Use of oncolytic viruses to generate therapeutic tumor¬ infiltrating lymphocytes for solid brain tumors

Abstract

Provided herein are methods of treating a solid brain tumor in a subject in need thereof using a combination of delta-24-RGD virotherapy with tumor-infiltrating lymphocytes (TIL) therapy. In certain embodiments, the methods comprise contacting donor lymphocytes with oncolytic adenovirus Delta-24-RGD and using MHC-peptide tetramers to filter out virus-targeting lymphocytes to provide tumor-targeting lymphocytes for TIL therapy.

Description

Atty. Dkt. No.: 642631-0064USE OF ONCOLYTIC VIRUSES TO GENERATE THERAPEUTIC TUMORINFILTRATING LYMPHOCYTES FOR SOLID BRAIN TUMORS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No.: 63 / 750,672, filed January 28, 2025, the entire contents of which are incorporated herein by reference.TECHNICAL FIELD

[0002] The present technology relates generally to methods of treating solid brain tumors by combining delta-24-RGD virotherapy with tumor-infiltrating lymphocytes (TIL) therapy. In some embodiments, the methods comprise contacting donor lymphocytes with oncolytic adenovirus Delta-24-RGD and using MHC-peptide tetramers to filter out virus-targeting lymphocytes to provide tumor-targeting lymphocytes for TIL therapy.BACKGROUND

[0003] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] Currently, there is no effective treatment for certain cancers, such as gliomas (e.g., glioblastomas and pediatric brain tumors) and pancreatic cancer, which are sometimes referred to as “cold tumors” or “immune desserts.” Adoptive T-cell transfer therapy has been effectively used to treat melanoma (considered a “hot” tumor), but has not been used to treat cold tumors because cold tumors tend to have low frequencies of tumor-infiltrating lymphocytes (TILs).

[0005] High-grade gliomas (HGGs, grades 3 and 4) are aggressive and malignant brain tumors originating from the glial cells, which perform supportive roles within the central nervous system (CNS). The prevalence of HGGs exhibits regional and age-dependent variability. In the United States, the annual age-adjusted incidence of malignant brain and CNS tumors was estimated to be 7.02 cases per 100,000 individuals during the years between 2015 and 2019. Of these, glioblastoma (GBM, grade 4), the most aggressive type of HGGs, constitutes about half of all primary malignant brain tumors and accounts for 3.26 annual incidences per 100,000 individuals. Notably, the rates escalate with advancing age and the incidence of GBM peaks at the ages between 75- and 84-years. The precise causes of HGGs remain elusive, but certain risk factors such as age, male gender, and genetic -1- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064predisposition have been attributed. Survival prospects are inversely correlated with age and tumor grade. For instance, the 5-year survival rate for GBM patients aged between 0-and 14-years hovers at 20%, which plunges to a mere 5.3% for individuals over 40-years. There were 84,264 deaths attributed to malignant brain and central nervous system malignancies during the years between 2015 and 2019.SUMMARY OF THE PRESENT TECHNOLOGY

[0006] The present technology relates generally to a method of treating “cold” tumors, such as primary brain tumors like glioblastomas and primary pediatric brain tumors using oncolytic virus therapy in combination with tumor-infiltrating lymphocyte (TIL) therapy. The methods may include locally injecting an oncolytic adenovirus, delta-24-RGD, within tumors to induce infiltration of CD8+ lymphocytes in sufficient quantities so that the lymphocytes may be harvested and expanded ex vivo, then sorted with MHC -peptide complexes to filter out virus-targeting lymphocytes to enrich for tumor-targeting lymphocytes for TIL therapy.

[0007] By combining oncolytic virotherapy to induce tumor infiltration of immune cells and selective ex vivo expansion of tumor-targeting lymphocytes, this technology provides adoptive cell therapy as a viable treatment option for patients with cold tumors.

[0008] In one aspect, the present disclosure provides a method for treating primary brain tumors in a subject in need thereof comprising (a) administering an effective amount of an oncolytic virus selected from among delta-24-RGD oncolytic virus, delta-24-RGD backbone, delta-24-RGDOX, delta-24-GREAT, delta-24-ACT, or a combination of any two or more thereof, to a primary brain tumor of a donor subject; (b) culturing tumor-infiltrating lymphocytes isolated from the primary brain tumor of the donor subject; and (c) administering an effective amount of the cultured tumor-infiltrating lymphocytes to a recipient subject with the primary brain tumor.

[0009] In another aspect, the present disclosure provides a method for preparing tumorinfiltrating lymphocytes for treatment of a primary brain tumor in a subject in need thereof comprising (a) administering an effective amount of an oncolytic virus selected from among delta-24-RGD oncolytic virus, delta-24-RGD backbone, delta-24-RGDOX, delta-24-GREAT, delta-24-ACT, or a combination of any two or more thereof, to a primary brain tumor of a donor subject; and (b) culturing tumor-infiltrating lymphocytes isolated from the primary brain tumor of the donor subject.-2- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064

[0010] In another aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof comprising (a) administering an effective amount of an oncolytic virus selected from among delta-24-RGD oncolytic virus, delta-24-RGD backbone, delta-24-RGDOX, delta-24-GREAT, delta-24-ACT, or a combination of any two or more thereof, to a solid tumor of a donor subject; (b) culturing tumor-infiltrating lymphocytes isolated from the solid tumor of the donor subject; (c) enriching for tumorinfiltrating lymphocytes by filtering out virus-targeting lymphocytes using virus-specific MHC-peptide tetramers; and (d) administering an effective amount of the enriched tumorinfiltrating lymphocytes to a recipient subject with a solid tumor.

[0011] In another aspect, the present disclosure provides a method for preparing tumorinfiltrating lymphocytes for adoptive cell therapy comprising (a) administering an effective amount of an oncolytic virus selected from among delta-24-RGD oncolytic virus, delta-24-RGD backbone, delta-24-RGDOX, delta-24-GREAT, delta-24-ACT, or a combination of any two or more thereof, to a solid tumor of a donor subject; (b) culturing tumor-infiltrating lymphocytes isolated from the solid tumor of the donor subject; and (c) enriching for tumorinfiltrating lymphocytes by filtering out virus-targeting lymphocytes using virus-specific MHC-peptide tetramers.In any and all embodiments of the methods disclosed herein, the step of administering the oncolytic virus may induce an immune response against the primary tumor (e.g., primary brain tumor) in the donor subject, or may enhance an ongoing immune response against the primary tumor (e.g., primary brain tumor) in the donor subject. The step of administering the oncolytic virus may increase CD8+ lymphocytes levels in the primary tumor (e.g., primary brain tumor) compared to CD8+ lymphocytes levels in the primary tumor (e.g., primary brain tumor) of a control subject that is not treated with oncolytic virus.

[0012] In any and all embodiments of the methods disclosed herein, the oncolytic virus may be administered via intratumoral, endovascular, and / or intravenous injection.

[0013] In any and all embodiments of the methods disclosed herein, the step of isolating the tumor-infiltrating lymphocytes may be isolated about 1 week to about 6 months or about 3 weeks to about 5 weeks post administration of the oncolytic virus. The tumor-infiltrating lymphocytes may be isolated via a tissue biopsy or surgical resection of the primary tumor (e.g., primary brain tumor).-3- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064

[0014] In any and all embodiments of the methods disclosed herein, the methods may further comprise enriching for tumor-infiltrating lymphocytes by filtering out virustargeting lymphocytes using virus-specific MHC-peptide tetramers. The virus-specific MHC-peptide tetramers may be selected from among 2Db / AdV5.El A MHC-peptide tetramer, HLA-A*02 / TLLYVLFEV (hexon) (SEQ ID NO: 1), HLA-A*01 / STDVASLNY (penton) (SEQ ID NO: 2), or a combination of any two or more thereof. The methods may further comprise expanding tumor-infiltrating lymphocytes ex vivo. Tumor-infiltrating lymphocytes may be expanded using IL-2, IL-7, IL- 15, or a combination of any two or more thereof.

[0015] In any and all embodiments of the methods disclosed herein, the tumor-infiltrating lymphocytes may be administered intravenously, intraperitoneally, subcutaneous, intramuscularly, or intratum orally injection. The donor subject and the recipient subject may be the same. The donor subject and the recipient subject may be different. The primary brain tumor may be selected from glioma, glioblastoma, and a pediatric primary brain tumor. The primary brain tumor may be a high-grade tumor.

[0016] In any and all embodiments of the methods disclosed herein, the methods may further include administering to the recipient subject an additional anti-cancer therapy. The additional anti-cancer therapy may be selected from chemotherapy, radiation therapy, immunotherapy, monoclonal antibodies, anti-cancer nucleic acids or proteins, anti-cancer microorganisms, and any combination of two or more thereof.

[0017] The solid tumor may be selected from adrenal cancers, bladder cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, intestinal cancers, kidney cancers, larynx cancers, acute and chronic liver cancers, lymph node cancers, lung cancers, melanomas, mesothelioma, nasopharynx cancers, neuroblastomas, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof. The solid tumor may be a brain tumor or pancreatic cancer.-4- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIGs. 1A-1F: Example of TILs harvesting from murine glioblastoma tumors treated with Delta-24-RGD and TILs sorting with virus-specific MHC-peptide tetramers. FIG. 1A: Procedure for harvesting TILs from murine glioblastoma tumors treated with Delta-24-RGD. FIG. IB: Fluorescence-activated cell sorting exclusion of TILs using H-2Db / AdV5.El A MHC-peptide tetramer. FIG. 1C: Procedure for TILs expansion using murine IL-2 and IL-7. FIG. ID: Number of TILs after a 6-day expansion protocol. FIG. IE: AdV5.ElA-specific T cells after a 6-day expansion protocol. FIG. IF:AdV5.ElA-specific T cells in MHC -tetramer sorted versus unsorted TILs after a 6-day expansion protocol.

[0019] FIGs. 2A-2C. Oncolytic adenovirus therapy narrows the T-cell repertoire, yet diversity predicts survival. FIG. 2A: Comparison of TCR diversity in tumors before and after treatment. FIG. 2B: Changes in clonal expansion between pre- and post-treatment tumors. FIG. 2C: Overall survival stratified by high versus low post-treatment TCR diversity.

[0020] FIGs. 3A-3C. Spatial proximity to adenoviruses shapes T-cell transcriptional states. FIGs. 3A-3B: Spatial distributions of adenoviral transcripts (gray, FIG. 3A) and T / NK cells (dark gray, FIG. 3B) within a post-treatment tumor section. FIG. 3C:Differential gene expression analysis comparing T / NK cells located adjacent (<50 pm) versus distant (>150 pm) from adenoviral transcripts.

[0021] FIGs. 4A-4C. Tumor infiltrating lymphocytes isolated from virus-treated tumors display anti-tumor efficacy. FIG. 4A: In vitro expansion of tumor-infiltrating lymphocytes (TILs) isolated from murine GL261 gliomas treated with Delta-24-RGD, either unsorted or sorted to exclude T cells recognizing adenoviral epitopes El A and E1B.FIG. 4B: Proportions of expanded T cells stained with tetramers for adenoviral epitopes (E1A, E1B) and the GL261 -specific epitope GARC1. FIG. 4C: Survival of GL261 gliomabearing mice treated with PBS or l><106TILs depleted of ElA / ElB-specific T cells.DETAILED DESCRIPTION

[0022] It is to be appreciated that certain aspects, modes, embodiments, variations, and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology. It is to be understood that the present disclosure is not limited to particular uses, methods, reagents, compounds,-5- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064compositions, or biological systems, which 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.

[0023] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel etal. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N. Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. ( \999) Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir ’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).Definitions

[0024] As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting and / or separating the subject matter described.

[0025] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.-6- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064

[0026] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0027] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0028] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration may be carried out by any suitable route, including but not limited to, orally, intranasally, intrathecally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intraocularly, intradermally, transmucosally, iontophoretically, or topically. Administration includes self-administration and the administration by another. Administration” of a cell or vector or other agent and compositions containing same may be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations may be carried out with the dose level and pattern being selected by the treating physician or in the case of animals, by the treating veterinarian. In some embodiments, administering or a grammatical variation thereof also refers to more than one doses with certain interval. In some embodiments, the interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer. In some embodiments, one dose is repeated for once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration may also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated,-7- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064and target cell or tissue. Non-limiting examples of route of administration include oral administration, intraperitoneal, infusion, nasal administration, inhalation, injection, and topical application. In some embodiments, the administration is an infusion (for example to peripheral blood of a subject) over a certain period of time, such as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours or longer.

[0029] As used herein “adoptive cell therapeutic composition” refers to any composition comprising cells suitable for adoptive cell transfer. In exemplary embodiments, the adoptive cell therapeutic composition comprises a cell type selected from a group consisting of a tumor infiltrating lymphocyte (TIL), TCR (i.e., heterologous T-cell receptor) modified lymphocytes (e.g., eTCR T cells and caTCR T cells) and CAR ( / .< ., chimeric antigen receptor) modified lymphocytes (e.g., CAR T cells). In another embodiment, the adoptive cell therapeutic composition comprises a cell type selected from a group consisting of T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells, regulatory T-cells, and peripheral blood mononuclear cells. In another embodiment, TILs, T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells, regulatory T-cells or peripheral blood mononuclear cells form the adoptive cell therapeutic composition. In one embodiment, the adoptive cell therapeutic composition comprises T cells.

[0030] The term “amino acid” refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refer to agents that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. In some embodiments, amino acids forming a polypeptide are in the D form. In some embodiments, the amino acids forming a polypeptide are in the L form. In some embodiments, a first plurality of amino acids-8- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064forming a polypeptide are in the D form, and a second plurality of amino acids are in the L form.

[0031] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter code.

[0032] As used herein, the term “analog” refers to a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.

[0033] As used herein, the term “antibody” collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, “antigen binding fragments,” which are antibody fragments capable of binding antigen such as Fab, Fv, single chain Fv (scFv), Fab’, and (Fab’)2, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits, and mice, as well as non-mammalian species, such as shark immunoglobulins. As used herein, “antibodies” (includes intact immunoglobulins) including “antigen binding fragments” specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 103M'1greater, at least 104M'1greater or at least 105M'1greater than a binding constant for other molecules in a biological sample). The term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rdEd., W.H. Freeman & Co., New York, 1997.

[0034] As used herein, the terms “cancer” or “tumor” are used interchangeably and refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell. As used herein, the term “cancer cells” includes precancerous (e.g., benign),-9- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064malignant, pre-metastatic, metastatic, and non-metastatic cells. Cancers of virtually every tissue are known to those of skill in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, pediatric primary brain tumors, melanomas, etc., and circulating cancers such as leukemias. Examples of cancer include, but are not limited to, ovarian cancer, breast cancer, colon cancer, lung cancer, prostate cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, and brain cancer. The phrase “cancer burden” or “tumor burden” refers to the quantity of cancer cells or tumor volume in a subject. Reducing cancer burden accordingly may refer to reducing the number of cancer cells, or the tumor volume in a subject. The term “cancer cell” refers to a cell that exhibits cancer-like properties, e.g., uncontrollable reproduction, resistance to anti- growth signals, ability to metastasize, and loss of ability to undergo programmed cell death (e.g., apoptosis) or a cell that is derived from a cancer cell, e.g., clone of a cancer cell.

[0035] A “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a nanoparticle, detectable agent, or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include carriers, such as pharmaceutically acceptable carriers. In some embodiments, the carrier (such as the pharmaceutically acceptable carrier) comprises, or consists essentially of, or yet further consists of a nanoparticle, such as a polymeric nanoparticle carrier or a lipid nanoparticle that may be used alone or in combination with another carrier, such as an adjuvant or solvent. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which may be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid components, which may also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D--10- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol. A composition as disclosed herein may be a pharmaceutical composition. A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0036] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control may be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0037] As used herein, the phrase “derived” means isolated, purified, mutated, or engineered, or any combination thereof. For example, a cell derived from a subject refers to the cell isolated from a biological sample obtained from the subject, and is optionally engineered.

[0038] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions may also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount may be given in one or more administrations.-11- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064

[0039] As used herein, the term “excipient” refers to a natural or synthetic substance formulated alongside the active ingredient of a medication, included for the purpose of long-term stabilization, bulking up solid formulations, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility.

[0040] As used herein, the term “immune cell” refers to any cell that plays a role in the immune response of a subject. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes.

[0041] As used herein, “immune response” refers to the action of one or more of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver or spleen (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells, metastatic tumor cells, etc. An immune response may include a cellular response, such as a T-cell response that is an alteration (modulation, e.g., significant enhancement, stimulation, activation, impairment, or inhibition) of cellular, z.e., T-cell function. A T-cell response may include generation, proliferation or expansion, or stimulation of a particular type of T-cell, or subset of T-cells, for example, effector CD4+, CD4+helper, effector CD8+, CD8+cytotoxic, or natural killer (NK) cells. Such T-cell subsets may be identified by detecting one or more cell receptors or cell surface molecules (e.g., CD or cluster of differentiation molecules). A T-cell response may also include altered expression (statistically significant increase or decrease) of a cellular factor, such as a soluble mediator (e.g., a cytokine, lymphokine, cytokine binding protein, or interleukin) that influences the differentiation or proliferation of other cells.

[0042] As used herein, the term “isolated,” “purified,” or “biologically pure” refers to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or polypeptide of the presently disclosed subject matter is purified if it is substantially free of cellular material, viral material, or-12- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” may denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that may be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which may be separately purified.

[0043] As used herein, the term “isolated cell” refers to a cell that is separated from the molecular and / or cellular components that naturally accompany the cell.

[0044] The term “lymphocyte” refers to all immature, mature, undifferentiated, and differentiated white blood cell populations that are derived from lymphoid progenitors including tissue specific and specialized varieties, and encompasses, by way of non-limiting example, B cells, T cells, NKT cells, and NK cells. In some embodiments, lymphocytes include all B cell lineages including pre-B cells, progenitor B cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, mature B cells, plasma B cells, memory B cells, B-l cells, B-2 cells, and anergic AN1 / T3 cell populations.

[0045] As used herein, “metastasis” refers to the spread of cancer from its primary site to neighboring tissues or distal locations in the body. Cancer cells (including cancer stem cells) may break away from a primary tumor, penetrate lymphatic and blood vessels, circulate through the bloodstream, and grow in normal tissues elsewhere in the body.Metastasis is a sequential process, contingent on tumor cells (or cancer stem cells) breaking off from the primary tumor, traveling through the bloodstream or lymphatics, and stopping at a distant site. Once at another site, cancer cells re-penetrate through the blood vessels or lymphatic walls, continue to multiply, and eventually form a new tumor (metastatic tumor). In some embodiments, this new tumor is referred to as a metastatic (or secondary) tumor.

[0046] As used herein, “oncolytic virus” refers to a virus that preferentially infects cancer cells, replicates in such cells, and induces lysis of the cancer cells through its replication process.

[0047] As used herein, the term “overall survival” or “OS” means the observed length of life from the start of treatment to death or the date of last contact.-13- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064

[0048] “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein. In some embodiments, a pharmaceutically acceptable carrier comprises, or consists essentially of, or yet further consists of a nanoparticle, such as a polymeric nanoparticle carrier or a lipid nanoparticle (LNP). Additionally or alternatively, pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

[0049] As used herein, “pharmaceutically acceptable excipient” refers to substances and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or a human. As used herein, the term includes all inert, nontoxic, liquid or solid fillers or diluents, as long as they do not react with the therapeutic substance of the present technology in an inappropriate negative manner, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, preservatives and the like, for example liquid pharmaceutical carriers e.g., sterile water, saline, sugar solutions, Tris buffer, ethanol and / or certain oils.

[0050] As used herein, “prevention,” “prevent,” or “preventing” of a disorder or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0051] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, z.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides, or oligomers,-14- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art.

[0052] As used herein, a “sample” or “biological sample” refers to a body fluid or a tissue sample isolated from a subject. In some cases, a biological sample may consist of or comprise whole blood, platelets, red blood cells, white blood cells, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and / or chorionic villi, cultured cells, endothelial cells, synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid and the like. The term "sample" may also encompass the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, cerebrospinal fluid (CSF), saliva, mucus, sputum, semen, sweat, urine, or any other bodily fluids. Samples may be obtained from a subject by any means including, but not limited to, venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art. A blood sample may be whole blood or any fraction thereof, including blood cells (red blood cells, white blood cells or leukocytes, and platelets), serum and plasma.

[0053] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0054] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0055] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0056] As used herein, “solid tumor” refers to all neoplastic cell growth and proliferation, and all pre-cancerous and cancerous cells and tissues, except for hematologic cancers such-15- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064as lymphomas, leukemias, and multiple myeloma. Examples of solid tumors include, but are not limited to: soft tissue sarcoma, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor and other bone tumors (e.g., osteosarcoma, malignant fibrous histiocytoma), leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, brain / CNS tumors (e.g., astrocytoma, glioma, glioblastoma, pediatric primary tumors, such as atypical teratoid / rhabdoid tumor, germ cell tumor, embryonal tumor, ependymoma) medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Some of the most common solid tumors for which the compositions and methods of the present disclosure would be useful include: head-and-neck cancer, rectal adenocarcinoma, glioma, medulloblastoma, urothelial carcinoma, pancreatic adenocarcinoma, uterine (e.g., endometrial cancer, fallopian tube cancer) ovarian cancer, cervical cancer prostate adenocarcinoma, non-small cell lung cancer (squamous and adenocarcinoma), small cell lung cancer, melanoma, breast carcinoma, ductal carcinoma in situ, renal cell carcinoma, and hepatocellular carcinoma, adrenal tumors (e.g., adrenocortical carcinoma), esophageal, eye (e.g., melanoma, retinoblastoma), gallbladder, gastrointestinal, Wilms’ tumor, heart, head and neck, laryngeal and hypopharyngeal, oral (e.g., lip, mouth, salivary gland), nasopharyngeal, neuroblastoma, peritoneal, pituitary, Kaposi’s sarcoma, small intestine, stomach, testicular, thymus, thyroid, parathyroid, vaginal tumor, and the metastases of any of the foregoing.

[0057] As used herein, the terms “subject,” “individual,” or “patient” are used interchangeably and refer to an individual organism, a vertebrate, or a mammal and may include humans, non-human primates, rodents, and the like (e.g., which is to be the recipient of a particular treatment, or from whom cells are harvested). In certain embodiments, the individual, patient, or subject is a human.-16- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064

[0058] “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0059] As used herein, “T cell receptor” or “TCR”, is a protein complex found on the surface of T cells, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex molecules. TCR is composed of two disulfide-linked protein chains. Cells expressing a TCR containing the highly variable alpha (a) and beta (P) chains are referred to as aP T cells. Cells expressing an alternate TCR, formed by variable gamma (y) and delta (8) chains, are referred to as y6 T cells. When the TCR engages with antigenic peptide and MHC (peptide / MHC), the T lymphocyte is activated through signal transduction, that is, a series of biochemical events mediated by associated enzymes, coreceptors, specialized adaptor molecules, and activated or released transcription factors. In some embodiments, the TCR is a native T cell receptor that is endogenous to the immune cells. In some embodiments, the TCR is an artificial receptor that mimics native TCR function, i.e., recognizing peptide antigens of key intracellular proteins in the context of MHC on the cell surface.

[0060] As used herein, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0061] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. Therapeutic effects of treatment include, without limitation, inhibiting recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By “treating a cancer” is meant that the symptoms associated with the cancer are, e.g., alleviated, reduced, cured, or placed in a state of remission. In some embodiments, “inhibiting,” means reducing or slowing the growth of a tumor. In some embodiments, the inhibition of tumor growth may be, for example, by 5% or more, 10% or more, 20% or-17- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. In some embodiments, the inhibition may be complete.

[0062] As used herein, “tumor immunity” refers to one or more processes by which tumors evade recognition and clearance by the immune system. Thus, as a therapeutic concept, tumor immunity is “treated” when such evasion is attenuated or eliminated, and the tumors are recognized and attacked by the immune system (the latter being termed herein “anti-tumor immunity”). An example of tumor recognition is tumor binding, and examples of tumor attack are tumor reduction (in number, size, or both) and tumor clearance.

[0063] As used herein “tumor-infiltrating ” immune cells refer to immune cells that have left the bloodstream and migrated into a tumor.

[0064] As used herein, “T-cell” refers to a thymus derived lymphocyte that participates in a variety of cell-mediated adaptive immune reactions.

[0065] As used herein, “helper T-cell” refers to a CD4+T-cell; helper T-cells recognize antigen bound to MHC Class II molecules. There are at least two types of helper T-cells, Thl and Th2, which produce different cytokines.

[0066] As used herein, “cytotoxic T-cell” refers to a T-cell that usually bears CD8 molecular markers on its surface (CD8+) and that functions in cell-mediated immunity by destroying a target cell having a specific antigenic molecule on its surface. Cytotoxic T-cells also release Granzyme, a serine protease that can enter target cells via the perforin-formed pore and induce apoptosis (cell death). Granzyme serves as a marker of cytotoxic phenotype. Other names for cytotoxic T-cell include CTL, cytolytic T-cell, cytolytic T lymphocyte, killer T-cell, or killer T lymphocyte. Targets of cytotoxic T-cells may include virus-infected cells, cells infected with bacterial or protozoal parasites, or cancer cells. Most cytotoxic T-cells have the protein CD8 present on their cell surfaces. CD8 is attracted to portions of the Class I MHC molecule. Typically, a cytotoxic T-cell is a CD8+cell.

[0067] It is also to be appreciated that the various modes of treatment of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.-18- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064Solid Tumors including High-Grade Gliomas

[0068] Solid tumor are abnormal masses of tissue that can be benign or malignant, and can form in many parts of the body. Solid tumors are characterized by neoplastic cell growth and proliferation. Examples of solid tumors include, but are not limited to: soft tissue sarcoma, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor and other bone tumors (e.g., osteosarcoma, malignant fibrous histiocytoma), leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, brain / CNS tumors (e.g., astrocytoma, glioma, glioblastoma, pediatric primary tumors, such as atypical teratoid / rhabdoid tumor, germ cell tumor, embryonal tumor, ependymoma) medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Some of the most common solid tumors for which the compositions and methods of the present disclosure would be useful include: head-and-neck cancer, rectal adenocarcinoma, glioma, medulloblastoma, urothelial carcinoma, pancreatic adenocarcinoma, uterine (e.g., endometrial cancer, fallopian tube cancer) ovarian cancer, cervical cancer prostate adenocarcinoma, non-small cell lung cancer (squamous and adenocarcinoma), small cell lung cancer, melanoma, breast carcinoma, ductal carcinoma in situ, renal cell carcinoma, and hepatocellular carcinoma, adrenal tumors (e.g., adrenocortical carcinoma), esophageal, eye (e.g., melanoma, retinoblastoma), gallbladder, gastrointestinal, Wilms’ tumor, heart, head and neck, laryngeal and hypopharyngeal, oral (e.g., lip, mouth, salivary gland), nasopharyngeal, neuroblastoma, peritoneal, pituitary, Kaposi’s sarcoma, small intestine, stomach, testicular, thymus, thyroid, parathyroid, vaginal tumor, and the metastases of any of the foregoing.

[0069] High-grade tumors (e.g., grades 3 or 4) are tumors that include cells that look abnormal under microscope and are poorly differentiated. High-grade tumors may grow-19- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064and spread faster than lower-grade tumors, and may have worse prognosis. Examples of high-grade tumors include high-grade gliomas. High-grade gliomas (HGGs, grades 3 and 4) are a type of solid tumors. They are aggressive and malignant brain tumors originating from the glial cells, which perform supportive roles within the central nervous system (CNS). The prevalence of HGGs exhibits regional and age-dependent variability. In the United States, the annual age-adjusted incidence of malignant brain and CNS tumors was estimated to be 7.02 cases per 100,000 individuals during the years between 2015 and 2019. Of these, glioblastoma (GBM, grade 4), the most aggressive type of HGGs, constitutes about half of all primary malignant brain tumors and accounts for 3.26 annual incidences per 100,000 individuals. Notably, the rates escalate with advancing age and the incidence of GBM peaks at the ages between 75- and 84-years. The precise causes of HGGs remain elusive, but certain risk factors such as age, male gender, and genetic predisposition have been attributed. Furthermore, ongoing investigations are exploring the interplay between neuronal activity and tumor progression, promising to shed light on the elusive origins of HGGs. Survival prospects are inversely correlated with age and tumor grade. For instance, the 5-year survival rate for GBM patients aged between 0- and 14-years hovers at 20%, which plunges to a mere 5.3% for individuals over 40-years. There were 84,264 deaths attributed to malignant brain and central nervous system malignancies during the years between 2015 and 2019.

[0070] Symptoms associated with HGGs often reflect heightened intracranial pressure, culminating in an array of debilitating manifestations. These include headaches characterized by their severity, persistence, and exacerbation in the morning or upon awakening. Cognitive decline, marked by memory impairment, diminished concentration, shortened attention spans, and cognitive confusion, is also common in HGG patients.Seizures manifest prominently, ranging from mild focal seizures that exhibit involuntary movements or localized sensory perturbations, to generalized seizures that entail loss of consciousness and convulsive muscle contractions. Neurological deficits, another hallmark of HGGs, encompass motor impairments such as weakness, paralysis, or compromised coordination, in addition to sensory anomalies like numbness or tingling sensations. Visual and auditory disturbances may manifest when the tumor exerts its influence on the visual or auditory regions of the brain. These symptoms contribute to increased pain and the lost quality of life for patients with HGGs.-20- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064

[0071] Despite our increasingly detailed understanding of the molecular characteristics and subtypes of HGGs, the prognosis for patients remains grim, particularly for patients with GBM that have a median survival period of 15 to 18 months after initial diagnosis. Alarmingly, over 90% of these patients experience disease recurrence or progression within the first year. The current standard treatment protocol for HGGs involves a combination of surgical intervention, radiation therapy, and chemotherapy, with an additional use of the tumor-treating field devices in some patients.Oncolytic Virotherapy for Solid Tumors

[0072] Oncolytic viruses (OVs) offer a new treatment modality for solid tumors that are highly resistant to conventional therapies. These viruses, whether naturally occurring or genetically engineered, are designed to specifically target and replicate within cancer cells. OVs capitalize on distinct genetic aberrations or signaling pathways commonly found in cancer cells to achieve cancer selectivity. As the infected cancer cells get lysed, they release additional viral particles that spread to neighboring cancer cells. This amplifies viral infection and intensifies tumor elimination. Beyond the direct lytic effect on cancer cells, OVs possess the capacity to invigorate the immune system's response against the tumor. Virus-infected tumor cells release virus- and tumor-specific antigens, which prompt the activation and recruitment of immune cells, such as T-cells and natural killer (NK) cells. This orchestrated response targets not only the virus-infected tumor cells but also extends to non-infected tumor cells. Consequently, OV infection leads to virus replication and direct cell lysis, as well as the establishment of a persistent and systemic immune response against the tumor.

[0073] Oncolytic virotherapy may provide a distinct advantage for patients whose tumors are not responsive to conventional therapies. In contrast to many chemotherapies, OVs possess a capacity to differentially infect and eliminate cancer cells while preserving the integrity of normal, healthy cells. Importantly, virotherapy may be integrated with various other treatment modalities, including radiation therapy, chemotherapy, targeted therapies, and immunotherapies, providing the potential for synergistic therapeutic outcomes. For instance, OVs have been shown to sensitize tumor cells to immune checkpoint inhibitors, predominantly by reshaping the tumor microenvironment and stimulating robust anti-tumor immune responses. The multifaceted mechanism underlying cancer cell destruction induced by OVs is notably diverse, encompassing direct cell lysis facilitated through virus replication, autophagy induction, and immune system activation.-21- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064This wide spectrum of cell death mechanisms significantly reduces the likelihood of cancer cells developing therapy resistance. The observation that many tumors have developed resistance to traditional treatments that primarily relied on the apoptosis pathway bolsters the importance of therapies with novel mechanisms of action. OVs may be further engineered to modulate angiogenesis, enhance autophagy, and ignite the immune system, thereby broadening their therapeutic potential. In summary, oncolytic virotherapy is emerging as a versatile and promising option for cancer treatment, offering innovative mechanisms for patients resistant to conventional therapies.Delta-24-RGD Oncolytic Virus

[0074] Delta-24-RGD oncolytic virus is an oncolytic adenovirus serotype 5 (Ad5).Without being bound by any theory, the virus has an RGD-4C coding sequence inserted in the fiber protein that provides an increased ability to infect integrin-expressing cancer cells. The virus also has a 24-base pair deletion in the El A gene that allows it to selectively replicate in cancer cells with the defective Rb-pathway.

[0075] Previous clinical trials have indicated that delta-24-RGD oncolytic virus is safe and efficacious for treating certain types of cancers. Results from these clinical trials and preclinical animal models suggested that local injection of delta-24-RGD oncolytic virus within tumors induced infiltration of CD8+ lymphocytes, as evidenced by immunohistochemistry analyses of patient tumor biopsies and flow cytometry analyses of murine tumors. These findings raise the potential for ACT with TILs, which thus far has been impractical for treating brain tumors like glioblastoma due to the low frequencies of TILs.

[0076] However, recent evidence suggested that virotherapy with delta-24-RGD oncolytic virus resulted in the generation of more TILs targeting viral antigens than TILs targeting tumor antigens. Data from clinical trials on oncolytic virotherapy suggested the development of two distinct immune responses, anti-viral immune responses, and antitumor immune response. In these trials, a significant number of patients treated with oncolytic viruses developed strong immune responses against the viruses, as evidenced by seroconversion in most patients and prompt viral clearance within a few weeks following administration. However, approximately 20% of the patients experienced durable antitumor immune responses, highlighting the disparity in the development of immune responses against viruses and tumors.-22- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064

[0077] The present technology addresses the problem of the larger production of TILS targeting viral antigens than those targeting tumor antigens by harvesting the TILS produced by virotherapy and enriching for tumor targeting lymphocytes from TILs by negatively sorting out T cells that recognize the dominant viral epitopes.

[0078] In addition to delta-24-RGD oncolytic virus, the backbone of delta-24-RGD oncolytic virus may be used alternatively or additionally to the delta-24-RGD oncolytic virus as described herein. The delta-24-RGD oncolytic virus may be genetically modified to express an immunomodulator. Genetically modified versions include delta-24-RGDOX expressing OX40L, delta-24-GREAT expressing GITRL, and delta-24-ACT expressing 411BL. The genetically modified versions may be used alternatively or additionally to the delta-24-RGD oncolytic virus as described herein.Immune Response

[0079] In addition to induction of the immune response by up-regulation of particular immune system activities (such as antibody and / or cytokine production, or activation of cell mediated immunity), immune responses may also include suppression, attenuation, or any other down-regulation of detectable immunity, so as to reestablish homeostasis and prevent excessive damage to the host’s own organs and tissues. In some embodiments, an immune response that is induced according to the methods of the present disclosure generates tumor infiltrating lymphocytes (TILs), including CD8+ cytolytic T cells, CD4+ helper T cells, regulatory T cells, B cells, natural killer (NK) cells, or a combination of two or more thereof that may bring about directly or indirectly the death, or loss of the ability to propagate, of a tumor cell.

[0080] Induction of an immune response by the compositions and methods of the present disclosure may be determined by detecting any of a variety of well-known immunological parameters (Takaoka et al., Cancer Set. 94:405-11 (2003); Nagorsen et al., Crit. Rev.Immunol. 22:449-62 (2002)). Induction of an immune response may therefore be established by any of a number of well-known assays, including immunological assays. Such assays include, but need not be limited to, in vivo, ex vivo, or in vitro determination of soluble immunoglobulins or antibodies; soluble mediators such as cytokines, chemokines, hormones, growth factors and the like as well as other soluble small peptide, carbohydrate, nucleotide and / or lipid mediators; cellular activation state changes as determined by altered functional or structural properties of cells of the immune system, for example cell-23- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064proliferation, altered motility, altered intracellular cation gradient or concentration (such as calcium); phosphorylation or dephosphorylation of cellular polypeptides; induction of specialized activities such as specific gene expression or cytolytic behavior; cellular differentiation by cells of the immune system, including altered surface antigen expression profiles, or the onset of apoptosis (programmed cell death); or any other criterion by which the presence of an immune response may be detected. For example, cell surface markers that distinguish immune cell types may be detected by specific antibodies that bind to CD4+, CD8+, or NK cells. Common methods for detecting the immune response include, but are not limited to flow cytometry, ELISA, immunohistochemistry. Procedures for performing these and similar assays are widely known and may be found, for example in Letkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, Current Protocols in Immunology, 1998).Pharmaceutical Compositions and Preparations of Oncolytic Virotherapies

[0081] Disclosed herein are pharmaceutical compositions comprising delta-24-RGD oncolytic virus that may contain a carrier or diluent, which may be a solvent or dispersion medium containing, for example, water, saline, tris buffer, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be affected by various antibacterial and antifungal agents and preservatives, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugars, or sodium chloride, and / or buffering agents are included. Prolonged absorption of the injectable compositions may be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin or carrier molecules. Other excipients may include wetting or emulsifying agents. In general, excipients suitable for injectable preparations may be included as apparent to those skilled in the art.

[0082] Pharmaceutical compositions and preparations delta-24-RGD oncolytic virus may be manufactured by means of conventional mixing, dissolving, granulating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical viral compositions may be formulated in conventional manner using one or more physiologically acceptable-24- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064carriers, diluents, excipients, or auxiliaries that facilitate formulating virus preparations suitable for in vitro, in vivo, or ex vivo use. The compositions may be combined with one or more additional biologically active agents (for example parallel administration of GM-CSF) and may be formulated with a pharmaceutically acceptable carrier, diluent, or excipient to generate pharmaceutical (including biologic) or veterinary compositions of the instant disclosure suitable for parenteral or intra-tumoral administration.

[0083] Many types of formulation are possible as is appreciated by those skilled in the art. The particular type chosen is dependent upon the route of administration chosen, as is well-recognized in the art. For example, systemic formulations will generally be designed for administration by injection, e.g., intravenous, as well as those designed for intratumoral delivery. In some embodiments, the systemic or intratumoral formulation is sterile.

[0084] Sterile injectable solutions are prepared by incorporating delta-24-RGD oncolytic virus in the required amount of the appropriate solvent with various other ingredients enumerated herein, as required, followed by suitable sterilization means. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the virus plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0085] In some embodiments, the delta-24-RGD oncolytic virus compositions of the present disclosure may be formulated in aqueous solutions, or in physiologically compatible solutions or buffers such as Hanks's solution, Ringer's solution, mannitol solutions or physiological saline buffer. In certain embodiments, any of the delta-24-RGD oncolytic virus compositions may contain formulator agents, such as suspending, stabilizing, penetrating or dispersing agents, buffers, lyoprotectants or preservatives such as polyethylene glycol, polysorbate 80, l-dodecylhexahydro-2H-azepin-2-one (laurocapran), oleic acid, sodium citrate, tris HC1, dextrose, propylene glycol, mannitol , polyoxyethylene (20) sorbitan monolaurate (Tween®-20), isopropyl myristate, benzyl alcohol, isopropyl alcohol, ethanol sucrose, trehalose and other such generally known in the art may be used in any of the compositions of the instant disclosure. (Pramanick et al., Pharma Times 45(3), 65-76 (2013)).-25- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064

[0086] The biologic or pharmaceutical compositions of the present disclosure may be formulated to allow the virus contained therein to be available to infect tumor cells upon administration of the composition to a subject. The level of virus in serum, tumors, and if desired other tissues after administration may be monitored by various well-established techniques, such as antibody-based assays (e.g., ELISA, immunohistochemistry, efc.).

[0087] The recombinant viruses of the present invention may be stored at -80°C with a titer of about 102- 109plaque forming units (pfu) per mL formulated in about 10 mM Tris, 140 mM NaCl at pH 7.7. For the preparation of vaccine shots, e.g., 102- 108or 102- 109viral particles may be lyophilized in 100 mL of phosphate-buffered saline (PBS) in the presence of 2% peptone and 1 % human albumin in an ampoule, preferably a glass ampoule.Alternatively, the injectable preparations may be produced by stepwise freeze-drying of the recombinant virus in a formulation. This formulation may contain additional additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other additives such as antioxidants or inert gas, stabilizers, or recombinant proteins (e.g., human serum albumin) suitable for in vivo administration. The glass ampoule is then sealed and may be stored between 4 °C and room temperature for several months. In some embodiments, the ampoule is stored at temperatures below -20 °C.

[0088] For therapy, the lyophilisate may be dissolved in an aqueous solution, such as physiological saline or Tris buffer, and administered either systemically or intratum orally. The mode of administration, the dose, and the number of administrations may be optimized by those skilled in the art.

[0089] The pharmaceutical composition according to the present disclosure may comprise an additional adjuvant. As used herein, an “adjuvant” refers to a substance that enhances, augments, or potentiates the host's immune response to tumor antigens. A typical adjuvant may be aluminum salts, such as aluminum hydroxide or aluminum phosphate, Quil A, bacterial cell wall peptidoglycans, virus-like particles, polysaccharides, toll-like receptors, nano-beads, etc. (Aguilar et al. (2007), Vaccine 25: 3752-3762).Effective Amount and Dosage of Delta-24-RGD Oncolytic Virus

[0090] In general, the subject is administered one or multiple dosages of delta-24-RGD oncolytic virus in the range of about 106to about 1012plaque forming units (pfu), although a lower or higher dose may be administered. In some embodiments, the dosage ranges from about 1010to about 1012pfu. In some embodiments, the dosage ranges from about 109to-26- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064about 1012pfu. In some embodiments, the dosage ranges from about 108to about 1012pfu. In some embodiments, the dosage ranges from about 107to about 1012pfu. In some embodiments, the dosage ranges from about 106to about 1012pfu. In some embodiments, the dosage ranges from about 105to about 1012pfu. In some embodiments, the dosage ranges from about 104to about 1012pfu. In some embodiments, the dosage ranges from about 103to about 1012pfu. In some embodiments, dosage is about 102to about 1012pfu. The equivalence of pfu to virus particles may differ according to the specific pfu titration method used. Generally, a pfu is equal to about 5 to 100 virus particles and 0.69 PFU is about 1 TCID50. A therapeutically effective amount of delta-24-RGD oncolytic virus may be administered in one or more divided doses for a prescribed period of time and at a prescribed frequency of administration.

[0091] For example, as is apparent to those skilled in the art, a therapeutically effective amount of delta-24-RGD oncolytic virus in accordance with the present disclosure may vary according to factors such as the disease state, age, sex, weight, and general condition of the subject, and the ability of delta-24-RGD oncolytic virus to elicit a desired immunological response in the particular subject (the subject’s response to therapy). In delivering delta-24-RGD oncolytic virus to a subject, the dosage will also vary depending upon such factors as the general medical condition, previous medical history, disease type and progression, tumor burden, the presence or absence of tumor infiltrating immune cells in the tumor, and the like.

[0092] In some embodiments, it may be advantageous to formulate compositions of the present disclosure in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form as used herein” refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically or veterinary acceptable carrier.Administration and Therapeutic Regimen of Delta-24-RGD Oncolytic Virus

[0093] Administration of the delta-24-RGD oncolytic virus may induce an immune response against the primary brain tumor in the donor subject, or may enhance an ongoing immune response against the primary brain tumor in the donor subject. Administering the delta-24-RGD oncolytic virus may increase CD8+ lymphocytes levels in the primary brain-27- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064tumor in the donor subject compared to CD8+ lymphocytes levels in primary brain tumors of a control subject that is not treated with delta-24-RGD oncolytic virus.

[0094] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Administration of delta-24-RGD oncolytic virus may be achieved using more than one route. Examples of routes of administration include, but are not limited to parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous), intratumoral, intrathecal, intranasal, systemic, transdermal, iontophoretic, intradermal, intraocular, endovascular, or topical administration. In one embodiment, delta-24-RGD oncolytic virus is administered directly into the tumor, e.g. by intratumoral injection, where a direct local reaction is desired. In an embodiment, delta-24-RGD oncolytic virus is administered intravenously, e.g. by intravenous injection. In an embodiment, delta-24-RGD oncolytic virus is administered endovascularly using carrier cells (e.g., mesenchymal cells). Additionally, administration routes of delta-24-RGD oncolytic virus may vary, e.g., first administration using an intratumoral injection, and subsequent administration via an intravenous injection, or any combination thereof. A therapeutically effective amount of delta-24-RGD oncolytic virus injection may be administered for a prescribed period of time and at a prescribed frequency of administration. In certain embodiments, delta-24-RGD oncolytic virus may be used in conjunction with other therapeutic treatments. For example, delta-24-RGD oncolytic virus may be administered in a neoadjuvant (preoperative) or adjuvant (postoperative) setting for subjects inflicted with bulky primary tumors. It is anticipated that such optimized therapeutic regimen will induce an immune response against the tumor, and reduce the tumor burden in a subject before or after primary therapy, such as surgery. Furthermore, delta-24-RGD oncolytic virus may be administered in conjunction with other therapeutic treatments such as chemotherapy or radiation.

[0095] In certain embodiments, the delta-24-RGD oncolytic virus is administered at least once weekly or monthly but may be administered more often if needed, such as two times weekly for several weeks, months, years or even indefinitely as long as benefits persist. More frequent administrations are contemplated if tolerated and if they result in sustained or increased benefits. Benefits of the present methods include but are not limited to the following: reduction of the number of cancer cells, reduction of the tumor size, eradication of tumor, inhibition of cancer cell infiltration into peripheral organs, inhibition or stabilization or eradication of metastatic growth, inhibition or stabilization of tumor growth,-28- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064and stabilization or improvement of quality of life. Furthermore, the benefits may include induction of an immune response against the tumor, activation of effector CD4+T-cells, an increase of effector CD8+T-cells, or reduction of regulatory CD4+cells. For example, in the context of glioma, a benefit may be a lack of recurrences or metastasis within one, two, three, four, five or more years of the initial diagnosis of glioma. Similar assessments may be made for other solid tumors.

[0096] In certain other embodiments, the tumor mass or tumor cells are treated with delta-24-RGD oncolytic virus in vivo, ex vivo, or in vitro. Ex vivo administration of delta-24-RGD oncolytic virus may include administration may include administration of delta-24-RGD oncolytic virus to an excised or resected solid tumor that has been removed from a patient. In vitro administration of delta-24-RGD oncolytic virus may include administration of delta-24-RGD oncolytic virus to solid tumor cells harvested from the patient.Adoptive Cell Therapy (ACT)

[0097] Adoptive cell transfer (ACT) is a very effective form of immunotherapy and involves the transfer of immune cells with antitumor activity into cancer patients. ACT is a treatment approach that involves the identification, in vitro, of lymphocytes with antitumor activity, the in vitro culturing of these cells to large numbers and their infusion into the cancer-bearing host. Lymphocytes used for adoptive transfer may be derived from the stroma of resected tumors (tumor infiltrating lymphocytes or TILs). They may also be derived or from blood if they are genetically engineered to express antitumor T cell receptors (TCRs) or chimeric antigen receptors (CARs), enriched with mixed lymphocyte tumor cell cultures (MLTCs), or cloned using autologous antigen presenting cells and tumor derived peptides. ACT in which the lymphocytes originate from the cancer-bearing host to be infused is termed autologous ACT. ACT may be autologous or non-autologous.

[0098] ACT with TILs is a promising form of T cell-based immunotherapy. Preparation of TILs involves surgical resection and culturing of TILs from tumors. Upon adequate TIL culturing expansion, patients may undergo lymphodepleting chemotherapy, and TIL adoptive transfer. ACT depends upon infiltration of T cells into tumors prior to harvest, successful ex vivo expansion of TILs, and potent anti-tumor effector function after transfer. The full potential of TIL therapy has been hampered by the inadequate activation and low persistence of TILs. For example, TIL therapy has been impractical for treating solid tumors like glioblastoma and pediatric brain tumors due to the low frequencies of TILs.-29- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064

[0099] Disclosed herein are compositions and methods for increasing activation and persistence of TILs using oncolytic virotherapy with delta-24-RGD oncolytic virus, surgical resection and culturing of TILs from virotherapy -treated tumors, and TIL adoptive transfer to a patient. Oncolytic virotherapy addresses previous problems with TIL therapy by generating sufficient TILs for subsequent ACT.

[0100] TILs may be harvested from tumors following virotherapy. In some embodiments, TILs are obtained from the stroma of resected tumors. TILs may be harvested and isolated at a predetermined time following administration of virotherapy, e.g., about 1 week to about 6 months post administration of the delta-24-RGD oncolytic virus, e.g., about 3 weeks to about 5 weeks post administration. For example, the TILs may be harvested 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks after virotherapy administration. For example, TILs may be harvested about 10 days to about 50 days, or about 20 to about 40 days, or about 25 days to about 35 days following administration of delta-24-RGD oncolytic virus. The TILs may be isolated via, for example, a tissue biopsy or surgical resection of the primary brain tumor.

[0101] Tumor samples may be obtained from patients and cell suspensions may be obtained. TILs may be isolated using any suitable manner, e.g., Percoll gradients, and cell suspensions may be obtained in any suitable manner, e.g., mechanically (disaggregating the tumor using, e.g., a gentleMACS(TM) Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase).

[0102] Recent evidence has further suggested that a substantial portion of TILs generated following virotherapy target viral, not tumor, antigens, as evidenced by T cell receptor sequencing analysis of patient tumor and enzyme-linked immunosorbent spot analyses of murine tumors. Therefore, the compositions and methods may further include sorting TILs to enrich for tumor-targeting TILs. Sorting TILS to enrich for tumor-targeting lymphocytes may include filtering out virus-targeting lymphocytes using virus-specific major histocompatibility complex molecules bound to peptide complex (MHC-peptide tetramers). MHC-peptide tetramers may bind to certain T-cells through specific molecular interactions with T-cell receptors (TCRs) that recognize the peptide epitopes. Sorting TILS to enrich for tumor-infiltrating lymphocytes may include using MHC-peptide tetramers with antibodies identifying T-cells (e.g., CD45 antibodies and CD3 antibodies). Antibodies may be generated using Good Manufacturing Practices (GMP). The antibodies may be mixed with TILs generated following virotherapy. The mixture of antibodies and TILs may be-30- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064incubated for a period of time (e.g., 30 minutes) at a temperature (e.g., 0°C to 4°C, 0°C to 10°C). Fluorescence-activated cell sorting (FACS) under sterile conditions may be used to identify and sort the desired subpopulations of TILs. For example, MHC-peptide tetramers may include 2Db / AdV5.El A MHC-peptide tetramer, HLA-A*02 / TLLYVLFEV (hexon) (SEQ ID NO: 1), HLA-A*01 / STDVASLNY (penton) (SEQ ID NO: 2), or a combination of any two or more thereof.

[0103] Before or after sorting, TILs may be cultured and expanded. Rapid expansion may provide an increase in the number of TILs of at least about 50-fold (e.g., 50-, 60-, 70-, 80-, 90-, or 100-fold, or greater) over a period of about 10 to about 14 days, or about 14 days. Rapid expansion may provide an increase of at least about 200-fold (e.g., 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or greater) over a period of about 10 to about 14 days, or about 14 days. Rapid expansion may provide an increase of at least about 1000-fold over a period of about 10 to about 14 days, or about 14 days. Rapid expansion may provide an increase of about 1000-fold over a period of about 14 days.

[0104] Culturing and expansion of TILs, e.g., T cells, may be accomplished by any of a number of methods as are known in the art. For example, T cells may be rapidly expanded using non-specific T-cell receptor stimulation in the presence of feeder lymphocytes and / or interleukin-2 (IL-2), IL-7, IL- 15, IL-21, or combinations thereof. For example, TILs may be cultures using murine-derived IL-2, IL-7, IL- 15, or a combination of any two or more thereof. For example, TILs may be cultures using murine-derived IL-2 and IL-7. The nonspecific T-cell receptor stimulus may, e.g., include around 30 ng / mL of OKT3, a mouse monoclonal anti-CD3 antibody. Alternatively, T cells may be rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMC) in vitro with one or more antigens (including antigenic portions thereof, such as epitope(s), or a cell of the cancer, which may be optionally expressed from a vector, such as an human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., approximately 0.3 pM MART-1 :26-35 (27 L) or gpl00:209-217 (210M)), in the presence of a T-cell growth factor, such as around 200-400 lU / mL, such as 300 lU / mL IL-2 or IL-15, with IL-2 being preferred. The in v / Z / 'o-induced T-cells may be rapidly expanded by restimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen- presenting cells. Alternatively, the T-cells may be restimulated with irradiated lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2, for example.-31- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064

[0105] Prior to administering ACT therapy, the patient may optionally be administered chemotherapy (e.g., nonmyeloablative lymphodepleting chemotherapy). The chemotherapy may be any suitable such therapy, which may be administered by any suitable route.Pharmaceutical Compositions and Preparations of TILs

[0106] Cultured TILs and compositions comprising the same may be conveniently provided as sterile liquid preparations, e.g, isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, may be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions may comprise carriers, which may be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

[0107] Sterile injectable solutions may be prepared by incorporating the TIL compositions in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions may also be lyophilized. The compositions may contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g, methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON1S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0108] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, may be added. Prevention of the action of microorganisms may be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.-32- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064According to the presently disclosed subject matter, however, any vehicle, diluent, or additive used would have to be compatible with the TILs of the presently disclosed subject matter.

[0109] The compositions may be isotonic, / .< ., they may have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of the presently disclosed subject matter may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is suitable particularly for buffers containing sodium ions.

[0110] Viscosity of the compositions, if desired, may be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose may be used because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of the thickener may depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. The choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel, or another liquid form, such as a time release form or liquid-filled form).[OHl] Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the TILs as described in the presently disclosed subject matter. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems may be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.Effective Amount and Dosage of TILs

[0112] One consideration concerning the therapeutic use of the TILs of the presently disclosed subject matter is the quantity of cells necessary to achieve an optimal effect. The quantity of cells to be administered will vary for the subject being treated. In certain embodiments, from about 102to about 1012, from about 103to about 1011, from about 104to about 1010, from about 105to about 109, or from about 106to about 108TILs of the presently disclosed subject matter are administered to a subject. More effective cells may-33- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064be administered in even smaller numbers. In some embodiments, at least about 1 x 108, about 2 x 108, about 3 x 108, about 4 x 108, about 5 x 108, about 1 x 109, about 5 x 109, about 1 x IO10, about 5 x IO10, about 1 x 1011, about 5 x 1011, about 1 x 1012or more TILs of the presently disclosed subject matter are administered to a human subject. The precise determination of what would be considered an effective dose may be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages may be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art. Generally, TILs are administered at doses that are nontoxic or tolerable to the patient.

[0113] The skilled artisan may readily determine the amount of cells and optional additives, vehicles, and / or carrier in compositions to be administered in methods of the presently disclosed subject matter. Typically, any additives (in addition to the active cell(s) and / or agent(s)) are present in an amount of from about 0.001% to about 50% by weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as from about 0.0001 wt % to about 5 wt %, from about 0.0001 wt% to about 1 wt %, from about 0.0001 wt% to about 0.05 wt%, from about 0.001 wt% to about 20 wt %, from about 0.01 wt% to about 10 wt %, or from about 0.05 wt% to about 5 wt %. For any composition to be administered to an animal or human, and for any particular method of administration, toxicity should be determined, such as by determining the lethal dose (LD) and LD50 in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. The timing for sequential administrations may be ascertained without undue experimentation.Administration and Therapeutic Regimen of TILs

[0114] TILs of the presently disclosed subject matter may be provided systemically or directly to a subject for treating cancer. In certain embodiments, TILs are directly injected into a tumor and / or an organ of interest. Additionally or alternatively, the TILs are provided indirectly to the tumor and / or organ of interest, for example, by administration into the circulatory system (e.g., the tumor vasculature) or into the tissue of interest (e.g., solid tumor). Expansion and differentiation agents may be provided prior to, during or after-34- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064administration of cells and compositions to increase production of the TILs either in vitro or in vivo.

[0115] TILs of the presently disclosed subject matter may be administered in any physiologically acceptable vehicle, systemically or regionally, normally intravascularly, intraperitoneally, subcutaneously, intramuscularly, intratumorally, intrathecally, or intrapleurally, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus). In certain embodiments, at least 1 x 105cells may be administered, eventually reaching 1 x IO10or more. In certain embodiments, at least 1 x 106cells may be administered. A cell population comprising TILs may comprise a purified population of cells. Those skilled in the art may readily determine the percentage of TILs in a cell population using various well-known methods, such as fluorescence activated cell sorting (FACS). The ranges of purity in cell populations comprising TILs may be from about 50% to about 55%, from about 55% to about 60%, about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%; from about 85% to about 90%, from about 90% to about 95%, or from about 95 to about 100%. Dosages may be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage). The TILs may be introduced by injection, catheter, or the like.

[0116] In certain embodiments, compositions of the presently disclosed subject matter comprise pharmaceutical compositions comprising TILs binding tumor antigens with a pharmaceutically acceptable carrier. Administration may be autologous or non-autologous. For example, TILs binding tumor antigens and compositions comprising the same may be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived T cells of the presently disclosed subject matter or their progeny (e.g., in vivo, ex vivo or in vitro derived) may be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a pharmaceutical composition comprising TILs, it may be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0117] For treatment, the amount of the TILs provided herein administered is an amount effective in producing the desired effect, for example, treatment of a cancer or one or more symptoms of a cancer. An effective amount may be provided in one or a series of-35- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064administrations of the TILs provided herein. An effective amount may be provided in a bolus or by continuous perfusion. For adoptive immunotherapy using antigen-specific T cells, cell doses in the range of about 106to about IO10are typically infused. Lower doses of the TILs may be administered, e.g., about 104to about 108.

[0118] The TILs of the presently disclosed subject matter may be administered by any methods known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration, intraperitoneal administration, and direct administration to the thymus. In certain embodiments, the TILs and the compositions comprising thereof are intravenously administered to the subject in need. Methods for administering cells for adoptive cell therapies, including, for example, donor lymphocyte infusion, and regimens for administration are known in the art and may be employed for administration of the TILs provided herein.

[0119] The presently disclosed subject matter provides various methods of using the TILs provided herein. In some embodiments, the TILs include cytotoxic (CD8+) T cells, helper (CD4+) T cells, B cells, NK cells, or any combination of two or more thereof. For example, the presently disclosed subject matter provides methods of reducing tumor burden in a subject. In one non-limiting example, the method of reducing tumor burden comprises administering an effective amount of the presently disclosed TILs to the subject, thereby inducing tumor cell death in the subject.

[0120] The presently disclosed TILs may reduce the number of tumor cells, reduce tumor size, and / or eradicate the tumor in the subject. In certain embodiments, the method of reducing tumor burden comprises administering an effective amount of TILs to the subject, thereby inducing tumor cell death in the subject. Non-limiting examples of suitable tumors include adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, acute and chronic leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non-Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid-36- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof. In some embodiments, the cancer is a relapsed or refractory cancer. In some embodiments, the cancer is resistant to one or more cancer therapies, e.g., one or more chemotherapeutic drugs.

[0121] The presently disclosed subject matter also provides methods of increasing or lengthening survival of a subject with cancer (e.g., a tumor). In one non-limiting example, the method of increasing or lengthening survival of a subject with cancer (e.g., a tumor) comprises administering an effective amount of the presently TILs to the subject, thereby increasing or lengthening survival of the subject. The presently disclosed subject matter further provides methods for treating or preventing cancer (e.g., a tumor) in a subject, comprising administering the presently disclosed TILs to the subject. Also provided herein are methods for treating or inhibiting tumor growth or metastasis in a subject comprising contacting a tumor cell with an effective amount of any of the TILs provided herein.

[0122] Cancers whose growth may be inhibited using the TILs of the presently disclosed subject matter include cancers typically responsive to immunotherapy. Non-limiting examples of cancers for treatment include breast cancer, endometrial cancer, ovarian cancer, colon cancer, lung cancer, stomach cancer, prostate cancer, renal cancer, pancreatic cancer, brain cancer, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), and metastases thereof.

[0123] Suitable human subjects for therapy typically comprise two treatment groups that may be distinguished by clinical criteria. Subjects with “advanced disease” or “high tumor burden” are those who bear a clinically measurable tumor. A clinically measurable tumor is one that may be detected on the basis of tumor mass (e.g., by palpation, CAT scan, sonogram, mammogram, or X-ray; positive biochemical or histopathologic markers on their own are insufficient to identify this population). A pharmaceutical composition embodied in the presently disclosed subject matter is administered to these subjects to elicit an antitumor response, with the objective of palliating their condition. Ideally, reduction in tumor mass occurs as a result, but any clinical improvement constitutes a benefit. Clinical improvement comprises decreased risk or rate of progression or reduction in pathological consequences of the tumor.

[0124] Another group of suitable subjects is known in the art as the “adjuvant group.” These are individuals who have had a history of neoplasia, but have been responsive to-37- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064another mode of therapy. The prior therapy may have included, but is not restricted to, surgical resection, radiotherapy, and traditional chemotherapy. As a result, these individuals have no clinically measurable tumor. However, they are suspected of being at risk for progression of the disease, either near the original tumor site, or by metastases. This group may be further subdivided into high-risk and low-risk individuals. The subdivision is made on the basis of features observed before or after the initial treatment. These features are known in the clinical arts, and are suitably defined for each different neoplasia. Features typical of high-risk subgroups are those in which the tumor has invaded neighboring tissues, or who show involvement of lymph nodes. Another group has a genetic predisposition to neoplasia but has not yet evidenced clinical signs of neoplasia. For instance, women testing positive for a genetic mutation associated with breast cancer, but still of childbearing age, may wish to receive one or more of the TILs described herein in treatment prophylactically to prevent the occurrence of neoplasia until it is suitable to perform preventive surgery.

[0125] The subjects may have an advanced form of disease, in which case the treatment objective may include mitigation or reversal of disease progression, and / or amelioration of side effects. The subjects may have a history of the condition, for which they have already been treated, in which case the therapeutic objective will typically include a decrease or delay in the risk of recurrence.Combination Therapy

[0126] The compositions of the present technology may be employed in conjunction with other therapeutic agents useful in the treatment of cancers. For example, the tumor antigentargeting TILs of the present technology may be separately, sequentially, or simultaneously administered with at least one additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy is selected from among a chemotherapy, a radiation therapy, an immunotherapy, a monoclonal antibody, an anti-cancer nucleic acid, an anti-cancer protein, an anti-cancer virus or microorganism, a cytokine, or any combination of two or more thereof.

[0127] Radiation therapy includes, but is not limited to, exposure to radiation, e.g., ionizing radiation, UV radiation, as known in the art. Exemplary dosages include, but are not limited to, a dose of ionizing radiation at a range from at least about 2 Gy to not more-38- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064than about 10 Gy or a dose of ultraviolet radiation at a range from at least about 5 J / m2to not more than about 50 J / m2, usually about 10 J / m2.

[0128] In some embodiments, the methods further comprise sequentially, separately, or simultaneously administering an immunotherapy to the subject. In some embodiments, the immunotherapy regulates immune checkpoints. In further embodiments, the immunotherapy comprises, or consists essentially of, or yet further consists of an immune checkpoint inhibitor, such as a Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4) inhibitor, or a Programmed Cell Death 1 (PD-1) inhibitor, or a Programmed Death Ligand 1 (PD-L1) inhibitor. In yet further embodiments, the immune checkpoint inhibitor comprises, or consists essentially of, or yet further consists of an antibody or an equivalent thereof recognizing and binding to an immune checkpoint protein, such as an antibody or an equivalent thereof recognizing and binding to CTLA4 (for example, Yervoy (ipilimumab), CP-675,206 (tremelimumab), AK104 (cadonilimab), or AGEN1884 (zalifrelimab)), or an antibody or an equivalent thereof recognizing and binding to PD-1 (for example, Keytruda (pembrolizumab), Opdivo (nivolumab), Libtayo (cemiplimab), Tyvyt (sintilimab), BGB-A317 (tislelizumab), JS001 (toripalimab), SHR1210 (camrelizumab), GB226 (geptanolimab), JS001 (toripalimab), AB 122 (zimberelimab), AK105 (penpulimab), HLX10 (serplulimab), BCD-100 (prolgolimab), AGEN2034 (balstilimab), MGA012 (retifanlimab), AK104 (cadonilimab), HX008 (pucotenlimab), PF-06801591 (sasanlimab), JNJ-63723283 (cetrelimab), MGD013 (tebotelimab), CT-011 (pidilizumab), or Jemperli (dostarlimab)), or an antibody or an equivalent thereof recognizing and binding to PD-L1 (for example, Tecentriq (atezolizumab), Imfinzi (durvalumab), Bavencio (avelumab), CS1001 (sugemalimab), orKN035 (envafolimab)).

[0129] In some embodiments, the methods further comprise sequentially, separately, or simultaneously administering a cytokine to the subject. In some embodiments, the cytokine is administered prior to, during, or subsequent to administration of the one or more TILs. In some embodiments, the cytokine is selected from the group consisting of interferon a, interferon P, interferon y, complement C5a, IL-2, TNFa, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12,-39- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2.

[0130] The methods for treating cancer may further comprise sequentially, separately, or simultaneously administering to the subject at least one chemotherapeutic agent, optionally selected from the group consisting of nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzyme inhibitors, epipodophyllotoxins, platinum coordination complexes, vinca alkaloids, substituted ureas, methyl hydrazine derivatives, adrenocortical suppressants, hormone antagonists, endostatin, taxols, camptothecins, SN-38, doxorubicin, doxorubicin analogs, antimetabolites, alkylating agents, antimitotics, anti-angiogenic agents, tyrosine kinase inhibitors, mTOR inhibitors, heat shock protein (HSP90) inhibitors, proteosome inhibitors, HD AC inhibitors, pro-apoptotic agents, methotrexate and CPT-11.Kits

[0131] The presently disclosed subject matter provides kits for the treatment of a disease, such as cancer. In certain embodiments, the kit comprises a therapeutic composition containing an effective amount of a delta-24-RGD oncolytic virus.

[0132] In some embodiments, the kit comprises a sterile container which contains a therapeutic delta-24-RGD oncolytic virus; such containers may be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0133] If desired, the delta-24-RGD oncolytic virus may be provided together with instructions for administering the virus to a subject having cancer. The instructions will generally include information about the use of the composition for the treatment or prevention of cancer. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of cancer or symptoms thereof; precautions; warnings; indications; counter-indications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (when-40- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0134] The delta-24-RGD oncolytic virus may be provided in the form of a prefilled syringe or autoinjection pen containing a sterile, liquid formulation or lyophilized preparation (e.g., Kivitz etal., Clin. Ther. 28:1619-29 (2006)).

[0135] A device capable of delivering the kit components through an administrative route may be included. Examples of such devices include syringes (for parenteral administration) or inhalation devices.

[0136] The kit components may be packaged together or separated into two or more containers. In some embodiments, the containers may be vials that contain sterile, lyophilized formulations of delta-24-RGD oncolytic virus that are suitable for reconstitution. A kit may also contain one or more buffers suitable for reconstitution and / or dilution of other reagents. Other containers that may be used include, but are not limited to, a pouch, tray, box, tube, or the like. Kit components may be packaged and maintained sterilely within the containers.EXAMPLES

[0137] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way. The examples herein are provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with preparing or using the methods of the present technology. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. The examples may include or incorporate any of the variations, aspects, or embodiments of the present technology described above. The variations, aspects, or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects, or embodiments of the present technology.Example 1: Materials and Methods

[0138] TILs harvesting from murine glioblastoma tumors treated with Delta-24-RGD Oncolytic Virus

[0139] FIG. 1A illustrates a procedure for harvesting TILs from murine glioblastoma tumors treated with Delta-24-RGD. Murine glioblastoma tumors treated with delta-24-RGD oncolytic virus are collected and TILs are isolated using the standard Percoll gradient-41- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064method and T cell isolation kits. GL261-5 murine glioblastoma cells (50,000 cells, refer to Jiang et al., Cancer Res. 2017 Jul 15;77(14):3894-3907) are implanted into the caudate nucleus (3.5 mm depth at 2.5 mm lateral and 1 mm anterior from the bregma, refer to Lal et al., JNeurosurg. 2000 Feb;92(2):326-33) of the brains of immunocompetent C57BL / 6 mice. One week after tumor implantation, the mice receive a single intratumoral injection of 107PFU of Delta-24-RGD. Two weeks after virus injection, the brains of the mice are collected, and TILs are isolated using physical (Miltenyi, Cat. No. 130-096-334) and enzymatic digestion of the brains, gradient centrifugation (Miltenyi, Cat. No. 130-109-398) and pan-T cell isolation kits (Miltenyi, Cat. No. 130-095-130).

[0140] TILs sorting with virus-specific MHC-peptide tetramers

[0141] Virus-targeting T cells are sorted from isolated TILs using virus-specific MHC-peptide tetramers, thereby increasing the concentration of tumor-targeting T cells.

[0142] Murine T cells targeting adenoviral epitope El A234-243 (SGPSNTPPEI) are sorted with the BD FACSAria II cell sorter (BD Biosciences) from isolated TILs using El A-specific H-2Db MHC-peptide tetramers (MHC tetramer production facility, Baylor College of Medicine), thereby increasing the concentration of tumor-targeting T cells. FIG. IB is a graph of fluorescence-activated cell sorting exclusion of TILs using H-2Db / AdV5.El A MHC-peptide tetramer.

[0143] After sorting, the remaining TILs are expanded ex vivo by activating the T cells with anti-mouse CD3 antibody at 1 pg / mL and anti-mouse CD28 antibody at 5 pg / mL (BioXCell, Cat. Nos. BE0001-1, BE0015-1), and culturing with recombinant murine IL-2 at 10 ng / mL and IL-7 at 63 ng / mL (Peprotech, Cat. No. 212-12, 212-17). See FIG. 1C. FIG. ID is a graph of the number of TILs after a 6-day expansion protocol. FIG. IE is a graph of AdV5. El A-specific T cells after a 6-day expansion protocol. FIG. IF is a graph of AdV5.ElA-specific T cells in MHC -tetramer sorted versus unsorted TILs after a 6-day expansion protocol.

[0144] TILs infusion into glioblastoma-bearing animals

[0145] Expanded TILs are infused into glioblastoma-bearing immunocompetent animals to examine the safety and efficacy of this treatment. Virus-specific MHC-peptide tetramers are generated for human patients following standard procedures.

[0146] Human patients treated with Delta-24-RGD Oncolytic Virus-42- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064

[0147] In clinical trials of human patients with gliomas, delta-24-RGD oncolytic virus was administered intratumorally, followed by partial or en-bloc surgical resection of tumors 1 month to 3 months following virus injection.

[0148] TILs were isolated from the patients’ resected tumors, and virus-targeting T cells were sorted out using virus-specific MHC-peptide tetramers as described herein.

[0149] Tumors were mechanically chopped into small pieces and digested with enzymes (e.g., collagenase) to release the cells. To isolate TILs, specialized cell sorting methods ( / .< ., fluorescence- or magnetic-activated cell sorting) were implemented using good manufacturing practices. Alternative methods consisting of expanding TILs within the tumor sections may also be used. Virus-targeting T cells were sorted out from TILs using virus-specific MHC-peptide tetramers.

[0150] TIL expansion and reinfusion into patients

[0151] After sorting, TILs were expanded ex vivo using high dose IL-2 or low dose IL-2 with anti-CD3 / anti-CD28 beads, or IL-2, IL-7, and optionally IL-15 in the presence of irradiated allogeneic peripheral blood mononuclear cells (PBMCs) as feeder cells or in the absence of feeder cells. The expanded TILs product was reinfused into the patient's tumor with IL-2 or other cytokines to maintain TILs activity. Prior to reinfusion, the TILs may be checked for quality controls, e.g., sterility, phenotype checking, and negativity for bloodborne disease. TILs were expanded following GMP standards.Example 2: Patient-derived information on the modulation of the T-cell receptor (TCR) repertoire after treatment with Delta-24-RGD

[0152] Patient gliomas were surgically resected two weeks after intra-tumoral administration of Delta-24-RGD. Genomic DNA was extracted from the tumors, and T-cell receptor (TCR) sequencing was performed on 6 pre-treatment and 9 post-treatment samples. Post-treatment tumors exhibited reduced TCR diversity and increased proportions of hyperexpanded clones, indicating that Delta-24-RGD therapy drives a focused T-cell immune response, likely targeting the virus (FIGs. 2A-2B). Importantly, higher posttreatment TCR diversity correlated with improved overall survival, suggesting that broader antigen recognition by T cells enhances therapeutic efficacy (FIG. 2C). Collectively, these findings imply that T-cell responses following Delta-24-RGD treatment may be dominated by anti-viral rather than anti-tumor immunity, but also suggest the recruitment of anti-tumor T cells that result in objective clinical responses.-43- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064

[0153] Spatial transcriptomic analysis of adjacent tumor sections revealed distinct molecular phenotypes based on proximity to adenoviral transcripts. T cells located near (<50 pm) adenoviral transcripts strongly upregulated genes associated with type I interferon-mediated anti-viral responses (including IFIT2, IFIT3, ISG15, and MX!) compared to T cells positioned farther away (>150 pm) (FIGs. 3A-3C). These observations support the concept that transcriptional signatures can help distinguish anti-viral from antitumor T-cell populations.

[0154] In a pilot study, treatment of syngeneic murine glioma models with Delta-24-RGD led to infiltration of lymphocytes into the gliomas, which could be harvested and expanded in vitro (FIG. 4A). Adenovirus-specific T cells could be identified and excluded from the tumor-infiltrating lymphocyte (TIL) populations using tetramer staining (FIG. 4B).Reinfusion of in vitro expanded TILs in treatment-naive, glioma-bearing mice resulted in long-term survival (>100 days post tumor injection) in 3 out of 6 mice (50%, median survival duration of 88.5 days) in animals treated with TILs depleted of adenovirus-epitope specific T cells, and in 1 out of 6 mice (17%, median survival duration of 58 days) in PBS-treated animals (FIG. 4C). Collectively, these results provide a strong rationale for using adoptive transfer of tumor-infiltrating lymphocytes following virotherapy as an effective treatment for residual tumors.

[0155] The present technology may include, but is not limited to, the features and combinations of features recited in the following lettered paragraphs, it being understood that the following paragraphs should not be interpreted as limiting the scope of the claims as appended hereto or mandating that all such features must necessarily be included in such claims:A. A method for treating primary brain tumors in a subject in need thereof comprising:(a) administering an effective amount of an oncolytic virus selected from among delta-24-RGD oncolytic virus, delta-24-RGD backbone, delta-24-RGDOX, delta-24-GREAT, delta-24-ACT, or a combination of any two or more thereof, to a primary brain tumor of a donor subject;(b) culturing tumor-infiltrating lymphocytes isolated from the primary brain tumor of the donor subject; and-44- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064(c) administering an effective amount of the cultured tumor-infiltrating lymphocytes to a recipient subject with a primary brain tumor.B. A method for preparing tumor-infiltrating lymphocytes for treatment of a primary brain tumor in a subject in need thereof comprising:(a) administering an effective amount of an oncolytic virus selected from among delta-24-RGD oncolytic virus, delta-24-RGD backbone, delta-24-RGDOX, delta-24-GREAT, delta-24-ACT, or a combination of any two or more thereof, to a primary brain tumor of a donor subject; and(b) culturing tumor-infiltrating lymphocytes isolated from the primary brain tumor of the donor subject.C. The method of paragraph A or B, wherein administration of the oncolytic virus induces an immune response against the primary brain tumor in the donor subject, or enhances an ongoing immune response against the primary brain tumor in the donor subject.D. The method of paragraph C, wherein administration of the oncolytic virus increases CD8+ lymphocytes levels in the primary brain tumor in the donor subject compared to CD8+ lymphocytes levels in a primary brain tumor of a control subject that is not treated with delta-24-RGD oncolytic virus.E. The method of any one of paragraphs A-D, wherein the oncolytic virus is administered via intratumoral, endovascular, and / or intravenous injection.F. The method of any one of paragraphs A-E, wherein the tumor-infiltrating lymphocytes are isolated about 1 week to about 6 months post administration of the delta-24-RGD oncolytic virus.G. The method of paragraph F, wherein the tumor-infiltrating lymphocytes are isolated about 3 weeks to about 5 weeks post administration of the oncolytic virus.H. The method of paragraph F, wherein the tumor-infiltrating lymphocytes are isolated about 1 month to about 6 months post administration of the oncolytic virus.-45- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064I. The method of any one of paragraphs A-H, wherein the tumor-infiltrating lymphocytes are isolated via a tissue biopsy or surgical resection of the primary brain tumor.J. The method of any one of paragraphs A-I, further comprising enriching for tumorinfiltrating lymphocytes by filtering out virus-targeting lymphocytes using virus-specific MHC-peptide tetramers.K. The method of paragraph J, wherein the virus-specific MHC-peptide tetramers is selected from among 2Db / AdV5.El A MHC-peptide tetramer, HLA-A*02 / TLLYVLFEV (hexon) (SEQ ID NO: 1), HLA-A*01 / STDVASLNY (penton) (SEQ ID NO: 2), or a combination of any two or more thereof.L. The method of any one of paragraphs A-K, further comprising expanding tumorinfiltrating lymphocytes ex vivo.M. The method of paragraph L, wherein the tumor-infiltrating lymphocytes are expanded using IL-2 and IL-7.N. The method of any one of paragraphs A or C-M, wherein the tumor-infiltrating lymphocytes are administered intravenously, intraperitoneally, subcutaneously, intramuscularly, or intratum orally.O. The method of any one of paragraph A or C-N, wherein the donor subject and the recipient subject are the same.P. The method of any one of paragraphs A or C-O, wherein the donor subject and the recipient subject are different.Q. The method any one of paragraphs A-P, wherein the primary brain tumor is selected from glioma, glioblastoma, and pediatric primary brain tumor.R. The method of any one of paragraphs A-Q, wherein the primary brain tumor is a high-grade tumor.S. The method of any one of paragraphs A-R, further comprising administering to the recipient subject an additional anti-cancer therapy.-46- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064T. The method of paragraph S, wherein the additional anti-cancer therapy is selected from among chemotherapy, radiation therapy, immunotherapy, monoclonal antibodies, anticancer nucleic acids or proteins, anti-cancer microorganisms, and any combination thereof.U. A method for treating a solid tumor in a subject in need thereof comprising:(a) administering an effective amount of an oncolytic virus selected from among delta-24-RGD oncolytic virus, delta-24-RGD backbone, delta-24-RGDOX, delta-24-GREAT, delta-24-ACT, or a combination of any two or more thereof, to a solid tumor of a donor subject;(b) culturing tumor-infiltrating lymphocytes isolated from a solid tumor of the donor subject;(c) enriching for tumor-infiltrating lymphocytes by filtering out virus-targeting lymphocytes using virus-specific MHC-peptide tetramers; and(d) administering an effective amount of the enriched tumor-infiltrating lymphocytes to a recipient subject with the solid tumor.V. A method for preparing tumor-infiltrating lymphocytes for adoptive cell therapy comprising:(a) administering an effective amount of an oncolytic virus selected from among delta-24-RGD oncolytic virus, delta-24-RGD backbone, delta-24-RGDOX, delta-24-GREAT, delta-24-ACT, or a combination of any two or more thereof, to a solid tumor of a donor subject;(b) culturing tumor-infiltrating lymphocytes isolated from a solid tumor of the donor subject; and(c) enriching for tumor-infiltrating lymphocytes by filtering out virus-targeting lymphocytes using virus-specific MHC-peptide tetramers.W. The method of paragraph U or V, wherein administration of the oncolytic virus induces an immune response against the solid tumor in the donor subject, or enhances an ongoing immune response against the solid tumor in the donor subject.X. The method of paragraph W, wherein administration of the oncolytic virus increases CD8+ lymphocytes levels in the solid tumor in the donor subject compared to-47- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064CD8+ lymphocytes levels in solid tumor of a control subject that is not treated with delta-24-RGD oncolytic virus.Y. The method of any one of paragraphs U-X, wherein the oncolytic virus is administered via intratumoral, endovascular, and / or intravenous injection.Z. The method of any one of paragraphs U-Y, wherein the tumor-infiltrating lymphocytes are isolated about 1 week to about 6 months post administration of the oncolytic virus.AA. The method of paragraph Z, wherein the tumor-infiltrating lymphocytes are isolated about 3 weeks to about 5 weeks post administration of the oncolytic virus.AB. The method of paragraph Z, wherein the tumor-infiltrating lymphocytes are isolated about 1 month to about 6 months post administration of the oncolytic virus.AC. The method of any one of paragraphs U-AB, wherein the tumor-infiltrating lymphocytes are isolated via a tissue biopsy or surgical resection of the solid tumor.AD. The method of any one of paragraphs U-AC, further comprising enriching for tumor-infiltrating lymphocytes by filtering out virus-targeting lymphocytes using virusspecific MHC -peptide tetramers.AE. The method of paragraph AD, wherein the virus-specific MHC -peptide tetramers are selected from among 2Db / AdV5.El A MHC-peptide tetramer, HLA- A*02 / TLLYVLFEV (hexon) (SEQ ID NO: 1), HLA-A*01 / STDVASLNY (penton) (SEQ ID NO: 2), or a combination of any two or more thereof.AF. The method of any one of paragraph U-AE, further comprising expanding tumorinfiltrating lymphocytes ex vivo.AG. The method of paragraph AF, wherein the tumor-infiltrating lymphocytes are expanded using IL-2, IL-7, IL-15, or a combination of any two or more thereof.AH. The method of any one of paragraphs U or W-AG, wherein the tumor-infiltrating lymphocytes are administered intravenously, intraperitoneally, subcutaneously, intramuscularly, or intratum orally.-48- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064Al. The method of any one of paragraphs U or W-AG, wherein the donor subject and the recipient subject are the same.AJ. The method of any one of paragraphs U or W-AG, wherein the donor subject and the recipient subject are different.AK. The method of any one of paragraphs U-AJ, wherein the solid tumor is selected from among adrenal cancers, bladder cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, intestinal cancers, kidney cancers, larynx cancers, acute and chronic liver cancers, lymph node cancers, lung cancers, melanomas, mesothelioma, nasopharynx cancers, neuroblastomas, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof.AL. The method of any one of paragraphs U-AK, wherein the solid tumor is a brain tumor or pancreatic cancer.AM. The method of any one of paragraphs U-AL, further comprising administering to the recipient subject an additional anti-cancer therapy.AN. The method of paragraph AM, wherein the additional anti-cancer therapy is selected from among chemotherapy, radiation therapy, immunotherapy, monoclonal antibodies, anticancer nucleic acids or proteins, anti-cancer microorganisms, and any combinations of two or more thereof.

[0156] Other embodiments are set forth in the following claims, along with the full scope of equivalents to which such claims are entitled.EQUIVALENTS

[0157] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present-49- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which 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.

[0158] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0159] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0160] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.-50- 4900-9231-5274.1

Claims

Atty. Dkt. No.: 642631-0064WHAT IS CLAIMED IS:

1. A method for treating primary brain tumors in a subject in need thereof comprising:(a) administering an effective amount of an oncolytic virus selected from among delta-24-RGD oncolytic virus, delta-24-RGD backbone, delta-24-RGDOX, delta-24-GREAT, delta-24-ACT, or a combination of any two or more thereof, to a primary brain tumor of a donor subject;(b) culturing tumor-infiltrating lymphocytes isolated from the primary brain tumor of the donor subject; and(c) administering an effective amount of the cultured tumor-infiltrating lymphocytes to a recipient subject with a primary brain tumor.

2. A method for preparing tumor-infiltrating lymphocytes for treatment of a primary brain tumor in a subject in need thereof comprising:(a) administering an effective amount of an oncolytic virus selected from among delta-24-RGD oncolytic virus, delta-24-RGD backbone, delta-24-RGDOX, delta-24-GREAT, delta-24-ACT, or a combination of any two or more thereof, to a primary brain tumor of a donor subject; and(b) culturing tumor-infiltrating lymphocytes isolated from the primary brain tumor of the donor subject.

3. The method of claim 1 or 2, wherein administration of the oncolytic virus induces an immune response against the primary brain tumor in the donor subject, or enhances an ongoing immune response against the primary brain tumor in the donor subject.

4. The method of claim 3, wherein administration of the oncolytic virus increases CD8+ lymphocytes levels in the primary brain tumor in the donor subject compared to CD8+ lymphocytes levels in a primary brain tumor of a control subject that is not treated with delta-24-RGD oncolytic virus.

5. The method of any one of claims 1-4, wherein the oncolytic virus is administered via intratumoral, endovascular, and / or intravenous injection.-51- 4900-9231-5274.1Atty. Dkt. No.: 642631-00646. The method of any one of claims 1-5, wherein the tumor-infiltrating lymphocytes are isolated about 1 week to about 6 months post administration of the delta-24-RGD oncolytic virus.

7. The method of claim 6, wherein the tumor-infiltrating lymphocytes are isolated about 3 weeks to about 5 weeks post administration of the oncolytic virus.

8. The method of claim 6, wherein the tumor-infiltrating lymphocytes are isolated about 1 month to about 6 months post administration of the oncolytic virus.

9. The method of any one of claims 1-8, wherein the tumor-infiltrating lymphocytes are isolated via a tissue biopsy or surgical resection of the primary brain tumor.

10. The method of any one of claims 1-9, further comprising enriching for tumorinfiltrating lymphocytes by filtering out virus-targeting lymphocytes using virus-specific MHC-peptide tetramers.

11. The method of claim 10, wherein the virus-specific MHC-peptide tetramers is selected from among 2Db / AdV5.El A MHC-peptide tetramer, HLA-A*02 / TLLYVLFEV (hexon) (SEQ ID NO: 1), HLA-A*01 / STDVASLNY (penton) (SEQ ID NO: 2), or a combination of any two or more thereof.

12. The method of any one of claims 1-11, further comprising expanding tumorinfiltrating lymphocytes ex vivo.

13. The method of claim 12, wherein the tumor-infiltrating lymphocytes are expanded using IL-2 and IL-7.

14. The method of any one of claims 1 or 3-13, wherein the tumor-infiltrating lymphocytes are administered intravenously, intraperitoneally, subcutaneously, intramuscularly, or intratum orally.

15. The method of any one of claims 1 or 3-14, wherein the donor subject and the recipient subject are the same.

16. The method of any one of claims 1 or 3-14, wherein the donor subject and the recipient subject are different.-52- 4900-9231-5274.1Atty. Dkt. No.: 642631-006417. The method any one of claims 1-16, wherein the primary brain tumor is selected from glioma, glioblastoma, and pediatric primary brain tumor.

18. The method of any one of claims 1-17, wherein the primary brain tumor is a highgrade tumor.

19. The method of any one of claims 1-18, further comprising administering to the recipient subject an additional anti-cancer therapy.

20. The method of claim 19, wherein the additional anti-cancer therapy is selected from among chemotherapy, radiation therapy, immunotherapy, monoclonal antibodies, anticancer nucleic acids or proteins, anti-cancer microorganisms, and any combination thereof.

21. A method for treating a solid tumor in a subject in need thereof comprising:(a) administering an effective amount of an oncolytic virus selected from among delta-24-RGD oncolytic virus, delta-24-RGD backbone, delta-24-RGDOX, delta-24-GREAT, delta-24-ACT, or a combination of any two or more thereof, to a solid tumor of a donor subject;(b) culturing tumor-infiltrating lymphocytes isolated from a solid tumor of the donor subject;(c) enriching for tumor-infiltrating lymphocytes by filtering out virus-targeting lymphocytes using virus-specific MHC-peptide tetramers; and(d) administering an effective amount of the enriched tumor-infiltrating lymphocytes to a recipient subject with the solid tumor.

22. A method for preparing tumor-infiltrating lymphocytes for adoptive cell therapy comprising:(a) administering an effective amount of an oncolytic virus selected from among delta-24-RGD oncolytic virus, delta-24-RGD backbone, delta-24-RGDOX, delta-24-GREAT, delta-24-ACT, or a combination of any two or more thereof, to a solid tumor of a donor subject;(b) culturing tumor-infiltrating lymphocytes isolated from a solid tumor of the donor subject; and-53- 4900-9231-5274.1Atty. Dkt. No.: 642631-0064(c) enriching for tumor-infiltrating lymphocytes by filtering out virus-targeting lymphocytes using virus-specific MHC-peptide tetramers.

23. The method of claim 21 or 22, wherein administration of the oncolytic virus induces an immune response against the solid tumor in the donor subject, or enhances an ongoing immune response against the solid tumor in the donor subject.

24. The method of claim 23, wherein administration of the oncolytic virus increases CD8+ lymphocytes levels in the solid tumor in the donor subject compared to CD8+ lymphocytes levels in solid tumor of a control subject that is not treated with delta-24-RGD oncolytic virus.

25. The method of any one of claims 21-24, wherein the oncolytic virus is administered via intratumoral, endovascular, and / or intravenous injection.

26. The method of any one of claims 21-25, wherein the tumor-infiltrating lymphocytes are isolated about 1 week to about 6 months post administration of the oncolytic virus.

27. The method of claim 26, wherein the tumor-infiltrating lymphocytes are isolated about 3 weeks to about 5 weeks post administration of the oncolytic virus.

28. The method of claim 26, wherein the tumor-infiltrating lymphocytes are isolated about 1 month to about 6 months post administration of the oncolytic virus.

29. The method of any one of claims 21-28, wherein the tumor-infiltrating lymphocytes are isolated via a tissue biopsy or surgical resection of the solid tumor.

30. The method of any one of claims 21-29, further comprising enriching for tumorinfiltrating lymphocytes by filtering out virus-targeting lymphocytes using virus-specific MHC-peptide tetramers.

31. The method of claim 30, wherein the virus-specific MHC-peptide tetramers are selected from among 2Db / AdV5.El A MHC-peptide tetramer, HLA-A*02 / TLLYVLFEV (hexon) (SEQ ID NO: 1), HLA-A*01 / STDVASLNY (penton) (SEQ ID NO: 2), or a combination of any two or more thereof.-54- 4900-9231-5274.1Atty. Dkt. No.: 642631-006432. The method of any one of claims 21-30, further comprising expanding tumorinfiltrating lymphocytes ex vivo.

33. The method of claim 32, wherein the tumor-infiltrating lymphocytes are expanded using IL-2, IL-7, IL-15, or a combination of any two or more thereof.

34. The method of any one of claims 21 or 23-32, wherein the tumor-infiltrating lymphocytes are administered intravenously, intraperitoneally, subcutaneously, intramuscularly, or intratum orally.

35. The method of any one of claims 21 or 23-33, wherein the donor subject and the recipient subject are the same.

36. The method of any one of claims 21 or 23-33, wherein the donor subject and the recipient subject are different.

37. The method of any one of claims 21-36, wherein the solid tumor is selected from among adrenal cancers, bladder cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, intestinal cancers, kidney cancers, larynx cancers, acute and chronic liver cancers, lymph node cancers, lung cancers, melanomas, mesothelioma, nasopharynx cancers, neuroblastomas, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof.

38. The method of any one of claims 21-36, wherein the solid tumor is a brain tumor or pancreatic cancer.

39. The method of any one of claims 21-38, further comprising administering to the recipient subject an additional anti-cancer therapy.

40. The method of claim 39, wherein the additional anti-cancer therapy is selected from among chemotherapy, radiation therapy, immunotherapy, monoclonal antibodies, anticancer nucleic acids or proteins, anti-cancer microorganisms, and any combinations of two or more thereof.-55- 4900-9231-5274.1

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