Methods and compositions for treating diseases using activated lymphocytes

Abstract

Described herein are methods and compositions for treating diseases, such as cancer, including solid tumors, inflammatory diseases, autoimmune diseases, or fibrosis. Provided are methods for treating a disease in a subject comprising administering to the subject a composition comprising an oncolytic virus and an immune cell wherein the immune cell is infected with an oncolytic virus.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 227,991, filed on July 30, 2021, and U.S. Provisional Application No. 63 / 320,129, filed on March 15, 2022, which are incorporated herein by reference in their entirety and for all purposes. Background Art

[0003] Cancer is a worldwide health emergency with at least 1 in 3 people personally affected by a cancer diagnosis during their lifetime. While the incidence of some cancers (lung) is decreasing, largely due to a decline in the use of tobacco products, the number of cancer cases overall has increased by 30% from 1973 to 2015.

[0004] Despite the fact that many therapeutic approaches currently exist and are used to successfully treat many types of cancer, there remains a huge unmet need for new paradigm-shifting therapies to address undruggable, untreatable, relapsed, or treatment-resistant cancers.

[0005] New approaches to treat hard-to-treat cancers are needed. Summary of the invention

[0006] Described herein are compositions and methods for treating a disease in a subject in need thereof by administering an oncolytic virus and immune cells to the subject. In embodiments, the disease is cancer. In embodiments, the disease is an infection, an autoimmune disease, a fibrotic or inflammatory disease.

[0007] In one aspect, provided herein is a composition containing an oncolytic virus and an immune cell, wherein the immune cell is infected with the oncolytic virus.

[0008] In one aspect, there is provided herein a composition containing an oncolytic virus and a T cell or NK cell (CAR-T cell or CAR-NK cell) expressing a chimeric antigen receptor (CAR). In embodiments, CAR is a bispecific CAR, an inducible CAR or a universal CAR.

[0009] In one aspect, provided herein is a composition comprising an oncolytic virus and a T cell expressing an exogenous T cell receptor (TCR).

[0010] In one aspect, provided herein is a pharmaceutical composition containing an oncolytic virus and immune cells and a pharmaceutically acceptable excipient, wherein the immune cells are infected with the oncolytic virus.

[0011] In one aspect, provided herein is a method for treating a disease in a subject, the method comprising administering to the subject a composition containing an oncolytic virus and an immune cell, wherein the immune cell is infected with the oncolytic virus.

[0012] In one aspect, provided herein is a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a composition containing an oncolytic virus and an immune cell, wherein the immune cell is infected with the oncolytic virus.

[0013] In one aspect, the present invention provides a method for preparing a composition containing an oncolytic virus and lymphocytes. In an embodiment, the method includes obtaining lymphocytes from a subject and contacting the lymphocytes with an oncolytic virus to form lymphocytes infected with the oncolytic virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 . Shown are chimeric antigen receptor (CAR)-redirected T cells (CAR T cells) before (left) and 48 hours (h) after infection with vaccinia virus (LIVP1.1.1).

[0015] Figure 2 The left panel shows the total viral infection area (μm) based on TurboFP365 expression over time (hours) for three groups of CAR T cells versus NT T cells generated from three patients. 2 / image). The right panel shows the total virus-infected area (μm) at the end of the time course (65 hours) for each condition. 2 / Image). Numbers indicate the patients from which the T cells were generated.

[0016] Figure 3 Total plaque forming units (PFU) of samples taken from conditioned media are shown. In the upper two panels, the Listeria strain (expressing TurboFP635 or ig-GFP variants, respectively) was used, and in the lower panel, the Copenhagen strain (expressing TurboFP635) was used. In each case, approximately 7x10 5 The total plaque forming units (PFU) were counted and plotted at 24, 48, 72, and 96 hours post infection.

[0017] Figure 4A-4QIt is a fluorescence micrograph showing infection and cell death progression of SH-SY5Y human neuroblastoma cells over time after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured with CAR T cells previously infected with vaccinia virus at 2:1 (SH-SY5Y cells: CAR T cells). Due to TurboFP635 expression, virus-expressing cells are red. SH-SY5Y cells were green (i.e., expressing GFP) before infection; yellow indicates co-expression of virus and GFP. Figure 4A : 1 h after co-culture. Figure 4B : 5 h after co-culture. Figure 4C : 10 h after co-culture. Figure 4D : 15h after co-culture. Figure 4E : 20 h after co-culture. Figure 4F : 1 day (d) after co-cultivation, zero 1 h. Figure 4G : 1d 5h after co-culture. Figure 4H : 1d 10h after co-culture. Fig. 4I : 1d 15h after co-culture. Figure 4J : 1d 20h after co-cultivation. Figure 4K : 1h after 2d of co-culture. Figure 4L : 2d 6h after co-culture. Figure 4M : 2d 11h after co-culture. Figure 4N : 2d 16h after co-culture. Fig.4O : 21h on day 2 of co-culture. Figure 4P : 1h after 3d of co-culture. Figure 4Q : 3d 3h after co-culture.

[0018] Figure 5 .Shows the ability of uninfected CAR T cells (top row) to kill SH-SY5Y neuroblastoma cells compared with CAR T cells infected with vaccinia virus (bottom row).SH-SY5Y cells were co-cultured with the CAR T cells shown at 2:1 (SH-SY5Y cells: CAR T cells), and fluorescence micrographs were taken at 0h (left) and 75h (right) after co-culture.Compared with tumor cells co-cultured with CART cells alone, tumor cells co-cultured with CAR T cells infected with vaccinia virus significantly enhanced tumor cell eradication during 75 hours of co-culture.SH-SY5Y cells are green (cells express GFP); vaccinia virus-infected cells are red.

[0019] Figure 6. A graph showing the total green object area (live SH-SY5Y cells) versus time (hours) indicates the amount of intact tumor cells cultured under the following conditions: SH-SY5Y alone, SH-SY5Y with vaccinia virus (0.01 MOI), SH-SY5Y with CAR T cells (2:1 co-culture), or SH-SY5Y with vaccinia virus-infected CAR T cells (2:1 co-culture). Fluorescence micrographs were taken at the indicated time points (e.g., Figure 5 middle) to determine the total green object area.

[0020] Figure 7 .Shows the number of vaccinia-infected T cells under the indicated conditions. The two figures on the left represent FACS analysis data (%) of polyclonal activated T cells infected with vaccinia; the two figures on the right show FACS analysis of CAR expression regulation after vaccinia virus infection. The right figure shows CAR expression under each condition as % CAR-positive cells. Compared with non-genetically modified T cells, vaccinia virus infection is more homogeneous in CAR-redirected T cells, and vaccinia infection has no negative impact on the level of CAR T cell expression. No infection-NT: uninfected T cells; No infection-CAR: uninfected CAR T cells; Infection-NT: vaccinia-infected T cells; Infection-CAR: vaccinia-infected CAR T cells.

[0021] Figure 8 .The left figure shows vaccinia virus-infected T cells, NK cells, and NK-T cells. FACS analysis of cell subsets present in patient-derived CAR gene-modified T cells. The left figure shows the percentage of T populations, NK populations, and NK-T populations in non-genetically modified (NT-untransduced) T cells and genetically modified CARs after vaccinia infection. On the right, the figure shows FACS analysis of CD8 and CD4 compartments in CD3+ populations with or without vaccinia infection in NT and CAR+ patient-derived lymphocytes. CAR detection: anti-CAR idiotype. T cell populations include: CD45 / CD3, NK-T: CD45 / CD3 / CD56, NK: CD45 / CD56+ / CD3-. The right figure shows CD4 and CD8 subsets of vaccinia virus-infected T cells. In this example, the percentage of infection in CD8 cells is greater than the percentage of infection in CD4 cells. The right panel shows from left to right the % positive cells compared to the following cells: VV+: only VV positive cells, CAR+: only CAR positive cells, + / +: cells positive for VV and CAR, - / -: cells double negative for VV and CAR.

[0022] Fig. 9A.Vaccinia infection induces T cell maturation mainly in the CD8 compartment, with a small number of naive cells and a large number of effector cells. Two graphs showing the % of positive VV-infected cells versus cell types are shown here. The cell types represented are naive = naive T cells, CM = central memory T cells, EM = effector memory T cells, and ET terminal effector memory T cells, and NT = non-transduced. The bar graph on the left shows a comparison of memory NT T cells (CD4+ / CD8+) and memory CAR T cells (CD4+ / CD8+) infected by VV. The bar graph on the right shows memory CAR T cells (CD4+ / CD8+) infected by VV.

[0023] Fig. 9B .Vaccinia infection induces T cell maturation mainly in the CD8 compartment, with a small number of naive cells and a large number of effector cells. Two graphs showing the % of positive VV-infected cells versus cell types are shown here. The cell types represented are naive = naive T cells, CM = central memory T cells, EM = effector memory T cells, and ET terminal effector memory T cells, and NT = non-transduced. The bar graph on the left shows a comparison of the maturation of memory CAR T cells (CD4+ / CD8+) that are not infected or infected with VV. The bar graph on the right shows a comparison of the maturation of CD4 / CD8- / -NT or CAR T cells that are not infected or infected with VV. DETAILED DESCRIPTION

[0024] After reading this specification, it will be apparent to those skilled in the art how to implement the present invention in various alternative embodiments and alternative applications. However, all various embodiments of the present invention will not be described herein. It should be understood that the embodiments presented here are presented only by way of example and not limitation. Therefore, these detailed descriptions of various alternative embodiments should not be interpreted as limiting the scope or breadth of the present invention as described below.

[0025] Before disclosing and describing the present invention, it is to be understood that the aspects described below are not limited to specific compositions, methods of making such compositions, or uses thereof, as these may of course vary. It is also to be understood that the terminology used herein is for describing particular aspects only and is not intended to be limiting.

[0026] The detailed description of the present invention is divided into different parts only for the convenience of the reader, and the disclosure found in any part can be combined with the disclosure in another part. For the convenience of the reader, titles or subtitles can be used in the specification, which are not intended to affect the scope of the present invention.

[0027] I. Definitions

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the present invention belongs. In this specification and the following claims, a number of terms will be mentioned, which should be defined as having the following meanings:

[0029] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" are intended to include the plural forms as well.

[0030] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0031] The term "about", when used in front of a numerical designation (e.g., temperature, time, amount, concentration, etc., including ranges), indicates an approximate value that may vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, the term "about", when used with respect to the amount of a dosage, means that the dosage may vary by + / - 10%.

[0032] "Comprising" is intended to mean that compositions and methods include the recited elements, but not excluding other elements. "Consisting essentially of" when used to define compositions and methods will mean excluding other elements having any essential significance to the combination for the stated use. Thus, a composition consisting essentially of the elements as defined herein will not exclude other materials or steps that do not materially affect one or more basic and novel characteristics of the claimed invention. "Consisting of" shall mean excluding elements and substantial method steps that exclude more than trace amounts of other ingredients. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0033] The term "disease" or "condition" refers to the state or health of a patient or subject that can be treated with the compounds or methods provided herein. The disease can be an autoimmune disease. The disease can be an inflammatory disease. The disease can be an infectious disease.

[0034] The term "treating" or "treatment" refers to any indicator of successful treatment or improvement of an injury, disease, lesion, or condition, including any objective or subjective parameter, such as elimination; relief; reduction of symptoms or making an injury, lesion, or condition more tolerable to the patient; slowing the rate of degeneration or decline; making the final point of degeneration less debilitating; improving the patient's physical and mental health. Treatment or improvement of symptoms can be based on objective or subjective parameters; including the results of physical examinations, neuropsychiatric tests, and / or psychiatric assessments. The term "treatment" and its variations can include preventing an injury, pathology, condition, or disease. In embodiments, treatment is prevention. In embodiments, treatment does not include prevention.

[0035] "Treating" or "treatment" as used herein (and as is well understood in the art) also broadly encompasses any method for obtaining a beneficial or desired result (including a clinical result) in a subject's condition. Beneficial or desired clinical results may include, but are not limited to, alleviation or improvement of one or more symptoms or symptoms, alleviation of the extent of the disease, stabilization (i.e., not worsening) of the disease state, prevention of disease transmission or spread, delay or slowing of disease progression, improvement or alleviation of the disease state, reduction of disease recurrence, and alleviation (whether partial or total, and whether detectable or undetectable). In other words, "treatment" as used herein includes any cure, improvement, or prevention of a disease. Treatment may prevent the occurrence of a disease; inhibit the spread of a disease; alleviate symptoms of a disease (e.g., eye pain, seeing halos, red eyes, extremely high intraocular pressure), completely or partially remove the underlying cause of the disease, shorten the duration of the disease, or a combination of these.

[0036] As used herein, "treating" and "treatment" include preventive treatment. The treatment method includes administering a therapeutically effective amount of an active agent to a subject. The administration step may consist of a single administration, or may include a series of administrations. The length of the treatment cycle depends on a variety of factors, such as the severity of the disorder, the age of the patient, the concentration of the active agent, the activity of the composition used for treatment, or a combination thereof. It should also be understood that the effective dose of the active agent used for treatment or prevention may increase or decrease over the course of a particular treatment or prevention regimen. Changes in dosage may occur and become apparent through standard diagnostic assays known in the art. In some cases, long-term administration may be required. For example, the composition is administered to the subject in an amount and duration sufficient to treat the patient. In many aspects, "treating" or "treatment" is not a preventive treatment.

[0037] The term "prevention" refers to reducing the occurrence (or recurrence) of disease symptoms in a patient. As indicated above, prevention can be complete (ie, no detectable symptoms) or partial, such that fewer symptoms are observed than would be possible without treatment.

[0038] "Patient", "subject" or "subject in need" refers to a living organism that suffers from or is susceptible to a disease or condition that can be treated by administering a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, pigs, deer and other non-mammals. In some embodiments, the patient is a human. In embodiments, the human is a pediatric patient. In embodiments, the patient is a domestic animal (e.g., goats, sheep, cattle, horses, etc.). In embodiments, the patient is a companion animal, including but not limited to dogs, cats, rodents (mice, rats, gerbils, hamsters, guinea pigs, chinchillas, etc.), rabbits, ferrets, etc.

[0039] An "effective amount" is an amount sufficient to achieve the stated purpose of a compound relative to the absence of the compound (e.g., to achieve the effect for which it is administered, to treat a disease, or to alleviate one or more symptoms of a disease or condition). An example of an "effective amount" is an amount sufficient to facilitate the treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount." "Allowance" of one or more symptoms (and grammatical equivalents of the phrase) means a reduction in the severity or frequency of the one or more symptoms, or the elimination of the one or more symptoms. The exact amount will depend on the purpose of the treatment, and will be determined by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (Volumes 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th edition, 2003, Gennaro ed., Lippincott, Williams & Wilkins).

[0040] As is well known in the art, therapeutically effective amounts for humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a dose that has been found to be effective in animals. As described herein, the dose in humans can be adjusted by monitoring effectiveness and adjusting the dose up or down. It is well within the capabilities of ordinary technicians to adjust the dose to achieve maximum efficacy in humans based on the methods described herein and other methods.

[0041] As used herein, the term "therapeutically effective amount" refers to the amount of a therapeutic agent sufficient to improve a condition as described above. For example, for a given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. The therapeutic efficacy can also be expressed as a "fold" increase or decrease. For example, a therapeutically effective amount can have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times, or more relative to a control.

[0042] The dosage may vary depending on the needs of the patient and the composition used. In the context of the present disclosure, the dosage administered to the patient should be sufficient to produce a beneficial therapeutic response in the patient over time. The size of the dosage will also be determined by the presence, nature and extent of any adverse side effects. The determination of the appropriate dosage for a specific situation is within the skill range of the practitioner. In some embodiments, treatment begins with a smaller dose that is lower than the optimal dose of the composition. Thereafter, the dosage is increased in small increments until the best effect is achieved in a variety of situations. The amount and interval of the dosage can be adjusted separately to provide an effective level of the administered composition for the specific clinical indication being treated. This will provide a treatment regimen commensurate with the severity of the subject's disease state.

[0043] As used herein, the term "administering" means oral administration, administration as a suppository, topical contact, intravenous administration, parenteral administration, intraperitoneal administration, intramuscular administration, intralesional administration, intrathecal administration, intraventricular administration, intrapleural administration, intraparenchymal administration, intranasal administration or subcutaneous administration, or implanting a sustained release device (e.g., micro-osmotic pump) to a subject. Administration is carried out by any route, including parenteral routes and transmucosal (e.g., buccal, sublingual, palate, gingival, nose, vagina, rectum or transdermal) routes. Parenteral administration includes, for example, intravenous, intramuscular, intraarteriole, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial. Other delivery modes include, but are not limited to, the use of liposome formulations, intravenous infusions, etc. Administration also includes direct administration, for example, direct administration to an inflammatory site. Direct administration can be delivered via guidance, such as magnetic resonance imaging (MRI) guided delivery. In an embodiment, administration does not include the administration of any activating agent other than the activating agent listed.

[0044] "Co-administration" means that the compositions described herein are administered at the same time, just before, or just after the administration of one or more additional therapies. The compositions provided herein can be administered alone or can be co-administered to a patient. Co-administration is intended to include the administration of the compositions alone or in combination (more than one composition) simultaneously or sequentially. Therefore, the formulations can also be combined with other active substances when necessary.

[0045] The terms "autologous", "autologous cells" or "autologous transplantation" as used herein in connection with cell transplantation indicate that the donor and recipient of the cells are the same individual. The terms "allogeneic", "allogeneic cells" or "allogeneic transplantation" as used herein in connection with cell transplantation indicate that the donor and recipient of the cells are different individuals of the same species.

[0046] As used herein, the term "lymphocyte" refers to an immune cell produced in the bone marrow and found in the blood and lymphatic tissues. Lymphocytes are a type of white blood cell. Other "immune cells" include, but are not limited to, neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes.

[0047] "B cells" or "B lymphocytes" refer to their standard use in the art. B cells are lymphocytes, a type of white blood cell (leukocyte), which develop into plasma cells ("mature B cells") that produce antibodies. "Immature B cells" are cells that can develop into mature B cells. Typically, pre-B cells undergo immunoglobulin heavy chain rearrangement to become pre-B precursor B cells, and further undergo immunoglobulin light chain rearrangement to become immature B cells. Immature B cells include T1 and T2 B cells.

[0048] As used herein, "T cells" or "T lymphocytes" are lymphocyte types (white blood cell subtypes) that play an important role in cell-mediated immunity. https: / / en.wikipedia.org / wiki / Cell-mediated_immunity They can be distinguished from other lymphocytes such as B cells and natural killer cells by the presence of T cell receptors on the cell surface. https: / / en.wikipedia.org / wiki / T-cell_receptorhttps: / / en.wikipedia.org / wiki / Cell_surface_receptor T cells include, for example, natural killer T (NKT) cells, cytotoxic T lymphocytes (CTLs), regulatory T (Treg) cells, and T helper cells. In embodiments, T cells are α / β T cells (expressing TCRα / β). In embodiments, T cells are γ / δ T cells (expressing TCRγ / δ). Different types of T cells can be distinguished by using T cell detection agents.

[0049] "Memory T cells" are T cells that have previously encountered and responded to their cognate antigen during a past infection, encounter with cancer, or previous vaccination. Upon encountering their cognate antigen a second time, memory T cells can replicate (divide) to produce an immune response that is faster and stronger than the immune system's first response to the pathogen.

[0050] "Regulatory T cells" or "suppressor T cells" are lymphocytes that regulate the immune system, maintain tolerance to self-antigens and prevent autoimmune diseases.

[0051] As used herein, "CAR-T" or "chimeric antigen receptor T cell" refers to a T cell that has been genetically engineered to contain an artificial T cell receptor.

[0052] "TCR" or "T cell receptor" refers to a protein complex found on the surface of a T cell or T lymphocyte.

[0053] "Oncolytic viruses" are viruses that can infect and kill cancer cells. Examples of oncolytic viruses include, but are not limited to, adenoviruses, herpes viruses, measles viruses, coxsackie viruses, polio viruses, reoviruses, pox viruses, vaccinia viruses, vesicular stomatitis viruses, seneca viruses, RIGVIR, semliki forrest viruses, maraba viruses, and Newcastle disease viruses.

[0054] As used herein, the term "cluster of differentiation protein" or "CD protein" or "named cluster" or "classification determinant" includes any cell surface protein identified as a target for an immunophenotypic cell. CD proteins can act as receptors or ligands involved in an immune response. CD proteins include, but are not limited to, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD44v6, CD123, CD138, CD171, CD4, CD8, or CD45RA+.

[0055] As used herein, the term "cancer" refers to all types of cancers, neoplasms or malignancies found in mammals (e.g., humans), including leukemias, lymphomas, carcinomas and sarcomas. Exemplary cancers that can be treated with the compounds or methods provided herein include brain cancer, gliomas, glioblastomas, neuroblastomas, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastomas, melanomas, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head cancer, Hodgkin's disease and non-Hodgkin's lymphomas. Exemplary cancers that can be treated with the compounds or methods provided herein include thyroid cancer, endocrine system cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, rectal cancer, gastric cancer and uterine cancer. Additional examples include thyroid cancer, bile duct cancer, pancreatic adenocarcinoma, skin melanoma, colon adenocarcinoma, rectal adenocarcinoma, stomach adenocarcinoma, esophageal adenocarcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, essential macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, endocrine or exocrine pancreatic tumors, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, or prostate cancer.

[0056] The term "leukemia" refers broadly to progressive malignant diseases of the blood-forming organs and is generally characterized by aberrant proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemias are usually clinically classified based on: (1) the duration and nature of the disease, whether acute or chronic; (2) the type of cells involved; myeloid (myeloid), lymphoid (lymphoid), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood - leukemic or non-leukemic (subleukemic). Exemplary leukemias that can be treated with the compounds or methods provided herein include, for example, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, leukemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myeloid leukemia, cutaneous leukemia, stem cell leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hematopoietic leukemia, hematoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, Leukopenic leukemia, lymphoid leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia, myelogranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, or undifferentiated cell leukemia.

[0057] As used herein, the term "lymphoma" refers to a group of cancers that affect hematopoietic and lymphoid tissues. It begins in lymphocytes, which are blood cells found primarily in lymph nodes, spleen, thymus, and bone marrow. The two main types of lymphoma are non-Hodgkin's lymphoma and Hodgkin's disease. Hodgkin's disease accounts for approximately 15% of all diagnosed lymphomas. This is a cancer associated with Reed-Sternberg malignant B lymphocytes. Non-Hodgkin's lymphoma (NHL) can be classified based on the growth rate of the cancer and the cell types involved. There are aggressive (high-grade) and indolent (low-grade) types of NHL. Based on the cell types involved, there are B-cell and T-cell NHL. Exemplary B cell lymphomas that can be treated with the compounds or methods provided herein include, but are not limited to, small lymphocytic lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, extranodal (MALT) lymphoma, nodular (monocytoid B cell) lymphoma, splenic lymphoma, diffuse large cell B lymphoma, Burkitt's lymphoma, lymphoblastic lymphoma, immunoblastic large cell lymphoma, or precursor B lymphoblastic lymphoma. Exemplary T cell lymphomas that can be treated with the compounds or methods provided herein include, but are not limited to, cutaneous T cell lymphoma, peripheral T cell lymphoma, pleomorphic large cell lymphoma, mycosis fungoides, and precursor T lymphoblastic lymphoma.

[0058] The term "sarcoma" generally refers to a tumor composed of material resembling embryonic connective tissue and usually consisting of tightly packed cells embedded in a fibrous or isomorphic substance. Sarcomas that can be treated with the compounds or methods provided herein include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemies sarcoma, liposarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chlorosarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, spontaneous multiple hemorrhage sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T cells, Janssen sarcoma, Kaposi's sarcoma, hepatic stellate cell sarcoma, angiosarcoma, white sarcoma, malignant mesenchymal sarcoma, extraperiosteal sarcoma, reticulum cell sarcoma of bone, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, or telangiectatic sarcoma.

[0059] The term "cancer" refers to a malignant new growth composed of epithelial cells that tends to infiltrate surrounding tissues and give rise to metastases. Exemplary cancers that can be treated with the compounds or methods provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar cell carcinoma, acinar carcinoma, adenoid carcinoma, adenoid cystic carcinoma, adenocarcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basoepithelial cell carcinoma, basaloid carcinoma, squamous basal cell carcinoma, bronchioloalveolar carcinoma, bronchiolar carcinoma, bronchial carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, choriocarcinoma, colloid carcinoma, comedo carcinoma, uterine corpus carcinoma, cribriform carcinoma, armor carcinoma, skin carcinoma, cylindrical carcinoma, cylindrical cell carcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epidermoid carcinoma, adenoid epithelial cell carcinoma, explant carcinoma, ulcerative carcinoma, fibrocarcinoma, gelatinous carcinoma, fibrous car ... carcinoma), gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, maternal carcinoma, hematopoietic carcinoma, hepatocellular carcinoma, schüttel cell carcinoma, clear cell carcinoma, adrenal carcinoma, embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, erosive ulcer, Kulczycki cell carcinoma, large cell carcinoma,Lenticular carcinoma, lenticular carcinoma, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanoma, soft carcinoma, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, acanthocyte carcinoma, mucoid carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatoid carcinoma, Schneider's carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, potato carcinoma, spheroid cell carcinoma, spindle cell carcinoma, medullary carcinoma spongiosum, squamous cell carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, or villous carcinoma.

[0060] The term "infection" or "infectious disease" refers to a disease or condition that can be caused by an organism (such as bacteria, viruses, fungi, or any other pathogenic microbial agent). In embodiments, infectious diseases are caused by pathogenic bacteria. Pathogenic bacteria are bacteria that cause disease (e.g., in humans). In embodiments, infectious diseases are bacteria-related diseases (e.g., tuberculosis, which is caused by Mycobacterium tuberculosis). Non-limiting bacteria-related diseases include pneumonia, which can be caused by bacteria such as Streptococcus and Pseudomonas; or foodborne diseases, which can be caused by bacteria such as Shigella, Campylobacter, and Salmonella. Bacteria-related diseases also include tetanus, typhoid, diphtheria, syphilis, and leprosy. In embodiments, the disease is bacterial vaginosis (i.e., bacteria that alter the vaginal microbiota caused by bacterial overgrowth that crowds out Lactobacillus species that maintain a healthy vaginal microbiota) (e.g., yeast infection or Trichomonas vaginalis); bacterial meningitis (i.e., bacterial inflammation of the meninges); bacterial pneumonia (i.e., bacterial infection of the lungs); urinary tract infection; bacterial gastroenteritis; or bacterial skin infection (e.g., impetigo or cellulitis). In embodiments, the infectious disease is Campylobacter jejuni, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Staphylococcus aureus, Streptococcus pneumoniae, or Vibrio cholerae infection.

[0061] As used herein, the terms "metastasis", "metastatic" and "metastatic cancer" are used interchangeably and refer to the spread of a proliferative disease or condition (e.g., cancer) from one organ or another non-adjacent organ or part of the body. "Metastatic cancer" is also referred to as "stage IV cancer". Cancer occurs at the site of origin, such as the breast, which is called a primary tumor, such as primary breast cancer. Some cancer cells in the primary tumor or site of origin acquire the ability to penetrate and infiltrate the surrounding normal tissues in the local area and / or the ability to penetrate the lymphatic system or the walls of the vascular system that circulates to other parts of the body and tissues through the system. The second clinically detectable tumor formed by the cancer cells of the primary tumor is called a metastatic tumor or secondary tumor. When cancer cells metastasize, it is presumed that the metastatic tumor and its cells are similar to the cells of the original tumor. Therefore, if lung cancer metastasizes to the breast, the secondary tumor at the breast site is composed of abnormal lung cells rather than abnormal breast cells. Secondary tumors in the breast refer to metastatic lung cancer. Therefore, the phrase metastatic cancer refers to a disease in which a subject suffers from or has suffered from a primary tumor and suffers from one or more secondary tumors. The phrase non-metastatic cancer or a subject with non-metastatic cancer refers to a disease in which a subject has a primary tumor but does not have one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject with a primary lung tumor or a history of a primary lung tumor and one or more secondary tumors at a second location or locations (e.g., in the breast).

[0062] The term "cutaneous metastasis" or "skin metastasis" refers to secondary malignant cell growth in the skin, where the malignant cells originate from the primary cancer site (e.g., breast). In cutaneous metastasis, cancer cells from the primary cancer site can migrate to the skin, where they divide and cause lesions. Cutaneous metastasis can be caused by the migration of cancer cells from a breast cancer tumor to the skin.

[0063] The term "visceral metastasis" refers to secondary malignant cell growth in an internal organ (e.g., heart, lung, liver, pancreas, intestine) or body cavity (e.g., pleura, peritoneum), wherein the malignant cells originate from a primary cancer site (e.g., head and neck, liver, breast). In visceral metastasis, cancer cells from the primary cancer site can migrate to internal organs, where they divide and cause lesions. Visceral metastasis can be caused by the migration of cancer cells from liver cancer tumors or head and neck tumors to internal organs. As used herein, the term "tumor" or "tumor cell" refers to any type of tumor, including solid tumors or non-solid tumors, diffuse tumors, metastatic or disseminated tumors, or tumor cells from any form of tumor.

[0064] The term "fibrosis" refers to any process of pathological wound healing or fibrous connective tissue development in response to damage or injury. Types of fibrosis include, but are not limited to, pulmonary fibrosis (replacement fibrosis, focal fibrosis, diffuse lung parenchymal disease, cystic fibrosis, fibrothorax, idiopathic pulmonary fibrosis, and radiation-induced lung injury), liver fibrosis (bridging fibrosis, cirrhosis, senescence of hepatic stellate cells), renal fibrosis (CYR61-induced cell senescence), glial scars, cardiac fibrosis (interstitial fibrosis, replacement fibrosis, and myocardial fibrosis), arteriosclerosis, athrofibrosis, chronic kidney disease, Crohn's disease, Dupuytren's contracture, keloids, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma / systemic sclerosis, and adhesive capsulitis.

[0065] The term "fibroblast" refers to a cell type that secretes extracellular matrix (ECM) components including, but not limited to, collagen, glycosaminoglycans, reticular fibers, elastic fibers, and ground substance.

[0066] The term "vaccine" as used herein refers to any type of biological preparation that contributes to or induces an active immune response to a specific disease or pathogen. Such biological preparations may include, but are not limited to, antigens derived from pathogenic agents or portions of antigens derived from pathogenic agents. Such biological preparations may also be in the form of live attenuated preparations, including live, or weakened or modified pathogenic agents or pathogens; or in the form of inactivated or killed pathogenic agents or pathogens. Alternative forms of such biological preparations also include, but are not limited to, subunits, toxoids, conjugates, DNA, and recombinant vectors, or any suitable form that may be developed in the future or may be used to induce an active immune response thereto.

[0067] It should be noted that in some embodiments, although the term "vaccine" is used herein, the vaccine need not provide significant immunity against smallpox (or any other pathogen), as long as it is effective against the diseases described herein. For example, a vaccine can be any immunogenic or infectious composition that treats a disease. In some cases, the term is used to identify certain materials or compositions, not necessarily the ability of the material or composition to provide immunity, such as against smallpox. The virus can be from any strain, including, for example, one or more listed below and elsewhere herein, including those strains that are not part of an approved or contemplated vaccine.

[0068] As used herein, "virus" refers to any one of a large group of entities called viruses. Viruses generally contain a protein shell surrounding the RNA or DNA core of genetic material, but do not contain a semipermeable membrane, and can only grow and proliferate in living cells. The viruses used in the methods provided herein include, but are not limited to, poxviruses, adenoviruses, herpes simplex viruses, Newcastle disease viruses, vesicular stomatitis viruses, mumps viruses, influenza viruses, measles viruses, reoviruses, human immunodeficiency viruses (HIV), hantaviruses, myxoma viruses, cytomegaloviruses (CMV), slow viruses, and any plant or insect viruses.

[0069] As used herein, "heterologous nucleic acid" refers to a nucleic acid, i.e., DNA or RNA, that has been introduced into a virus or cell (or an ancestor of a cell). Such heterologous nucleic acids may comprise the sequence of a gene and operable regulatory elements. For example, a heterologous nucleic acid may comprise a selectable marker gene, a suicide gene, or a gene that expresses a useful protein product that is not endogenously expressed or is endogenously expressed at a low level.

[0070] As used herein, the term "concurrently" referring to the administration of oncolytic viruses and cells refers to administration within 48 hours of each other. In some embodiments, the oncolytic viruses and cells are administered within 36 hours of each other, within 24 hours of each other, within 12 hours of each other, within 10 hours of each other, within 8 hours of each other, within 6 hours of each other, within 4 hours of each other, within two hours of each other, and within 1 hour of each other.

[0071] The terms "autologous", "autologous cells" or "autologous transplantation" as used herein in connection with cell transplantation indicate that the donor and recipient of the cells are the same individual. The terms "allogeneic", "allogeneic cells" or "allogeneic transplantation" as used herein in connection with cell transplantation indicate that the donor and recipient of the cells are different individuals of the same species.

[0072] "Pharmaceutically acceptable excipients" and "pharmaceutically acceptable carriers" refer to substances that facilitate administration of active agents to subjects and / or facilitate absorption by subjects, and the substances can be included in the compositions of the present disclosure without causing significant adverse toxicological effects to patients. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, physiological saline solutions, lactated Ringer's solutions, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solutions), alcohols, oils, gelatin, carbohydrates such as lactose, straight-chain starch or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and pigments, etc. Such preparations can be sterilized and, if necessary, can be mixed with adjuvants (such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, coloring substances and / or aromatic substances, etc.), which do not react harmfully with the compounds of the present disclosure. Those skilled in the art will recognize that other pharmaceutical excipients can also be used in the present disclosure.

[0073] II. Oncolytic viruses

[0074] Oncolytic viruses are viruses that target cancer cells. Oncolytic viruses can be genetically engineered to selectively replicate in cancer cells and lyse cancer cells while avoiding healthy cells. Oncolytic viruses destroy cancer cells by 1) directly infecting cancer cells and lysing them, or 2) recruiting and directing a renewed host immune response to cancer cells.

[0075] The potential of oncolytic viruses as cancer treatments was first discovered after patients with myeloid leukemia were infected with influenza and entered spontaneous remission. Viruses from many categories have been genetically engineered to be oncolytic, including but not limited to: adenovirus, herpes virus, measles virus, coxsackie virus, polio virus, reovirus, pox virus, vaccinia virus, vesicular stomatitis virus, Seneca virus, RIGVIR, Semliki Forest virus, Maraba virus and Newcastle disease virus. Viral components are targets for genetic engineering and include: addition of tumor-specific promoters, viral gene knockout, viral capsid modification and expression of immune system activators (including antibodies, cytokines and co-stimulatory molecules).

[0076] Variola virus is the cause of smallpox. Unlike variola virus, vaccinia virus does not usually cause systemic disease in immunocompetent individuals, so it has been used as a live vaccine to immunize against smallpox. Due to successful vaccination against vaccinia virus worldwide, smallpox has been eradicated as a natural disease. Except for people at higher risk of poxvirus infection (e.g., laboratory workers), routine smallpox vaccination has been stopped for many years. Although routine childhood immunization against smallpox was stopped in the United States in 1972, the use of smallpox vaccine is generally considered safe for pediatric use.

[0077] Attenuated strains derived from pathogenic viruses can be used to produce live vaccines. Non-limiting examples of virus strains that have been used as smallpox vaccines include, but are not limited to, Listeria (also known as Elstree), New York City Health Department strain ("NYCBH strain"), Dalian strain, Ikeda strain, LC16M8 strain, Western Reserve (WR) strain, Copenhagen strain, Tashkent strain, Tiantan strain, Wyeth strain, IHD-J and IHD-W strains, Brighton strain, Ankara strain, MVA strain, Dalian type I strain, LIPV, LC16MO, LIVP, WR 65-16, EM63, and Connaught strain. In some embodiments, the smallpox vaccine as disclosed herein is an attenuated New York City Health Department (NYCBOH) strain of vaccinia virus. In some embodiments, the NYCBOH strain of vaccinia virus can be ATCC VR-118 or CJ-MVB-SPX.

[0078] Surprisingly, oncolytic viruses can infect immune cells, and infected immune cells can be used to target and kill tumor cells. In embodiments, the oncolytic virus is a poxvirus.

[0079] In embodiments, the poxvirus is a vaccinia virus. In some embodiments, the vaccinia virus is selected from the group consisting of Dryvax strain, ACAM1000 strain, ACAM2000 strain, Listerine strain, EM63 strain, LIVP strain, Tiantan strain, Copenhagen strain, Western Reserve strain, Modified Ankara Vaccinia (MVA) strain, New York City Health Department strain, Dalian strain, Ikeda strain, LC16M8 strain, Tashkent strain, Wyeth strain, IHD-J strain, IHD-W strain, Brighton strain, Dalian type I strain, and Connaught strain. In some embodiments, the vaccinia virus strain is Drayvax. In some embodiments, the vaccinia virus strain is ACAM1000. In some embodiments, the vaccinia virus strain is ACAM2000. In some embodiments, the vaccinia virus strain is Listerine strain. In some embodiments, the vaccinia virus strain is EM63. In some embodiments, the vaccinia virus strain is LIVP. In some embodiments, the vaccinia virus strain is Tiantan strain. In some embodiments, the vaccinia virus strain is a Copenhagen strain. In some embodiments, the vaccinia virus strain is a Western Reserve strain. In some embodiments, the vaccinia virus strain is a modified Ankara vaccinia (MVA). In some embodiments, the vaccinia virus strain is an Ikeda strain. In some embodiments, the vaccinia virus strain is a New York City Health Department strain. In some embodiments, the vaccinia virus strain is a Dalian strain. In some embodiments, the vaccinia virus strain is LC16M8. In some embodiments, the vaccinia virus strain is a Tashkent strain. In some embodiments, the vaccinia virus strain is a Wyeth strain. In some embodiments, the vaccinia virus strain is an IHD-J. In some embodiments, the vaccinia virus strain is an IHD-W. In some embodiments, the vaccinia virus strain is a Brighton strain. In some embodiments, the vaccinia virus strain is a Dalian I type strain. In some embodiments, the vaccinia virus strain is a Connaught strain.

[0080] In embodiments, poxvirus is oncolytic. In some embodiments, vaccinia virus is oncolytic. In some embodiments, oncolytic vaccinia virus is selected from Dryvax strain, ACAM1000 strain, ACAM2000 strain, Lister strain, EM63 strain, LIVP strain, Tiantan strain, Copenhagen strain, Western Reserve strain, Modified Ankara vaccinia (MVA) strain, New York City Health Department strain, Dalian strain, Ikeda strain, LC16M8 strain, Tashkent strain, Wyeth strain, IHD-J strain, IHD-W strain, Brighton strain, Dalian type I strain and Connaught strain. In some embodiments, vaccinia virus Dryvax strain is oncolytic. In some embodiments, vaccinia virus Dryvax strain is ACAM1000. In some embodiments, vaccinia virus ACAM2000 strain is oncolytic. In some embodiments, vaccinia virus Listerine strain is oncolytic. In some embodiments, vaccinia virus EM63 strain is oncolytic. In some embodiments, the vaccinia virus LIVP strain is oncolytic. In some embodiments, the vaccinia virus Tiantan strain is oncolytic. In some embodiments, the vaccinia virus Copenhagen strain is oncolytic. In some embodiments, the vaccinia virus Western Reserve strain is oncolytic. In some embodiments, the vaccinia virus modified Ankara vaccinia strain (MVA) is oncolytic. In some embodiments, the vaccinia virus New York City Health Department strain is oncolytic. In some embodiments, the vaccinia virus Dalian strain is oncolytic. In some embodiments, the vaccinia virus Ikeda strain is oncolytic. In some embodiments, the vaccinia virus LC16M8 strain is oncolytic. In some embodiments, the vaccinia virus Tashkent strain is oncolytic. In some embodiments, the vaccinia virus Wyeth strain is oncolytic. In some embodiments, the vaccinia virus IHD-J strain is oncolytic. In some embodiments, the vaccinia virus IHD-W strain is oncolytic. In some embodiments, the vaccinia virus Brighton strain is oncolytic. In some embodiments, the vaccinia virus Dalian type I strain is oncolytic. In some embodiments, the vaccinia virus Connaught strain is oncolytic.

[0081] In embodiments, oncolytic viruses do not lyse immune cells. In some embodiments, oncolytic viruses do not lyse allogeneic immune cells. In some embodiments, oncolytic viruses do not lyse autologous immune cells. In some embodiments, oncolytic viruses do not lyse lymphocytes. In embodiments, "no lysis" indicates that the virus does not lyse cells for at least 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 86 hours, 98 hours or more after infecting cells with the virus.

[0082] US Patent No. 10,105,436, which is incorporated herein by reference in its entirety for all that it teaches, describes poxviruses that can be used in the methods and compositions described herein, including smallpox vaccines.

[0083] III. Immune cells

[0084] Any method for preparing and using CAR, CAR T cells or CAR-NK cells can be used with the compositions and methods described herein. Methods for preparing and using CAR-T cells are well known in the art, such as disclosed in U.S. Pat. No. 9,328,156, which is incorporated herein by reference in its entirety for all that is taught therein.

[0085] In one aspect, provided herein is a composition containing an oncolytic virus and an immune cell, wherein the immune cell is infected with the oncolytic virus.

[0086] In embodiments, immune cells are lymphocytes. In some embodiments, lymphocytes are selected from: T cells, α / βT cells, γ / δT cells, B cells, NK cells, NK-T cells, myeloid cells or lymphoid dendritic cells. In some embodiments, lymphocytes are α / βT cells. In some embodiments, lymphocytes are γ / δT cells. In some embodiments, lymphocytes are B cells. In some embodiments, lymphocytes are NK cells. In some embodiments, lymphocytes are NK-T cells. In some embodiments, lymphocytes are myeloid cells. In some embodiments, lymphocytes are lymphoid dendritic cells.

[0087] In embodiments, the immune cells are derived from the subject to be treated with the composition (autologous). In embodiments, the immune cells are allogeneic to the subject to be treated with the composition.

[0088] In embodiments, immune cells are genetically modified. In some embodiments, immune cells are genetically modified to target target cells. In some embodiments, the target cells are cancer cells, pathogens, or cells infected by pathogens. In some embodiments, the target cells are cancer cells. In some embodiments, the target cells are pathogens. In some embodiments, the target cells are cells infected by pathogens. In some embodiments, the target cells are autologous immune cells.

[0089] In one aspect, provided herein are compositions comprising an oncolytic virus and an immune cell expressing a chimeric antigen receptor (CAR). In embodiments, the immune cell is a lymphocyte. In one aspect, provided herein is a composition comprising an oncolytic virus and a T cell or NK cell (CAR-T cell or CAR-NK cell) expressing a chimeric antigen receptor (CAR). In embodiments, CAR is a bispecific CAR, an inducible CAR or a universal CAR.

[0090] In one aspect, provided herein is a composition comprising an oncolytic virus and a T cell expressing an exogenous T cell receptor (TCR).

[0091] In some embodiments, the T cells are autologous. In some embodiments, the T cells are allogeneic.

[0092] In embodiments, chimeric antigen receptor (CAR)-T cells are also administered to a subject. In embodiments, CAR targets antigens associated with a disease. In some embodiments, CAR-T cells are autologous. In some embodiments, CAR-T cells are allogeneic.

[0093] In embodiments, chimeric antigen receptor (CAR)-T cells are also administered to a subject. In embodiments, CAR targets an antigen associated with a disease. In embodiments, CAR-T cells are autologous. In embodiments, CAR-T cells are allogeneic.

[0094] In one aspect, provided herein is a composition containing an oncolytic virus and an immune cell, wherein the immune cell is infected with the oncolytic virus.

[0095] In embodiments, the oncolytic virus is a poxvirus. In embodiments, the oncolytic virus is a vaccinia virus. In some embodiments, the oncolytic vaccinia virus is selected from Dryvax strains, ACAM1000 strains, ACAM2000 strains, Lister strains, EM63 strains, LIVP strains, Tiantan strains, Copenhagen strains, Western Reserve strains, Modified Ankara Vaccinia (MVA) strains, New York City Health Department strains, Dalian strains, Ikeda strains, LC16M8 strains, Tashkent strains, Wyeth strains, IHD-J strains, IHD-W strains, Brighton strains, Dalian I strains and Connaught strains.

[0096] In embodiments, oncolytic viruses do not lyse immune cells. In embodiments, the virus does not lyse immune cells for at least 1 day to at least 10 days after infection. In embodiments, the virus does not lyse immune cells for at least 1 day to at least 5 days after infection. In embodiments, the virus does not lyse immune cells for at least 1 day after infection. In embodiments, the virus does not lyse immune cells for at least 2 days after infection. In embodiments, the virus does not lyse immune cells for at least 3 days after infection. In embodiments, the virus does not lyse immune cells for at least 4 days after infection. In embodiments, the virus does not lyse immune cells for at least 5 days after infection. In embodiments, the virus does not lyse immune cells for at least 6 days after infection. In embodiments, the virus does not lyse immune cells for at least 7 days after infection. In embodiments, the virus does not lyse immune cells for at least 8 days after infection. In embodiments, the virus does not lyse immune cells for at least 9 days after infection. In embodiments, the virus does not lyse immune cells for at least 10 days after infection. The length of time can be any value or sub-range within the listed range.

[0097] How to use

[0098] Cancer Treatment

[0099] In embodiments, T cells, such as CAR-T cells, are administered in combination with oncolytic viruses. In some embodiments, oncolytic viruses infect T cells before administration. In some embodiments, oncolytic virus-infected T cells are administered. Methods for administering T cells, such as CAR-T cells, are well known in the art and can be determined by skilled clinicians.

[0100] The methods and compositions disclosed herein can be used to treat any solid tumor or hematological malignancy. Tumors that can be treated by the methods disclosed herein include, but are not limited to, bladder tumors, breast tumors, prostate tumors, carcinomas, basal cell carcinomas, bile duct cancer, bladder cancer, bone cancer, brain cancer, CNS cancer, gliomas, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasia, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian ... cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, colorectal cancer, kidney cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer and urinary system cancer, such as lymphosarcoma, osteosarcoma, breast tumors, mast cell tumors, brain tumors, melanoma, adenosquamous carcinoma, carcinoid lung tumors, bronchial adenoma, bronchiolar adenocarcinoma, small cell lung cancer, non-small cell lung cancer, fibroma, myxochondroma, lung sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing sarcoma, Wilm's tumor, Burkitt's lymphoma, microglioma, neuroblastoma, osteoclastoma, oral neoplasia, fibrosarcoma, osteosarcoma and rhabdomyosarcoma, genital squamous cell carcinoma, infectious venereal tumors, testicular tumors, seminoma, Sertoli cell tumor tumor), hemangiopericytoma, histiocytoma, chloroma, granulocytic sarcoma, corneal papilloma, corneal squamous cell carcinoma, angiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, gastric tumor, adrenal cancer, oral papillomatosis, hemangioendothelioma, cystadenoma, follicular lymphoma, intestinal lymphosarcoma, fibrosarcoma and squamous cell carcinoma of the lung, leukemia, hemangiopericytoma, eye neoplasia, foreskin fibrosarcoma, ulcerative squamous cell carcinoma, foreskin carcinoma, connective tissue Tissue neoplasia, mastocytoma, hepatocellular carcinoma, lymphoma, pulmonary adenomatosis, pulmonary sarcoma, Rous sarcoma, reticuloendothelial hyperplasia, fibrosarcoma, Wilms tumor, B-cell lymphoma, lymphoid leukemia, retinoblastoma, hepatic neoplasia, lymphosarcoma, plasmacytoid leukemia, porcine bladder sarcoma (in fish), caseous lymphadenitis, lung cancer, insulinoma, lymphoma, sarcoma, salivary gland tumors, neuroma, islet cell tumor, gastric MALT lymphoma, and gastric adenocarcinoma.

[0101] In some embodiments, the tumor is selected from metastatic melanoma; esophageal and gastric adenocarcinoma; bile duct cancer (any stage); pancreatic cancer (any stage); gallbladder cancer (any stage); high-grade mucinous appendiceal cancer (any stage); high-grade gastrointestinal neuroendocrine cancer (any stage); mesothelioma (any stage); soft tissue sarcoma; prostate cancer; renal cell carcinoma; small cell lung cancer; non-small cell lung cancer; head and neck squamous cell carcinoma; colorectal cancer; ovarian cancer; hepatocellular carcinoma; and glioblastoma.

[0102] In some embodiments, the tumor is selected from the group consisting of glioblastoma, breast cancer, lung cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, central nervous system tumors, and melanoma.

[0103] In some embodiments, the tumor or cancer that can be treated is a child or pediatric tumor or cancer. For example, the tumor or cancer can be a leukemia, lymphoma, sarcoma, etc. Non-limiting examples of leukemia include acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Non-limiting examples of the type of lymphoma include Hodgkin's disease (or Hodgkin's lymphoma) and non-Hodgkin's lymphoma (such as B and T cell lymphoma). Non-limiting examples of solid tumors or cancers in pediatric patients include brain tumors, Ewing's sarcoma, eye cancer (retinoblastoma), germ cell tumors, kidney tumors (for example, Wilms tumor), liver cancer, neuroblastoma, osteosarcoma, rhabdomyosarcoma, skin cancer (for example, melanoma), soft tissue sarcoma and thyroid cancer. In some embodiments, the subject is a person. In some embodiments, the subject is a pediatric patient. In some embodiments, the subject is a newborn. In some embodiments, the subject is an infant. In some embodiments, the subject is a child. In some embodiments, the subject is a teenager. In some embodiments, the subject is older than 12 months. In some embodiments, the subject is younger than 18 years old.

[0104] In embodiments, the method further comprises administering to the subject a known cancer treatment, such as a chemotherapeutic agent, a therapeutic antibody, a cancer vaccine, etc. In embodiments, the oncolytic virus comprises a recombinant polynucleotide, wherein the recombinant polynucleotide encodes a therapeutic molecule. In embodiments, the therapeutic molecule treats cancer.

[0105] U.S. Patent No. 10,105,436, which is incorporated herein by reference in its entirety, describes methods of administering, preparing, storing, and using compositions comprising poxviruses and cells that can be used in the methods and compositions described herein.

[0106] Treatment of other diseases

[0107] In one aspect, a method for treating a disease or condition in a subject in need thereof is provided, wherein the disease or condition is not cancer. The method comprises administering an oncolytic virus and an immune cell to the subject. In embodiments, the method further comprises treating the disease or condition. In embodiments, treating the disease or condition comprises administering a known treatment for the disease or condition to the subject.

[0108] In one aspect, a method for treating a disease characterized by chronic inflammation is provided. The method comprises administering an oncolytic virus and an immune cell to a subject. In embodiments, the method further comprises treating chronic inflammation. In embodiments, treating chronic inflammation comprises administering a known treatment for chronic inflammation to a subject.

[0109] In one aspect, a method for converting chronic inflammation into acute inflammation in a subject in need thereof is provided. The method comprises administering an oncolytic virus and immune cells to the subject.

[0110] In one aspect, a method for treating infection is provided. The method comprises administering an oncolytic virus and an immune cell to a subject. In embodiments, the method further comprises treating infection. In embodiments, treating chronic inflammation comprises administering a known treatment for infection to a subject. In embodiments, the infection is a pathogen infection.

[0111] In one aspect, a method for treating an autoimmune disease is provided. The method comprises administering an oncolytic virus and an immune cell to a subject. In embodiments, the method further comprises treating an autoimmune disease. In embodiments, treating an autoimmune disease comprises administering a known treatment for an autoimmune disease to a subject.

[0112] In one aspect, a method for treating fibrosis is provided. The method comprises administering an oncolytic virus and an immune cell to a subject. In embodiments, the method further comprises treating fibrosis. In embodiments, treating fibrosis comprises administering a known treatment for an autoimmune disease to the subject.

[0113] In embodiments, the oncolytic virus comprises a recombinant polynucleotide, wherein the recombinant polynucleotide encodes a therapeutic molecule. In embodiments, the therapeutic molecule treats the disease. In embodiments, the therapeutic molecule is an anti-inflammatory molecule.

[0114] In embodiments, the disease is a chronic inflammatory disease. In embodiments, the chronic inflammatory disease is an autoimmune disease. In embodiments, the chronic inflammatory disease is asthma, chronic peptic ulcer, tuberculosis, arthritis, periodontitis, ulcerative colitis, Crohn's disease, sinusitis, active hepatitis, atherosclerosis, dermatitis, inflammatory bowel disease (IBS), systemic lupus, fibromyalgia, type 1 diabetes, psoriasis, multiple sclerosis, Addison's disease, Graves' disease, Sjögren's syndrome ( syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, or celiac disease.

[0115] In embodiments, the inflammatory disease is transplant rejection, Dupytren's contracture, peyronies, periodontitis, endometriosis, hepatitis, glomerulonephritis, atherosclerosis, cardiovascular disease, arthritis (e.g., osteoarthritis, rheumatoid arthritis, or psoriatic arthritis), inflammatory encephalopathy (including sequelae of stroke, encephalitis), atherosclerosis, traumatic injury, infection, and / or shock. In one embodiment, the inflammatory disease is chronic obstructive pulmonary disease (COPD), such as emphysema, chronic bronchitis, or refractory (irreversible) asthma.

[0116] In embodiments, the inflammatory disease is an intestinal fistula, chronic radiation injury (which results in inflammatory tissue defects such as radiation cystitis or radiation enteritis), a duodenal ulcer, or a chronic inflammatory disease of the central nervous system such as post-stroke neuroinflammation, schizophrenia, autism, addiction, chronic traumatic encephalopathy, or vaccine-induced neurotoxicity.

[0117] In one embodiment, the autoimmune disease is myasthenia gravis (MG), Hashimoto's thyroiditis, vasculitis, Graves' disease, psoriasis, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome, type 1 diabetes, lupus, multiple sclerosis, rheumatoid arthritis, Addison's disease, Sjögren's syndrome, celiac disease, myositis, ankylosing spondylitis, or scleroderma.

[0118] In embodiments, the fibrosis is pulmonary fibrosis (replacement fibrosis, focal fibrosis, diffuse parenchymal lung disease, cystic fibrosis, fibrothoracis, idiopathic pulmonary fibrosis, and radiation-induced lung injury), liver fibrosis (bridging fibrosis, cirrhosis, senescence of hepatic stellate cells), renal fibrosis (CYR61-induced cellular senescence), glial scars, cardiac fibrosis (interstitial fibrosis, replacement fibrosis, and myocardial fibrosis), atherosclerosis, athrofibrosis, chronic kidney disease, Crohn's disease, Dupuytren's contracture, keloids, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma / systemic sclerosis, and adhesive capsulitis.

[0119] In one embodiment, the infectious disease is caused by bacteria, viruses or fungi. In one embodiment, the infectious disease is caused by a virus. In one embodiment, the virus is a rhinovirus, a coronavirus, influenza or a respiratory syncytial virus. In one embodiment, the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0120] In one embodiment, the infectious disease causes or is capable of causing a cytokine storm in a subject. In one embodiment, the inflammatory disease causes or is capable of causing a cytokine storm in a subject.

[0121] In embodiments, the therapeutic molecule treats the disease. In embodiments, the therapeutic molecule is a cytokine, a therapeutic antibody, a therapeutic fusion protein, an RNA, a peptide or a polypeptide. In embodiments, the cytokine is an anti-inflammatory cytokine. In embodiments, the cytokine is selected from interleukin (IL) -1 receptor antagonist, IL-4, IL-6, IL-10, IL-11, IL-13, IFN-α and transforming growth factor-β. In an embodiment, the therapeutic molecule is selected from abatacept (Orencia), adalimumab (Humira), anakinra (Kineret), certolizumab pegol (Cimzia), etanercept (Enbrel), golimumab (Simponi), infliximab (Remicade), ixekizumab (Taltz), natalizumab (Tysabri), rituximab (Rituxan), secukinumab (Cosentyx), tocilizumab (Actemra), ustekinumab (Stelara), vedolizumab (Entyvio), basiliximab (Simulect), daclizumab (Zinbryta), and muromonab (Orthoclone OKT3).

[0122] In embodiments, the therapeutic molecule improves the treatment of the disease. For example, the therapeutic molecule can be a receptor that promotes the uptake of the therapeutic agent by cells expressing the therapeutic molecule. In another example, the therapeutic molecule can be an antigen recognized by the therapeutic agent. In another example, the therapeutic molecule can be an enzyme used by cells to produce a therapeutic agent (e.g., a steroid). In embodiments, the therapeutic agent is a medicament for treating a disease.

[0123] In embodiments, the method further includes administering a therapeutic agent to the subject. In embodiments, the therapeutic agent is in the same composition as the oncolytic virus and the immune cell. In embodiments, the therapeutic agent is administered separately from the oncolytic virus and the immune cell. In embodiments, the therapeutic agent is a medicament for treating a disease. In embodiments, the therapeutic agent is selected from abatacept (Orencia), adalimumab (Humira), anakinra (Kineret), certolizumab (Cimzia), etanercept (Enbrel), golimumab (Simponi), infliximab (Remicade), tiazumab (Taltz), natalizumab (Tysabri), rituximab (Rituxan), sujin monoclonal antibody (Cosentyx), tocilizumab (Actemra), ustekinumab (Stelara), vedolizumab (Entyvio), basiliximab (Simulect), daclizumab (Zinbryta) and muromonab (Orthoclone OKT3).

[0124] In embodiments, the oncolytic virus is administered in a therapeutically effective amount, e.g., an amount sufficient to treat the disease. In embodiments, the immune cells are administered in a therapeutically effective amount, e.g., an amount sufficient to treat the disease. In embodiments, the oncolytic virus and the immune cells are in the same (single) composition.

[0125] The amount of poxvirus administered to an average-sized adult human can be, for example, 1 x 10 2 Up to 1x10 10 Plaque forming units, 1x10 3 Up to 1x10 8 Plaque forming units, 1x10 4 Up to 1x10 6 plaque forming units, or any value or subrange therebetween. As a specific example, about 2.5 x 10 5 plaque forming units.

[0126] PCT Patent Publication No. WO 2020 / 247385 is hereby incorporated by reference in its entirety for all that is taught therein.

[0127] IV. Composition

[0128] In one aspect, the present invention provides a composition comprising an oncolytic virus (e.g., a poxvirus, such as smallpox, such as a smallpox vaccine) and an immune cell. In an embodiment, the oncolytic virus comprises a recombinant polynucleotide, wherein the recombinant polynucleotide encodes a therapeutic molecule.

[0129] In some embodiments, the compositions disclosed herein include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to solvents, diluents, preservatives, dispersion or suspension aids, isotonic agents, thickeners or emulsifiers, solid binders, and lubricants suitable for a particular dosage form. Those skilled in the art are aware of the various different carriers that can be used to formulate pharmaceutical compositions, and know their preparation techniques (see Remington's Pharmaceutical Sciences, edited by Gennaro, Mack Publishing, Easton, Pa., 1995; which is incorporated herein by reference in its entirety). Pharmaceutically acceptable carriers may include, but are not limited to, Ringer's solution, isotonic saline, starch, potato starch, sugar, glucose, powdered tragacanth, malt, gelatin, talc, cellulose and its derivatives, ethylcellulose, sodium carboxymethylcellulose, cellulose acetate excipients, cocoa butter, suppository wax, agar, alginic acid, oil, cottonseed oil, peanut oil, safflower oil, sesame oil, olive oil, soybean oil, corn oil, glycol, propylene glycol, esters, ethyl laurate, ethyl oleate, buffer, aluminum hydroxide, magnesium hydroxide, phosphate buffer solution, pyrogen-free water, ethanol, other nontoxic compatible lubricants, sodium lauryl sulfate, magnesium stearate, coloring agents, release agents, coating agents, sweeteners, flavoring agents, and aromatics. Pharmaceutically acceptable carriers may also include preservatives and antioxidants. One or more of the above materials may be explicitly excluded from the compositions and methods of some embodiments.

[0130] The effective dose of each treatment modality disclosed herein may vary according to various factors, including but not limited to the specific treatment, the compound or pharmaceutical composition employed, the mode of administration, the condition being treated, and / or the severity of the condition being treated. Therefore, the dosage regimen of the combination of the present invention is selected according to a variety of factors, including the route of administration and the renal and liver function of the patient. A physician, clinician, or veterinarian of ordinary skill can easily determine and prescribe the effective amount of a single active ingredient required to prevent, resist, or prevent the progression of a condition. Achieving optimal precision in the concentration of the active ingredient within a range that produces efficacy without toxicity requires a regimen based on the kinetics of the availability of the active ingredient to the target site.

[0131] Methods for preparing pharmaceutical compositions containing the relevant therapeutics disclosed herein are known in the art and will be apparent from the art, from known standard reference texts such as Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18th Edition (1990), which is incorporated herein by reference in its entirety.

[0132] It should be understood that the embodiments described herein are not limited to vaccination or vaccination itself, but also involve generating an immune response or reaction to an antigen associated with a disease. Although the words "vaccine", "vaccination" or other similar terms are used for convenience, it should be understood that such embodiments also involve immune compositions, immunogenic compositions, immune response generation, immunization, etc., wherein absolute preventive immunity is not required or generated. For example, embodiments referring to vaccination can also involve generating or helping to cause an immunogenic response or immune response to an antigen, regardless of whether the response results in absolute eradication or immunity to the disease to be treated.

[0133] U.S. Patent No. 10,105,436, which is incorporated herein by reference in its entirety, describes methods of administering, preparing, storing, and using compositions comprising poxviruses and cells that can be used in the methods and compositions described herein.

[0134] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in view of this will be suggested to those skilled in the art and are included within the spirit and scope of this application and the scope of the appended claims. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0135] Example

[0136] Example 1: Preparation of patient-derived CAR T cells

[0137] As previously reported (Quintarelli et al., Oncoimmunology. 2018;7(6):e1433518, incorporated herein by reference in its entirety), patient-derived cells were expanded and cultured in the presence of IL7 and IL15; the same procedure is used in ongoing Phase I / II clinical studies (NCT03373097 and NCT03373071).

[0138] Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from healthy donors using lymphocyte separation medium. T lymphocytes were activated with immobilized OKT3 (1 μg / ml) and anti-CD28 (1 μg / ml) monoclonal antibodies (mAbs) in the presence of recombinant human interleukin-2 (IL2, 100 U / ml), or with a combination of recombinant human interleukin-7 (IL7, 10 ng / ml) and 15 (IL15, 5 ng / ml). On day 3, activated T cells were transduced using supernatants and the above-mentioned specific cytokines in plates pre-coated with recombinant human RetroNectin. On day 5 after transduction, T cells were expanded in a medium containing 45% RPMI1640 and 45% Click medium (Sigma-Aldrich, Co.; USA), supplemented with 10% FBS and 2 mM Glutamax, and supplemented twice a week.

[0139] Example 2: Vaccinia virus infection of CAR T cells

[0140] exist Figure 1-9B T cells used in the studies shown were infected in the presence of culture medium in each experiment. Exemplary infections included 2.5 x 10 6 VV PFU (MOI 5) infected about 5x10 5 Cells / ml were maintained for 2 to 3 days. Infected cells were washed three times before any co-culture experiments.

[0141] Vaccinia virus can infect polyclonal activated T cells and chimeric antigen receptor (CAR) T cells. In addition, vaccinia virus can replicate inside T cells to produce new viral progeny. Figure 1 Fluorescence micrographs of CAR T cells before infection with vaccinia virus (LIVP1.1.1) (left panel) and 48 hours (h) after infection (right panel) are shown.

[0142] Example 3: Cellular assay of CAR T cells infected with vaccinia virus

[0143] Figure 2 Two graphs are shown indicating that CAR T cells are more susceptible to vaccinia virus infection than non-transduced (NT) T cells. The left graph shows the total virus-infected area (μm) over time (hours) for three groups of CAR cells versus NT cells generated from three patients. 2 / image). The right panel shows the total viral infection area (μm) at the end of the time course (65 hours) for each condition (patients 1, 2, and 3 CART and NT samples). 2 / image).

[0144] Figure 3.Shows that for two vaccinia virus strains, previously infected CAR T / NT cells spontaneously release low levels of virus over 96 hours. In the upper two panels, the Listeria strain (variants TurboFP635 and ig-GFP) was used, and in the lower panel, the Copenhagen strain (TurboFP635 variant) was used. In each case, a total of ~7x10 5 The total plaque forming units (PFU) were counted and plotted at 24, 48, 72, and 96 hours.

[0145] Figure 4. Shows that vaccinia virus released by previously infected T cells can infect tumor cells in vitro. It was also found that vaccinia-infected T cells remain functional and that vaccinia virus is released from infected T cells only after the CAR receptor or exogenous T cell receptor engages the target tumor cell. Vaccinia-infected tumor cells are lysed and new vaccinia virus particles are released.

[0146] Figure 5 .Show that CAR T cells infected with vaccinia virus eradicate more SH-SY5Y neuroblastoma cells than CAR T cells alone. SH-SY5Y cells were co-cultured with CAR T cells (top row) or CART cells previously infected with vaccinia virus (bottom row) at a 2:1 ratio. Compared with tumor cells co-cultured with CAR T cells alone, tumor cell eradication was significantly enhanced during 75 hours in tumor cells co-cultured with CAR T cells infected with vaccinia virus. These data represent in vitro co-cultures that are closer to in vivo conditions, with more tumor cells than T cells in the co-cultures.

[0147] Figure 6 . A graph showing the total green object area (live cells) versus time (hours) indicates the amount of intact tumor cells in cultures alone, +vaccinia virus (0.01 MOI), +CAR T cells (2:1 co-culture), or containing vaccinia virus-infected CAR T cells.

[0148] Figure 7 .Shows the number of vaccinia-infected T cells under the indicated conditions. The two figures on the left represent FACS analysis data (%) of polyclonal activated T cells infected with vaccinia, and the two figures on the right show FACS analysis of CAR expression regulation after vaccinia virus infection. The right figure shows CAR expression under each condition as % CAR-positive cells. Compared with non-genetically modified T cells, vaccinia virus infection is more homogeneous in CAR-redirected T cells, and vaccinia infection has no negative impact on the level of CAR T cell expression. No infection-NT: uninfected T cells; No infection-CAR: uninfected CAR T cells; Infection-NT: vaccinia-infected T cells; Infection-CAR: vaccinia-infected CAR T cells.

[0149] Figure 8 .The left figure shows vaccinia virus-infected T cells, NK cells, and NK-T cells. FACS analysis of cell subsets present in patient-derived CAR gene-modified T cells. The left figure shows the percentage of T populations, NK populations, and NK-T populations in non-genetically modified (NT-untransduced) T cells and genetically modified CARs after vaccinia infection. On the right, FACS analysis of CD8 and CD4 compartments in CD3+ populations with or without vaccinia infection in NT and CAR+ patient-derived lymphocytes. CAR detection: anti-CAR idiotype. T cell populations include: CD45 / CD3, NK-T: CD45 / CD3 / CD56, NK: CD45 / CD56+ / CD3-. The right figure shows CD4 and CD8 subsets of vaccinia virus-infected T cells. In this example, the percentage of infection in CD8 cells is greater than the percentage of infection in CD4 cells. The right panel shows from left to right the % positive cells compared to the following cells: VV+: only VV positive cells, CAR+: only CAR positive cells, + / +: cells positive for VV and CAR, - / -: cells double negative for VV and CAR.

[0150] Fig. 9A Vaccinia infection induces T cell maturation primarily in the CD8 compartment, with a small number of naive cells and a large number of effector cells. Fig. 9B Vaccinia infection induces T-cell maturation predominantly in the CD8 compartment, with a small number of naive cells and a large number of effector cells. Fig. 9A and Fig. 9B It was shown that in the CD8 compartment, after VV infection, the amounts of naive and memory T cell subsets were reduced compared to non-vaccinia infected cells.

[0151] Example 4: Administration of vaccinia virus-infected CAR T cells

[0152] T cells are removed from the patient's blood by apheresis or leukapheresis. The T cells are optionally genetically engineered to express a chimeric antigen receptor of interest. The T cells can be expanded in vitro.

[0153] T cells are infected with vaccinia virus at an MOI of about 2. The infected T cells are administered to the patient via intravenous injection.

Claims

1. A composition comprising an oncolytic virus and immune cells, wherein the immune cells are infected by the oncolytic virus.

2. The composition of claim 1, wherein the immune cells are lymphocytes.

3. The composition of claim 2, wherein the lymphocyte is a T cell, a B cell, a NK cell, a NK-T cell or a myeloid cell, including a lymphoid dendritic cell.

4. The composition of any one of claims 1 to 3, wherein the immune cells are derived from a subject to be treated with the composition.

5. The composition of any one of claims 1 to 3, wherein the immune cells are allogeneic to the subject to be treated with the composition.

6. The composition of any one of claims 1 to 5, wherein the oncolytic virus is a poxvirus.

7. A composition as described in any one of claims 1 to 6, wherein the immune cell is genetically modified.

8. The composition of claim 7, wherein the immune cells are genetically modified to target target cells.

9. The composition of claim 8, wherein the target cell is a cancer cell, a fibroblast, a pathogen, or a cell infected by a pathogen.

10. The composition of any one of claims 1 to 9, wherein the virus does not lyse the immune cells for at least 5 days after infection.

11. A composition comprising a poxvirus and a T cell or NK cell expressing a chimeric antigen receptor (CAR) (CAR-T cell or CAR-NK cell).

12. A composition comprising a poxvirus and a T cell expressing an exogenous T cell receptor (TCR).

13. The composition of claim 11 or 12, wherein the T cells are derived from lymphocytes obtained from a subject to be treated with the composition.

14. The composition of claim 11 or 12, wherein the T cells are derived from lymphocytes obtained from an unrelated donor.

15. The composition of claim 13 or 14, wherein the lymphocytes are T cells, NK cells or NK-T cells.

16. The composition of claim 13 or 14, wherein the lymphocytes are T cells or NK cells.

17. The composition of any one of claims 11 to 16, wherein the CAR targets an antigen associated with a disease.

18. The composition of claim 17, wherein the disease is cancer.

19. The composition of claim 17, wherein the disease is an infectious disease.

20. The composition of claim 17, wherein the disease is fibrosis.

21. The composition of any one of claims 1 to 20, wherein the poxvirus is a vaccinia virus.

22. The composition of claim 21, wherein the vaccinia virus is selected from the group consisting of Dryvax, ACAM1000, ACAM2000, Listeria, EM63, LIVP, Tiantan, Copenhagen, Western Reserve, Modified Vaccinia Ankara (MVA), New York City Health Department, Dalian, Ikeda, LC16M8, Copenhagen Western Reserve, Tashkent, Tiantan, Wyeth, IHD-J and IHD-W, Brighton, Dalian I and Connaught.

23. The composition of claim 22, wherein the vaccinia virus is a Listeria strain.

24. The composition of claim 22, wherein the vaccinia virus is a Copenhagen strain.

25. The composition of any one of claims 1 to 24, wherein the poxvirus is oncolytic.

26. A composition as described in any one of claims 11 to 25, wherein the CAR targets CD5, CD7, CD19, CD20, CD22, CD30, CD33, CD44v6, CD123, CD138, CD171, B7-H3, BCMA, CEA, CSPG4, EGFR, EGFRvIII, EphA2, FAP, FLT3, GD2, Glypican 3, Igκ, Igλ, IL13, Her2, Her3, LeY, Mesothelin, PD-L1, PSMA, ROR1, SLAMF7.

27. The composition of any one of claims 11 to 25, wherein the exogenous TCR targets a tumor-associated antigen.

28. The composition of any one of claims 11 to 25, wherein the exogenous TCR targets a fibrosis-associated antigen.

29. The composition of claim 27, wherein the tumor-associated antigen is PRAME, WT1, survivin, MAGE-A3, MART-1, gp-100, p53, or NY-ESO1.

30. A pharmaceutical composition comprising the composition of any one of claims 1 to 29 and a pharmaceutically acceptable excipient.

31. A method for treating a disease in a subject, the method comprising administering to the subject a composition as claimed in any one of claims 1 to 30.

32. The method of claim 31, wherein the disease is an infection, an autoimmune disease, a fibrotic disease, or an inflammatory disease.

33. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject the composition of any one of claims 1 to 30.

34. The method of claim 33, wherein the cancer is selected from the group consisting of high grade glioma, low grade glioma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, B-cell and T-cell acute lymphoblastic leukemia, mantle cell lymphoma, multiple myeloma, acute myeloid leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, sarcoma, neuroblastoma, retinoblastoma, Wilms' tumor, medulloblastoma, germ cell tumors, desmoid tumors, juvenile myelomonocytic leukemia, lung cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, kidney cancer and bladder cancer, head and neck cancer, melanoma, uterine cancer and prostate cancer.

35. The method of claim 34, wherein the cancer is glioblastoma.

36. The method of any one of claims 31 to 35, wherein the composition is administered to the subject by intravenous, intraperitoneal, intrathecal, intracerebroventricular, intrapleural, intraparenchymal, intraventricular, intraarticular, or intraocular injection.

37. The method of any one of claims 31 to 35, wherein the composition is administered directly to the area affected by the disease.

38. The method of any one of claims 31 to 37, wherein the composition is administered by MRI-guided delivery.

39. The method of any one of claims 31 to 38, wherein the subject is a human.

40. The method of any one of claims 31 to 39, wherein the immune cell is derived from the subject.

41. The method of any one of claims 31 to 39, wherein the immune cells are allogeneic to the subject.

42. The method of any one of claims 31 to 41, wherein the infected immune cell releases the poxvirus upon binding to a target cell.

43. A method for preparing a composition comprising a poxvirus and lymphocytes, the method comprising: (a) obtaining leukocytes from a subject; as well as (b) contacting the leukocytes with a poxvirus to form poxvirus-infected leukocytes.

44. The method of claim 43, wherein the leukocytes are isolated prior to step b).

45. The method of claim 43 or 44, wherein the leukocytes are isolated by leukapheresis, peripheral blood or bone marrow.

46. ​​The method of any one of claims 43 to 45, wherein the leukocytes comprise α / β-T cells, γ / δ-T cells, NK-T cells, NK cells.

47. The method of claim 41, wherein the T cells are further polarized.

48. The method of claim 41, wherein the T cells are CD4 or CD8 or CD45RA+, naive T cells, memory T cells, stem cell-like memory T cells.

49. The method of any one of claims 43 to 48, wherein the T cell comprises a chimeric antigen receptor (CAR) (CAR-T cell) and an exogenous TCR.

50. The method of any one of claims 43 to 49, wherein the poxvirus is a vaccinia virus.

51. The method of claim 50, wherein the vaccinia virus is selected from the group consisting of Dryvax, ACAM1000, ACAM2000, Listeria, EM63, LIVP, Tiantan, Copenhagen, Western Reserve, Modified Vaccine Ankara (MVA), New York City Health Department, Dalian, Ikeda, LC16M8, Copenhagen Western Reserve, Tashkent, Tiantan, Wyeth, IHD-J and IHD-W, Brighton, Dalian I and Connaught.

52. The method of claim 50, wherein the vaccinia virus is a Listeria strain.

53. The method of claim 50, wherein the vaccinia virus is a Copenhagen strain.

54. The method of any one of claims 43 to 53, wherein the lymphocytes are infected with the poxvirus at a titer > 0.5 MOI.

55. The method of claim 54, wherein the lymphocytes are infected with the poxvirus at a titer > 1 MOI.

56. The method of any one of claims 43 to 55, wherein the lymphocytes are incubated with the virus for about 1 day to about 5 days.

57. The method of claim 56, wherein the lymphocytes are incubated with the virus for about 3 days.

58. The method of any one of claims 43 to 57, wherein the CAR or the exogenous TCR targets an antigen associated with a disease.

59. A method as claimed in claim 58, wherein the CAR targets CD5, CD7, CD19, CD20, CD22, CD30, CD33, CD44v6, CD123, CD138, CD171, B7-H3, BCMA, CEA, CSPG4, EGFR, EGFRvIII, EphA2, FAP, FLT3, GD2, Glypican 3, Igκ, Igλ, IL13, Her2, Her3, LeY, mesothelin, PD-L1, PSMA, ROR1, SLAMF7.

60. The method of claim 58, wherein the exogenous TCR targets a tumor-associated antigen.

61. The method of claim 60, wherein the tumor-associated antigen is PRAME, WT1, survivin, MAGE-A3, MART-1, gp-100, p53, or NY-ESO1.

62. The method of any one of claims 58 to 61, wherein the disease is cancer.

63. The method of any one of claims 58 to 61, wherein the disease is an infection, an autoimmune disease, a fibrotic or an inflammatory disease.

64. The method of any one of claims 43 to 63, wherein the poxvirus does not lyse the lymphocytes.

65. The method of any one of claims 43 to 64, wherein the lymphocytes are derived from a subject to be treated with the composition.

66. A composition as described in any one of claims 43 to 64, wherein the lymphocytes are allogeneic to the subject to be treated with the composition.

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