoye, NC-37, 20-B8, EB2, 1G2, EB1, EB3, 2B8, GA-10 clone 20, HKB-11 / kidney-B cell hybrid); diffuse large B cell lymphoma cells (e.g., Toledo, Pfeiffer); mantle cell lymphoma cells (e.g., JeKo-1, JMP-1, PF-1, JVM-2, REC-1, Z-138, Mino, MAVER-1); AML cells (e.g., AML-193, BDCM, KG-1, KG-1a, Kasumi-6, HL-60 / S4); CML cells (e.g., K562, K562-r, K562-s, LAMA84-r, LAMA84-s, AR230-r, AR230-s); ALL cells (e.g., N6 / ADR, RS4;11, NALM6 clone G5, Luucy, SUP-B15, CCRF-SB); erythroleukemia cells (e.g., IDH2-mutant-TF-1 isogenic cell line); myelomonoblastic leukemia cells (e.g., GDM-1) Examples include malignant non-Hodgkin NK lymphoma cells (e.g., NK-92, NK-92MI); myeloma / plasmacytoma cells (e.g., U266B1 / U266, HAA1, SA13, RPMI8226, NCI-H929, MC / CAR); multiple myeloma cells (e.g., MM.1R, IM-9, MM.1S); and macrophage cell lines (e.g., MD, SC, WBC264-9C).

[0166] Commercially available allogeneic cell lines include, for example, mesenchymal stem cells such as APCETH-201, APCETH-301 (APCETH), Cx601 (TIGENIX), TEMCELL, MSC-100-IV, and Prochymal (MESOBLAST); induced pluripotent stem cells (iPSCs) such as ToleraCyte (Fate Therapeutics); fibroblasts such as CCD-16Lu and WI-38; tumor-associated fibroblasts such as Malme-3M, COLO 829, HT-144, Hs 895.T, and hTERT PF179T CAF; endothelial cells such as HUVEC, HUVEC / TERT 2, and TIME; and embryonic epithelial cells such as HEK-293, HEK-293 STF, 293T / 17, and 293T / 17. This includes SF, HEK-293.2sus; embryonic stem cells, e.g., hESC BG01V; and epithelial cells, e.g., NuLi-1, ARPE-19, VK2 / E6E7, Ect1 / E6E7, RWPE-2, WPE-stem, End1 / E6E7, WPMY-1, NL20, NL20-TA, WT 9-7, WPE1-NB26, WPE-int, RWPE2-W99, and BEAS-2B.

[0167] Autologous or allogeneic whole tumor cell vaccines include, for example, GM-CSF secreting whole tumor cell vaccines (GVAX) such as GVAX Prostate (based on PC3 / LNCaP); GVAX Pancreas; GVAX Lung; and GVAX Renal Cell from Cell Genesys / BioSante / Aduro Biotech.

[0168] Allogeneic human tumor cell lines include, for example, the NCI-60 panel (BT549, HS 578T, MCF7, MDA-MB-231, MDA-MB-468, T-47D, SF268, SF295, SF539, SNB-19, SNB-75, U251, Colo205, HCC 2998, HCT-116, HCT-15, HT29, KM12, SW620, 786-O, A498, ACHN, CAKI, RXF) 393, SN12C, TK-10, UO-31, CCRF-CEM, HL-60, K562, MOLT-4, RPMI-8226, SR, A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-H522, LOX IMVI, M14, MALME-3M, MDA-MB-435, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3, NCI-ADR-RES, DU145, PC-3). Other allogeneic human tumor cell lines include, for example, fibrosarcoma cell line (HT-1080); hepatocarcinoma cell line (Hih-7); prostate cancer cell lines (LAPC4, LAPC9, VCaP, LuCaP, MDA PCa 2a / 2b, C4, C4-2, PTEN-CaP8, PTEN-P8); breast cancer cell lines (HCC1599, HCC1937, HCC1143, MDA-MB-468, HCC38, HCC70, HCC1806, HCC1187, DU4475, BT-549, Hs 578T, MDA-MB-231, MDA-MB-436, MDA-MB-157, MDA-MB-453, HCC1599, HCC1937, HCC1143, MDA-MB-468, HCC38, HCC70, HCC1806, HCC1187, DU4475, BT-549, Hs 578T, MDA-MB-231, MDA-MB-436, MDA-MB-157, MDA-MB-453, BT-20, HCC1395, MDA-MB-361, EMT6, T-47D, HCC1954); Head and neck cancer cell lines (A-253, SCC-15, SCC-25, SCC-9, FaDu, Detroit 562);Lung cell lines (NCI-H2126, NCI-H1299, NCI-H1437, NCI-H1563, NCI-H1573, NCI-H1975, NCI-H661, Calu-3, NCI-H441); Sac cell lines (Capan-2, Panc 10.05, CFPAC-1, HPAF-II, SW1990, BxPC-3, AsPC-1, MIA PaCa-2, Hs 766T, Panc 05.04, PL45); Ovary cell lines (PA-1, Caov-3, SW626, SK-OV-3); Bone cell lines (HOS, A-673, SK-PN-DW, U-2) OS, Saos-2; colon cell lines (SNU-C1, SK-CO-1, SW1116, SW948, T84, LS123, LoVo, SW837, SNU-C1, SW48, RKO, COLO) 205, SW1417, LS411N, NCI-H508, HT-29, Caco-2, DLD-1; gastric cell lines (KATOIII, NCI-N87, SNU-16, SNU-5, AGS, SNU-1); gynecological cell lines (SK-LMS-1, HT-3, ME-180, Caov-3, SW626, MES-SA, SK-UT-1, KLE, AN3-CA, HeLa); sarcoma cell lines (SW684, HT-1080, SW982, RD, GCT, SW872, SJSA-1, MES-SA / MX2, MES-SA, SK-ES-1, SU-CCS-1, A-673, VA-ES-BJ, Hs 822.T, RD-ES, HS 132.T, Hs 737.T, Hs 863.T, Hs 127.T, Hs 324.T, Hs 821.T, Hs 706.T, Hs 707(B).Ep, LL 86 / LeSa, Hs 57.T, Hs 925.T, GCT, KHOS-312H, KHOS / NP R-970-5, SK-LMS-1, HOS);Black tumor cell lines (SK-MEL-1, A375, G-361, SK-MEL-3, SH-4, SK-MEL-24, RPMI-7951, CHL-1, Hs 695T, A2058, VMM18, A375.S2, Hs 294T, VMM39, A375-P, VMM917, VMM5A, VMM15, VMM425, VMM17, VMM1, A375-MA1, A375-MA2, SK-MEL-5, Hs 852.T, LM-MEL-57, A101D, LM-MEL-41, LM-MEL-42, MeWo, LM-MEL-53, MDA-MB-435S, C32, SK-MEL-28, SK-MEL-2, MP38, MP41, C32TG, NM2C5, LM-MEL-1a, A7 / M2A7; squamous cell carcinoma cell lines (SiHa, NCI-H520, SCC-15, NCI-H226, HCC1806, SCC-25, FaDu, SW 954, NCI-H2170, SCC-4, SW 900, NCI-H2286, NCI-H2066, SCC-9, SCaBER, SW579, SK-MES-1, 2A3, UPCI:SCC090, UPCI:SCC152, CAL 27, RPMI 2650, UPCI:SCC154, SW756, NCI-H1703, ME-180, SW962; Hepatocellular carcinoma cell lines (Hep G2, Hep 3B2.1-7 / Hep 3B, C3A, Hep G2 / 2.2.1, SNU-449, SNU-398, SNU-475, SNU-387, SNU-182, SNU-423, PLC / PRF / 5; bladder cell lines (5637, HT-1197, HT-1376, RT4, SW780, T-24, TCCSUP, UM-UC-3); kidney cell lines (ACHN, 786-O / 786-0, 769-P, A-498, Hs 891.T, Caki-2, Caki-1); embryonal carcinoma / spermatorrhea cell lines (NTERA-2 cl.D1, NCCIT, Tera-2, Tera-1, Cates-1B); glioma cell lines (LN-229, U-87). MG, T98G, LN-18, U-118 MG, M059K, M059J, U-138 MG, A-172; astrocytoma cell lines (SW1088, CCF-STTG1, SW1783, CHLA-03-AA); brain cell lines (PFSK-1, Daoy);This includes thyroid cancer cell lines (TT, MDA-T68, MDA-T32, MDA-T120, MDA-T85, MDA-T41) and mesothelioma cell lines (NCI-H28, NCI-H226, NCI-H2452, NCI-H2052, MSTO-211H).

[0169] Compatible cells Any cells used in the systems provided herein, for example, the cells described above, can be tested for matching suitability with the target to which the virus and / or viral therapy is to be applied, i.e., the ability of the cells to overcome or improve the innate and / or adaptive immune responses to the cells and / or associated viruses. Selecting cells that are well-suited to the target can further increase the therapeutic efficacy of the systems provided herein. Optimal cell-virus combinations (e.g., the ability of cells to promote viral amplification) and methods for screening cells suitable for the target are described in U.S. Provisional Patent Application No. 62 / 680,570 and U.S. Patent Application No. 16 / 536,073. Modified cell media sensitized and / or manipulated in one or more ways for improved cell delivery are also provided herein (see, for example, the sections below).

[0170] Modified cell media with improved delivery and / or matching capabilities Also provided herein are cell media (carrier cells) whose properties are modified to facilitate the delivery of oncolytic viruses to targets and / or to improve matching with targets. Any of the cell media provided herein (e.g., stem cells, immune cells, cancer cells) can be modified in this manner. Such properties may include, but are not limited to, improved promotion of viral amplification in the cell delivery medium, improved ability to evade immune responses to the cell media and / or viruses, and / or improved immunosuppression. In embodiments, the immunomodulatory capacity (e.g., evasion of immune responses, suppression of immune responses) may be local and / or transient and present to the extent necessary to facilitate delivery, accumulation, and infection of viruses in tumors or other cancerous cells.

[0171] In some embodiments, the modified cell media provided herein can be screened using matching assays such as those described in U.S. Provisional Patent Application No. 62 / 680,570 and U.S. Patent Application No. 16 / 536,073 to confirm their suitability as cell media for the delivery of oncolytic viruses to specific targets and / or specific cancer / tumor types. In embodiments, multiple / panel modified cell media can be screened by the matching assays described in U.S. Provisional Patent Application No. 62 / 680,570 and U.S. Patent Application No. 16 / 536,073 and ranked in order of their matching ability. In some examples, the panel of cell media may include unmodified cell media.

[0172] In short, as described in U.S. Provisional Patent Application No. 62 / 680,570 and U.S. Patent Application No. 16 / 536,073, a method for adapting carrier cells to a target includes performing one or more of the following steps:

[0173] 1. In a co-culture containing a cell medium, oncolytic virus, and cells derived from the subject, the following: (a) A decrease in the level of one or more markers for T cell activation compared to conditions that are otherwise equivalent except for the absence of a cellular medium; (b) A decrease in the level of one or more markers for NK cell activation compared to conditions otherwise equivalent except in the absence of a cell medium; and (c) A step of determining whether a cell medium overcomes the immune barrier in a subject by detecting one or more decreased levels of one or more markers for NKT cell activation compared to conditions that are otherwise equivalent except in the absence of the cell medium, wherein the cell medium is suitable for the subject if one or more of (a), (b), and (c) are satisfied.

[0174] 2. (a) A step of measuring the amount of viral amplification obtained when the virus and cell medium are incubated together with cells from which the target originates; (b) A step of measuring the amount of viral amplification obtained when the virus and cell medium are incubated under equivalent conditions, except in the absence of cells from the subject; and (c) A step of comparing the amounts measured in (a) and (b), wherein the cell medium is suitable for the subject if the amplification amount measured in (a) is at least 20% of the amplification amount measured in (b).

[0175] 3. A step of identifying identical alleles in a cell medium and a target at one or more major histocompatibility complex (MHC) and / or killer cell inhibitor receptor (KIR) gene loci, and a step of identifying a cell medium as compatible with a target if, for example, 50% or more of the alleles are identical.

[0176] The modified cell media provided herein may include one or more modifications as described below in this section and elsewhere in this specification:

[0177] (i) Cell media sensitized / protected for improved viral amplification and / or immunomodulation Modified cell media (carrier cells) for generating and using the CAVES system provided herein may include one or more embodiments. In these embodiments, the cell media may be sensitized to enhance its viral amplification capacity by pretreatment / loading with one or more of the following: IL-10, TGFβ, VEGF, FGF-2, PDGF, HGF, IL-6, GM-CSF, growth factors, RTK / mTOR agonists, wnt protein ligands, and GSK3 inhibitors / antagonists (e.g., tideglucib, valporic acid). In other embodiments, for example, the cell medium can be sensitized to block the induction of an antiviral state by pre-treating / loading it with small molecule or protein inhibitors that interfere with IFN type I / II receptors and / or downstream signaling, including but not limited to IFNAR1 / IFNAR2 signaling, IFNGR1 / IFNGR2 signaling, STAT1 / 2 signaling, Jak1 signaling (e.g., tofacitinib, ruxolitinib, baracitinib), Jak2 signaling (e.g., SAR302503, LY2784544, CYT387, NS-018, BMS-911543, AT9283), IRF3 signaling, IRF7 signaling, IRF9 signaling, TYK2 signaling (e.g., BMS-986165), and TBK1 signaling (e.g., BX795, CYT387, AZ13102909).

[0178] In some embodiments, the cell medium may be pre-treated / loaded with HDAC inhibitors to interfere with / de-distort IFN signaling / responsiveness; such inhibitors may include, but are not limited to, vorinostat, romidepsin, thidamide, panobinostat, bellinostat, valporic acid, mosetinostat, abexinostat, entinostat, SB939, resminostat, gibinostat, xinostat, HBI-8000, kebetrin, CUDC-101, AR-42, CHR-2845, CHR-3996, 4SC-202, CG200745, ACY-1215, ME-344, sulforaphane, and / or trichostatin. In other embodiments, the cell medium may be pre-treated / loaded with antagonists of virus sensing and / or antiviral defense pathways mediated by STING, PKR, RIG-1, MDA-5, OAS-1 / 2 / 3, AIM2, MAVS, RIP-1 / 3, DAI(ZBP1); such antagonists may include, but are not limited to, one or more of the following: K1, E3L, K3L proteins (vaccinia), NS1 / NS2 proteins (influenza), NS3-4A (hepatitis C), NP and Z proteins (arenavirus), VP35 (Ebola virus), US11, ICP34.5, ICP0 (HSV), M45 (MCMV), and X protein (BDV: Borna disease virus).In the embodiment, cells are infected with viral MHC antagonists, such as A40R MHC antagonist (vaccinia), Nef and TAT (HIV), E3-19K (adenovirus), ICP47 (HSV-1 / 2), CPXV012 and CPXV203 (cowpox), ORF66 (VZV), EBNA1, BNLF2a, BGLF5, BILF1 (EBV), US2 / gp24, US3 / gp23, US6 / gp21, US10, US11 / gp33 (hCMV), Rh178 / VIHCE (RhCMV), U21 (HHV-6 / 7), LANA 1. Cell media can be protected from alloinactivation / rejection determinants by pretreatment / loading with one or more of the following: ORF37 / SOX, kK3 / MIR1, kK5 / MIR2 (KSHV), mK3 (MHV-68), UL41 / vhs (α-herpesvirus, HSV, BHV-1, PRV), UL49.5 (Varicerovirus, BHV-1, EHV-1 / 4, PRV), and m4 / gp34, m6 / gp48, m27, m152 / gp40 (mCMV).

[0179] In one embodiment, the modified cell medium can be pre-treated / loaded with a virus-derived B2M antagonist, such as UL18 (HCMV). In another embodiment, the cell medium can be pre-treated / loaded with an antagonist of MIC-A and MIC-B (NKG2D ligand), such as kK5 (KHSV). In some embodiments, the cell medium may be pre-treated / loaded with one or more viral immunosuppressive factors, including, but not limited to, immuno-FAS / TNF / granzyme B-induced apoptosis inhibitors (e.g., Ectromelia / Vaccinia virus SP1-2 / CrmA), IL-1 / NFκB / IRF3 antagonists (e.g., Vaccinia virus encoding N1), IL-1 and TLR antagonists (e.g., IL-18 binding proteins, A46R, A52R), IL-1β antagonists (e.g., B15R / B16R), TNFα blockers (e.g., Vaccinia virus CmrC / CmrE), IFNα / β blockers (e.g., Vaccinia virus B18R / B19R), and IFNγ blockers (e.g., Vaccinia virus B8R). In embodiments, the cell medium may be pre-treated / loaded with small molecule inhibitors of TAP1 / 2 and / or tapacin.

[0180] In embodiments, the modified cell medium can be protected from complement by, for example, pre-treating / loading the cell medium with small molecule inhibitors of complement factors (e.g., C1, C2, C3, C4, C5, MBL); such inhibitors may include, but are not limited to, VCP (vaccinia virus complement regulatory protein), B5R (vaccinia virus complement inhibitor), scFv anti-CD1q / CD1r / CD1s, anti-C3, anti-C5 (e.g., eculizumab), complement family peptide C3 inhibitors (e.g., Cp40), human soluble membrane (s / m) proteins (e.g., s / mCR1 (CD35), s / mCR2 (CD21), s / mCD55, s / mCD59), human complement factor H and derivatives, cobra venom factor, and one or more derivatives having complement inhibitory activity.

[0181] In the embodiments described above, instead of loading or treating cells with these factors, the virus can be modified to express these products, or, by the methods herein, the virus expresses these products in carrier cells when incubated with them.

[0182] Sensitized cell media can be prepared by methods known in the art. For example, the cell medium can be pre-treated with a sensitizer, such as a protein or small molecule agonist / antagonist, by incubation for 10 minutes to 48 hours or more before cell banking, viral infection, or administration to a subject, for example, about or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes or about or at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours or more. To enhance the loading of protein / lipid-insoluble small molecules into cells, lipofectamine or alternative protein transfection reagents such as Xfect (Takara), Pierce Pro-Ject (ThermoFisher), Pro-DeliverIN (OZ Biosciences), and TurboFect (Fermentas) can be used.

[0183] (ii) Sensitized for resistance to virus-borne killing (for extended survival time and improved local immunosuppression) In some embodiments, the modified cell medium can be pre-treated / loaded with one or more agents to make the cell medium resistant to virus-mediated killing. For example, in some embodiments, the cell medium can be pre-treated with type I and / or type II interferons. In other embodiments, the cell medium can be pre-treated with antiviral agonist / inducer substances (e.g., STING, PKR, RIG-I, MDA-5). To produce such a "protected" cell medium, any autologous or allogeneic cell medium can be pre-treated with interferon type I (e.g., IFNα / β) and / or type II (e.g., IFNγ) and / or STING, PKR, RIG-I, MDA-5, OAS-1 / 2 / 3, AIM-2, MAVS, RIP-1 / 3, or agonists of the DAI(ZBP1) pathway, without viral infection or with viral infection. Before infection, processing can be performed for 30 minutes to a maximum of 48 hours or more, for example, about or at least 30 minutes or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 hours or more.

[0184] These “protected” cell media can be administered simultaneously as separate compositions with matching / sensitized / manipulated cell media that are not thus protected and contain viruses; the protected cell media can provide extended survival time and / or improved local immunosuppression. In some embodiments, the protected cell media can be administered, for example, about or at least 10, 15, 20, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours before or after administration of matching / sensitized / manipulated cell media that are not thus protected and contain viruses, or within 1, 2, 3, 4, or 5 days.

[0185] (iii) Cell medium manipulated to improve viral amplification and / or immunomodulation In some embodiments, modified cell media for use in the systems and methods provided herein are manipulated for transient or permanent gene expression or repression, thereby promoting improved viral amplification and / or immunomodulation. The cell media can be manipulated in one or more of the following embodiments. Any of the cell media provided herein can be modified using one or a combination of the embodiments for sensitizing, protecting, and / or manipulating the cell media provided herein.

[0186] In some embodiments, the cell medium can be manipulated to be unresponsive to an antiviral state induced by interferon (IFN). For example, the cell medium can be manipulated for transient or permanent repression (e.g., locus excision) of IFN type I / II receptor and / or downstream signaling, such as repression of one or more of the following: type I / II interferon receptor expression; IFNα / β, IFNγ receptor expression; IFNAR1 / IFNAR2 receptor expression; IFNGR1 / IFNGR2 receptor expression; STAT1 / 2 receptor expression; Jak1 / 2 receptor expression; IRF3 receptor expression; IRF7 receptor expression; IRF9 receptor expression; TYK2 kinase expression and TBK1 kinase expression.

[0187] In the embodiments, the cell medium can be manipulated for transient or permanent suppression of elements of cytosolic viral DNA / RNA sensing and antiviral defense mechanisms, including, but not limited to, PKR, RIG-I, MDA-5, cGAS, STING, TBK1, IRF3, OAS-1 / 2 / 3, AIM2, MAVS, RIP-1 / 3, and DAI(ZBP1). In other embodiments, the cell medium can be manipulated for transient or persistent expression of antagonists of virus sensing and antiviral defense pathways mediated by, for example, STING, PKR, RIG-1, MDA-5, OAS-1 / 2 / 3, AIM2, MAVS, RIP-1 / 3, DAI(ZBP1); these may include, but are not limited to, one or more of K1, E3L, K3L (vaccinia); NS1 / NS2 (influenza A); NS3-4A (hepatitis C); NP, Z protein (arenavirus); VP35 (Ebola virus); US11, ICP34.5, ICP0 (HSV); M45 (MCMV); and X protein (BDV: Borna disease virus).

[0188] In some embodiments, modified cell media can be manipulated to evade allorecognition by T cells and NKT cells. For example, cell media can be manipulated for transient or permanent repression of the following expressions: MHC class I molecules (HLA-A, B, C); MHC class II molecules (HLA-DP, DQ, DR); MHC-like molecules (CD1a / b / c / d); or regulators of transcription or expression of MHC class I, MHC class II, or MHC-like molecules (e.g., TAP1 / 2, tapasin, beta-2 microglobulin, CIITA, RFXANK, RFX5, and RFXAP). In other examples, cell media can be manipulated for transient or permanent expression of the following: viral B2M antagonists (e.g., UL18(HCMV); and / or viral MHC antagonists (e.g., A40R). MHCI (Vaccinia); Nef, TAT (HIV); E3-19K (Adenovirus); ICP47 (HSV-1 / 2); CPXV012, CPXV203 (Cowpox); EBNA1, BNLF2a, BGLF5, BILF1 (EBV); ORF66 (VZV); US2 / gp24, US3 / gp23, US6 / gp21, US10, US11 / gp33 (hCMV); rh178 / VIHCE (RhCMV) ;U21(HHV-6 / 7);LANA1, ORF37 / SOX, kK3 / MIR1, kK5 / MIR2(KHSV);mK3(MHV-68);UL41 / vhs(α-herpesvirus, HSV, BHV-1, PRV);UL49.5(Varicerovirus, BHV-1, EHV-1 / 4, PRV);and one or more of m4 / gp34, m6 / gp48, m27, m152 / gp40(mCMV)).

[0189] In embodiments, the cell medium can be manipulated to avoid allorecognition by NK cells. For example, the cell medium can be manipulated to transiently or permanently suppress the expression of one or more of the following: membrane-bound MICA / B (NKG2D ligand); membrane-bound PVR (DNAM-1 ligand); membrane-bound nectin-2 (DNAM-1 ligand). In other examples, cell media can be manipulated for transient or persistent expression of the following: antagonists of MIC-A and MIC-B (NKG2D ligands) (e.g., kK5 (KHSV)); antagonists of the NKG2D receptor (e.g., cowpox OMCP); antagonists of NCRs targeting NKp30, NKp44, and NKp46 receptors (e.g., HA (hemagglutinin - vaccinia and other viruses)); ligands of NK inhibitory receptors (KIRs) (e.g., HLA-Bw4; HLA-C2); and ligands of NK inhibitory receptors (NKG2a / CD94) (e.g., HLA-E and derivatives, either alone or by creating HLA-E binding peptides and combining them with 21M HLA-B ligands to stabilize HLA-E surface expression).

[0190] In certain embodiments, cellular media can be manipulated to express human or viral immunosuppressive factors (e.g., to prevent / inhibit allogeneic anticellular media or antiviral immune responses). This can be achieved by encoding in the cellular genome and / or viral genome. Human-derived factors include, but are not limited to, IDO, arginase, TRAIL, iNOS, IL-10, TGFβ, VEGF, FGF-2, PDGF, HGF, IL-6, sMICA, sMICB, sHLA-G, HLA-E, PD-L1, FAS-L, B7-H4, and single-chain antibodies (scFv) that target or deplete NK and / or NKT cells. Viral-derived factors include, but are not limited to, Ectromelia / Vaccinia virus SPI-2 / CrmA (an inhibitor of immune FAS / TNF / granzyme B-induced apoptosis); N1 encoded by vaccinia virus (an IL-1 / NFκB / IRF3 antagonist); HA (an NCR antagonist targeting NKp30, NKp44, and NKp46); IL-18 binding proteins; A40R; A46R; A52R; B15R / B16R; TNFα blockers (e.g., vaccinia virus CmrC / CmrE); IFNα / β blockers (e.g., vaccinia virus B18R / B19R); IFNγ blockers (e.g., vaccinia virus B8R); and other IL-1 / IL-1β / NFκB / IRF3 / NCR / MHCI / TLR / NKG2D antagonists.

[0191] In some embodiments, the cell medium can be manipulated to express cancer or stem cell-derived factors that otherwise would be unacceptable to the cell medium and / or promote viral infection of tumor cells. For example, the cell medium can be manipulated to express one or more of the following: cancer-related antigens (e.g., oncotesticular antigens (MAGE-A1, MAGE-A3, MAGE-A4, NY-ESO-1, PRAME, CT83, SSX2, BAGE family, CAGE family); carcinoembryonic antigens (AFP, CEA); oncogenes / tumor suppressors (myc, Rb, Ras, p53, telomerase); differentiation antigens (MELAN, tyrosinase, TRP-1 / 2, gp100, CA-125, MUC-1, ETA); GM-CSF; IL-10; TGFβ; VEGF; FGF-2; PDGF; HGF; IL-6; growth factors; RTK / mTOR agonists; and wnt protein ligands.

[0192] In embodiments, the modified cell medium may be manipulated to express factors that interfere with the function of complement and / or neutralizing antibodies, including, but not limited to, one or more of the following: protein antagonists of complement factors (C1, C2, C3, C4, C5, MBL); vaccinia virus complement regulatory proteins (VCP); vaccinia virus complement inhibitors (B5R); scFv anti-CD1q / CD1r / CD1s; anti-C3, anti-C5 (e.g., eculizumab); compstatin family peptide C3 inhibitors (e.g., Cp40); human soluble membrane (s / m) proteins (e.g., s / mCR1(CD35), s / mCR2(CD21), s / mCD55, s / mCD59); human complement factor H and derivatives, as well as cobra venom factor and derivatives having complement inhibitory activity.

[0193] (iv) Cell medium engineered to express angiogenesis inhibitors for vascular normalization / tumor angiogenesis reprogramming In some embodiments, the cell medium can be engineered to encode angiogenesis inhibitors (for example, to reprogram / repair, stabilize, and / or normalize tumor vascular systems). Details of such inhibitors are described in the following section, "2. Viruses," which describes such modifications of viruses that are components of CAVES provided herein. As described herein, angiogenesis inhibitors can induce vascular normalization and repair tumor vascular systems (tumor vascular reprogramming) by restoring balance to a cascade of signals initiated by the interaction of tumor cells with their local cellular environment. This can result in enhanced tumor perfusion and reduced intratumoral hypoxia, which in turn can result in improved reduction of primary tumor growth, ascites, and metastasis.

[0194] The CAVES compositions provided herein, as well as related methods of use and treatment, may include cellular media encoding molecules that inhibit angiogenesis, including those that downregulate pro-angiogenic factors and / or upregulate anti-angiogenic factors. Alternatively, the CAVES compositions provided herein may be administered in combination with angiogenic inhibitors and / or viruses encoding angiogenic inhibitors.

[0195] (v) Cell medium engineered to express transgenes for conditional cell immortalization In some embodiments, the cell media for the CAVES compositions and related methods provided herein can be manipulated to encode a transgene for conditional cell immortalization (see, for example, the review by Wall et al., Cell Gene Therapy Insights, 2(3):339-355 (2016), which is incorporated herein in its entirety by reference, and the references cited therein). Conditional immortalization uses inducible transgene techniques to produce cells that, when the transgene is active, can be grown to clinically viable quantities in a stable and consistent manner to obtain target cells of interest, for example, under the control of an operator, but can be inactivated as needed so that they return to a normal post-mitotic state. This enables the safe delivery of consistently reproducible, stable, and scalable cell formulations (e.g., CAVES) to subjects requiring treatment, while minimizing or eliminating the risk of cancer from administering constitutively immortalized cells. The ability to obtain a large number of cell media of consistent quality in a scalable, cost-effective, and safe manner for administration to a target is desirable for developing “ready-made” allogeneic cells for clinical use in cell-based compositions such as CAVES provided in this specification.

[0196] Conditional immortalization of cells can be carried out using methods known to those skilled in the art, and can be varied, for example, depending on the type of cell and the species from which it is obtained. For example, stress activation of the p53 and pRB pathways is a common cause of aging in mice, and stress can be mitigated by inhibiting or silencing these genes by growing mouse cells under optimal culture and oxygen conditions. Human cells, on the other hand, require telomere maintenance, for example, by telomerase rearrangement, in addition to silencing the p53 and pRB genes. Exemplary genes that can be controlled (conditionally) activated / inactivated for cell division include, for example, oncogenes and telomerase. Exemplary techniques for conditional immortalization include, but are not limited to, the following:

[0197] E6 / E7 Tumor proteins derived from human papillomavirus type 16 (HPV16), E6, and E7 cooperate in mediating cell immortalization by inactivating tumor suppressor factors such as p53 and pRB. Therefore, conditional immortalization can be achieved by conditionally inactivating these tumor suppressor factors when proliferation is desired, and then by activating them when proliferation is to be stopped (e.g., before administration as a treatment; see, e.g., Storey et al., Oncogene, 11:653-661 (1995)).

[0198] Myc gene The c-myc gene, along with its viral homolog v-myc, exerts regulatory control over various cellular functions. In particular, it drives cell cycle entry and cell division, making it an attractive target for creating stable immortalized cell lines. Mutations in the myc gene that constitutively express the myc gene are associated with oncogenic transformation and lead to cancer. Therefore, controlled expression of c-myc, preferably under operator control, is desired for conditional immortalization.

[0199] Conditional immortalization technology c-MycER TAM c-myc is a fusion gene encoding a chimeric protein containing the N-terminal cleaved hormone-binding domain of the mutant mouse estrogen receptor (G525R). The mutant G525R can no longer bind to 17β-estradiol and estrogen, but responds to activation in the presence of the synthetic estrogen-like agonist 4-hydroxytamoxifen (4-OHT). Therefore, when cells are cultured in the presence of 4-OHT, c-myc activity and subsequent cell division are promoted, but in the absence of 4-OHT, the cells revert to an inactivated state and can undergo maturation / differentiation like normal cells.

[0200] c-MycER TAMThe synthesis of this material does not affect the cellular phenotype, and this conditional immortalization technique has been used to develop human stem cell lines from cortical neuroepithelium, which have been investigated in preclinical animal studies for ischemic stroke and limb ischemia, and are currently being investigated in clinical trials as a treatment for stroke injury (Phase 1 and Phase 2) and Phase 1 trials for clinical limb ischemia.

[0201] Among myc oncogenes, the avian virus homolog v-myc has also been shown to effectively immortalize human neural stem cells (hNSCs). The p110gag-myc protein, encoded by the avian myelomatosis virus genome, is spontaneously downregulated after differentiation. Similar to its cellular counterpart, the growth and differentiation of v-myc-transduced hNSCs depend on pro-mitotic stimulation by growth factors. Spontaneous downregulation of avian v-myc was observed 24–48 hours after engraftment in neonatal mice, demonstrating its potential as a conditionally immortalized cell medium. Established v-myc hNSC cell lines have shown potential as delivery vehicles for selective gene therapy due to their tumor-targeting properties. Preclinical studies of genetically modified hNSC lines (HB1, F3, CD) expressing cytosine deaminase resulted in tumor site transformation of 5-fluorocytosine to the chemotherapeutic agent 5-fluorouracil. Currently, a Phase 1 clinical trial is underway to study the dosage and side effects of this anti-cancer strategy (ID: 13401 NCI-2013-02346 13401).

[0202] Thermosensitive Simian virus SV40 T antigen SV40 is a double-stranded DNA virus derived from rhesus monkeys. SV40 possesses numerous antigens, including a large tumor antigen (Tag). Tag modulates a cellular signaling pathway that induces cells to enter the S phase and undergo a DNA damage response that promotes viral DNA replication. Tag also binds to and inactivates p53 and pRB family proteins, potent tumor suppressors involved in cell cycle progression and apoptosis, creating an ideal environment for viral replication. Early studies on rodent cells showed that Tag immortalizes these cells, granting them unlimited proliferative capacity. Subsequent inactivation of Tag resulted in a rapid and irreversible loss of proliferative capacity during the G1 and G2 phases of the cell cycle, demonstrating that Tag is continuously required to maintain the proliferative state. These characteristics make Tag an ideal candidate for developing controllable cell lines.

[0203] Tag inactivation was achieved using a temperature-sensitive mutant of large Tag (SV40 tsA58), first isolated in 1975, which was found to behave as wild-type at tolerable temperatures (33.5°C) but was biologically inactive at the non-tolerable temperature of 39°C. Therefore, conditional immortalization can be achieved by promoting differentiation by growing cells at tolerable temperatures and then raising the cell temperature to the non-tolerant temperature. Preclinical studies by ReNeuron Ltd. (UK) / University College London are being conducted for the treatment of retinitis pigmentosa using human embryonic retinal cell lines (hRPCs) conditionally immortalized with the SV40 large tumor antigen.

[0204] Telomerase In human somatic cells, the gradual shortening of telomeres, short repeat sequences at the ends of chromosomes, associated with each cell division, has been proposed as the mitotic clock. Human telomeres contain multiple tandem repeat sequences of TTTAGG located at the ends of chromosomes. Human telomeres rely on the enzyme telomerase to maintain their length, but human somatic cells do not express telomerase at levels sufficient to maintain telomeres, resulting in a shortening of approximately 50 base pairs with each cell division. In summary, telomere loss associated with a lack of telomerase activity is the mitotic clock responsible for limiting the number of divisions before aging.

[0205] The catalytic subunit hTERT of human telomerase reverse transcriptase catalyzes the synthesis of 6bp repeats, thereby extending telomeres. Since the basal level of telomerase in primary human cells is insufficient for unlimited lifespan, transduction of exogenous hTERT can lead to life extension.

[0206] While it was initially proposed that telomerase activity reconstitution using hTERT would be sufficient for immortalizing primary human cells, it was found that telomerase reconstitution alone is sometimes insufficient. In these cases, secondary inactivation of regulatory pathways such as p16 and pRB was necessary.

[0207] While the above studies evaluated constitutive activation, telomerase has been shown to successfully support conditional immortality in combination with other conditional transgenes (O'Hare et al., Proc. Natl Acad. Sci. USA, 98(2):646-651 (2001)). For example, the SV40 U19 Tag mutant lacked the ability to bind to the SV40 origin of replication and, when delivered with a recombinant retrovirus encoding the U19 Tag, was more efficient at immortalizing rodent cells than the wild-type Tag. Vectors incorporating both tsA58 (temperature-sensitive Tag mutant, see description above) and the U19 mutant were constructed to create mouse oligodendrocyte progenitor cell lines capable of in vitro differentiation (Almazan et al., Brain Res., 579:234-245 (1992)). The U19tsA58 tag was found to be able to create conditionally immortalized cell lines from rat neonate optic nerves that can differentiate into oligodendrocytes (Barnett et al., Eur.J.Neurosci., 5:1247-1260 (1993)). The U19tsA58 tag was also used to study the heterogeneity of candidate regenerated olfactory nerve sheath cells from the olfactory bulb and lamina propria (Franceschini et al., Dev.Biol., 173(27):327-343 (1996)). A study by O'Hare et al. showed that ectopic expression of hTERT or U19tsA58 tag alone was insufficient for immortalizing newly isolated human cells, but the combination of genes, regardless of the order in which they were introduced, resulted in the efficient generation of immortalized cell lines (O'Hare et al., Proc.Natl Acad.Sci.USA, 98(2):646-651 (2001)).

[0208] Cre-loxP series Bacteriophage p1 Cre is an enzyme that promotes recombination at a specific site called loxP. When two 33bp loxP sequences are oriented, recombination occurs, resulting in the cleavage and removal of intervening sequences. The application of Cre-loxP-mediated reversible immortalization is promising for both autologous and allogeneic cell therapies. Biopsies and primary cultures can be immortalized with recombinant oncogenes adjacent to the loxP site. Subsequent Cre transfection results in the excision of the immortalization gene. After oncogene removal, the cells should be identical to the primary culture population, but their number is increased.

[0209] The Cre-loxP system has been applied to rat adrenal cells possessing hTERT and Tag as immortalization genes, as well as to human hepatocytes and myogenic cells (see discussion above). To exclude any cells that do not necessarily need to have the transgene deleted, the herpes simplex virus 1-thymidine kinase (HSV-TK) suicide gene was included as a negative control for recombination to kill a small portion of refractory immortalized cells in the presence of ganciclovir (GCV) after Cre transfection. Tamoxifen-dependent Cre recombinase was also incorporated to achieve controlled excision of oncogenes.

[0210] Tet-On and Tet-Off Conditional immortalization has also been achieved through the use of transcriptional regulatory systems. The most widely used is one induced using the prokaryotic tetracycline repression system. This transcriptional regulatory system utilizes a tet repressor (tetR) protein that binds to a sequence called the tetracycline operator (tetO) in the absence of an antibiotic (tetracycline or doxycycline). In the presence of an antibiotic, it binds to the repressor, thereby inhibiting the repressor's binding to tetO.

[0211] The first system available for conditional immortalization is called "Tet-Off" and was developed in HeLa cells. In this system, the tet repressor binding site is inserted between the promoter and the transcription start site so that the repressor binding sterically blocks transcription. However, this steric hindrance is overcome by the addition of small amounts of tetracycline and doxycycline to prevent tetR from binding to tetO, thereby inducing reporter gene expression.

[0212] As an alternative, the "Tet-On" system was created by fusing tetR with the C-terminal activating domain of viral particle protein 16 (VP16) derived from herpes simplex virus (HSV), thereby generating a hybrid transcription activator (tTA) that stimulates a promoter fused to the tetO sequence. Modification of four amino acids yielded a reverse tetracycline transactivator (rtTA) that binds to tetO only in the presence of tetracycline or doxycycline. Oncogenes (c-Myc and Tag) and telomerase (hTERT) were initially tested in mouse embryonic fibroblasts (MEFs), mouse kidney cells (293T), mouse embryonic stem cells, and human endothelial cells using a Tet-based immortalization system. Furthermore, mesenchymal stromal cells (MSCs) were immortalized in a tetracycline-inducible system. Tetracycline-inducible hTERT-expressing MSC cell lines were found to retain pluripotency, and immortalization was dependent on telomere elongation. Conditionally immortalized MSC strains were generated by lentiviral transfection of Tag-hTERT combined with a doxycycline / tetracycline induction (Tet-On) system (Koch et al., Genome Res., 2013;23:248-259 (2013)). These cells were used to study changes in senescence-related DNA methylation (SA-DNAm) and could be maintained in culture for 80 days without signs of senescence. Removal of doxycycline from the culture medium immediately halted growth and increased expression of senescence-related β-galactosidase. When cells were exposed to antibiotics and unaffected by SA-DNAm, telomere length significantly increased.

[0213] Methods for modifying cell media Several methods for manipulating cells, such as for creating the above-mentioned manipulated cell media, are known in the art. Such methods include, but are not limited to, the following:

[0214] (a) CRISPR-CAS9 targeted repression (permanent gene / locus deletion). The cell medium can be transfected with a DNA plasmid expressing both the CAS9 protein and a guide RNA (gRNA) specific to the gene of interest. The gRNA-CAS9 mediated break in the genome can be repaired using a donor DNA plasmid, which specifically deletes the targeted gene and permanently and completely loses the protein encoded by the gene. The loss of protein expression can be verified using PCR (at the DNA level), Northern blot / FISH (at the RNA level), or any protein assay such as Western blot or flow cytometry.

[0215] (b) CRISPR-CAS9 targeted expression (permanent gene / locular insertion) This method allows for the insertion of a target gene at a specific location in the cell medium genome. The cell medium can be transfected with a DNA plasmid expressing both the CAS9 protein and a guide RNA (gRNA) specific to the particular insertion site. The gRNA-CAS9 mediated break in the genome can be repaired using a donor DNA plasmid, which contains the target insertion gene with sequences of the cell medium genome flanking the DNA break / double-strand break site on both sides, resulting in homologous recombination-mediated insertion of the target gene at a specific genomic location, rather than randomly. Successful insertion and protein expression can be verified using PCR (at the DNA level), Northern blot / FISH (at the RNA level), or any protein assay such as Western blot or flow cytometry.

[0216] (c) RNA interference (retroviral / lentiviral / transposon-mediated transduction of shRNA / microRNA) (permanent gene repression): ShRNA / microRNAs targeting specific genes / proteins of interest can be designed and cloned into retroviral / lentiviral / transposon vectors for stable integration into cell medium genomes. Cell medium can be transduced with the vector, and transduced cells can be selected using selection markers encoded in the vector. ShRNA-mediated repression of the target gene can be evaluated using, for example, Northern blotting and protein assays.

[0217] (d) Lentivirus / γ-retrovirus-mediated random / multicopy gene insertion For stable, random integration into cell-mediated genomes, specific genes / proteins of interest can be designed and / or cloned into retroviral or lentiviral vectors. Cell-mediated transduction can be performed with viral vectors, and transduced cells can be selected using selection markers encoded in the vectors. ShRNA-mediated repression of the gene of interest is evaluated using Northern blotting, as well as any protein assay such as Western blotting and flow cytometry.

[0218] (e) Transposon-mediated random / multicopy gene insertion Specific genes / proteins of interest can be designed and / or cloned into mammalian transposon vector systems such as PiggyBac (SBI System Biosciences) or equivalents. Cellular media can be cotransfected with transposon vectors containing the target gene (cDNA) flanked by a reverse-terminal repeat (ITR) sequence and a transposase vector. The transposase enzyme can mediate the transfer of the target gene to the TTAA chromosome integration site. Transduced cells can optionally be selected using a selection marker encoded in the vector. Insertion and protein expression can be verified using PCR (at the DNA level), Northern blotting / FISH (at the RNA level), or any protein assay such as Western blotting or flow cytometry.

[0219] (f) Transient gene repression of the expression of the target protein can be achieved, for example, via RNA interference. siRNA / microRNA can be transfected into a cellular medium by any of the established methodologies known in the art, e.g., calcium chloride transfection; lipofection; Xfect; electroporation; sonoporation and cell compression (for example, to introduce siRNA).

[0220] (g) Transient gene expression can be achieved, for example, by cloning the gene of interest into a suitable mammalian plasmid expression vector that can be transfected into a cell medium having plasmid DNA encoding the desired product. Alternatively, mRNA encoding the gene / protein of interest can be directly transfected into a cell medium. Transfection can be carried out using any of the established methodologies, e.g., calcium chloride transfection; lipofection; Xfect; electroporation; sonoporation and cell compression (for example, to introduce siRNA).

[0221] 2. Virus The carrier cells selected and / or modified as described above can be used in viral therapies using any virus identified as having oncolytic properties. Exemplary oncolytic viruses that can be used in the methods, combinations, and compositions provided herein are as follows:

[0222] Types of viruses Oncolytic viruses are primarily characterized by tumor cell-specific replication, resulting in tumor cell lysis and efficient tumor regression. Oncolytic viruses treat tumor cells by colonizing or accumulating in tumor cells, including metastatic tumor cells such as circulating tumor cells, and replicating there. Methods, compositions, and combinations can be carried out using any anti-cancer vaccine or virus. For example, oncolytic viruses can be any naturally occurring or engineered recombinant viruses, including, but are not limited to, vaccinia virus, poxvirus, herpes simplex virus, adenovirus, adeno-associated virus, measles virus, reovirus, varicella stomatitis virus (VSV), coxsackievirus, Semryki forest virus, Seneca Valley virus, Newcastle disease virus, Sendai virus, dengue virus, picornavirus, poliovirus, parvovirus, retrovirus, lentivirus, alphavirus, flavivirus, rhabdovirus, papillomavirus, influenza virus, mumps virus, gibbon leukemia virus, and Sindbis virus. In many cases, tumor selectivity is an inherent characteristic of viruses such as vaccinia viruses and other oncolytic viruses. Generally, oncolytic viruses perform their treatment by replicating in tumors or tumor cells and causing lysis.

[0223] In some embodiments, attenuated strains derived from pathogenic viruses are used in the production of live vaccines. Non-exclusive examples of vaccinia viruses include, but are not limited to, Lister (also known as Elstree), New York City Department of Health (NYCBH), Dairen, Ikeda, LC16M8, Western Reserve (WR), Copenhagen (Cop), Tashkent, Tian Tan, Wyeth, Dryvax, IHD-J, IHD-W, Brighton, Ankara, Modified Vaccinia Ankara (MVA), Dairen I, LIPV, LC16M0, LIVP, WR 65-16, EM63, Bern, Paris, CVA382, NYVAC, ACAM2000, ACAM1000, and Connaught strains. The viruses may be clonal strains of oncolytic viruses. A sequence of nucleotides encoding a chromophore-producing enzyme can be inserted into or in place of a non-essential gene or region within the genome of an unmodified oncolytic virus, or into or in place of a nucleic acid encoding a heterologous gene product within the genome of an unmodified oncolytic virus.

[0224] In some embodiments, the vaccinia virus used with the cells and in the method in the present invention is an attenuated New York City Department of Health (NYCBOH) strain. In some embodiments, the NYCBOH strain of vaccinia virus may be ATCC VR-118 or CJ-MVB-SPX.

[0225] In some embodiments, the vaccinia virus is selected from Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, or modified vaccinia Ankara (MVA). In some embodiments, the oncolytic virus does not lack any of the genes present in one or more of these strains.

[0226] In some embodiments, the virus or vaccine is a virus capable of replication. In some embodiments, the virus or vaccine is replication-deficient. In some embodiments, the virus or vaccine is not attenuated. In other embodiments, the virus or vaccine is attenuated.

[0227] Other unmodified oncolytic viruses include any known to those skilled in the art, selected from viruses known as GLV-1h68, JX-594, JX-954, ColoAd1, MV-CEA, MV-NIS, ONYX-015, B18R, H101, OncoVEX GM-CSF, Reolysin, NTX-010, CCTG-102, Cavatak, Oncorine, and TNFerade.

[0228] The oncolytic viruses for use in the methods provided herein include several well known to those skilled in the art, for example, vesicular stomatitis virus, e.g., U.S. Patent Nos. 7,731,974, 7,153,510, 6,653,103 and U.S. Patent Application Publications 2010 / 0178684, 2010 / 0172877, 2010 / 0113567, 2007 / 0098743, 20050260601, and 20050220818. See also European Patent Nos. 1385466, 1606411 and 1520175; for example, U.S. Patent Nos. 7,897,146, 7731,952, 7,550,296, 7,537,924, 6,723,316, 6,428,968 and U.S. Patent Application Publications 2011 / 0177032, 2011 / 0158948, 2010 / 0092515, 2009 / 0274728, and 2 See patents 009 / 0285860, 2009 / 0215147, 2009 / 0010889, 2007 / 0110720, 2006 / 0039894 and 20040009604; retroviruses, e.g., U.S. Patent Nos. 6,689,871, 6,635,472, 6,639,139, 5,851,529, 5,716,826, 5,716,613 and U.S. Patent Application Publication No. 20110212530. See also; and adeno-associated viruses, including U.S. Patent Nos. 8,007,780, 7,968,340, 7,943,374, 7,906,111, 7,927,585, 7,811,814, 7,662,627, 7,241,447, 7,238,526, 7,172,893, 7,033,826, 7,001,765, 6,897,045 and 6,632,670.

[0229] Newcastle disease virus Newcastle disease virus (NDV) is an avian paramyxovirus with a negative polarity single-stranded RNA genome that infects poultry and is generally non-pathogenic to humans, but can cause influenza-like symptoms (Tayeb et al. (2015) Oncolytic Virotherapy 4:49-62; Cheng et al. (2016) J.Virol.90:5343-5352). Due to its cytoplasmic replication, lack of host genome integration and recombination, and high genomic stability, NDV and other paramyxoviruses offer a safer and more attractive alternative to other oncolytic viruses such as retroviruses or some DNA viruses (Matveeva et al. (2015) Molecular Therapy-Oncolytics 2,150017). NDV exhibits tumor selectivity, replicating 10,000 times more rapidly in tumor cells than in normal cells, and causing tumor lysis through direct cytopathic effects and induction of immune responses (Tayeb et al. (2015); Lam et al. (2011) Journal of Biomedicine and Biotechnology, Article ID 718710). The mechanism of NDV tumor selectivity is not fully understood, but defects in interferon production and responses to IFN signaling in tumor cells enable the virus to replicate and spread (Cheng et al. (2016); Ginting et al. (2017) Oncolytic Virotherapy 6:21-30). The high affinity of paramyxoviruses for cancer cells can also be attributed to the overexpression of viral receptors containing sialic acid on the surface of cancer cells (Cheng et al. (2016); Matveeva et al. (2015); Tayeb et al. (2015)).

[0230] Unmodified NDV strains are classified as long-latency (non-pathogenic), subpathogenic (moderately pathogenic), or short-latency (pathogenic) based on their pathogenicity in chickens. Short-latency and subpathogenic strains can replicate (and lyse) multiple human cancer cells, while long-latency strains cannot (Cheng et al. (2016); Matveeva et al. (2015)). NDV strains are also classified as lytic or non-lytic, with only lytic strains being able to produce viable and infectious offspring (Ginting et al. (2017); Matveeva et al. (2015)). On the other hand, the oncolytic effect of non-lytic strains is mainly due to their ability to stimulate an immune response that results in antitumor activity (Ginting et al. (2017) Oncolytic Virotherapy 6:21-30). Commonly used subpathogenic lysates in tumor therapy include PV701 (MK107), MTH-68 / H, and 73-T, while commonly used long-latency non-lysates include HUJ, Ulster, and Hitchner-B1 (Tayeb et al. (2015); Lam et al. (2011); Freeman et al. (2006) Mol.Ther. 13(1):221-228).

[0231] The use of NDV as an oncolytic virus was first reported in the early 1950s when adenovirus and NDV were injected directly into uterine cancer cells, causing partial necrosis. Tumor regrowth was observed, likely due to the suppression of oncolytic activity by the production of neutralizing antibodies against the virus (Lam et al. (2011) Journal of Biomedicine and Biotechnology, Article ID 718710). Recently, the NDV strain PV701 demonstrated activity against colorectal cancer in a phase 1 trial (Laurie et al. (2006) Clin. Cancer Res. 12(8):2555-2562), and the NDV strain 73-T demonstrated in vitro oncolytic activity against various human cancer cell lines, including fibrosarcoma, osteosarcoma, neuroblastoma, and cervical cancer, as well as in vivo therapeutic efficacy in mice with several cancer xenografts, including human neuroblastoma, fibrosarcoma xenografts, and colon, lung, breast, and prostate cancer xenografts (Lam et al. (2011)). The NDV strain MTH-68 / H resulted in significant regression of tumor cell lines, including PC12, MCF7, HCT116, DU-145, HT-29, A431, HELA, and PC3 cells, demonstrating a favorable response in patients with advanced cancer when administered by inhalation (Lam et al. (2011)). The non-lytic strain Ulster demonstrated cytotoxic effects against colon cancer, and the lytic strain Italian effectively killed human melanoma (Lam et al. (2011)). The long-latency NDV strain HUJ demonstrated oncolytic activity against recurrent glioblastoma multiforme when administered intravenously to patients, and the long-latency strain LaSota extended survival in patients with colorectal cancer (Lam et al. (2011); Freeman et al. (2006) Mol.Ther.13(1):221-228). It was also able to infect and kill non-small cell lung cancer (A549), glioblastoma (U87MG and T98G), mammary adenocarcinoma (MCF7 and MDA-MB-453), and hepatocellular carcinoma (Huh7) cell lines (Ginting et al. (2017) Oncolytic Virotherapy 6:21-30).

[0232] Genetically modified NDV strains have also been evaluated for oncolytic therapy. For example, the influenza NS1 gene, an IFN antagonist, was introduced into the genome of the NDV strain Hitchner-B1, resulting in enhanced oncolytic effects in various human tumor cell lines and mouse models of B16 melanoma (Tayeb et al. (2015)). The antitumor / immunostimulatory effects of NDV were enhanced by introducing IL-2 or GM-CSF genes into the viral genome (Lam et al. (2011)).

[0233] In addition to the use of free viruses, studies have evaluated the use of NDV oncolytic products, NDV-infected cell-based media, and combination therapies with other non-cancer agents for cancer treatment. In several clinical trials, NDV oncolytic products demonstrated oncolytic activity against malignant melanoma (Lam et al. (2011)). The use of NDV-infected cell-based media has also been demonstrated. Autologous tumor cell lines infected with NDV have been used against colorectal cancer, breast cancer, ovarian cancer, kidney cancer, head and neck cancer, and glioblastoma (Lam et al. (2011)). MSCs derived from bone marrow, fat, and umbilical cord infected with the NDV strain MTH-68 / H delivered the virus to co-cultured A172 and U87 glioma cells and glioma stem cells, resulting in dose-dependent cell death in glioma cells, low levels of apoptosis and inhibition of autoregeneration in glioma stem cells, and higher levels of apoptosis than direct infection with naked virus (Kazimirsky et al. (2016) Stem Cell Research & Therapy 7:149). Combination therapy using intratumoral NDV injection and systemic CTLA-4 antibody administration resulted in efficient rejection of pre-established distant tumors (Matveeva et al. (2015)).

[0234] Maraba virus The maraba virus was first isolated from the Amazonian phlebotomine in Brazil and has not been detected outside of South America. Phylogenetically, the maraba virus belongs to the genus Vesiculovirus in the family Rhabdoviridae and is genetically distinct from, but shares some homology with, the protozoan vesicular stomatitis virus (VSV). (See Pol et al., Oncolytic Virother., 7:117-128 (2018) and the references cited therein (their contents are incorporated herein in their entirety by reference)).

[0235] Among the 20 rhabdovirus strains screened for oncolytic marabaviruses, this virus exhibited the broadest tumor affinity. The virus was the only candidate to complete the lytic cycle in all human and mouse cell lines derived from various cancer types tested (i.e., breast, brain, colon, skin, lung, ovarian, prostate, and kidney cancers). Marabaviruses (such as VSV) utilize, but are not limited to, the ubiquitous low-density lipoprotein receptor (LDLR) for entry into target cells, providing one explanation for a broad range of infected malignant cell hosts. Consistent with this, reduced LDLR expression was associated with decreased susceptibility to marabavirus entry and death in several cell lines derived from ascites fluid of ovarian cancer patients.

[0236] To enhance marabavirus replication in malignant cells, its genome was genetically engineered. Two single mutations were introduced, which translate to L123W and Q242R substitutions in the M and G protein sequences, respectively. In vitro, the resulting strain (named MG1) showed faster replication, larger burst size, and increased killing efficacy in tumor cells compared to wild-type (wt) and other marabavirus mutants. Conversely, MG1 was strongly attenuated in normal primary cells, confirming its cancer selectivity (Brun et al., Mol.Ther.18(8):1440-1449(2010)).

[0237] In vitro, the oncolytic activity of MG1 has been validated against multiple adherent cancer cell lines of human, canine, and mouse origin (e.g., origins of central nervous system cancer, sarcoma, breast cancer, and colon cancer). Furthermore, MG1 has been found to infect, replicate, and induce cell death in ovarian cancer cells regardless of stage (Tong et al., Mol. Ther. Oncolytics, 2():15013). Ex vivo, MG1 strains have been shown to exhibit proliferative infection and significant cytopathic effects against resected tissues derived from prostate cancer, head and neck squamous cell carcinoma, or sarcoma. In vivo, MG1 can be safely delivered systemically, enabling treatment of both localized and disseminated cancerous lesions. The oncolytic activity of MG1 has been confirmed in multiple syngeneic mouse tumor models and in xenograft models using human cancer cell lines or patient-derived tumors transplanted into immunodeficient mice (e.g., in mice: colon cancer (CT26, CT26lacZ), leukemia (L1210), lung cancer (TC1), mammary cancer (EO771, EMT6, 4T1), prostate cancer (TRAMP-C2), sarcoma (S180), skin cancer (B16F10, B16F10Ova, B16lacz); in humans: breast cancer (HCI-001, HCI-003) and ovarian cancer (ES2, OVCAR4).

[0238] The activity of the MG1 strain of maraba vesicurovirus depends not only on direct cytotoxicity but also on the induction of both innate and adaptive anti-tumor immunity. Therefore, maraba viruses can function as both selective tumor-destroying oncolytic viruses and immune-stimulating T-cell vaccines. Leaving healthy cells unaffected, the Maraba platform directly attacks cancer cells, altering the tumor microenvironment and making cancer more susceptible to targeted vaccine-induced immune responses. This technology was developed by Turnstone Biologics (Ottawa, Ontario), a clinical-stage immuno-oncology company, which recently entered into a study, option, and license agreement with AbbVie (North Chicago, Illinois) for exclusive options to license up to three Turnstone next-generation oncolytic virus immunotherapies.

[0239] Parvovirus H-1 parvovirus (H-1PV) is a small, non-enveloped, single-stranded DNA virus belonging to the Parvoviridae family, and its natural host is the rat (Angelova et al. (2017) Front. Oncol. 7:93; Angelova et al. (2015) Frontiers in Bioengineering and Biotechnology 3:55). H-1PV is non-pathogenic to humans and is attractive as an oncolytic virus due to its favorable safety profile, lack of existing H-1PV immunity in humans, and lack of integration into host cell genomes (Angelova et al. (2015)). H-1PV has demonstrated broad tumor-suppressing activity against both solid tumors, including preclinical forms of breast, gastric, cervical, brain, pancreatic, and colorectal cancers, and hematological malignancies, including lymphomas and leukemias (Angelova et al. (2017) Front.Oncol.7:93; Angelova et al. (2015) Frontiers in Bioengineering and Biotechnology 3:55). H-1PV stimulates antitumor responses through increased presentation of tumor-associated antigens, dendritic cell maturation, and release of pro-inflammatory cytokines (Moehler et al. (2014) Frontiers in Oncology 4:92). H-1PV also exhibits tumor selectivity, which is thought to be due to the availability of cell replication and transcription factors, overexpression of cellular proteins that interact with the NS1 parvovirus protein, and activation of metabolic pathways involved in the functional regulation of NS1 in tumor cells rather than normal cells (Angelova et al. (2015) Frontiers in Bioengineering and Biotechnology 3:55). Due to the harmless nature of H-1PV, wild-type strains are often used, negating the need for genetically modified attenuation (Angelova et al. (2015)).

[0240] Studies have shown that infection of human glioma cells with oncolytic H-1PV leads to efficient cell killing, and that high-grade glioma stem cell models are tolerant to lytic H-1PV infection. Enhanced glioma cell killing was observed when the virus was administered immediately after tumor cell irradiation, suggesting that this protocol may be useful for unresectable recurrent glioblastoma (Angelova et al. (2017) Front. Oncol. 7:93; Angelova et al. (2015) Frontiers in Bioengineering and Biotechnology 3:55). Intracerebral or systemic H-1PV injection resulted in glioma regression without toxic side effects in immunocompetent rats with orthotopic RG-2 tumors, as well as in immunodeficient animals transplanted with human U87 gliomas (Angelova et al. (2015) Frontiers in Bioengineering and Biotechnology 3:55). Del H-1PV is a highly infectious, compatible mutant strain that spreads to human transformed cell lines and demonstrated in vivo oncolytic activity in xenograft models of pancreatic and cervical cancer (Geiss et al. (2017) Viruses 9,301). H-1PV also demonstrated oncolytic activity against a panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) (Geiss et al. (2017) Viruses 9,301) and human melanoma cells (SK29-Mel-1, SK29-Mel-1.22) (Moehler et al. (2014) Frontiers in Oncology 4:92). In another study, nude rats with cervical cancer xenografts demonstrated dose-dependent tumor growth arrest and regression after treatment with H-1PV (Angelova et al. (2015) Frontiers in Bioengineering and Biotechnology 3:55). Intratumoral and intravenous administration of H-1PV also demonstrated significant growth suppression in human breast cancer xenografts in immunodeficient mice (Angelova et al. (2015) Frontiers in Bioengineering and Biotechnology 3:55).Intratumoral H-1PV injection into mice with human gastric cancer or human Burkitt lymphoma resulted in tumor regression and growth inhibition (Angelova et al. (2015) Frontiers in Bioengineering and Biotechnology 3:55).

[0241] The first Phase I / IIa clinical trial of oncolytic H-1PV (ParvOryx01) in patients with relapsed glioblastoma multiforme was completed in 2015 (Clinical Trial NCT01301430), demonstrating favorable progression-free survival, clinical safety, and patient tolerability via intratumoral or intravenous injection (Angelova et al. (2017); Geiss et al. (2017) Viruses 9,301; Geletneky et al. (2017) Mol.Ther. 25(12):2620-2634). This study demonstrated the ability of H-1PV to dose-dependently cross the blood-brain barrier and establish an immunogenic antitumor response characterized primarily by leukocyte infiltration by CD8+ and CD4+ T lymphocytes, as well as the ability to detect several markers of immune cell activation, including perforin, granzyme B, IFNγ, IL-2, CD25, and CD40L, in locally treated tumors (Geletneky et al. (2017) Mol.Ther.25(12):2620-2634).

[0242] H-1PV has also demonstrated efficient killing of highly aggressive pancreatic ductal adenocarcinoma (PDAC) cells in vitro, including gemcitabine-resistant cells, and intratumoral injection of H-1PV resulted in tumor regression and extended animal survival in an orthotopic rat model of PDAC (Angelova et al. (2017); Angelova et al. (2015)). Similar results, including selective tumor targeting and lack of toxicity, were observed in an immunodeficient nude rat model of PDAC (Angelova et al. (2015)). Combinations of H-1PV with cell proliferation inhibitors (cisplatin, vincristine) or targeted agents (sunitinib) result in synergistic induction of apoptosis in human melanoma cells (Moehler et al. (2014)). The combination of H-1PV and the HDAC inhibitor valproic acid resulted in synergistic cytotoxicity against cervical and pancreatic cells (Angelova et al. (2017)), and the therapeutic efficacy of gemcitabine was significantly improved when combined with H-1PV in a two-step protocol (Angelova et al. (2015)). Like other viruses, H-1PV can be engineered to express anti-cancer molecules. For example, studies have shown that parvovirus H1-derived vectors expressing apoptin have a greater ability to induce apoptosis than wild-type H-1PV (Geiss et al. (2017)).

[0243] As with other oncolytic viruses, the therapeutic potential of parvoviruses is limited by nonspecific uptake due to the ubiquitous expression of cell surface receptors that recognize parvoviruses, and by the development of neutralizing antibodies after repeated administration. H-1PV, when combined with a cell-based medium, has demonstrated antitumor effects while circumventing these potential challenges. In one study, autologous MH3924A rat hepatoma cells were used for targeted delivery of H-1PV and suppression of metastases formed by the same cells (Raykov et al. (2004) Int. J. Cancer 109:742-749). The hepatoma cells were inactivated by gamma irradiation 24 hours after H-1PV infection, but this reduced the yield of progeny viruses by less than half. Compared to direct viral injection, medium-based therapy resulted in improved suppression of metastases and less production of neutralizing antibodies, supporting the use of carrier cells for systemic delivery of oncolytic parvoviruses (Raykov et al. (2004)).

[0244] Measles virus Measles virus (MV) is an enveloped single-stranded RNA virus with a negative sense genome belonging to the Paramyxoviruses family (Aref et al. (2016) Viruses 8:294; Hutzen et al. (2015) Oncolytic Virotherapy 4:109-118). Its unsegmented genome is stable, with a low risk of mutation and reversion to a pathogenic form, and replication in the cytoplasm eliminates the risk of insertion DNA mutagenesis in infected cells (Aref et al. (2016); Hutzen et al. (2015)). MV was first isolated in 1954 from a patient named Edmonston and developed into a live vaccine with an excellent safety profile, which, through attenuation after multiple in vitro passages, has protected more than one billion individuals worldwide over 50 years (Aref et al. (2016) Viruses 8:294; Hutzen et al. (2015) Oncolytic Virotherapy 4:109-118). The derivative of this strain, designated MV-Edm, is the most commonly used MV strain in oncolytic therapy research. The Schwarz / Moraten measles vaccine strain is less virulent and more immunogenic than the Edm derivative, making it safer and more immunomodulatory (Veinalde et al. (2017) Oncoimmunology 6(4):e1285992). The oncolytic effect of wild-type MV was described in the 1970s, and improvements have been reported in patients with acute lymphoblastic leukemia, Burkitt lymphoma, and Hodgkin lymphoma (Aref et al. (2016)).

[0245] MVs use three main receptors to enter target cells: CD46, nectin-4, and signaling lymphocyte-activating molecules (SLAMs) (Aref et al. (2016); Hutzen et al. (2015)). While SLAMs, expressed in activated B and T cells, immature thymocytes, monocytes, and dendritic cells, are the main receptors in wild-type strains, attenuated tumor-selective MV-Edm strains primarily target the CD46 receptor, a complement activation regulator overexpressed in many tumor cells (Aref et al. (2016); Hutzen et al. (2015); Jacobson et al. (2017) Oncotarget 8(38):63096-63109; Msaouel et al. (2013) Expert Opin. Biol. Ther. 13(4)). Nectin-4, primarily expressed in respiratory epithelium, is utilized by both wild-type and attenuated MV strains (Aref et al. (2016); Msaouel et al. (2013) Expert Opin. Biol. Ther. 13(4)). As with other oncolytic viruses, defects in the IFN antiviral response of tumor cells also contribute to MV's tumor selectivity (Aref et al. (2016); Jacobson et al. (2017) Oncotarget 8(38):63096-63109). MV has been studied in clinical trials for the treatment of several cancers, including multiple myeloma (NCT02192775, NCT00450814), head and neck cancer (NCT01846091), mesothelioma (NCT01503177), and ovarian cancer (NCT00408590, NCT02364713).

[0246] MVs have been genetically engineered to express immunostimulatory and immunomodulatory genes, including, for example, genes encoding IL-13, INF-β, GM-CSF, and Helicobacter pylori neutrophil-activating protein (NAP) (Aref et al. (2016), Hutzen et al. (2015); Msaouel et al. (2013) Expert Opin. Biol. Ther. 13(4)). Combination therapy with oncolytic MVs and anti-CTLA4 and anti-PD-L1 antibodies has been shown to be effective in melanoma mouse models (Aref et al. (2016); Hutzen et al. (2015)). Due to widespread vaccination or previous natural infection, most patients have prior immunity to MVs, which hinders their therapeutic potential. To circumvent this, MVs are delivered to tumors via carrier cells such as mesenchymal stem cells, effectively evading host neutralizing antibodies and demonstrating efficacy in preclinical models of acute lymphoblastic leukemia, hepatocellular carcinoma, and ovarian cancer (Aref et al. (2016)). Several other cell carriers, including the U-937 monocyte cell line, immature and mature primary dendritic cells, PMBCs, activated T cells, primary CD14+ cells, multiple myeloma MM1 cell line, and blood outgrowth endothelial cells, have demonstrated success in MV delivery (Msaouel et al. (2013) Expert Opin. Biol. Ther. 13(4)). A clinical trial (NCT02068794) has studied the use of oncolytic MV-infected mesenchymal stem cells in the treatment of patients with recurrent ovarian cancer. Another strategy to overcome existing immune responses includes a combination of MV therapy and immunosuppressants such as cyclophosphamide (Hutzen et al. (2015)).

[0247] MV-CEA MV-CEA, genetically engineered to express the tumor marker carcinoembryonic antigen (CEA), releases CEA into the patient's bloodstream after infection with cancer cells, enabling the detection of CEA levels and thus tracking of in vivo viral infection (Aref et al. (2016); Hutzen et al. (2015)). The therapeutic potential of MV-CEA has been preclinically demonstrated and investigated in a Phase I clinical trial (NCT00408590) for the treatment of ovarian cancer.

[0248] MV-NIS MV-NIS is another traceable oncolytic MV in the Edmonston vaccine lineage that has been engineered to express the sodium-iodine cotransporter (NIS), and exhibits superior viral amplification compared to MV-CEA due to the placement of the NIS transgene downstream of the hemagglutinin (H) gene in the MV genome, instead of upstream of the nucleocapsid (N) gene as in the MV-CEA construct (Aref et al. (2016); Galanis et al. (2015) Cancer Res. 75(1):22-30). 123 I, 124 I, 125 I, 131 I and 99m Radioisotopes such as Tc are transported via NIS expressed in MV-NIS-infected cells, enabling non-invasive imaging using, for example, PET, SPECT / CT, and gamma cameras (Msaouel et al. (2013) Expert Opin. Biol. Ther. 13(4)). NIS expression can also improve the efficacy of oncolytic MV by promoting the entry of β-emitting radioisotopes such as I-131 into tumor cells for radioviral therapy, and the results have been preclinically demonstrated in multiple myeloma, glioblastoma multiple, head and neck cancer, anaplastic thyroid cancer, and prostate cancer models (Aref et al. (2016); Hutzen et al. (2015); Msaouel et al. (2013)). Several Phase I / II clinical trials were conducted to investigate the use of MV-NIS in multiple myeloma (NCT00450814, NCT02192775), mesothelioma (NCT01503177), head and neck cancer (NCT01846091), and ovarian cancer (NCT02068794) using virus-infected MSCs.

[0249] Reovirus Respiratory entero-orphan viruses, commonly known as reoviruses, are non-pathogenic, non-enveloped double-stranded RNA viruses belonging to the Reoviridae family. Wild-type reoviruses are ubiquitous in the environment and result in 70-100% seropositivity in the general population (Gong et al. (2016) World J. Methodol. 6(1):25-42). There are three serotypes of reovirus: type 1 Lang, type 2 Jones, type 3 Abney, and type 3 Dearing (T3D). T3D is the most commonly used naturally occurring oncolytic reovirus serotype in preclinical and clinical research.

[0250] Oncolytic reoviruses are tumor-selective due to activated Ras signaling characteristic of cancer cells (Gong et al. (2016); Zhao et al. (2016) Mol. Cancer Ther. 15(5):767-773). Activation of the Ras signaling pathway disrupts the cell's antiviral response by inhibiting the phosphorylation of dsRNA-dependent protein kinase (PKR), a protein normally responsible for preventing viral protein synthesis (Zhao et al. (2016)). Ras activation also enhances viral uncoating and deassembly, increases viral progeny production and infectivity, and promotes progeny release through enhanced apoptosis (Zhao et al. (2016)). It is estimated that approximately 30% of all human tumors exhibit abnormal Ras signaling (Zhao et al. (2016)). For example, most malignant gliomas possess an activated Ras signaling pathway, and reoviruses have demonstrated antitumor activity in 83% of malignant glioma cells in vitro, in vivo in a human malignant glioma model, and in 100% of glioma specimens ex vivo (Gong et al. (2016) World J. Methodol. 6(1):25-42). Furthermore, pancreatic adenocarcinoma has a very high incidence of Ras mutations (approximately 90%), and reoviruses showed potent cytotoxicity in 100% of pancreatic cell lines tested in vitro and induced regression in 100% of subcutaneous tumor mouse models in vivo (Gong et al. (2016)).

[0251] Reoviruses have demonstrated broad preclinically broad anticancer activity across a range of malignancies, including colon cancer, breast cancer, ovarian cancer, lung cancer, skin cancer (melanoma), neurological cancer, hematological cancer, prostate cancer, bladder cancer, and head and neck cancer (Gong et al. (2016)). Reovirus therapy has now been tested in combination with radiotherapy, chemotherapy, immunotherapy, and surgery. The combination of reovirus and radiotherapy has been shown to be beneficial in treating head and neck, colorectal, and breast cancer cell lines in vitro, as well as in vivo colorectal cancer and melanoma models (Gong et al. (2016)). The combination of reovirus and gemcitabine, as well as reovirus, paclitaxel, and cisplatin, has been shown to be successful in mouse tumor models (Zhao et al. (2016)). Preclinical studies of the B16 melanoma mouse model have shown that the combination of oncolytic reovirus and anti-PD-1 therapy improves anticancer efficacy compared to reovirus alone (Gong et al. (2016); Zhao et al. (2016); Kemp et al. (2015) Viruses 8,4).

[0252] Numerous clinical trials using reoviruses have been conducted. Reolysin® (Oncolytics Biotech Inc.) has shown anticancer activity against malignancies both on its own and in combination with other therapeutic agents. For example, a Phase I clinical trial of Reolysin® (NCT00528684) for the treatment of recurrent malignant gliomas showed that the reovirus was well-tolerated, and a Phase I / II trial demonstrated that Reolysin® could kill tumor cells without damaging normal cells in patients with ovarian epithelial cancer, primary peritoneal cancer, and fallopian tube cancer who had not responded to platinum-based chemotherapy (NCT00602277). A Phase II clinical trial of Reolysin® (NCT00503295) showed that it was safe and effective in treating patients with metastatic bone and soft tissue sarcomas in the lungs. Reolysin® reovirus in combination with FOLFIRI and bevacizumab is in clinical trials in patients with metastatic colorectal cancer (NCT01274624). A Phase II clinical trial of Reolysin® reovirus in combination with the chemotherapy drug gemcitabine was conducted in patients with advanced pancreatic adenocarcinoma (NCT00998322), a Phase II clinical trial investigated the therapeutic effect of Reolysin® reovirus in combination with docetaxel in patients with metastatic castration-resistant prostate cancer (NCT01619813), and a Phase II clinical trial investigated the combination of Reolysin® reovirus and paclitaxel in patients with advanced / metastatic breast cancer (NCT01656538). In a Phase III clinical trial, the efficacy of Reolysin® reovirus in combination with paclitaxel and carboplatin in platinum-refractory head and neck cancer was investigated (NCT01166542). A Phase II clinical trial using this combination therapy was conducted in patients with non-small cell lung cancer (NCT00861627) and metastatic melanoma (NCT00984464). A Phase I clinical trial of Reolysin® reovirus in combination with carfilzomib and dexamethasone in patients with relapsed or refractory multiple myeloma is currently underway (NCT02101944).

[0253] Systemic administration of reovirus has limited therapeutic efficacy due to the presence of neutralizing an...

Claims

1. A composition or combination, (a) Oncolytic viruses and, (b) comprising isolated epidermal adipose stromal cells (SA-ASC; CD235a- / CD45- / CD34+ / CD146- / CD31-) or isolated pericytes (CD235a- / CD45- / CD34- / CD146+ / CD31-), A composition or combination in which one is a mixture of isolated cells and viruses, and the combination is two separate compositions, one of which contains viruses and the other composition contains isolated cells.

2. The composition or combination according to claim 1, wherein the oncolytic virus is selected from poxvirus, adenovirus, herpes simplex virus, Newcastle disease virus, varicella stomatitis virus, measles virus, reovirus, cytomegalovirus (CMV), and lentivirus.

3. The composition or combination according to claim 2, wherein the oncolytic virus is vaccinia virus.

4. The composition or combination according to claim 3, wherein the vaccinia virus is a poxvirus selected from the strains of Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Western Reserve, Modified Vaccinia Ankara (MVA), New York City Board of Health, Dairen, Ikeda, LC16M8, Copenhagen, Tashkent, Wyeth, IHD-J, IHD-W, Brighton, Dairen I, and Connaught.

5. A composition or combination according to any one of claims 1 to 4, wherein both oncolytic virus and superembryonic adipocytes are formulated.

6. The combination according to any one of claims 1 to 4, wherein the oncolytic virus and the superembryonic adipocytes are present in separate compositions.

7. A composition or combination according to any one of claims 1 to 6 for use in treating solid tumors or hematological malignancies.

8. The solid tumors or hematological malignancies include bladder tumors, breast tumors, prostate tumors, carcinomas, basal cell carcinomas, biliary tract cancers, bladder cancers, bone cancers, brain cancers, central nervous system (CNS) cancers, gliomas, cervical cancers, choroidal cancers, colon and rectal cancers, connective tissue cancers, digestive system cancers, endometrial cancers, esophageal cancers, eye cancers, head and neck cancers, stomach cancers, carcinomas in situ, kidney cancers, laryngeal cancers, leukemias, liver cancers, lung cancers, lymphomas, and Hodgkin's lymphomas. Tumors, non-Hodgkin lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, kidney cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, urinary tract cancer, lymphosarcoma, osteosarcoma, mammary gland tumors, mast cell tumors, brain tumors, adenosquamous cell carcinoma, carcinoid lung tumors, bronchial gland tumors, bronchial gland cancers, small cell lung cancer, non-small cell lung cancer, fibroma, myxoid cartilage Tumors, pulmonary sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma, Wilms' tumor, Burkitt lymphoma, microglia, osteoclastoma, oral neoplasms, fibrosarcoma, genital squamous cell carcinoma, sexually transmitted disease tumors, testicular tumors, seminoma, Sertoli cell tumor, hemangioepoid tumor, histiocytoma, chloroplastoma, granulocytic sarcoma, corneal papilloma, corneal squamous cell carcinoma, angiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, gastric tumor, adrenal cancer, oral papillomatosis The composition or combination according to claim 7, selected from hemangioendothelioma, cystadenoma, follicular lymphoma, intestinal lymphosarcoma, pulmonary squamous cell carcinoma, leukemia, hemangioendothelioma, ocular neoplasm, prepistolary fibrosarcoma, ulcerative squamous cell carcinoma, prepistolary carcinoma, connective tissue neoplasm, mast cell tumor, hepatocellular carcinoma, pulmonary adenomatosis, pulmonary sarcoma, Rous sarcoma, reticuloendotheliopathy, nephroblastoma, B-cell lymphoma, lymphocytic leukemia, retinoblastoma, hepatic neoplasm, lymphosarcoma, plasmacytoid leukemia, swim bladder sarcoma (fish), caseous lymphadenitis, lung cancer, insulinoma, neuroma, pancreatic islet cell tumor, gastric MALT lymphoma, and gastric adenocarcinoma.

9. A combination for use in the treatment of solid tumors or hematological malignancies in the subject, (a) Stem cells, The aforementioned stem cells contain an oncolytic virus, The stem cells are adipose-derived stromal cells selected from isolated epidermal adipocytes (SA-ASC; CD235a- / CD45- / CD34+ / CD146- / CD31-) and isolated pericytes (CD235a- / CD45- / CD34- / CD146+ / CD31-), (b) A combination comprising a composition containing a substance that activates the T cell response in the subject, which contains a blocking antibody against a negative costimulatory molecule or an agonist antibody against an activating costimulatory molecule.

10. The combination according to claim 9, wherein the substance comprises a blocking antibody against a negative costimulatory molecule in (b).

11. The combination according to claim 9, wherein the substance comprises an inhibitor of the PD-1 pathway in (b).

12. The combination according to any one of claims 9 to 11, wherein the oncolytic virus is a vaccinia virus.

13. A cell-assisted viral expression system (CAVES) for use in cancer treatment, The present invention comprises a carrier cell, an oncolytic virus, and at least one immunomodulatory protein or recombinant therapeutic protein encoded by the virus and expressed on the surface of the cell. The carrier cells are adipocytes selected from isolated superembryonic adipocytes (SA-ASC; CD235a- / CD45- / CD34+ / CD146- / CD31-) and isolated pericytes (CD235a- / CD45- / CD34- / CD146+ / CD31-); The carrier cells contain an oncolytic virus, and the oncolytic virus infects the cells with an infection multiplicity (MOI) of 0.001 to 10; The aforementioned virus causes the carrier cells in CAVES to contain or display at least one immunomodulatory protein or recombinant therapeutic protein on their surface; The carrier cell ex vivo expresses at least one immunomodulatory protein or recombinant therapeutic protein encoded by the virus and expressed by the association of the virus and the carrier cell; and, The expression of the immunomodulatory protein and / or recombinant therapeutic protein encoded by the virus is achieved by incubating the carrier cells with the virus after infection for a sufficient time to achieve the expression of the immunomodulatory protein and / or recombinant therapeutic protein. Cell-assisted viral expression system.

14. The composition or combination according to any one of claims 1 and 3 to 12, wherein the oncolytic virus is a vaccinia virus comprising the nucleic acid sequence shown in Sequence ID No. 71 or a sequence having at least 95% sequence identity therewith.

15. Isolated cells comprising an oncolytic virus, selected from isolated epiadipocytes (SA-ASCs; CD235a- / CD45- / CD34+ / CD146- / CD31-) and isolated pericytes (CD235a- / CD45- / CD34- / CD146+ / CD31-).

16. The isolated cell according to claim 15, wherein the virus is a vaccinia virus.

17. A composition comprising the isolated cells described in claim 16 in a pharmaceutically acceptable medium.

18. The composition according to claim 17, for use in treating solid tumors or hematological malignancies.

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