Methods and compositions for using activated lymphocytes in the treatment of disease

Abstract

Described herein are methods and compositions for treating a disease, such as cancer (including solid tumors), an inflammatory disease, an autoimmune disease, or a fibrotic disease. A method 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 the oncolytic virus.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 227,991, filed July 30, 2021, and U.S. Provisional Application No. 63 / 320,129, filed March 15, 2022, each of which is incorporated by reference in its entirety for all purposes. [Background technology]

[0002] Cancer is a global health emergency, with at least one in three people personally affected by a cancer diagnosis during their lifetime. Although the incidence of primary cancer (lung) has declined, due primarily to reduced use of tobacco products, the number of overall cancer cases increased by 30% between 1973 and 2015.

[0003] Although many treatments currently exist and are used successfully to treat many types of cancer, there remains a significant unmet need for paradigm-shifting new therapies to address hard-to-drug, untreatable, recurrent or treatment-unresponsive cancers.

[0004] New methods for treating intractable cancers are needed. Summary of the Invention

[0005] Described herein are compositions and methods for treating a disease in a subject in need thereof by administering an oncolytic virus and an immune cell to the subject. In embodiments, the disease is cancer. In embodiments, the disease is an infectious disease, an autoimmune disease, a fibrotic disease, or an inflammatory disease.

[0006] In one aspect, provided herein is a composition comprising an oncolytic virus and an immune cell, wherein the immune cell is infected with the oncolytic virus.

[0007] In one aspect, provided herein is a composition comprising an oncolytic virus and a T cell or NK cell expressing a chimeric antigen receptor (CAR) (CAR-T cell or CAR-NK cell). In an embodiment, the CAR is a bispecific CAR, an inducible CAR, or a universal CAR.

[0008] In one aspect, provided herein is a composition comprising an oncolytic virus and a T cell expressing an exogenous T cell receptor (TCR).

[0009] In one aspect, provided herein is a pharmaceutical composition comprising an oncolytic virus and an immune cell, and a pharma- ceutical acceptable excipient, wherein the immune cell is infected with the oncolytic virus.

[0010] In one aspect, provided herein is a method for treating a disease in a subject, the method comprising administering to the subject a composition comprising an oncolytic virus and an immune cell, wherein the immune cell is infected with the oncolytic virus.

[0011] In one aspect, provided herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject a composition comprising 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 making a composition comprising an oncolytic virus and a lymphocyte. 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]

[0013] [Figure 1]Chimeric antigen receptor (CAR)-redirected T cells (CAR T cells) are shown before (left panel) and 48 hours (h) after infection with vaccinia virus (LIVP1.1.1). [Diagram 2] The ability of vaccinia to infect non-transduced T cells (those not expressing CAR; "NT") compared to CAR T cells is shown. The plot on the left shows the total viral infected area (μm2 / image) over time (hours) based on TurboFP365 expression for three sets of CAR T cells vs. NT T cells generated from three patients. The plot on the right shows the total viral infected area (μm2 / image) for each condition at the final time point (65 hours). Numbers refer to the patient from whom the T cells were generated. [Diagram 3] Total plaque forming units (PFU) of samples from conditioned media are shown. In the top two plots, the Lister strain (mutants expressing TurboFP635 or ig-GFP, respectively) was used, and in the bottom plot, the Copenhagen strain (expressing TurboFP635) was used. In both cases, approximately 7x105 cells were infected at an MOI of approximately 1.7 and washed after 2 hours. Total plaque forming units (PFU) were counted and plotted at 24, 48, 72, and 96 hours post-infection. [Figure 4A] Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. 1 hour after co-culture. [Figure 4B]Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. Five hours after co-culture. [Figure 4C] Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. 10 hours after co-culture. [Figure 4D] Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. 15 hours after co-culture. [Figure 4E] Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. 20 hours after co-culture. [Figure 4F] Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. After 1 day (d) 1 hour of co-culture. [Figure 4G] Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. 1 day 5 hours after co-culture. [Figure 4H] Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. After 1 day 10 hours of co-culture. [Figure 4I]Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. After 1 day and 15 hours of co-culture. [Figure 4J] Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. After 1 day and 20 hours of co-culture. [Figure 4K] Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. 2 days 1 hour after co-culture. [Figure 4L] Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. 2 days 6 hours after co-culture. [Figure 4M] Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. 2 days 11 hours after co-culture. [Figure 4N] Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. 2 days 16 hours after co-culture. [Figure 4O] Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. 2 days 21 hours after co-culture. [Figure 4P]Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. 3 days 1 hour after co-culture. [Figure 4Q] Fluorescence micrographs showing the progression of infection and cell death over time in SH-SY5Y human neuroblastoma cells after co-culture with vaccinia-infected CAR T cells. SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with CAR T cells pre-infected with vaccinia virus. Virus-expressing cells are red due to TurboFP635 expression. SH-SY5Y cells before infection are green (i.e., GFP expression) and yellow indicates virus and GFP co-expression. 3 days 3 hours after co-culture. [Diagram 5] The ability of uninfected CAR T cells to kill SH-SY5Y neuroblastoma cells (top) is shown compared to CAR T cells infected with vaccinia virus (bottom). SH-SY5Y cells were co-cultured 2:1 (SH-SY5Y cells:CAR T cells) with the indicated CAR T cells, and fluorescence microscopy images were taken at 0 (left) and 75 (right) hours of co-culture. Tumor cells co-cultured with vaccinia virus-infected CAR T cells showed significantly enhanced eradication of tumor cells over 75 hours of co-culture compared to tumor cells co-cultured with CAR T cells alone. SH-SY5Y cells are green (cells express GFP) and vaccinia-infected cells are red. [Figure 6]Plots of green object total area (live SH-SY5Y cells) over time (hours) showing the amount of intact tumor cells in cultures of SH-SY5Y alone, SH-SY5Y with vaccinia virus (0.01 MOI), SH-SY5Y with CAR T cells (2:1 coculture), or SH-SY5Y with vaccinia virus-infected CAR T cells (2:1 coculture) under the following conditions: Fluorescence micrographs (e.g., similar to Figure 5) were taken at the indicated time points to determine the green object total area. [Figure 7] The number of vaccinia-infected T cells under the indicated conditions is shown. The two left plots represent FACS analysis data (%) of vaccinia-infected polyclonal activated T cells, and the two right plots show FACS analysis of the modulation of CAR expression after vaccinia virus infection. The right plot shows CAR expression in each condition as a percentage (%) of CAR-positive cells. Vaccinia virus infection is more uniform in CAR-redirected T cells compared to T cells without genetic modification, and vaccinia infection does not adversely affect the expression level of CAR T cells. Uninfected-NT: uninfected T cells; Uninfected-CAR: uninfected CAR T cells; Infected-NT: vaccinia-infected T cells; Infected-CAR: vaccinia-infected CAR T cells. [Figure 8]The left plot shows that vaccinia virus infects T cells, NK cells, and NK-T cells. FACS analysis of cell subpopulations present in patient-derived CAR-modified T cells. The left panel shows the percentages of T, NK, and NK-T populations in non-modified (NT-non-transduced) T cells and modified CARs after vaccinia infection. The right graph shows FACS analysis of CD8 and CD4 compartments in the CD3+ population in both NT and CAR+ patient-derived lymphocytes with or without vaccinia infection. CAR detection: anti-CAR idiotype. T cell populations include CD45 / CD3, NK-T: CD45 / CD3 / CD56, NK: CD45 / CD56+ / CD3-. The right plot shows that vaccinia virus infects CD4 and CD8 subpopulations of T cells. The percentage of infection in CD8 cells is greater than CD4 cells in this example. The right panel shows the percentage (%) of positive cells from left to right: VV+: VV positive cells only, CAR+: CAR positive cells only, + / +: cells positive for both VV and CAR, - / -: cells double negative for VV and CAR. [Figure 9A] Vaccinia infection induces maturation of T cells primarily in the CD8 compartment, resulting in fewer naive cells and more effector cells. Shown here are two plots of the percentage (%) of VV-infected positive cells versus cell type. 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 left bar graph shows a comparison of memory NT T cells (CD4+ / CD8+) and memory CAR T cells (CD4+ / CD8+) infected with VV. The right bar graph shows memory CAR T cells (CD4+ / CD8+) infected with VV. [Figure 9B]Vaccinia infection induces maturation of T cells mainly in the CD8 compartment, leading to less naive cells and more effector cells. Shown here are two plots of the percentage (%) of VV-infected positive cells versus cell type. 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 left bar graph shows a comparison of maturation of memory CAR T cells (CD4+ / CD8+) uninfected or infected with VV. The right bar graph shows a comparison of maturation of CD4 / CD8- / - NT or CAR T cells uninfected or infected with VV. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] After reading this description, it will become apparent to those skilled in the art how to implement the present invention in various alternative embodiments and applications. However, not all of the various embodiments of the present invention are described herein. It should be understood that the embodiments shown herein are provided by way of example only and are not limiting. Therefore, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the present invention as set forth below.

[0015] Before the present invention is disclosed and described, it is to be understood that the embodiments described below are not limited to particular compositions, methods of preparing such compositions, or uses thereof, which, as such, can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0016] The detailed description of the present invention is divided into various sections for the convenience of the reader only, and the disclosure in any section may be combined with the disclosure in another section. Headings or subheadings may be used herein for the convenience of the reader, but are not intended to affect the scope of the present invention.

[0017] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0018] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0019] "Optionally" or "optionally" means that the event or circumstance described below may or may not occur, and the description includes both cases where the event or circumstance occurs and cases where the event or circumstance does not occur.

[0020] The term "about" when used prior to numerical designations, such as temperature, time, amount, concentration, and others, including ranges, indicates an approximation that may vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, the term "about" used in reference to a dose means that the dose may vary by + / - 10%.

[0021] "Comprising" or "comprising" means that the compositions and methods include the recited elements, but are not intended to exclude others. "Consisting essentially of" when used to define compositions and methods means excluding other elements that are of essential importance to the combination for the purposes described. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not substantially affect the basic and novel feature(s) of the claimed invention. "Consisting of" means excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each term in these transitional phrases are within the scope of the invention.

[0022] The term "disease" or "condition" refers to a state or condition 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.

[0023] The term "treating" or "treatment" refers to any indication of success in treating or ameliorating an injury, disease, condition, or state, including any objective or subjective parameter, such as remission; relief; reduction of symptoms or making the injury, condition, or state tolerable to the patient; slowing the rate of degeneration or debilitation; reducing the final stage of degeneration; improving the patient's physical or mental well-being. Treatment or amelioration of symptoms can be based on subjective or objective parameters, including the results of a physical exam, neuropsychological exam, and / or psychiatric evaluation. The term "treating" and its conjugations can include prevention of injury, condition, state, or disease. In an embodiment, treating is prevention. In an embodiment, treating does not include prevention.

[0024] "Treating" or "treatment" as used herein (as is well understood in the art) also broadly includes any approach to obtain beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether partial or complete, whether detectable or undetectable, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), prevention of disease spread or spread, delay or slowing of disease progression, improvement or palliation of the disease state, reduction in recurrence of disease, and remission. In other words, "treatment" as used herein includes any cure, amelioration, or prevention of disease. Treatment may be to prevent the onset of disease, to stop the spread of disease, to relieve symptoms of disease (e.g., eye pain, seeing halos around lights, bloodshot eyes, very high eye pressure), to completely or partially eliminate the underlying cause of disease, to shorten the duration of disease, or a combination thereof.

[0025] As used herein, "treating" and "treatment" include prophylactic treatment. The treatment method includes administering a therapeutically effective amount of an active agent to a subject. The administering step may consist of a single administration or may include a series of administrations. The length of the treatment period depends on various factors, such as the severity of the condition, the age of the patient, the concentration of the active agent, the activity of the composition used for treatment, or a combination thereof. It is also understood that the effective dosage of the agent used for treatment or prevention may increase or decrease during the course of a particular treatment or prevention regimen. Changes in dosage may occur and be evident by standard diagnostic assays known in the art. In some cases, chronic administration may be required. For example, the composition is administered to the subject in an amount and for a period sufficient to treat the patient. In an embodiment, the treating or treatment is not a prophylactic treatment.

[0026] The term "prevent" refers to reducing the occurrence (or recurrence) of disease symptoms in a patient. As noted above, prevention may be complete (no detectable symptoms) or partial, with milder symptoms observed than would occur in the absence of treatment.

[0027] A "patient," "subject," or "subject in need thereof" refers to a living organism suffering from or susceptible to a disease or condition that can be treated by administration of a pharmaceutical composition provided herein. Non-limiting examples include humans, other mammals, cows, 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, cows, horses, etc.). In embodiments, the patient is a companion animal, including but not limited to dogs, cats, rodents (e.g., mice, rats, gerbils, hamsters, guinea pigs, chinchillas, etc.), rabbits, ferrets, etc.

[0028] An "effective amount" is an amount sufficient for the compound to achieve a stated purpose (e.g., achieve the effect of administration, treat a disease, or reduce one or more symptoms of a disease or condition) compared to the absence of the compound. An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or reduction of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount." A "reduction" of one or more symptoms (and grammatical equivalents of this phrase) means that the severity or frequency of the symptom(s) is reduced, or that the symptom(s) is eliminated. The exact amount will vary depending on the purpose of the treatment, and can be ascertained by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 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).

[0029] As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, dosages for humans can be derived to achieve doses that have been found to be effective in animals. Dosages in humans can be adjusted by monitoring efficacy and adjusting dosages upward or downward as described herein. Based on the methods described herein and other methods, it is well within the capabilities of one of ordinary skill in the art to adjust dosages to achieve maximum efficacy in humans.

[0030] The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent sufficient to improve a disorder, as described above. For example, for a given parameter, a therapeutically effective amount indicates an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. The therapeutic effectiveness can also be expressed as an increase or decrease of "-fold". For example, a therapeutically effective amount can have an effect of at least 1.2-fold, 1.5-fold, 2-fold, 5-fold or more compared to a control.

[0031] Dosage may vary depending on the patient's requirements and the composition used. The dose administered to the patient should be sufficient to provide the patient with a beneficial therapeutic response over time in the context of this disclosure. The size of the dose will also be determined by the existence, nature, and extent of any adverse side effects. Determining the appropriate dosage for a particular situation is within the skill of the physician. In some embodiments, treatment is initiated with a dosage that is less than the optimum amount of the composition. Thereafter, depending on the situation, the dosage is gradually increased until the optimal effect is achieved. Dosage and administration intervals can be individually adjusted to provide a level of the administered composition that is effective for the particular clinical indication being treated. This provides a treatment regimen that is commensurate with the severity of the individual's disease state.

[0032] As used herein, the term "administering" refers to oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intraventricular, intrapleural, intraparenchymal, intranasal or subcutaneous administration to a subject, or implantation of a sustained release device, e.g., a mini-osmotic pump. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, and the like. Administration also includes direct administration, e.g., administration directly to the site of inflammation. Direct administration may be via guided delivery, e.g., magnetic resonance imaging (MRI) guided delivery. In an embodiment, administration does not include administration of any active agent other than the listed active agents.

[0033] "Concomitant administration" means that the compositions described herein are administered simultaneously, immediately before, or immediately after the administration of one or more additional therapeutic methods. The compositions provided herein can be administered to a patient alone or in combination. Concomitant administration means that the compositions are administered individually or in combination (two or more compositions), simultaneously or sequentially. Thus, the preparation can be combined with other active substances if desired.

[0034] As used herein with respect to cell transplantation, the terms "autologous," "autologous cells," or "autologous transplant" indicate that the donor and recipient of cells are the same individual. As used herein with respect to cell transplantation, the terms "allogeneic," "allogeneic cells," or "allogeneic transplant" indicate that the donor and recipient of cells are different individuals of the same species.

[0035] The term "lymphocyte" as used herein refers to an immune cell that is made in the bone marrow and is present in the blood and lymphoid 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.

[0036] "B cell" or "B lymphocyte" refers to standard usage in the art. B cells are lymphocytes, a type of white blood cell, that develop into antibody-producing plasma cells ("mature B cells"). "Immature B cells" are cells that can develop into mature B cells. Generally, pro-B cells undergo rearrangement of immunoglobulin heavy chains to become pro-B pre-B cells, and further rearrangement of immunoglobulin light chains to become immature B cells. Immature B cells include T1 and T2 B cells.

[0037] As used herein, a "T cell" or "T lymphocyte" is a type of lymphocyte (a subtype of white blood cell) that plays a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of a T cell receptor on the cell surface. T cells include, for example, natural killer T (NKT) cells, cytotoxic T lymphocytes (CTLs), regulatory T (Treg) cells, and T helper T cells. In an embodiment, the T cell is an α / β T cell (expressing TCR α / β). In an embodiment, the T cell is a γ / δ T cell (expressing TCR γ / δ). The various types of T cells can be distinguished by the use of a T cell detection agent.

[0038] "Memory T cells" are T cells that have previously encountered and responded to their cognate antigen during a previous infection, cancer encounter, or previous vaccination. When memory T cells encounter their cognate antigen a second time, they can replicate (divide) and mount a faster and stronger immune response than they did the first time the immune system responded to the pathogen.

[0039] "Regulatory T cells" or "suppressor T cells" are lymphocytes that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease.

[0040] As used herein, "CAR-T" or "chimeric antigen receptor T cells" refers to T cells that have been genetically engineered to contain an artificial T cell receptor.

[0041] "TCR" or "T cell receptor" refers to the protein complex found on the surface of T cells or T lymphocytes.

[0042] "Oncolytic virus" refers to a virus that can infect and kill cancer cells. Examples of oncolytic viruses include, but are not limited to, adenovirus, herpes virus, measles virus, coxsackie virus, poliovirus, reovirus, poxvirus, vaccinia virus, vesicular stomatitis virus, Seneca virus, RIGVIR, Semliki Forest virus, Maraba virus and Newcastle disease virus.

[0043] The term "cluster of differentiation proteins" or "CD proteins" or "cluster of differentiation antigens" or "typing determinants" as used herein includes any of the cell surface proteins identified as targets for classifying cells into immunophenotypes. CD proteins may function as receptors or ligands involved in immune responses. 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+.

[0044] As used herein, the term "cancer" refers to all types of cancer, neoplasms, or malignant tumors found in mammals (e.g., humans), including leukemia, lymphoma, carcinoma, and sarcoma. Exemplary cancers that may be treated with the compounds or methods provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colon cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head cancer, Hodgkin's disease, and non-Hodgkin's lymphoma. Exemplary cancers that may be treated with the compounds or methods provided herein include thyroid, endocrine, brain, breast, cervix, colon, head and neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, and uterine cancer. Additional examples include thyroid cancer, bile duct cancer, pancreatic adenocarcinoma, cutaneous melanoma, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, squamous cell carcinoma of the head and neck, invasive breast cancer, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesion, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical carcinoma, pancreatic endocrine or exocrine tumors, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, or prostate cancer.

[0045] The term "leukemia" refers broadly to progressive malignant diseases of the blood-forming organs and is generally characterized by the abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow. Leukemias are generally classified clinically based on (1) the duration and nature of the disease: acute or chronic, (2) the type of cells involved: bone marrow (myeloid), lymph (lymphoid), or monocytic, and (3) the increase or absence of an increase in the number of abnormal cells in the blood: leukemic or non-leukemic (subleukemic). Exemplary leukemias that may be treated with the compounds or methods provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, leukemic leukemia, basophilic leukemia, blastic leukemia, bovine leukemia, chronic myeloid leukemia, leukemia cutis, embryonic leukemia, eosinophilic leukemia, Gross' leukemia, hairy cell leukemia, erythroblastic leukemia, hematoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelogenous leukemia, myelogranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia, or anaplastic cell leukemia.

[0046] As used herein, the term "lymphoma" refers to a group of cancers that affect the hematopoietic and lymphatic tissues. Lymphomas begin in lymphocytes, which are blood cells found primarily in lymph nodes, spleen, thymus, and bone marrow. There are two main types of lymphoma: non-Hodgkin's lymphoma and Hodgkin's disease. Hodgkin's disease accounts for approximately 15% of all lymphoma diagnoses. It is a Reed-Sternberg malignant B-lymphocyte-related cancer. Non-Hodgkin's lymphoma (NHL) can be classified based on how fast the cancer grows and the type of cells involved. There are aggressive and indolent types of NHL. Based on the type of cells involved, there are B-cell NHL and T-cell NHL. Exemplary B-cell lymphomas that may 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, nodal (monocytoid B-cell) lymphoma, splenic lymphoma, diffuse large cell B-cell lymphoma, Burkitt's lymphoma, lymphoblastic lymphoma, immunoblastic large cell lymphoma, or precursor B-lymphoblastic lymphoma. Exemplary T-cell lymphomas that may be treated with the compounds or methods provided herein include, but are not limited to, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma, mycosis fungoides, and precursor T-lymphoblastic lymphoma.

[0047] The term "sarcoma" generally refers to tumors composed of embryonic connective tissue-like material and generally consists of closely packed cells embedded in a fibrous or homogeneous substance. Sarcomas that may be treated with the compounds or methods provided herein include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy sarcoma, liposarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastoma, botryoid sarcoma, chlorosarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibro ...sarcoma, These include blastic cell sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multifocal pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cell, lymphoma, immunoblastic sarcoma of T cell, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemia sarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulum cell sarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, or telangiectatic sarcoma.

[0048] The term "carcinoma" refers to a malignant new growth composed of epithelial cells that tend to invade surrounding tissues and give rise to metastases. Exemplary carcinomas that may be treated with the compounds or methods provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, lobular carcinoma, adenoid cystic carcinoma, adenomatous carcinoma, adrenal cortical carcinoma, alveolar cell carcinoma, basal cell carcinoma, basaloid cell carcinoma, basal squamous cell carcinoma, bronchioloalveolar carcinoma, bronchiolocarcinoma, bronchogenic carcinoma, encephalomyeloma, cholangiocarcinoma, trophoblastic tumor, and colloid carcinoma. , comedocarcinoma, uterine cancer, cribriform carcinoma, armor carcinoma, skin cancer, columnar carcinoma, columnar cell carcinoma, ductal carcinoma, scirrhous carcinoma, embryonal carcinoma, cerebro-like carcinoma, epithelioid carcinoma, glandular epithelial carcinoma, exophytic carcinoma, ulcerative carcinoma, fibrocarcinoma, gelatinous carcinoma, mucinous carcinoma, giant cell carcinoma, adenocarcinoma, granulosa cell carcinoma, hair matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hürthle cell carcinoma, hyaline carcinoma, adrenal-like carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Chrompecher carcinoma, Kruczykki cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipoma-like carcinoma, lymphoepithelial carcinoma, medullary carcinoma carcinoma), melanoma, soft carcinoma, mucinous carcinoma, mucosecreting carcinoma, mucous cell carcinoma, mucoepidermoid carcinoma, mucosal carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, squamous cell carcinoma, soft carcinoma, renal cell carcinoma, reserve cell carcinoma, sarcomatoid carcinoma, Schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, solanoid carcinoma, spheroid cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, beaded carcinoma, telangiectatic carcinoma, telangiectatic carcinoma, transitional cell carcinoma, nodular carcinoma, nodular carcinoma, verrucous carcinoma, or choriocarcinoma.

[0049] The term "infectious disease" or "infectious disease" refers to a disease or condition that may be caused by an organism such as a bacterium, a virus, a fungus, or any other pathogenic microbial agent. In an embodiment, the infectious disease is caused by a pathogenic bacterium. A pathogenic bacterium is a bacterium that causes disease (e.g., in humans). In an embodiment, the infectious disease is a bacteria-related disease (e.g., tuberculosis caused by Mycobacterium tuberculosis). Non-limiting bacteria-related diseases include pneumonia, which may be caused by bacteria such as Streptococcus and Pseudomonas, or food poisoning, which may 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 the proliferation of bacteria that displace Lactobacilli species that maintain a healthy vaginal microbial population) (e.g., yeast infection, or Trichomonas vaginalis); bacterial meningitis (i.e., bacterial inflammation of the meninges); bacterial pneumonia (i.e., bacterial infection of the bladder; urinary tract infection; bacterial gastroenteritis; or bacterial skin infection (e.g., impetigo, or cellulitis). In embodiments, the infectious disease is an infection with Campylobacter jejuni, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitides, Staphylococcus aureus, Streptococcus pneumonia, or Vibrio cholera.

[0050] As used herein, the terms "metastasis," "metastatic," and "metastatic cancer" can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. "Metastatic cancer" is also referred to as "Stage IV cancer." Cancer begins at a site of origin, e.g., the breast, which is referred to as the primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or site of origin acquire the ability to penetrate and invade normal tissues surrounding the local area, and / or penetrate the walls of the lymphatic or vascular system circulating through that system to other sites and tissues in the body. A clinically detectable secondary tumor formed from cancer cells of the primary tumor is referred to as a metastatic tumor or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, when lung cancer metastasizes to the breast, the secondary tumor at the breast site is composed of abnormal lung cells, not abnormal breast cells. Secondary tumors in the breast are referred to as metastatic lung cancer. Thus, the term metastatic cancer refers to a disease in which a subject has or has had a primary tumor and has one or more secondary tumors. The term non-metastatic cancer or a subject with a 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 which a subject has or has had a primary lung tumor and has one or more secondary tumors in a second location or multiple locations, such as the breast.

[0051] The term "skin metastasis" or "skin metastasis" refers to the growth of secondary malignant cells in the skin, where the malignant cells originate from a primary cancer site (e.g., breast). In skin metastasis, cancerous cells from the primary cancer site migrate to the skin, where they can divide and cause lesions. Skin metastasis can result from the migration of cancer cells from a breast cancer tumor to the skin.

[0052] The term "visceral metastasis" refers to the growth of secondary malignant cells in internal organs (e.g., heart, lung, liver, pancreas, intestine) or body cavities (e.g., pleura, peritoneum) where the malignant cells originate from the primary cancer site (e.g., head and neck, liver, breast). In visceral metastasis, cancerous cells originating from the primary cancer site migrate to internal organs where they can divide and cause lesions. Visceral metastasis can result from the migration of cancer cells from liver cancer tumors or head and neck tumors to internal organs. The term "tumor" or "tumor cell" as used herein refers to any type of tumor, including solid or non-solid tumors, dispersed tumors, metastatic or disseminated tumors, or tumor cells originating from any form of tumor.

[0053] The term "fibrosis" refers to any process of pathological wound healing or development of fibrous connective tissue in response to injury or damage. Types of fibrosis include, but are not limited to, pulmonary fibrosis (replacement fibrosis, focal fibrosis, diffuse parenchymal lung disease, cystic fibrosis, fibrothorax, idiopathic pulmonary fibrosis, and radiation-induced lung injury), liver fibrosis (bridging fibrosis, liver cirrhosis, hepatic stellate cell senescence), kidney fibrosis (CYR61-induced cellular senescence), glial scar, cardiac fibrosis (interstitial fibrosis, replacement fibrosis, and myocardial fibrosis), arteriosclerosis, arthrofibrosis, chronic kidney disease, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma / systemic sclerosis, and adhesive capsulitis.

[0054] The term "fibroblast" refers to a type of cell that secretes components of the extracellular matrix (ECM), including, but not limited to, collagen, glycoaminoglycans, reticular fibers, elastic fibers, and ground substance.

[0055] The term "vaccine" as used herein refers to any type of biological preparation that contributes to or induces an active immune response against a particular disease or pathogen. Such biological preparations may include, but are not limited to, antigens derived from disease-causing agents or portions of antigens derived from disease-causing agents. Such biological preparations may also be in the form of attenuated live preparations, including live or attenuated or modified disease-causing agents or pathogens, or in the form of inactivated or killed disease-causing agents or pathogens. Alternative forms of such biological preparations include, but are not limited to, subunit, toxoid, conjugate, DNA and recombinant vector forms, or any suitable form that may be developed or made available in the future to induce an active immune response against them.

[0056] In some embodiments, the term "vaccine" is used herein, but it should be noted that a vaccine need not provide significant immunity against smallpox (or any other pathogen) as long as it is effective against the disease 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 a particular material or composition, but does not necessarily identify the ability of the material or composition to provide immunity against, for example, smallpox. The virus can be from any virus strain, including one or more of those listed below and elsewhere herein, including those that are not part of an approved or contemplated vaccine.

[0057] As used herein, "virus" refers to any of the large group of entities referred to as viruses. Viruses typically contain a protein coat surrounding an RNA or DNA core of genetic material, but do not have a semipermeable membrane and are only capable of growing and propagating in living cells. Viruses for use 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), lentiviruses, and any plant or insect viruses.

[0058] As used herein, "heterologous nucleic acid" refers to a nucleic acid, DNA or RNA, that has been introduced into a virus or cell (or an ancestor of a cell). Such heterologous nucleic acid may include sequences and operable regulatory elements related to genes. For example, heterologous nucleic acid may include a selectable marker gene, a suicide gene, or a gene that expresses a useful protein product that is not endogenously expressed or has low endogenous expression levels.

[0059] As used herein, the term "concurrently" with respect to administration of an oncolytic virus and cells refers to administration within 48 hours of each other. In some embodiments, the oncolytic virus 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 2 hours of each other, or within 1 hour of each other.

[0060] As used herein with respect to cell transplantation, the terms "autologous," "autologous cells," or "autologous transplant" indicate that the donor and recipient of cells are the same individual. As used herein with respect to cell transplantation, the terms "allogeneic," "allogeneic cells," or "allogeneic transplant" indicate that the donor and recipient of cells are different individuals of the same species.

[0061] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to substances that aid in the administration and / or absorption of an active agent by a subject and can be included in the compositions of the present disclosure without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and coloring agents. Such preparations are sterilized and can be mixed, if desired, with auxiliary substances that do not adversely react with the compounds of the present disclosure, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, coloring agents, and / or aromatic substances. Those of ordinary skill in the art will recognize other pharmaceutical excipients that are useful in the present disclosure.

[0062] II. Oncolytic viruses Oncolytic viruses are viruses that target cancer cells. They can be engineered to selectively replicate in and lyse cancer cells while avoiding healthy cells. Oncolytic viruses destroy cancer cells by either 1) directly infecting and lysing the cancer cells, or 2) recruiting and inducing a new host immune response against the cancer cells.

[0063] The potential of oncolytic viruses as a treatment for cancer was initially discovered when a patient with myeloid leukemia was infected with influenza and went into spontaneous remission. Many classes of viruses have been engineered to be oncolytic, including but not limited to adenovirus, herpesvirus, measles virus, coxsackievirus, poliovirus, reovirus, poxvirus, vaccinia virus, vesicular stomatitis virus, Seneca virus, RIGVIR, Semliki Forest virus, Maraba virus, and Newcastle disease virus. Viral components are targets for engineering, including the addition of tumor-specific promoters, knocking out viral genes, modifying the viral capsid, and expressing immune system activators including antibodies, cytokines, and costimulatory molecules.

[0064] Variola virus is the causative agent of smallpox. Unlike variola virus, vaccinia virus does not usually cause systemic disease in immunocompetent individuals and is therefore used as a live vaccine to immunize against smallpox. Smallpox has been eradicated as a natural disease due to successful worldwide vaccination with vaccinia virus. Routine smallpox vaccination has been discontinued for many years, except for individuals at high risk for poxvirus infection (e.g., laboratory workers). The United States discontinued routine childhood immunization against smallpox in 1972, but the use of the smallpox vaccine is generally considered safe for use in children.

[0065] Attenuated strains derived from pathogenic viruses can be used to produce live vaccines. Non-limiting examples of virus strains used as smallpox vaccines include, but are not limited to, Lister (also known as Elstree), New York City Board of Health ("NYCBH strain"), Dairen, Ikeda, LC16M8, Western Reserve (WR), Copenhagen, Tashkent, Tian Tan, Wyeth, IHD-J and IHD-W, Brighton, Ankara, MVA, Dairen I, LIPV, LC16MO, LIVP, WR 65-16, EM63, and Connaught strains. In some embodiments, the smallpox vaccine disclosed herein is an attenuated vaccinia virus strain of the New York City Board of Health (NYCBOH). In some embodiments, the NYCBOH strain of vaccinia virus can be ATCC VR-118 or CJ-MVB-SPX.

[0066] Surprisingly, oncolytic viruses can infect immune cells, which can be used to target and kill tumor cells, hi embodiments, the oncolytic virus is a poxvirus.

[0067] In some embodiments, the poxvirus is a vaccinia virus. In some embodiments, the vaccinia virus is selected from the following strains: Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, Modified Vaccinia Ankara (MVA), New York City Board of Health, Dairen, Ikeda, LC16M8, Tashkent, Wyeth, IHD-J, IHD-W, Brighton, Dairen I, and Connaught. 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 Lister. 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 Tian Tan. In some embodiments, the vaccinia virus strain is Copenhagen. In some embodiments, the vaccinia virus strain is Western Reserve. In some embodiments, the vaccinia virus strain is Modified Vaccinia Ankara (MVA). In some embodiments, the vaccinia virus strain is Ikeda. In some embodiments, the vaccinia virus strain is New York City Board of Health. In some embodiments, the vaccinia virus strain is Dairen. In some embodiments, the vaccinia virus strain is LC16M8. In some embodiments, the vaccinia virus strain is Tashkent. In some embodiments, the vaccinia virus strain is Wyeth. In some embodiments, the vaccinia virus strain is IHD-J. In some embodiments, the vaccinia virus strain is IHD-W. In some embodiments, the vaccinia virus strain is Brighton.In some embodiments, the vaccinia virus strain is Dairen I. In some embodiments, the vaccinia virus strain is Connaught.

[0068] In some embodiments, the poxvirus is oncolytic. In some embodiments, the vaccinia virus is oncolytic. In some embodiments, the oncolytic vaccinia virus is selected from the following strains: Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, Modified Vaccinia Ankara (MVA), New York City Board of Health, Dairen, Ikeda, LC16M8, Tashkent, Wyeth, IHD-J, IHD-W, Brighton, Dairen I, and Connaught. In some embodiments, the vaccinia virus strain Dryvax is oncolytic. In some embodiments, the vaccinia virus strain Dryvax is ACAM1000. In some embodiments, the vaccinia virus strain ACAM2000 is oncolytic. In some embodiments, the vaccinia virus strain Lister is oncolytic. In some embodiments, the vaccinia virus strain EM63 is oncolytic. In some embodiments, the vaccinia virus strain LIVP is oncolytic. In some embodiments, the vaccinia virus strain Tian Tan is oncolytic. In some embodiments, the vaccinia virus strain Copenhagen is oncolytic. In some embodiments, the vaccinia virus strain Western Reserve is oncolytic. In some embodiments, the vaccinia virus strain Modified Vaccinia Ankara (MVA) is oncolytic. In some embodiments, the vaccinia virus strain New York City Board of Health is oncolytic. In some embodiments, the vaccinia virus strain Dairen is oncolytic. In some embodiments, the vaccinia virus strain Ikeda is oncolytic. In some embodiments, the vaccinia virus strain LC16M8 is oncolytic. In some embodiments, the vaccinia virus strain Tashkent is oncolytic.In some embodiments, the vaccinia virus strain Wyeth is oncolytic. In some embodiments, the vaccinia virus strain IHD-J is oncolytic. In some embodiments, the vaccinia virus strain IHD-W is oncolytic. In some embodiments, the vaccinia virus strain Brighton is oncolytic. In some embodiments, the vaccinia virus strain Dairen I is oncolytic. In some embodiments, the vaccinia virus strain Connaught is oncolytic.

[0069] In embodiments, the oncolytic virus does not lyse immune cells. In some embodiments, the oncolytic virus does not lyse allogeneic immune cells. In some embodiments, the oncolytic virus does not lyse autoimmune cells. In some embodiments, the oncolytic virus does not lyse lymphocytes. In embodiments, "does not lyse" refers to the virus not lysing the cells for at least 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 86 hours, 98 hours or more after infecting the cells with the virus.

[0070] U.S. Pat. No. 10,105,436 describes poxviruses, including smallpox vaccines, that can be used in the methods and compositions described herein, and is incorporated by reference in its entirety for all that it teaches.

[0071] III. Immune cells Any method for making and using CARs, CAR T cells, or CAR-NK cells can be used with the compositions and methods described herein. Methods for making and using CAR-T cells are well known in the art and are disclosed, for example, in U.S. Patent No. 9,328,156, the entire teaching of which is incorporated herein by reference.

[0072] In one aspect, provided herein is a composition comprising an oncolytic virus and an immune cell, wherein the immune cell is infected with the oncolytic virus.

[0073] In embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is selected from a T cell, an alpha / beta T cell, a gamma / delta T cell, a B cell, a NK cell, a NK-T cell, a myeloid cell, or a lymphoid dendritic cell. In some embodiments, the lymphocyte is an alpha / beta T cell. In some embodiments, the lymphocyte is a gamma / delta T cell. In some embodiments, the lymphocyte is a B cell. In some embodiments, the lymphocyte is a NK cell. In some embodiments, the lymphocyte is a NK-T cell. In some embodiments, the lymphocyte is a myeloid cell. In some embodiments, the lymphocyte is a lymphoid dendritic cell.

[0074] In an embodiment, the immune cells are derived from the subject being treated with the composition (autologous). In an embodiment, the immune cells are allogeneic to the subject being treated with the composition.

[0075] In embodiments, the immune cells are genetically modified. In some embodiments, the immune cells are genetically modified to target a target cell. In some embodiments, the target cell is a cancer cell, a pathogen, or a cell infected with a pathogen. In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is a pathogen. In some embodiments, the target cell is a cell infected with a pathogen. In some embodiments, the target cell is an autoimmune cell.

[0076] In one aspect, provided herein is a composition comprising an oncolytic virus and an immune cell expressing a chimeric antigen receptor (CAR). In an embodiment, 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 expressing a chimeric antigen receptor (CAR) (CAR-T cell or CAR-NK cell). In an embodiment, the CAR is a bispecific CAR, an inducible CAR, or a universal CAR.

[0077] In one aspect, provided herein is a composition comprising an oncolytic virus and a T cell expressing an exogenous T cell receptor (TCR).

[0078] In some embodiments, the T cells are autologous. In some embodiments, the T cells are allogeneic.

[0079] In some embodiments, chimeric antigen receptor (CAR)-T cells are also administered to the subject. In some embodiments, the CAR targets an antigen associated with the disease. In some embodiments, the CAR-T cells are autologous. In some embodiments, the CAR-T cells are allogeneic.

[0080] In an embodiment, chimeric antigen receptor (CAR)-T cells are also administered to the subject. In an embodiment, the CAR targets an antigen associated with the disease. In an embodiment, the CAR-T cells are autologous. In an embodiment, the CAR-T cells are allogeneic.

[0081] In one aspect, provided herein is a composition comprising an oncolytic virus and an immune cell, wherein the immune cell is infected with the oncolytic virus.

[0082] 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 the following strains: Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, Modified Vaccinia Ankara (MVA), New York City Board of Health, Dairen, Ikeda, LC16M8, Tashkent, Wyeth, IHD-J, IHD-W, Brighton, Dairen I, and Connaught.

[0083] In embodiments, the oncolytic virus does 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 subrange within the recited range.

[0084] How to use Cancer Treatment In some embodiments, T cells, for example CAR-T cells, are administered in combination with oncolytic virus.In some embodiments, oncolytic virus is administered before infecting T cells.In some embodiments, oncolytic virus is administered to infect T cells.The method for administering T cells, for example CAR-T cells, is well known in the art and can be determined by a skilled clinician.

[0085] 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 bladder tumor, breast tumor, prostate tumor, carcinoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer, CNS cancer, glial tumor, cervical cancer, choriocarcinoma, colon and rectal 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, tumour, oral cancer, ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, kidney cancer, cancer of the respiratory system, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, cancer of the urinary system, e.g. lymphosarcoma, osteosarcoma, breast tumor, mast cell tumor, brain tumor, melanoma, adenosquamous carcinoma, carcinoid lung tumor, bronchial adenocarcinoma, bronchiolar adenocarcinoma, small cell lung cancer, non-small cell lung cancer, fibroma, myxochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma, Wilms' tumor, Burke's tumor, lymphoma, microglioma, neuroblastoma, osteoclastoma, oral neoplasm, fibrosarcoma, osteosarcoma and rhabdomyosarcoma, genital squamous cell carcinoma, transplantable genital tumors, testicular tumor, seminoma, Sertoli cell tumor, hemangiopericytoma, histiocytoma, chloroma, granulocytic sarcoma, corneal papilloma, corneal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, gastric tumor, adrenal carcinoma, oral papillomatosis, hemangioendothelioma, cystadenoma, follicular lymphoma, intestinal lymphosarcoma, fibrosarcoma, and pulmonary squamous cell carcinoma, leukemia, hemangiopericytoma, ocular neoplasm These include, but are not limited to, foreskin fibrosarcoma, ulcerating squamous cell carcinoma, foreskin carcinoma, connective tissue neoplasms, mast cell tumors, hepatocellular carcinoma, lymphoma, pulmonary adenomatosis, pulmonary sarcoma, Rous sarcoma, reticuloendotheliosis, fibrosarcoma, nephroblastoma, B cell lymphoma, lymphocytic leukemia, retinoblastoma, liver neoplasms, lymphosarcoma, plasma cell leukemia, sarcoma of the swim bladder (fish), caseous lymphadenitis, lung cancer, insulinoma, lymphoma, sarcoma, salivary gland tumors, neuroma, pancreatic islet cell tumors, gastric MALT lymphoma, and gastric adenocarcinoma.

[0086] In some embodiments, the tumor is selected from metastatic melanoma, esophageal and gastric adenocarcinoma, bile duct carcinoma (any stage), pancreatic adenocarcinoma (any stage), gallbladder carcinoma (any stage), high-grade mucinous carcinoma of the appendix (any stage), high-grade gastrointestinal neuroendocrine carcinoma (any stage), mesothelioma (any stage), soft tissue sarcoma, prostate cancer, renal cell carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, squamous cell carcinoma of the head and neck, colorectal carcinoma, ovarian carcinoma, hepatocellular carcinoma, and glioblastoma.

[0087] In some embodiments, the tumor is selected from glioblastoma, breast cancer, lung cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, central nervous system tumors, and melanoma.

[0088] In some embodiments, the tumor or cancer that can be treated is an infant 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 types of lymphoma include Hodgkin's disease (or Hodgkin's lymphoma) and non-Hodgkin's lymphoma (e.g., B-cell and T-cell lymphoma). Non-limiting examples of solid tumors or cancer in pediatric patients include brain tumors, Ewing's sarcoma, eye cancer (retinoblastoma), germ cell tumors, kidney tumors (e.g., Wilms' tumor), liver cancer, neuroblastoma, osteosarcoma, rhabdomyosarcoma, skin cancer (e.g., melanoma), soft tissue sarcoma, and thyroid cancer. In some embodiments, the subject is a human. 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 an adolescent. In some embodiments, the subject is over 12 months of age. In some embodiments, the subject is under 18 years of age.

[0089] In embodiments, the method further comprises administering to the subject a known treatment for cancer, such as a chemotherapeutic agent, a therapeutic antibody, a cancer vaccine, etc. In embodiments, the oncolytic virus comprises a recombinant polynucleotide, the recombinant polynucleotide encoding a therapeutic molecule. In embodiments, the therapeutic molecule treats the cancer.

[0090] U.S. Pat. No. 10,105,436 describes methods of administering, making, storing, and using compositions containing poxviruses and cells that can be used in the methods and compositions described herein, and is incorporated by reference in its entirety.

[0091] Treatment of other disorders In one aspect, a method is provided for treating a disease or condition in a subject in need thereof, where the disease or condition is not cancer. The method comprises administering an oncolytic virus and an immune cell to the subject. In an embodiment, the method further comprises treating the disease or condition. In an embodiment, treating the disease or condition comprises administering a known treatment for the disease or condition to the subject.

[0092] 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 an embodiment, the method further comprises treating the chronic inflammation. In an embodiment, treating the chronic inflammation comprises administering a known treatment for chronic inflammation to the subject.

[0093] In one aspect, a method is provided for altering chronic inflammation to acute inflammation in a subject in need thereof, the method comprising administering to the subject an oncolytic virus and an immune cell.

[0094] In one aspect, a method for treating an infectious disease is provided. The method comprises administering an oncolytic virus and an immune cell to a subject. In an embodiment, the method further comprises treating the infectious disease. In an embodiment, treating the infectious disease comprises administering to the subject a known treatment for chronic inflammation. In an embodiment, the infectious disease is an infection caused by a pathogen.

[0095] 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 an embodiment, the method further comprises treating the autoimmune disease. In an embodiment, treating the autoimmune disease comprises administering a known treatment for the autoimmune disease to the subject.

[0096] 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 an embodiment, the method further comprises treating the fibrosis. In an embodiment, treating the fibrosis comprises administering to the subject a known treatment for an autoimmune disease.

[0097] In embodiments, the oncolytic virus comprises a recombinant polynucleotide, and the recombinant polynucleotide encodes a therapeutic molecule. In embodiments, the therapeutic molecule treats a disease. In embodiments, the therapeutic molecule is an anti-inflammatory molecule.

[0098] In an embodiment, the disease is a chronic inflammatory disease.In an embodiment, the chronic inflammatory disease is an autoimmune disease.In an embodiment, 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, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, or celiac disease.

[0099] In embodiments, the inflammatory disease is transplant rejection, Dupuytren's contracture, Peyronie's disease, periodontitis, endometriosis, hepatitis, glomerulonephritis, atherosclerosis, cardiovascular disease, arthritis (e.g., osteoarthritis, rheumatoid arthritis, or psoriatic arthritis), inflammatory brain disease (including post-stroke encephalitis), atherosclerosis, traumatic injury, infection, and / or shock. In one embodiment, the inflammatory disease is chronic obstructive pulmonary disease (COPD), e.g., emphysema, chronic bronchitis, or refractory (irreversible) asthma.

[0100] In embodiments, the inflammatory disease is an intestinal fistula, chronic radiation injury (leading to inflammatory tissue defects such as radiation cystitis or radiation enteritis), duodenal ulcer, or a chronic inflammatory disease of the central nervous system, e.g., neuroinflammation after stroke, schizophrenia, autism, intoxication, chronic traumatic encephalopathy, or vaccine-induced neurotoxins.

[0101] 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 mellitus, lupus, multiple sclerosis, rheumatoid arthritis, Addison's disease, Sjogren's syndrome, celiac disease, myositis, ankylosing spondylitis, or scleroderma.

[0102] In one embodiment, the fibrosis is pulmonary fibrosis (replacement fibrosis, focal fibrosis, diffuse parenchymal lung disease, cystic fibrosis, fibrothorax, idiopathic pulmonary fibrosis, and radiation-induced lung injury), hepatic fibrosis (bridging fibrosis, cirrhosis, hepatic stellate cell senescence), renal fibrosis (CYR61 induced cellular senescence), glial scar, cardiac fibrosis (interstitial fibrosis, replacement fibrosis, and myocardial fibrosis), arteriosclerosis, arthrofibrosis, 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.

[0103] In one embodiment, the infectious disease is caused by a bacterium, a virus, or a fungus. 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).

[0104] In one embodiment, the infectious disease causes or is capable of causing a cytokine storm in the subject. In one embodiment, the inflammatory disease causes or is capable of causing a cytokine storm in the subject.

[0105] In embodiments, the therapeutic molecule treats a 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-alpha, and transforming growth factor-beta. In embodiments, the therapeutic molecule is selected from abatacept (Orencia), adalimumab (Humira), anakinra (Kineret), certolizumab (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).

[0106] In embodiments, the therapeutic molecule improves the treatment of a disease. For example, the therapeutic molecule can be a receptor that facilitates uptake of a therapeutic agent by cells expressing the therapeutic molecule. In another example, the therapeutic molecule can be an antigen that is recognized by a therapeutic agent. In another example, the therapeutic molecule can be an enzyme that is used by cells to produce a therapeutic agent (e.g., a steroid). In embodiments, the therapeutic agent is an agent that treats a disease.

[0107] In embodiments, the method further comprises administering a therapeutic agent to the subject. In embodiments, the therapeutic agent is in the same composition as the oncolytic virus and the immune cells. In embodiments, the therapeutic agent is administered separately from the oncolytic virus and the immune cells. In embodiments, the therapeutic agent is an agent that treats a disease. In embodiments, the therapeutic agent is selected from abatacept (Orencia), adalimumab (Humira), anakinra (Kineret), certolizumab (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).

[0108] 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.

[0109] The amount of poxvirus administered to an average-sized adult is, for example, 1 × 10 2 ~1×10 10 plaque-forming units, 1 × 10 3 ~1×10 8plaque-forming units, 1 × 10 4 ~1×10 6 plaque forming units, or any value or subrange therebetween. In one specific example, about 2.5×10 5 may be used.

[0110] PCT Patent Publication No. WO2020 / 247385 is hereby incorporated by reference in its entirety for all of its teachings.

[0111] IV. Composition In one aspect, provided herein is a composition comprising an oncolytic virus (e.g., a poxvirus, e.g., a smallpox, e.g., a smallpox vaccine) and an immune cell. In embodiments, the oncolytic virus comprises a recombinant polynucleotide, and the recombinant polynucleotide encodes a therapeutic molecule.

[0112] In some embodiments, the compositions disclosed herein comprise pharmaceutically acceptable carriers.As used herein, the term "pharmaceutically acceptable carriers" refers to solvents, diluents, preservatives, dispersion or suspension aids, isotonicity agents, thickening or emulsifying agents, solid binders, and lubricants that are suitable for specific dosage forms.Those skilled in the art will recognize the various different carriers that can be used when formulating pharmaceutical compositions, and also know the preparation techniques thereof (see Remington's Pharmaceutical Sciences Ed. by Gennaro, Mack Publishing, Easton, Pa., 1995; the entirety of which is incorporated herein by reference). Pharmaceutically acceptable carriers 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, ethyl cellulose, 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, buffers, aluminum hydroxide, magnesium hydroxide, phosphate buffer, pyrogen-free water, ethyl alcohol, other non-toxic compatible lubricants, sodium lauryl sulfate, magnesium stearate, coloring agents, release agents, coating agents, sweeteners, flavoring agents, and odorants. Pharmaceutically acceptable carriers may include preservatives and antioxidants. One or more of the above materials may be specifically excluded from the compositions and methods of some embodiments.

[0113] The effective dosage of each of the therapeutic methods disclosed herein may vary depending on 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. Thus, the administration regimen of the combination of the present invention is selected according to various factors, including the route of administration and the renal and hepatic function of the patient. A physician, clinician, or veterinarian of ordinary skill in the art can easily determine and prescribe the effective amount of the single active ingredient required to prevent, combat, or stop the progression of a condition. Achieving with optimal precision the concentration of the active ingredient within the range that produces efficacy without toxicity requires a regimen based on the kinetics of the availability of the active ingredient to the target site.

[0114] Methods for preparing pharmaceutical compositions containing the associated treatments disclosed herein are known in the art and can be found in the art, known standard references, such as Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18th edition (1990), which is incorporated herein by reference in its entirety.

[0115] It is to be understood that the embodiments described herein are not limited to vaccination or vaccination per se, but also relate to generating an immune response or reaction against an antigen associated with a disease. The terms "vaccine", "vaccination" or other similar terms are used for convenience, and it is to be understood that such embodiments also relate to immune compositions, immunogenic compositions, immune response generation, immunization, etc., where absolute prophylactic immunity is not required or generated. For example, embodiments relating to vaccination may also relate to helping generate or induce an immunogenic or immune response against an antigen, whether or not that response results in absolute eradication or immunization against the disease being treated.

[0116] U.S. Pat. No. 10,105,436 describes methods of administering, making, storing, and using compositions containing poxviruses and cells that can be used in the methods and compositions described herein, and is incorporated by reference in its entirety.

[0117] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations therein will be suggested to those skilled in the art and are 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. EXAMPLES

[0118] Example 1: Preparation of patient-derived CAR T cells Patient-derived cells were expanded in the presence of IL7 and IL15 as previously reported (Quintarelli et al., Oncoimmunology. 2018;7(6):e1433518, incorporated herein by reference in its entirety.) The same procedure is being used in ongoing Phase I / II clinical trials (NCT03373097 and NCT03373071).

[0119] 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 antibody (mAb) in the presence of recombinant human interleukin-2 (IL2, 100 U / ml) or a combination of recombinant human interleukin-7 (IL7, 10 ng / ml) and 15 (IL15, 5 ng / ml). Transduction of activated T cells was performed on day 3 on recombinant human RetroNectin pre-coated plates using the supernatant and specific cytokines as listed above. On day 5 after transduction, T cells were expanded in medium containing 45% RPMI1640 and 45% Click medium (Sigma-Aldrich, Co.; USA) supplemented with 10% FBS and 2 mM Glutamax. The medium was replenished twice a week.

[0120] Example 2: Infection of CAR T cells with vaccinia virus T cells used in the studies shown in Figures 1-9B were infected in each experiment in the presence of culture medium. An example of an infection was approximately 5 x 10 5 2.5 x 10 cells / ml 6 The cells were infected with 100% VV PFU (MOI of 5) for 2–3 days at 37 °C. Infected cells were washed three times before any co-culture experiments.

[0121] Vaccinia virus can infect both polyclonal activated T cells and chimeric antigen receptor (CAR) T cells. In addition, vaccinia virus can replicate in T cells and produce viral progeny. Figure 1 shows fluorescent micrographs of CAR T cells before (left panel) and 48 hours (h) after (right panel) infection with vaccinia virus (LIVP1.1.1).

[0122] Example 3: Cellular assay with CAR T cells infected with vaccinia virus Figure 2 shows two plots showing that CAR T cells are more permissive to infection by vaccinia virus than non-transduced (NT) T cells. The plot on the left shows the total area infected with virus (μm2) over time (hours) for three sets of CAR cells versus NT cells generated from three patients. 2 The plot on the right shows the total viral infected area (μm2) for each condition (CART and NT samples from patients 1, 2, and 3) at the final time point (65 hours). 2 / Image) is shown.

[0123] Figure 3 shows low levels of spontaneous virus release from pre-infected CAR T / NT cells over a 96-hour period for two vaccinia virus strains. The top two plots used the Lister strain (mutants TurboFP635 and ig-GFP), while the bottom plot used the Copenhagen strain (TurboFP635 mutant). In both cases, a total of approximately 7x10 5 Cells were infected at an MOI of approximately 1.7 and washed 2 hours later. Total plaque forming units (PFU) were counted and plotted at 24, 48, 72, and 96 hours.

[0124] Figure 4 shows that the vaccinia virus released by pre-infected T cells can infect tumor cells in vitro.It is also found that the vaccinia-infected T cells remain functional, and the vaccinia virus is released from the infected T cells only after the CAR receptor or exogenous T cell receptor binds to the target tumor cells.The vaccinia-infected tumor cells lyse and release new vaccinia particles.

[0125] Figure 5 shows that CAR T cells infected with vaccinia virus eradicate more SH-SY5Y neuroblastoma cells over time than CAR T cells alone. SH-SY5Y cells were co-cultured 2:1 with CAR T cells (top row) or CAR T cells pre-infected with vaccinia virus (bottom row). Tumor cells co-cultured with vaccinia virus-infected CAR T cells showed significantly enhanced eradication of tumor cells over 75 hours compared to tumor cells co-cultured with CAR T cells alone. These data represent in vitro co-cultures that approximate in vivo conditions, with more tumor cells than T cells in the co-culture.

[0126] Figure 6 shows plots of green object total area (live cells) over time (hours) showing the amount of intact tumor cells in culture with either alone, + vaccinia virus (0.01 MOI), + CAR T cells (2:1 co-culture), or vaccinia virus-infected CAR T cells.

[0127] FIG. 7 shows the number of vaccinia-infected T cells under the indicated conditions. The two plots on the left represent the FACS analysis data (%) of vaccinia-infected polyclonal activated T cells, and the two plots on the right show the FACS analysis of the modulation of CAR expression after vaccinia virus infection. The plot on the right shows the CAR expression in each condition as a percentage (%) of CAR-positive cells. Vaccinia virus infection is more uniform in CAR-redirected T cells compared to T cells without genetic modification, and vaccinia infection does not adversely affect the expression level of CAR T cells. Uninfected-NT: uninfected T cells; Uninfected-CAR: uninfected CAR T cells; Infected-NT: vaccinia-infected T cells; Infected-CAR: vaccinia-infected CAR T cells.

[0128] Figure 8. Left plot shows that vaccinia virus infects T cells, NK cells, and NK-T cells. FACS analysis of cell subpopulations present in patient-derived CAR-modified T cells. Left panel shows percentages of T, NK, and NK-T populations in non-modified (NT-non-transduced) T cells and modified CARs after vaccinia infection. Right: FACS analysis of CD8 and CD4 compartments in the CD3+ population in both NT and CAR+ patient-derived lymphocytes with or without vaccinia infection. CAR detection: anti-CAR idiotype. T cell populations include CD45 / CD3, NK-T: CD45 / CD3 / CD56, NK: CD45 / CD56+ / CD3-. Right plot shows that vaccinia virus infects CD4 and CD8 subpopulations of T cells. The percentage of infection in CD8 cells is greater than CD4 cells in this example. The right panel shows the percentage (%) of positive cells from left to right: VV+: VV positive cells only, CAR+: CAR positive cells only, + / +: cells positive for both VV and CAR, - / -: cells double negative for VV and CAR.

[0129] Figure 9A. Vaccinia infection induces maturation of T cells primarily in the CD8 compartment, leading to fewer naive cells and more effector cells. Figure 9B. Vaccinia infection induces maturation of T cells primarily in the CD8 compartment, leading to fewer naive cells and more effector cells. Figures 9A and 9B show that following VV infection, the abundance of naive and memory T cell subpopulations is reduced in the CD8 compartment compared to non-vaccinia infected cells.

[0130] Example 4: Administration of CAR T cells infected with vaccinia virus 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 may be expanded in vitro.

[0131] T cells are infected with vaccinia virus at an MOI of approximately 2. The infected T cells are administered intravenously to the patient.

Claims

1. A composition comprising a oncolytic virus and immune cells, wherein the immune cells are infected with the oncolytic virus.

2. The composition according to claim 1, wherein the immune cells are lymphocytes, T cells, B cells, NK cells, NK-T cells, or myeloid cells including lymphoid dendritic cells.

3. The immune cells are (i) derived from a subject to be treated with the composition; (ii) allogeneic to the subject to be treated with the composition; and / or (iii) genetically modified to target target cells; The composition according to claim 1 or 2.

4. The composition according to claim 1, wherein the oncolytic virus is a poxvirus.

5. The composition according to claim 3, wherein the target cells are cancer cells, fibroblasts, pathogens, or cells infected with pathogens.

6. The composition according to claim 1, wherein the oncolytic virus does not lyse immune cells for at least 5 days after infection.

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

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

9. The T cells are (i) lymphocytes obtained from a subject to be treated with the composition; or (ii) lymphocytes obtained from an unrelated donor; The composition according to claim 7 or 8, which is derived from.

10. The composition according to claim 9, wherein the lymphocytes are T cells, NK cells or NK-T cells.

11. The composition according to claim 7, wherein the CAR-T cells or CAR-NK cells target an antigen associated with a disease.

12. The composition according to claim 11, wherein the disease is cancer, an infectious disease, or fibrosis.

13. The composition according to any one of claims 4, 7 and 8, wherein the poxvirus is a vaccinia virus.

14. The composition according to claim 13, wherein the vaccinia virus is selected from the strains of Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, modified vaccinia Ankara (MVA), New York City Board of Health, Dairen, Ikeda, LC16M8, Western Reserve Copenhagen, Tashkent, Tian Tan, Wyeth, IHD-J and IHD-W, Brighton, Dairen I and Connaught.

15. The composition according to claim 4, 7 or 8, wherein the poxvirus is oncolytic.

16. The composition according to claim 7, 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.

17. The composition according to claim 8, wherein the exogenous TCR targets a tumor-associated antigen or a fibrosis-associated antigen.

18. The composition according to claim 17, wherein the tumor-associated antigen is PRAME, WT1, survivin, MAGE-A3, MART-1, gp-100, p53, or NY-ESO1.

19. A pharmaceutical composition comprising the composition according to any one of claims 1, 7 and 8 and a pharmaceutically acceptable excipient.

20. A pharmaceutical composition for use in the treatment of a disease, comprising the composition according to any one of claims 1, 7 and 8 and a pharmaceutically acceptable excipient.

21. The pharmaceutical composition according to claim 20, wherein the disease is an infectious disease, an autoimmune disease, fibrosis, or an inflammatory disease.

22. A pharmaceutical composition for use in the treatment of cancer, comprising the composition according to any one of claims 1, 7 and 8 and a pharmaceutically acceptable excipient.

23. The pharmaceutical composition according to claim 22, wherein the cancer is selected from glioblastoma multiforme, low-grade glioma, non-Hodgkin lymphoma, Hodgkin lymphoma, B-cell and T-cell acute lymphoblastic leukemia, mantle cell lymphoma, multiple myeloma, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, sarcoma, neuroblastoma, retinoblastoma, nephroblastoma, medulloblastoma, germ cell tumor, desmoid tumor, juvenile myelomonocytic leukemia, lung cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, kidney and bladder cancer, head and neck cancer, melanoma, uterine cancer, and prostate cancer.

24. The pharmaceutical composition according to claim 20 or 22, formulated for administration by intravenous, intraperitoneal, intrathecal, intraventricular, intrapleural, intrasubstantial, intraventricular, intra-articular, or intraocular injection.

25. The pharmaceutical composition according to claim 20 or 22, formulated for administration by MRI-guided delivery.

26. A method for producing a composition comprising leukocytes infected with poxvirus, comprising: (a) obtaining leukocytes from a subject; and (b) contacting the leukocytes with poxvirus to form leukocytes infected with poxvirus. The above method.

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