Regulation of B-cell translocation gene 1 (BTG1) for adoptive cell therapy

Abstract

Embodiments of the present disclosure include methods and compositions relating to cells modified for expression of B-cell translocation gene 1 (BTG1) and methods for their use. In certain embodiments, the modified cells are used in adoptive cell therapy for certain disease states. In some embodiments, the modified cells are immune cells, such as T cells, and have reduced expression of BTG1, which are utilized to enhance the treatment of cancer and / or infectious diseases. In some embodiments, the modified cells have increased inducible expression of BTG1, which are utilized to treat autoimmune diseases. In some embodiments, expression of BTG1 in endogenous cells is increased in individuals with autoimmune diseases.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 374,447, filed September 2, 2022, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to at least the fields of immunology, cell biology, molecular biology, and medicine (including at least cancer medicine). Summary of the Invention [Problem to be solved by the invention]

[0003] T cell exhaustion is an active process characterized by the gradual loss of effector function and proliferative capacity following prolonged antigen stimulation in chronic infections and cancer. Therapeutic immune cells, such as T cells and NKT cells, engineered to express tumor-specific chimeric antigen receptors (CARs) also become exhausted, limiting their antitumor efficacy and associated with tumor evasion and disease progression / recurrence. Specific gene expression and epigenetic changes have been implicated in the T cell exhaustion process, but the precise mechanisms underlying the hyporesponsiveness of exhausted cells remain poorly understood.

[0004] The present disclosure provides a solution to a long-standing need in the art for improved cell therapy. [Means for solving the problem]

[0005] Embodiments of the present disclosure include methods and compositions for use with any type of therapeutic cell, including immune effector cells. Therapeutic cells can be tailored and utilized for specific therapeutic applications, such as cancer treatment, and such cells of the present disclosure can be modified to enhance one or more of the cell's activities. In various embodiments, the present disclosure generally relates to modulating an individual's endogenous cells to (1) prevent or reduce unwanted cell wasting and / or control excessive activation with associated toxicity of therapeutic cells used in the individual (e.g., cancer patients), or (2) suppress the activity of autoimmune cells, such as in patients with autoimmune diseases.

[0006] In some embodiments, BTG1 expression and / or activity is modulated in cells used in adoptive cell therapy for any purpose, with the type of modulation being related to the intended therapeutic use of the cells modified accordingly. In various embodiments, modulation can be downregulating expression of the endogenous BTG1 gene in the cells used for therapy and / or reducing the activity of the BTG1 protein in the cells. In other embodiments, modulation can be upregulating expression of the endogenous BTG1 gene in the cells and / or introducing heterologous BTG1 into the cells (including by overexpression) and / or increasing the activity of the BTG1 protein in the cells.

[0007] In certain embodiments, cells for cell therapy are prone to exhaustion or otherwise exhibit poor persistence, poor activation, poor proliferation, poor homing, and / or poor cytotoxicity. Such cells may be subject to modulation of BTG1 expression and / or activity to improve one or more of these properties. Some immune cells may benefit from modulation of BTG1 expression and / or activity to improve one or more of these properties. In certain embodiments, endogenous BTG1 expression and / or activity is reduced in cells used in cell therapies such as cancer and chronic infections, which may or may not be specific types of immune cells. In various embodiments, any type of T cell (CD8 T cell, CD4 T cell, abT cell, gamma / delta T cell, virus-specific T cell (e.g., Epstein-Barr virus, cytomegalovirus, BK virus, human herpesvirus, adenovirus, respiratory syncytial virus, influenza, parainfluenza virus 3, human metapneumovirus, etc.)), NKT cell, MAIT cell, cytokine-induced killer cell, NK cell, macrophage, or mixture thereof, is engineered to have reduced expression and / or activity of endogenous BTG1, such that the cells exhibit reduced or no exhaustion compared to the same type of cell in the absence of such modulation. Such reduction in endogenous BTG1 expression and / or activity may result in the regulated cells exhibiting reduced exhaustion and improved persistence, activation, proliferation, homing, and / or cytotoxicity compared to the same type of cell that has not been so engineered.

[0008] In certain embodiments, endogenous BTG1 expression and / or activity is increased in cells used in cell therapy, or heterologous BTG1 is introduced into the cells, which may be other specific cell types. In certain embodiments, it is desirable for cells for cell therapy to be exhaustible or otherwise exhausted, to have poor persistence, poor activation, poor proliferation, poor homing, and / or poor cytotoxicity.

[0009] In certain embodiments, upregulation of BTG1 is useful in endogenous autoimmune cells, such as agents that promote upregulation of BTG1.

[0010] In some embodiments, increasing or regulating BTG1 expression is useful in controlling the toxicity of therapeutic effector cells in cancer and infectious diseases (chronic or acute), e.g., can be used as an "off switch." In some embodiments, BTG1 expression is controlled by an inducible promoter, allowing inhibition or killing of any type of therapeutic cell encompassed herein as needed, such as during a cytokine storm.

[0011] Embodiments of the present disclosure include methods of enhancing individual cell therapy, comprising reducing the expression and / or activity of B-cell translocation gene 1 (BTG1) in cells for cell therapy.

[0012] Embodiments of the present disclosure include a method of enhancing cell therapy for an individual, comprising reducing the expression and / or activity of B-cell translocation gene 1 (BTG1) in a cell, if the cell is not a T cell.

[0013] In certain embodiments, the cells are immune cells, stem cells, one or more of their derivatives, or a mixture thereof, optionally with the derivative being iPSC-derived T cells, NKT cells, or NK cells. The cells may be CD8 T cells, CD4 T cells, natural killer T (NKT) cells, MAIT cells, gamma / delta T cells, virus-specific T cells, cytokine-induced killer cells, NK cells, macrophages, or a mixture thereof. In certain embodiments, the cells are modified to express one or more heterologous genes, while in other cases, the cells are not modified to express one or more heterologous genes. In certain embodiments, the heterologous genes may include one or more modified receptors, antibodies, cytokines, suicide genes, costimulatory factors, regulatory factors, or combinations thereof. The modified receptor may be an antigen receptor, a chemokine receptor, or a cytokine receptor, or the cells may be one or more of these types. The antigen receptor may be a chimeric antigen receptor (CAR) or a T cell receptor. The CAR may comprise one, two, or more costimulatory domains, such as CD28, 4-1BB, OX40, CD2, DAP10, CD40, ICOS, CD27, TLR, MYD88; 2B4, NKG2D, or a combination thereof. In certain embodiments, the antigen receptor may target GD2, CD19, GPC3, B7-H3, CD20, BCMA, CD30, CD38, CD5, CD7, HER2, PSMA, mesothelin, EGFR, IL13RA2, or a combination thereof. The CAR may comprise one or more activation domains, such as CD3ζ, DAP12, 2B4, or a combination thereof. In certain cases, the cytokine is IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-33, or a combination thereof. In certain embodiments, the antibody is a monospecific antibody, a bispecific antibody, a trispecific antibody, or a mixture thereof. The antibody may be a bispecific T cell engager or a trispecific T cell engager. In some embodiments, the reducing step uses one or more agents to reduce expression of the endogenous BTG1 gene in the cell.The one or more agents may include nucleic acids, peptides, and / or polypeptides. The one or more agents may include CRISPR agents, siRNAs, shRNAs, transposons, or mixtures thereof. In some cases, the reducing step uses one or more agents that reduce the activity of BTG1 protein in cells, and the one or more agents may include one or more small molecules or one or more antibodies that target BTG1. The method may further include administering a therapeutically effective amount of the cells to an individual in need thereof. In some cases, the individual has cancer or an acute or chronic infection.

[0014] Embodiments of the present disclosure may include a plurality of any of the cells encompassed herein, which may be included in a pharmaceutically acceptable excipient.

[0015] In some embodiments, non-cancerous cells engineered to contain reduced BTG1 expression and / or activity are provided, where the cells express one or more heterologous genes. The heterologous genes can include one or more engineered receptors, antibodies, cytokines, suicide genes, costimulatory factors, regulatory factors, or combinations thereof. The engineered cells can be immune cells or stem cells. In certain embodiments, the cells are CD8 T cells, CD4 T cells, NKT cells, MAIT cells, gamma / delta T cells, virus-specific T cells, cytokine-induced killer cells, NK cells, macrophages, or a combination thereof. In some embodiments, the reduced expression is achieved by one or more CRISPR agents, siRNAs, shRNAs, transposons, or a combination thereof. The reduced activity can be achieved by one or more small molecules. In some embodiments, the engineered receptor is an antigen receptor or cytokine receptor, as described elsewhere herein. In some embodiments, the reduced BTG1 activity is achieved by one or more small molecules or one or more antibodies targeting BTG1.

[0016] Specific examples of the present disclosure include genetically engineered CD8 T cells, CD4 T cells, NKT cells, MAIT cells, gamma / delta T cells, virus-specific T cells, cytokine-induced killer cells, NK cells, macrophages, or mixtures thereof, which have been genetically engineered to contain reduced expression and / or activity of BTG1. The cells may express one or more genes, such as one or more altered receptors, antibodies, cytokines, suicide genes, costimulatory factors, regulatory factors, or combinations thereof.

[0017] Embodiments of the present disclosure include methods of treating cancer and / or acute or chronic infection in an individual, the method comprising administering to the individual a therapeutically effective amount of any one of a plurality of cells of the present disclosure. In some embodiments, the plurality of cells comprises NK cells, NK T cells, and / or macrophages comprising a GD2 CAR, a GPC3 CAR, a CD19 CAR, and / or a B7-H3 CAR. The cells may be allogeneic or autologous to the individual. In certain embodiments, the acute or chronic infection is human immunodeficiency virus, tuberculosis, herpes, viral hepatitis, or COVID.

[0018] Embodiments of the present disclosure include methods for treating an autoimmune disease in an individual, the methods comprising administering to the individual a therapeutically effective amount of cells comprising increased inducible BTG1 expression and / or activity and / or administering to the individual a therapeutically effective amount of an agent that increases BTG1 expression and / or activity in endogenous cells of the individual. In some embodiments, the cells are immune cells, stem cells, one or more of their derivatives, or a mixture thereof. The increased expression is due to expression of BTG1 on a vector in the cells, which may be an extrachromosomal vector or an integrating vector. In some embodiments, the increased expression is due to introduction of a heterologous promoter in the regulatory region of the endogenous BTG1 gene in the cells. The increased activity of BTG1 may be due to introduction of a small molecule into the cells. The cells may or may not express one or more heterologous genes. In certain cases, the autoimmune disease is type 1 diabetes, lupus, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, glomerulonephritis, granulomatosis with polyangiitis, Graves' disease, or Guillain-Barré syndrome. Idiopathic thrombocytopenic purpura, juvenile idiopathic arthritis, myasthenia gravis, myocarditis, multiple sclerosis, pemphigus / bullous pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, or vitiligo. Cells may be allogeneic or autologous to an individual.

[0019] Embodiments of the present disclosure include engineered cells in which the cells have been engineered to have increased expression of endogenous BTG1 or to contain a vector expressing heterologous BTG1. The vector may be an exochromosomal vector or an integrating vector. Increased expression may be achieved by introducing a heterologous promoter into the regulatory region of the endogenous BTG1 gene in the cell. Increased BTG1 activity may be achieved by introducing a small molecule into the cell, and the cell may express one or more heterologous genes.

[0020]

[0003] Embodiments include methods of controlling the activity and / or toxicity of cell therapy, including increasing BTG1 expression and / or activity in cells of the cell therapy. Increased expression of BTG1 may be achieved by an inducible promoter.

[0004] Embodiments may also include methods of reducing expression of the endogenous BTG1 gene in cells, such as by using one or more agents comprising a CRISPR agent, miRNA, siRNA, shRNA, transposon, or a mixture thereof.

[0021] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0022] The following drawings form part of this specification and are included to further describe certain aspects of the present disclosure, and the present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0023] [Figures 1A-1E] BTG1 expression is elevated in exhausted chimeric antigen receptor (CAR)-natural killer T cells (NKT). [Figure 1A] This study design involves a repeated tumor co-culture system to induce CAR-NKT exhaustion through chronic antigen exposure. Manufactured CAR-NKT cells are replated with fresh CHLA255 NB tumor cells every 5 days and cultured for multiple cycles. [Figure 1B] The cytotoxic activity of CAR-NKT during repeated co-cultures was measured at the indicated time points. [Figure 1C]Uniform Manifold Approximation (UMAP) projections of single-cell RNA sequencing (scRNAseq) results of manufactured CAR-NKT infusion products (IP), CAR-NKT after five cycles of repeated co-culture with tumor cells (5RcC), and CAR-NKT isolated from peripheral blood (PB) after infusion. [Figure 1D] UMAP projection of CAR-NKT gene expression obtained by scRNAseq from pre-infusion and post-5RcC samples. [Figure 1E] Volcano plot showing differences in expressed genes in CAR-NKT after 5RcC and pre-infusion products. [Figures 2A-2E] Overexpression of BTG1 reduces total RNA expression and proliferative capacity in NKT cells. [Figure 2A] Design of retroviral constructs encoding a BTG1-green fluorescent protein fusion (BTG1.GFP) or GFP and firefly luciferase (GFP control) to assess the effects of overexpression (OE) of BTG1 in NKT cells. [Figure 2B] BTG1 expression in NKT cells expressing the GFP.BTG1 construct compared to expression in wild-type (WT) NKT cells by qPCR. [Figure 2C] Western blot comparison of BTG1 expression in NKT cells expressing the GFP.BTG1 construct with expression in wild-type (WT) NKT cells. [Figure 2D] Pathway enrichment analysis showing gene programs with increased expression in BTG1 OE NKT. [Figure 2E] Comparison of BTG1 OE NKT and GP control NKT. Evaluated by six independent subjects. [Figure 3A-3C] Assessment of the role of BTG1 in regulating the antitumor properties of NKT. [Figure 3A] BTG1 protein expression determined by Western blotting at the indicated time points in a repeated tumor challenge assay (RTC). [Figure 3B]BTG1 protein expression measured by Western blotting after activation with CD3 / CD28-specific monoclonal antibodies [Figure 3C] BTG1 mRNA expression determined by qPCR at the indicated time points. [Figure 4A-4B] BTG1 expression in activated T cells: Peripheral blood T cells were stimulated with plate-bound CD3 / CD28 antibodies and cultured in the presence of IL2. [Figure 4A] BTG1 protein expression measured by Western blotting at the indicated time points. [Figure 4B] Absolute numbers of BTG1 OE versus GFP control T cells after in vitro culture. [Figures 5A-5C] BTG1 KD in GD2-CAR-NKTs. A microRNA targeting BTG1 and a scrambled control were cloned downstream of GD2-CAR into an MMuLV-based gammaretroviral construct. [Figure 5A] Design of retroviral constructs for BTG1 knockdown. [Figure 5B-5C] BTG1 transcript and protein expression in NKT cells expressing the indicated constructs was quantified by qPCR and Western blotting, respectively. [Figures 6A-6K] BTG1 knockdown (KD) enhances the antitumor activity of GD2-CAR-NKT.NKT cells were transduced with a retroviral vector encoding CAR + / - interleukin-15 (IL15) and / or artificial microRNA (amiR) specific for BTG1 or a scrambled control. [Figure 6A] Fold increase in NKT expressing a particular construct after transduction. [Figure 6B] CD62L frequency after transduction in CAR.15 NKT with and without BTG1 KD. [Figure 6C] CD62L expression in CAR.15.amiR-BTG1 NKTs gated on CAR+ and CAR- populations. [Figure 6D]Frequency of PD-1+CAR.15 NKT with or without BTG1 KD. [Figure 6E] Cytolytic activity of CAR.15.amiR.BTG1 and scrambled control NKT against GD2-high-expressing CHLA255 and GD2-low-expressing CHLA136 NB cell lines assessed at the indicated coculture times. [Figure 6F] Residual tumor cell frequencies after 5 days of coculture of the indicated NKT groups with CHLA255 cells at E:T ratios of 1–5. [Figure 6G] Fold expansion of CAR-NKT cells after six cycles of co-culture with NB cells. [Figure 6H] Experimental design for in vivo evaluation of CAR-NKT antitumor activity in an aggressive metastatic NB xenograft model. [Figure 6I] Bioluminescence images of tumor-implanted mice at specific time points. [Figure 6J] Changes in tumor burden based on bioluminescence imaging over time. [Figure 6K] Kaplan-Meier survival curves for mice in the indicated groups. 10 mice per treatment group, survival comparison by Gehan-Breslow-Wilcoxon test. [Figures 7A-7B] BTG1 KD enhances the antitumor activity of GD2-CAR T cells. [Figure 7A] Doubling rate of GD2-CAR T cells after three rounds of co-culture with CHLA255 neuroblastoma (NB) cells (E:T=1:1, N=6, two-tailed paired t-test). [Figure 7B] Changes in CAR percentage of T cells before and after three rounds of coculture with CHLA255 NB cells (N=6, two-tailed paired t-test). [Figures 8A-8F] BTG1 KD enhances the antitumor activity of GD2-CAR T cells in an in vivo metastatic neuroblastoma xenograft model. [Figure 8A] Experimental design for in vivo evaluation of the antitumor activity of GD2-CAR T cells in an aggressive metastatic neuroblastoma xenograft model. [Figure 8B]Schematic of retroviral constructs for BTG1 KD and scrambled miRNA control. LTR, long terminal repeat. scFv, single-chain variable fragment. H-TM, hinge-transmembrane. [Figure 8C] Bioluminescence images of tumor-bearing mice at specific time points. [Figure 8D] Changes in tumor burden based on bioluminescence images in Figure 8C over time. [Figure 8E] Kaplan-Meier survival curves for 10 mice per group, and survival comparisons using the Gehan-Breslow-Wilcoxon test. [Figure 8F] Quantification of human T cells (human CD45+) in the total cell population taken from mouse blood on day 10. [Figures 9A-9D] Deletion of BTG1 in T cells increases the frequency of memory T cells. [Figure 9A] Experimental design for inserting a CD34-Q8 tag into the BTG1 locus using CRISPR technology. [Figure 9B] Representative flow cytometry of CD34-Q8 tag expression in T cells 5 days after CRISPR insertion. [Figure 9C] BTG1 protein expression in T cells by Western blot at day 7 after CRISPR KI. [Figure 9D] Representative plots and summary showing expression of memory markers CD45RA and CCR7 in T cells with CD34-Q8 tagged KI and Cas9-only control (N=4) at the BTG1 locus. DETAILED DESCRIPTION OF THE INVENTION

[0024] [I. Definition Example] Following traditional patent law practice, the words "a" and "an" when used herein in conjunction with the word "comprising," including the claims, mean "one or more." Some embodiments of the present disclosure may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. Any method or composition described herein can be implemented with respect to any other method or composition described herein, and different embodiments can be combined.

[0025] Throughout this specification, unless the context otherwise requires, the words "comprise," "comprises," and "comprising" mean the inclusion of a particular step, element, or group of steps or elements, but not the exclusion of other steps, elements, or group of steps or elements. The phrase "consisting of" means the inclusion of and is limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are required, and that no other elements may be present. The phrase "consisting essentially of" means the inclusion of the subsequent recited elements, and is limited to other elements that do not interfere with or facilitate the activity or function specified in the disclosure of the recited elements. Thus, the phrase "consisting essentially of" means that the recited elements are required, but that other elements are optional and may or may not be present depending on whether they affect the activity or behavior of the recited elements.

[0026] Reference herein to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "an additional embodiment," or "a further embodiment," or any combination thereof, means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places herein are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0027] As used herein, the terms "or" and "and / or" are used to describe a combination or mutually exclusive case of multiple elements. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z may be specifically excluded from an implementation.

[0028] In this application, the term "about" is used in accordance with its plain and ordinary meaning within the field of cell and molecular biology to indicate that a value includes the standard deviation error of the device or method being employed to determine the value.

[0029] "Autoimmune disease" refers to a disorder in which the immune system mounts an immune response (e.g., a B-cell or T-cell response) against antigens that are part of the normal host (i.e., self-antigens), resulting in tissue damage. Self-antigens can originate from host cells or from resident bacteria, such as microorganisms that normally colonize mucosal surfaces (known as commensal flora).

[0030] As used herein, "reduction of expression" or "disruption" or "alteration" refers to the complete or partial elimination or reduction of expression of one or more gene products encoded by a gene of interest, compared to the expression level of the gene product in the absence of the alteration. Exemplary gene products include the mRNA and protein products encoded by the gene. Some mutations are transient or reversible, while others are permanent. Some mutations result in functional or full-length proteins or mRNAs, while truncated or non-functional products may be produced. In some embodiments herein, gene activity or function is inhibited, as opposed to expression. Genetic alterations are generally caused by artificial methods, i.e., the addition or introduction of compounds, molecules, complexes, or compositions, and / or alterations of the nucleic acids of or associated with a gene, e.g., alterations at the DNA level. Exemplary methods of genetic alteration include genetic alteration techniques such as gene silencing, knockdown, knockout, and / or gene editing. Examples include antisense technologies (such as RNAi, siRNA, shRNA, and / or ribozymes), which generally result in a transient reduction in expression, and gene editing technologies, which inactivate or modify targeted genes by inducing cleavage and / or homologous recombination. Examples include insertions, mutations, and deletions. Such genetic modifications typically result in the suppression and / or complete absence of expression of the normal, or "wild-type," product encoded by the gene. Examples of such genetic mutations include insertions, frameshift and missense mutations, deletions, knock-ins (including knock-ins with markers such as CD34-Q8 tags, GFP, or other selectable markers), and knockouts (deletions) of entire or partial genes. Such mutations occur in coding regions, e.g., one or more exons, and preclude the production of a full-length product, a functional product, or any product, such as by inserting a stop codon.Such mutations can also occur through mutations in promoters or enhancers, or other regions that affect transcriptional activation, and can prevent gene transcription. Genetic mutations include gene targeting, which involves inactivating a targeted gene by homologous recombination.

[0031] As used herein, the term "engineered" refers to something produced by the hand of man, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, the engineered item is synthetic and includes elements not found in nature or constructed using the methods utilized in this disclosure. In certain embodiments, vectors are engineered by recombinant nucleic acid techniques and cells are engineered by gene transfer or transduction with a gene transfer vector. Cells may be engineered to express a heterologous protein that the cell does not naturally express, either because the heterologous protein is recombinant or synthetic, or because the cell does not naturally express that protein. Engineered organisms do not exist in nature.

[0032] As used herein, the terms "exhausted" or "exhaustion" refer to immune cells, including T cells, that become dysfunctional due to reduced effector function, persistent expression of inhibitory receptors, and a transcriptional state that differs from that of functional effector or memory T cells. Exhaustion can occur during chronic infection, autoimmune disease, or cancer and can interfere with optimal control of infection and tumors, respectively.

[0033] The term "exogenous," when used in reference to a protein, gene, nucleic acid, or polynucleotide in a cell or organism, refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means; when used in reference to a cell, the term refers to a cell that has been isolated and then introduced into another cell or organism by artificial or natural means. A foreign nucleic acid can be from a different organism or cell, or it can be one or more additional copies of a nucleic acid that is naturally present in the organism or cell. A foreign cell can be from a different organism or from the same organism. As non-limiting examples, a foreign nucleic acid can be in a chromosomal location that is different from its location in a native cell or it can be flanked by other nucleic acid sequences not found in nature.

[0034] As used herein, "natural killer T (NKT) cells" refers to a subset of innate immune-like T lymphocytes that recognize glycolipids presented by the monomorphic MHC-like molecule CD1d. Unlike T cells, NKT cells do not recognize HLA class I or class II molecules. Type I, or invariant, NKT cells express the invariant T cell receptor (TCR) α chain Vα24-Jα18, which in certain embodiments is paired with Vβ11. This NKT subset can be identified using the monoclonal antibody clone 6B11 or by reactivity to the synthetic glycolipid α-galactosylceramide. In specific embodiments, NKT cells express one or more artificial antigen receptors, such as one or more CARs.

[0035] The phrases "pharmaceutical or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, such as a human. The formulation of pharmaceutical compositions containing antibodies or additional active ingredients will be well known to those of skill in the art in light of the present disclosure. Furthermore, it is understood that for animal (e.g., human) administration, the formulation should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.

[0036] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes various materials and combinations thereof well known to those skilled in the art, such as aqueous solvents such as water, alcohol / water mixtures, and saline; intravenous vehicles such as sodium chloride and Ringer's sugar solution; non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate; dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, inert gases), isotonicity agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, coloring agents, fluid or nutrient replenishers, etc. The pH and exact concentration of each component in a pharmaceutical composition are adjusted based on known parameters.

[0037] The term "subject," as used herein, generally refers to an individual who has or is suspected of having cancer, or who has or is suspected of having an acute or chronic infectious disease and / or autoimmune disease. A "subject" is any living organism or animal to which the method or substance is applied, including mammals (e.g., humans, laboratory animals (primates, rats, mice, rabbits, etc.), livestock (cows, sheep, goats, pigs, turkeys, chickens, etc.), domestic animals (dogs, cats, rodents, etc.), horses, transgenic non-human animals, etc.). A "subject" may be a patient, such as an individual suffering from or suspected of suffering from a benign or malignant neoplasm, cancer, or other disease (sometimes referred to as a "medical condition"). A subject may be undergoing or have already undergone treatment. A subject may also be asymptomatic. A subject may also be healthy, including those seeking prevention of cancer, infectious diseases, or autoimmune diseases. The term "individual" may be used synonymously with "subject," at least in some contexts. A "subject" or "individual" may or may not be an inpatient in a medical institution, or may be treated as an outpatient in a medical institution. They may also receive one or more pharmaceutical compositions via means such as the internet. Such individuals may be human or non-human animals and include individuals of all ages, including adults, minors (i.e., children), infants, and fetuses. The term "subject" does not necessarily imply the existence of medical need and may be a willing or involuntary participant in an experiment as part of a clinical trial or basic scientific research. A subject may also be participating in a clinical trial.

[0038] As used herein, the terms "treatment" or "treating" include any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include, for example, even a minimal reduction in one or more measurable indicators in the treatment of cancer, infectious diseases, or autoimmune diseases. "Treatment" may optionally include the alleviation or amelioration of one or more symptoms of the disease or pathological condition, or the slowing of the progression of the disease or pathological condition. The term "treatment" does not necessarily imply a complete elimination or cure of the disease or pathological condition or its associated symptoms. "Treating" may mean alleviating at least one symptom of the disease or pathological condition.

[0039] As used throughout this specification, the term "therapeutically effective" refers to anything that promotes or improves the well-being of a subject in connection with the medical treatment of that disease. This term includes, but is not limited to, reducing the frequency or severity of one or more signs or symptoms of the disease. For example, treating cancer can include shrinking a tumor, reducing the invasiveness of a tumor, slowing the rate of cancer growth, or inhibiting metastasis. Additionally, treating cancer can also include extending the subject's survival and / or improving their quality of life.

[0040] II. General Embodiments In various embodiments, cell therapy for an individual in need thereof is prepared such that the cells have modulated expression and / or activity of endogenous BTG1, or in other cases, have exogenous BTG1 introduced into the cells.

[0041] The present disclosure encompasses embodiments in which a cell therapy for a disease is generated based on the disease being treated. In some embodiments, an individual has a medical condition in which cells for treatment of that condition need to be persistent and avoid exhaustion. In certain embodiments, when cancer or chronic viral infections (e.g., HIV, chronic viral hepatitis) are being treated (by way of example only), it is beneficial for cells in the cell therapy to avoid exhaustion or to reduce the degree of exhaustion. As shown elsewhere herein, for example, BTG1 is involved in the exhaustion of NKT cells and T cells, and its knockdown, knockout, or reduction in activity is beneficial for the persistence of these cells. Such cells are effective in treating cancer or chronic viral infections.

[0042] However, in some embodiments, for example when autoimmune diseases are being treated, it is beneficial for the cells of the cell therapy to be exhausted, or to have an increased or accelerated degree of exhaustion.

[0043] Embodiments of the present disclosure include methods of enhancing cell therapy in an individual, the methods comprising decreasing BTG1 expression and / or activity in cells for cell therapy, or increasing BTG1 expression and / or activity in cells for cell therapy, which may or may not be T cells as the case may be.

[0044] Embodiments of the present disclosure include methods of producing cells for cell therapy in an individual, the method comprising reducing BTG1 expression and / or activity in the cells, optionally including cases where the cells are not T cells. In certain embodiments, provided herein are methods of producing cells for cell therapy in an individual, the method comprising increasing BTG1 expression and / or activity in the cells or in endogenous cells of the individual.

[0045] In certain embodiments, the present disclosure provides methods comprising reducing the expression and / or activity of BTG1 in the cell, optionally including when the cell is not a T cell. In some embodiments, the present disclosure provides methods comprising increasing the expression and / or activity of BTG1 in the cell or in endogenous cells of the individual.

[0046] [III. Reduction of BTG1 Expression and / or Activity] In certain embodiments, cells used to treat cancer or acute or chronic infections are required to have reduced or no exhaustion, for example, compared to their natural counterparts in vivo or cells that have not been artificially modified with BTG1. In certain cases, the cells may be immune cells, including at least any type of T cell (including CD8 T cells, CD4 T cells, αβ T cells, γδ T cells, and virus-specific T cells), NKT cells, MAIT cells, cytokine-induced killer cells, NK cells, macrophages, or a mixture thereof. In certain embodiments, the cells are any type of T cell or NKT cell. In various embodiments, cells for cancer or acute or chronic infections have regulated BTG1 expression, regulated BTG1 activity, or both. In certain embodiments, regulated expression includes disruption or reduction of expression of the endogenous BTG1 gene compared to when the regulated expression is absent. In certain embodiments, regulated activity includes reduction of the activity of the endogenous BTG1 protein compared to when the regulated expression is absent.

[0047] In certain embodiments, expression of the endogenous BTG1 gene is reduced in cells for cancer treatment or treatment of acute or chronic infections. The expression can be engineered to be artificially reduced by any suitable method.

[0048] In some embodiments, reduction of BTG1 gene expression is achieved by causing a disruption in the gene, such as a knockout, insertion, missense or frameshift mutation (e.g., biallelic frameshift mutation), deletion of all or part of the gene (e.g., one or more exons or parts thereof), and / or knock-in. For example, altered BTG1 gene expression can be caused by sequence-specific or targeted nucleases, including DNA-binding targeted nucleases (such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)) and RNA-guided nucleases such as CRISPR-associated nucleases (Cas), designed to specifically target the sequence of the BTG1 gene or parts thereof.

[0049] In some embodiments, alteration of the expression, activity, and / or function of the BTG1 gene is achieved by disrupting the gene, which is modified to reduce expression by at least about 20%, 30%, or 40%, typically at least about 50%, 60%, 70%, 80%, 90%, or 95%, compared to expression in the absence of the genetic modification or the components introduced to effect the modification.

[0050] In some embodiments, the modification is transient or reversible, and expression of the gene is restored at a later time point. In other embodiments, the modification is neither reversible nor transient, but may be, for example, permanent.

[0051] In some embodiments, gene modification is carried out by inducing one or more double-strand breaks and / or one or more single-strand breaks in the BTG1 gene, usually in a targeted manner.In some embodiments, the double-strand or single-strand breaks are generated by a nuclease, such as an endonuclease, for example, a gene-targeting nuclease.In some embodiments, the breaks are induced in the coding region of the gene, for example, in an exon.For example, in some embodiments, the induction occurs near the N-terminal portion of the coding region, for example, in the first exon, the second exon, or any subsequent exon.

[0052] In some embodiments, the double-stranded or single-stranded break is repaired via a cellular repair process, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). In some embodiments, the repair process is error-prone, resulting in a disruption of the gene, such as a frameshift mutation, e.g., a biallelic frameshift mutation. This can result in a complete knockout of the gene. For example, in some embodiments, the disruption includes the introduction of a deletion, mutation, and / or insertion. In some embodiments, the disruption results in the appearance of a premature stop codon. In some embodiments, the presence of an insertion, deletion, translocation, frameshift mutation, and / or premature stop codon results in disruption of the expression, activity, and / or function of the gene.

[0053] In some embodiments, genetic modification is achieved using antisense technology, e.g., RNA interference (RNAi), short interfering RNA (siRNA), short hairpin (shRNA), and / or ribozymes are used to selectively suppress or inhibit expression of the BTG1 gene. siRNA technology is RNAi, which uses double-stranded RNA molecules that have a sequence homologous to and a sequence complementary to the nucleotide sequence of the mRNA transcribed from the gene. The siRNA generally is homologous / complementary to a region of the mRNA transcribed from the gene, or may be an siRNA containing multiple RNA molecules homologous / complementary to different regions. In some embodiments, the siRNA is included in a polycistronic construct, e.g., included together with an expression construct that produces an exogenous gene product.

[0054] (A.1 ZFPs and ZFNs) In some embodiments, the DNA targeting molecule comprises a BTG1-binding protein, such as one or more zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs), fused to an effector protein, such as an endonuclease. Examples include ZFNs, TALEs, and TALENs.

[0055] In some embodiments, the BTG1-targeting molecule comprises one or more zinc finger proteins (ZFPs) or domains thereof that bind sequence-specifically to the BTG1 gene. A ZFP or domain thereof is a protein or domain within a larger protein that binds sequence-specifically to DNA via one or more zinc fingers, i.e., amino acid sequence regions within the binding domain whose structure is stabilized by the coordination of zinc ions. The term zinc finger DNA-binding protein is often abbreviated as zinc finger protein or ZFP. ZFPs include artificial ZFP domains that target specific DNA sequences, typically 9-18 nucleotides in length, which are generated by combining individual fingers.

[0056] ZFPs have a finger domain approximately 30 amino acids in length, containing an α-helix containing two invariant histidine residues coordinated via zinc to two cysteine ​​residues in a β-turn, and may have two, three, four, five, or six fingers. Generally, the sequence specificity of a ZFP can be altered by making amino acid substitutions at four helical positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Thus, in some embodiments, the ZFP or ZFP-containing molecule is non-naturally occurring, e.g., designed to bind to a selected target site.

[0057] In some embodiments, the BTG1-targeting molecule is or includes a zinc-finger DNA-binding domain fused to a DNA cleavage domain, forming a zinc-finger nuclease (ZFN). In some embodiments, the fusion protein includes a cleavage domain (or cleavage half-domain) derived from at least one Type 1iS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. In some embodiments, the cleavage domain is derived from the Type 1iS restriction endonuclease, Fok I. Fok I catalyzes double-stranded cleavage of DNA, typically 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand.

[0058] Many gene-specifically designed zinc fingers are commercially available. For example, Sangamo Biosciences (Richmond, Calif., USA), in partnership with Sigma-Aldrich (St. Louis, Mo., USA), has developed a platform for zinc finger construction (CompoZr), allowing researchers to completely bypass the construction and validation of zinc fingers and providing specifically targeted zinc fingers for thousands of proteins (Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405). In some embodiments, commercially available zinc fingers are used or custom-designed. (See, e.g., Sigma-Aldrich product catalog numbers CSTZFND, CSTZFN, CTil-1KT, and PZD0020.)

[0059] (B. TALs, TALEs, and TALENs) In some embodiments, molecules that target BTG1 include transcription activator-like protein (TAL) DNA-binding domains, such as those found in transcription activator-like effector protein (TALE) proteins, which may be naturally occurring or engineered (non-naturally occurring). See, e.g., U.S. Patent Publication No. 2011 / 0301073, which is incorporated herein by reference in its entirety.

[0060] A TALE DNA-binding domain, or TALE, is a polypeptide containing one or more TALE repeat domains / units. The repeat domain is responsible for the binding of the TALE to its corresponding target DNA sequence. A "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length and shares at least some sequence homology with other TALE repeat sequences in naturally occurring TALE proteins. Each TALE repeat unit contains one or two DNA-binding residues that constitute a Repeat Variable Diresidue (RVD), usually located at positions 12 and / or 13 of the repeat. Canonical codes for DNA recognition by these TALEs have been identified; for example, the HD sequence at positions 12 and 13 binds to cytosine (C), NG to thymine (T), NI to adenine (A), NN to guanine (G) or adenine (A), and NO to thymine (T) and non-canonical RVDs. In some embodiments, TALEs can target any gene by designing TAL sequences with specificity for the target DNA sequence, which generally begins with a thymidine.

[0061] In some embodiments, the molecule is a DNA-binding endonuclease, such as a TALE nuclease (TALEN). In some embodiments, the TALEN is a fusion protein comprising a DNA-binding domain derived from a TALE and a nuclease catalytic domain for cleaving a nucleic acid target sequence.

[0062] In some embodiments, the TALEN recognizes and cleaves a target sequence within the BTG1 gene. In some embodiments, the DNA cleavage causes a double-strand break. In some embodiments, the cleavage promotes the rate of homologous recombination or non-homologous end joining (NHEJ). Generally, NHEJ is an imperfect repair process, often resulting in changes to the DNA sequence at the cleavage site. In some embodiments, the repair mechanism involves rejoining the remaining portions of the two DNA ends via direct ligation or so-called microhomology-mediated end joining. In some embodiments, NHEJ-mediated repair results in small insertions or deletions, which can be used to disrupt and thereby silence genes. In some embodiments, the modification can be a substitution, deletion, or addition of at least one nucleotide. In some embodiments, cells in which a cleavage-induced mutation event, i.e., a mutation event subsequent to an NHEJ event, has occurred can be identified and / or selected by methods well known to those skilled in the art.

[0063] In some embodiments, TALE repeats are assembled to target specific genes (Gaj et al., 2013). A TALEN library targeting 18,740 human protein-coding genes has been constructed (Kim et al., 2013). Custom-designed TALE sequences are commercially available through Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, Ky., USA), and Life Technologies (Grand Island, NY, USA). In particular, TALENs targeting CD38 are commercially available (see Gencopoeia, product catalog numbers HTN222870-1, HTN222870-2, and HTN222870-3). Exemplary molecules are described, for example, in U.S. Patent Publication Nos. US 2014 / 0120622 and 2013 / 0315884.

[0064] In some embodiments, the TALEN is introduced as a transgene encoded by one or more plasmid vectors, which may include a selection marker for identifying and / or selecting cells that have received the vector.

[0065] (C.RGENs (CRISPR / Cas system)) In some embodiments, the modification is performed using one or more DNA-binding nucleic acids, for example, modification by RNA-guided endonuclease (RGEN). For example, the modification can be performed using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. Generally, the term "CRISPR system" refers to the transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, including the sequence encoding the Cas gene, the tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or active partial tracrRNA), the tracr-mate sequence (including the "direct repeat" in the context of the endogenous CRISPR system and the partial direct repeat processed by tracrRNA), the guide sequence (also referred to as "spacer" in the context of the endogenous CRISPR system), and / or other sequences and transcripts from the CRISPR locus.

[0066] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide RNA) that binds to DNA in a sequence-specific manner and a Cas protein (e.g., Cas9) with nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a Type I, Type II, or Type III CRISPR system. For example, it can be derived from a specific organism with an endogenous CRISPR system, such as Streptococcus pyogenes.

[0067] In some embodiments, a Cas nuclease and a gRNA (comprising a fusion of a target sequence-specific crRNA and an anchored tracrRNA) are introduced into cells. Generally, a target site at the 5' end of the gRNA guides the Cas nuclease to the target site, e.g., a gene, using complementary base pairing. The target site may be selected based on its location immediately 5' to a protospacer adjacent motif (PAM) sequence, typically NGG or NAG. In this regard, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Generally, CRISPR systems are characterized by elements that promote the formation of a CRISPR complex at the site of the target sequence. Typically, "target sequence" refers to a sequence to which the guide sequence is designed to be complementary, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Absolute complementarity is not required, just sufficient complementarity to result in hybridization and formation of a CRISPR complex.

[0068] The CRISPR system can induce a double-strand break (DSB) at the target site, followed by disruption or modification as described herein. In other embodiments, a Cas9 mutant called a "nickase" is used to nick one strand at the target site. Paired nickases can be used, for example, to improve specificity; guided by two different gRNAs, each targeting a different sequence, the nicks are introduced simultaneously to form 5' overhangs. In other embodiments, catalytically inactive Cas9 is fused to an exogenous effector domain, such as a transcriptional repressor or activator, to affect gene expression.

[0069] The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, for example, within a cellular organelle. Generally, a sequence or template that can be used for recombination into a target locus that includes a target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence." In some embodiments, an exogenous template polynucleotide may be referred to as an editing template. In some embodiments, the recombination is homologous recombination.

[0070] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) causes cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 base pairs or more of the target sequence). A tracr sequence comprising or consisting of all or a portion of a wild-type tracr sequence (e.g., about or about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides) can also form part of a CRISPR complex by hybridizing along all or a portion of a tracr mate sequence operably linked to a guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to be able to hybridize and participate in the formation of a CRISPR complex, e.g., 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity over the entire length of the tracr mate sequence when optimally aligned.

[0071] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into cells, and expression of the elements of the CRISPR system can direct the formation of CRISPR complexes at one or more target sites. Components can be delivered to cells as proteins and / or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined into a single vector, and one or more additional vectors can provide any components of the CRISPR system not included in the first vector. A vector can contain one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, one expression construct can target CRISPR activity to multiple different corresponding target sequences within a cell.

[0072] One or more vectors driving the expression of one or more CRISPR system elements can be introduced into cells, such that expression of the CRISPR system elements directs the formation of CRISPR complexes at one or more target sites. Components can be delivered to cells as proteins and / or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined into a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. A vector can contain one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, the one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to direct CRISPR activity to multiple different corresponding target sequences within a cell.

[0073] The CRISPR enzyme may be Cas9 (e.g., derived from S. pyogenes or S. pneumoniae). CRISPR enzymes can direct cleavage of one or both strands at the location of a target sequence, e.g., within the target sequence and / or within the complementary strand of the target sequence. The vector can encode a CRISPR enzyme having a mutation compared to the corresponding wild-type enzyme, where the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of S. pyogenes Cas9 converts Cas9, a nuclease that cleaves both strands, into a nickase that cleaves a single strand. In some embodiments, Cas9 nickase may be used in combination with two guide sequences, e.g., targeting the sense and antisense strands of a DNA target, respectively. This combination results in cleavage of both strands and can be used to induce NHEJ or HDR.

[0074] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a specific cell, such as a eukaryotic cell. The eukaryotic cell may be derived from or derived from a specific organism, such as a mammal (but not limited to a human, mouse, rat, rabbit, dog, or non-human primate). Generally, codon optimization refers to the process of modifying a nucleic acid sequence by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell, while retaining the native amino acid sequence, to enhance expression in the target host cell. Different biological species exhibit unique biases toward specific codons for specific amino acids. Codon bias (differences in codon usage between biological species) is often correlated with the translation efficiency of messenger RNA (mRNA), which is further thought to depend on the nature of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of a specific tRNA in a cell generally reflects the codon most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a desired organism based on codon optimization.

[0075] Generally, guide sequence refers to any polynucleotide sequence that has sufficient complementarity with target polynucleotide sequence, and hybridizes with the target sequence to direct the sequence-specific binding of CRISPR complex to the target sequence.In some embodiments, the degree of complementarity between guide sequence and its corresponding target sequence is about or about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0076] Optimal alignment can be determined by any suitable algorithm used to align sequences, including, but not limited to, the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0077] CRISPR enzymes can be part of fusion proteins containing one or more foreign protein domains. CRISPR enzyme fusion proteins can contain any additional protein sequence and can optionally contain a linker sequence between any two domains. Examples of protein domains that can be fused to CRISPR enzymes include, but are not limited to, epitope tags, reporter gene sequences, and protein domains with one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional dissociation factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza virus hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, autofluorescent proteins including green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes can be fused to gene sequences encoding proteins or fragments thereof that bind to DNA molecules or other intracellular molecules. These include, but are not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can be part of fusion proteins containing CRISPR enzymes are described in U.S. Patent Publication No. US 20110059502, which is incorporated herein by reference.

[0078] In other embodiments, the activity of endogenous BTG1 protein is reduced in cells for cancer treatment or acute or chronic infection treatment. This may be in addition to or instead of reducing expression of the endogenous BTG1 gene. Protein activity can be reduced by any suitable means, such as one or more small molecules, one or more antibodies, or a combination thereof. Small molecules can be selected from libraries, for example, based on their ability to reduce activity in vitro, and then used with cells for cancer treatment. In some embodiments, one or more antibodies that bind to BTG1 can at least partially, or in some cases completely, inhibit its activity. The antibodies can be of any type, such as monoclonal or polyclonal. BTG1 antibodies are commercially available or can be produced using standard techniques.

[0079] Increased BTG1 Expression and / or Activity In certain embodiments, cells used in cell therapy may benefit from increased BTG1 expression and / or activity, or endogenous cells in a recipient individual may have increased BTG1 expression and / or activity. In certain embodiments, such cells are, for example, cells used to treat one or more autoimmune diseases. The cells may have increased expression and / or activity by any suitable means. In certain embodiments, the cells have increased expression of the BTG1 gene, increased activity of endogenous BTG1 protein, or both; in certain embodiments, the expression may be inducible to avoid potential toxicity. In cells with increased protein expression, the expression may be due to expression of an exogenous BTG1 gene on a vector. In some embodiments, the increased expression of the BTG1 gene is due to increased expression of the endogenous BTG1 gene in the cell, achieved, for example, by introducing a promoter into a regulatory region or other region positioned to control the endogenous BTG1 gene. For example, a constitutive promoter can be introduced into the regulatory region of the endogenous BTG1 gene, and examples of such promoters include the SV40, CMV, UBC, EF1A, and PGK promoters.

[0080] When exogenous BTG1 is introduced into cells via a vector, those skilled in the art have sufficient knowledge and skills to construct the vector using standard recombinant techniques, including, but not limited to, plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Vectors include retroviral vectors (e.g., those derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., those derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenoviral (Ad) vectors (including replication-competent, replication-defective, and "gutless" forms), adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesiculovirus vectors, Maraba virus vectors, and the like.

[0081] In certain embodiments, the vector is a multicistronic vector, in which case a single vector may encode the BTG1 gene and one or more exogenous proteins, such as one or more CARs and / or TCRs, suicide genes, one or more cytokines, etc.

[0082] Certain embodiments of the present disclosure may provide viral vectors encoding one or more gene products encompassed herein. In generating recombinant viral vectors, non-essential genes are typically replaced with genes or coding sequences for foreign (or non-native) proteins. Viral vectors are a type of expression construct that utilizes viral sequences to introduce nucleic acids and, in some cases, proteins into cells. The ability of certain viruses to infect cells, enter cells via receptor-mediated endocytosis, and integrate into the host cell genome to stably and efficiently express viral genes makes them promising candidates for introducing foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that may be used to deliver nucleic acids in certain embodiments of the present invention include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors.

[0083] [V. Foreign Proteins] In certain embodiments, any cell of the present disclosure is modified to express one or more exogenous proteins. In some embodiments, the exogenous proteins can promote the activity of the cell in any way, including at least cell activation, persistence, proliferation, homing, and / or cytotoxicity. The modulation of the cell to include one or more exogenous proteins can occur before, during, or after modulation of BTG1 expression and / or activity. In some embodiments, the one or more exogenous proteins can be introduced on the same or different vector as one or more factors that cause a decrease in BTG1 expression or activity or one or more factors that cause an increase in BTG1 expression or activity.

[0084] A. Bispecific or Multispecific Antibodies In some embodiments, the cells are engineered to express one or more bispecific or multispecific antibodies, while in other embodiments, the cells do not express antibodies, but antibodies are used in conjunction with the cells.

[0085] If the cells are engineered to express an antibody, the antibody may serve as an engager, bridging specific immune effector cells with specific target cells and causing their destruction. In certain embodiments, the engineered cells are used in conjunction with standard T cell engagers (BiTEs). This is because, in certain embodiments, the cells are engineered to express CD3, which is often the T cell antigen to which BiTE engagers bind. In such cases, the BiTE may target a cancer antigen or a viral antigen, which can be tailored to the recipient individual's disease state. For example, the BiTE may be engineered to bind to a cancer antigen characteristic of the individual's cancer cells.

[0086] In some embodiments, the cells may be NK cells engineered to express one or more bispecific NK engagers (BiKEs), or may be used in combination with the cells without expressing the BiKEs. The BiKEs may contain antibodies that bind to surface proteins on NK cells (including surface proteins naturally expressed on NK cells) and may also contain antibodies that bind to a desired target antigen. The BiKEs may target NK cells via antibodies against NK surface proteins, such as CD16, CS1, CD56, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR, or KIR. In such cases, the BiKEs used in the present disclosure may also target cancer antigens or viral antigens, which can be tailored to the recipient's condition. For example, the BiKEs may be engineered to bind to cancer antigens characteristic of cancer cells of the individual's cancer.

[0087] B. Modified Receptors In certain embodiments, the cells are engineered to express one or more engineered receptors. In some embodiments, the engineered receptors may be engineered antigen receptors that target any type of cancer or viral antigen. The receptors may be tailored to target desired antigens based on the cells associated with the recipient individual's disease state.

[0088] <1. Chimeric Antigen Receptor> In some embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR). The cells may be engineered to encode at least one CAR, which may be a first-generation, second-generation, or third- or later-generation CAR. A CAR may or may not be bispecific for two or more different antigens. A CAR may include one or more costimulatory domains. Each costimulatory domain may include, for example, one or more costimulatory domains of a member of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD27, NKG2D, 2B4M, CD40, ICOS, TLR, MYD88, 2B4, or a combination thereof. In certain embodiments, the CAR lacks one or more specific costimulatory domains, for example, the CAR may lack 4-1BB and / or may lack CD28.

[0089] In certain embodiments, the intracellular CAR polypeptide comprises an extracellular spacer domain, which may also be referred to as a "hinge," that links the antigen-binding domain and the transmembrane domain. The extracellular spacer domain may include, but is not limited to, an Fc fragment of an antibody or a fragment or derivative thereof, a hinge region of an antibody or a fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, or a combination thereof. Examples of extracellular spacer domains include, but are not limited to, an artificial spacer composed of a polypeptide such as a CD8-alpha hinge, CD28, Gly3, or the CH1 and CH3 domains of an IgG (e.g., human IgG1 or IgG4). In particular examples, the extracellular spacer domain can comprise (i) the hinge, CH2, and CH3 regions of IgG4, (ii) the hinge region of IgG4, (iii) the hinge and CH2 of IgG4, (iv) the hinge region of CD8-alpha or CD4, (v) the hinge, CH2, and CH3 regions of IgG1, (vi) the hinge region of IgG1, or (vii) the hinge and CH2 of IgG1, (vii) the hinge region of CD28, or a combination thereof. In certain embodiments, the hinge is derived from IgG1, and in particular aspects, the CAR polypeptide comprises a particular IgG1 hinge amino acid sequence or is encoded by a particular IgG1 hinge nucleic acid sequence.

[0090] The transmembrane domain in the CAR may be of natural or synthetic origin. If natural, in some embodiments, the domain is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., including at least the transmembrane region of) the α, β, or ζ chain of the T cell receptor, CD28, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules (e.g., DAP10 or DAP12). Alternatively, in some embodiments, the transmembrane domain may be synthetic. In some embodiments, the synthetic transmembrane domain may be composed primarily of hydrophobic residues such as leucine and valine. In some embodiments, a three-residue motif of phenylalanine, tryptophan, and valine may be present at each end of the synthetic transmembrane domain.

[0091] In certain embodiments, the CAR comprises one or more activation domains, such as CD3ζ, DAP12, 2B4, or a combination thereof.

[0092] In some embodiments, the modified receptor utilizes one or more homing receptors (e.g., those that can home to a target without necessarily emitting a signal, such as adhesion molecules) and / or one or more chemokine receptors. Examples of chemokine receptors include CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors. In certain examples, the chemokine receptor is a receptor for CCR2, CCR3, CCR5, CCR8, CCR7, CXCR3, L-selectin (CD62L), CXCR1, CXCR2, or CX3CR1.

[0093] 2. T cell receptor (TCR) In some embodiments, the modified antigen receptor comprises a recombinant TCR and / or a TCR cloned from a naturally occurring T cell. "T cell receptor" or "TCR" refers to a molecule that comprises variable alpha and beta chains (also referred to as TCRα and TCRβ, respectively) or variable gamma and delta chains (also referred to as TCRγ and TCRδ, respectively) and is capable of specifically binding to an antigenic peptide bound to an MHC receptor. In some embodiments, the TCR is of the alpha / beta type.

[0094] TCRs, which typically exist in α / β and γ / δ forms, are generally structurally similar, although the T cells that express them may differ in anatomical location or function. TCRs can exist on the cell surface or in soluble form. Generally, TCRs are present on the surface of T cells (or T lymphocytes), where they typically recognize antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, TCRs may contain a constant domain, a transmembrane domain, and / or a short cytoplasmic domain. For example, in some embodiments, each chain of a TCR may have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a C-terminal short cytoplasmic domain. In some embodiments, TCRs are associated with invariant proteins of the CD3 complex, which are involved in mediating signal transduction. Unless otherwise specified, the term "TCR" is understood to encompass functional TCR fragments thereof. The term also encompasses full-length or full-length TCRs, including α / β or γ / δ TCRs.

[0095] Thus, as used herein, reference to a TCR includes any TCR or functional fragment thereof, such as the antigen-binding site of a TCR that binds to a specific antigenic peptide bound to an MHC molecule, i.e., an MHC-peptide complex. An "antigen-binding site" or "antigen-binding fragment" (which may be used interchangeably) refers to a molecule that comprises a portion of the structural domain of a TCR and binds to an antigen (e.g., an MHC-peptide complex) bound by the intact TCR. In some instances, the antigen-binding site comprises the variable domains of the TCR, such as the variable α and β chains of the TCR, which are sufficient to form a binding site for binding to a specific MHC-peptide complex. For example, each chain typically comprises three complementarity-determining regions.

[0096] In some embodiments, the variable domains of the TCR chains associate to form immunoglobulin-like loops, or complementarity-determining regions (CDRs), which confer antigen recognition and form the binding site of the TCR molecule, thereby determining peptide specificity. Generally, as in immunoglobulins, the CDRs are separated by framework regions (FRs). In some embodiments, CDR3 is the CDR primarily responsible for recognizing processed antigens, while CDR1 of the α chain has been shown to interact with the N-terminal portion of antigenic peptides, and CDR1 of the β chain has been shown to interact with the C-terminal portion of peptides. CDR2 is thought to recognize MHC molecules. In some embodiments, the variable region of the β chain may further comprise a hypervariable region (HV4).

[0097] In some embodiments, a TCR chain comprises a constant domain. For example, similar to an immunoglobulin, the extracellular portion of a TCR chain (e.g., an α chain, a β chain) comprises two immunoglobulin domains: an N-terminal variable domain (e.g., Vα or Vβ; typically amino acids 1-116 according to Kabat numbering, see Kabat et al., "Sequences of Proteins of Immunological Interest," U.S. Dept. Health and Human Services, Public Health Service National Institutes of Health); Health, 1991, 5th ed.), and includes one constant domain adjacent to the cell membrane (e.g., an α chain constant domain or Cα, typically amino acids 117-259 according to Kabat; a β chain constant domain or Cβ, typically amino acids 117-295 according to Kabat). For example, in some instances, the extracellular portion of a two-chain TCR includes two constant domains near the cell membrane and two variable domains comprising the CDRs distal to the cell membrane. The constant domain of the TCR includes a short linking sequence in which cysteine ​​residues form disulfide bonds to link the two chains. In some embodiments, the TCR may have additional cysteine ​​residues in each of the α and β chains, such that the TCR includes two disulfide bonds in the constant domains.

[0098] In some embodiments, the TCR chain can include a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some instances, the TCR chain includes a cytoplasmic domain. In some instances, this structure allows the TCR to associate with other molecules, such as CD3. For example, a TCR that includes a constant domain with a transmembrane region can anchor the protein to the cell membrane and associate with the invariant subunit of the CD3 signaling apparatus or complex.

[0099] Generally, CD3 is a multiprotein complex, and in mammals, it can have three distinct chains (γ, δ, and ε) and a ζ chain. For example, in mammals, the complex can contain a homodimer of CD3γ, CD3δ, two CD3ε, and a CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are cell surface proteins belonging to the immunoglobulin superfamily, each containing one immunoglobulin domain, and are highly homologous. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a feature that allows them to associate with positively charged T cell receptor chains. The intracellular domains of the CD3γ, CD3δ, and CD3ε chains each contain one conserved motif, an immunoreceptor tyrosine-based activation motif (ITAM), while each CD3ζ chain has three ITAMs. Generally, ITAMs are involved in the signaling activity of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in propagating signaling from the TCR into the cell. The CD3 chain and the ζ chain, together with the TCR, form a structure known as the T cell receptor complex.

[0100] In some embodiments, the TCR may be a heterodimer of two chains, i.e., α and β (or optionally γ and δ), or may be a single-chain TCR construct. In some embodiments, the TCR is a heterodimer consisting of two independent chains (α and β or γ and δ), which may be linked by a disulfide bond or the like. In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into a cell. In some embodiments, nucleic acid encoding the TCR may be obtained from various sources, for example, by polymerase chain reaction (PCR) amplification of a known TCR DNA sequence. In some embodiments, the TCR is obtained from a biological source, for example, a cell such as a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other known source. In some embodiments, the T cell may be an in vivo isolated cell. In some embodiments, a high-affinity T cell clone is isolated from a patient and the TCR is isolated. In some embodiments, the T cell may be a cultured T cell hybridoma or clone. In some embodiments, TCR clones against target antigens are generated in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system, or HLA). For tumor antigens, see, e.g., Parkhurst et al., 2009 and Cohen et al., 2005. In some embodiments, phage display is used to isolate TCRs against target antigens (see, e.g., Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, the TCR or antigen-binding portion thereof can be synthetically generated based on sequence information of the TCR.

[0101] (C. Cytokines) In some embodiments, the cells are modified to express one or more exogenous cytokines or to upregulate the normal expression of one or more exogenous cytokines, which may or may not be transduced or transfected with the one or more cytokines on the same vector as other exogenous genes (e.g., CARs).

[0102] One or more cytokines can be coexpressed from the vector as separate polypeptides from any component of the exogenous gene product. For example, interleukin-15 (IL-15) is tissue-specific and is only observed at certain levels in serum or systemically under pathological conditions. IL-15 has several properties that make it suitable for adoptive immunotherapy. IL-15 is a homeostatic cytokine that induces the development and proliferation of innate immune cells, promotes the elimination of established tumors by alleviating the functional suppression of tumor-intrinsic cells, and suppresses activation-induced cell death (AICD). In addition to IL-15, other cytokines are also contemplated. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells used in human applications. IL-15-expressing cells have the ability to provide sustained supportive cytokine signaling, which is beneficial for cell survival after infusion.

[0103] In certain embodiments, the cells express one or more exogenously supplied cytokines. In one example, the cytokines are IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, GMCSF, or a combination thereof. Cytokines can be exogenously supplied to NK cells by expression from an expression vector within the cells. In another example, endogenous cytokines within the cells are upregulated by manipulating the expression control of the cytokine, e.g., by genetic modification of the promoter site. When a cytokine is supplied to the cells on an expression construct, the cytokine may be encoded from the same vector as one or more components of the CD3 complex, with or without the TCR complex.

[0104] (D. Antigen) The modified antigen receptors and antibodies encompassed by the present disclosure can target one or more specific antigens. Antigens targeted by antibodies and / or modified antigen receptors include those expressed in the context of the disease, condition, or cell type targeted through adoptive cell therapy. Diseases and conditions include proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, hematological cancers, and cancers of the immune system (e.g., lymphomas, leukemias, and / or myelomas, e.g., B, T, and myeloid leukemias, lymphomas, and multiple myeloma). In some embodiments, the antigen is selectively expressed or overexpressed in cells of the disease or condition, e.g., tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells or on modified cells.

[0105] In this method, any suitable antigen can be targeted.In some cases, the antigen may be associated with cancer cells, but not associated with non-cancer cells.Representative antigens include, but are not limited to, antigen molecules from infectious agents, self / autoantigens, tumor / cancer-associated antigens, and tumor neoantigens.In certain embodiments, the antigen targeted by modified receptor is GD2, CD19, GPC3, and / or B7-H3.

[0106] In certain embodiments, the antigen is selected from the group consisting of NY-ESO, CD19, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, CD22, CD70, CD38, CD123, CLL1, carcinoembryonic antigen, alpha fetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, CD 33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutant p53, Ras, mutant Ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf, C-Raf, cyclin-dependent kinase), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, Gp100, PSA, PSM, tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphatidylinositol 3-kinases (PI3Ks), TRK receptors, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signaling transfer factor 1 (TACSTD1), TACSTD2, receptor tyrosine kinase (e.g., epidermal growth factor receptor (EGFR) (especially EGFRvIII),Platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), VEGFR2, cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), transcription factors and activators (STAT3, STAT5, STAT6), hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor-κB (NF-B), Notch receptors (e.g., Notch 1-4), NY ESO1, c-Met, mammalian target of rapamycin (mTOR), WNT extracellular signal-regulated kinases (ERKs) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation fragment, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2) ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysaccharide sialic acid, MYCN, RhoC, GD3, fucosyl-GM1, mesothelin, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, phos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1.

[0107] Tumor-associated antigens may be derived from cancers such as prostate, breast, colon, lung, pancreas, kidney, mesothelioma, ovary, liver, brain, bone, stomach, spleen, testis, cervix, anus, gallbladder, thyroid, or melanoma. Representative tumor-associated antigens or antigens derived from tumor cells include MAGE1, 3, and MAGE4 (or other MAGE antigens described in International Patent Publication WO99 / 40188), PRAME, BAGE, RAGE, Lage (also known as NY ESO1), SAGE, HAGE, or GAGE. Non-limiting examples of these tumor antigens are expressed in a wide range of tumor types, including melanoma, lung cancer, sarcoma, and bladder cancer. See, e.g., U.S. Patent No. 6,544,518. Prostate cancer tumor-associated antigens include, e.g., prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphatase, NKX3.1, and six-transmembrane epithelial antigen of the prostate (STEAP).

[0108] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. Additionally, tumor antigens can be self-peptide hormones, for example, 10-amino acid-long peptides such as full-length gonadotropin-releasing hormone (GnRH) that are useful in the treatment of various cancers.

[0109] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. Additionally, tumor antigens can be self-peptide hormones, for example, 10-amino acid-long peptides such as full-length gonadotropin-releasing hormone (GnRH) that are useful in the treatment of various cancers.

[0110] Antigens include epitope regions or peptides derived from genes mutated in tumor cells or transcribed at different levels in tumor cells compared with normal cells. Examples include telomerase enzyme, survivin, mesothelin, mutated Ras, BCR / ABL rearrangements, Her2 / neu, mutant or wild-type p53, cytochrome P4501B1, and aberrantly expressed intronic sequences (e.g., N-acetylglucosaminyltransferase-V). Clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myeloma and B-cell lymphoma; tumor antigens containing epitope regions or peptides derived from oncoviral processes (e.g., human papillomavirus proteins E6 and E7, Epstein-Barr virus protein LMP2); and tumor-selectively expressed non-mutated oncofetal proteins (e.g., carcinoembryonic antigen and alpha-fetoprotein).

[0111] (E. Suicide gene) In certain embodiments, suicide genes are used in conjunction with cell therapy to control its use and allow for termination of cell therapy at a predetermined event and / or time. Suicide genes are used in cells into which they are introduced, as needed, to cause the death of the cell. Cells of the present disclosure that have been modified to contain one or more vectors encompassed by the disclosure may contain one or more suicide genes. In some embodiments, the term "suicide gene," as used herein, is defined as a gene whose gene product is converted into a compound that causes the host cell to die upon administration of a prodrug or other drug. In other embodiments, the suicide gene encodes a gene product that, when desired, is targeted by a drug (e.g., an antibody) that targets the suicide gene product.

[0112] In some instances, if an individual undergoing and / or receiving cell therapy exhibits one or more symptoms of one or more adverse events, such as cytokine release syndrome, neurotoxicity, anaphylaxis / allergy, and / or on-target / off-target toxicity (among other examples), or is deemed to be at risk for imminent development of such symptoms, the cell therapy may be subject to the use of one or more suicide genes of any type. The use of a suicide gene may be part of a planned protocol of treatment, or may be used only when the need for its use is recognized. In some instances, cell therapy is terminated by the use of an agent targeting the suicide gene or its gene product because treatment is no longer required.

[0113] The use of suicide genes can be initiated when an individual experiences one or more adverse events, which can be recognized by any means, including routine monitoring, which may or may not be continuous from the start of cell therapy. Adverse events can be detected by physical examination and / or testing. If an individual has cytokine release syndrome (also known as cytokine storm), they may exhibit, for example, elevated levels of inflammatory cytokines (by way of example only: interferon-γ, granulocyte-macrophage colony-stimulating factor, IL-10, IL-6, TNF-α), fever, fatigue, hypotension, hypoxia, tachycardia, nausea, capillary leak, cardiac, renal, or hepatic dysfunction, or a combination thereof. If an individual experiences neurotoxicity, they may exhibit confusion, delirium, aplasia, and / or convulsions. In some instances, tests are performed for markers associated with the onset and / or severity of cytokine release syndrome, such as C-reactive protein, IL-6, TNF-α, and / or ferritin.

[0114] Examples of suicide gene / prodrug combinations that can be used include herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase-thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. Another example is the so-called suicide gene Escherichia coli purine nucleoside phosphorylase, which converts the prodrug 6-methylpurine deoxyriboside into the toxic purine 6-methylpurine. Other suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome P450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-α,γ-lyase (MET), EGFRv3, and thymidine phosphorylase (TP).

[0115] [VI. Infectious diseases] In some embodiments, cell therapy cells are engineered to contain reduced expression of BTG1, and these cells are then used to treat one or more chronic or acute infectious diseases. The type of infectious disease may be caused by bacteria, such as tuberculosis; viruses, such as human immunodeficiency virus (HIV), viral hepatitis, human papillomavirus (HPV), or herpes simplex virus (HSV); fungi; or parasites. In some embodiments, chronic diseases with infectious origins are also included, such as cervical cancer (human papillomavirus: HPV) and liver cancer (hepatitis B and C viruses). In one embodiment, the infectious disease is COVID-19.

[0116] In certain embodiments, an individual with an acute or chronic infection is administered a therapeutically effective amount of cells, e.g., specific immune cells, in which BTG1 is inducible and / or whose activity is enhanced by the administration of a drug. In some embodiments, the cells are also modified to express one or more exogenous proteins, such as modified antigen receptors.

[0117] [VII. Autoimmune diseases] In some embodiments, there are individuals in need of treatment for an autoimmune disease, e.g., individuals who would benefit from cells with enhanced BTG1 expression and / or activity. In some examples, modified cells that have increased BTG1 expression and / or activity relative to cells not modified in a similar manner are administered in a therapeutically effective amount to an individual with an autoimmune disease (including an individual with one or more symptoms of an autoimmune disease). In certain embodiments, BTG1 expression can be induced for reasons such as to prevent toxicity. In other examples, BTG1 expression and / or activity is increased in an individual with an autoimmune disease, e.g., by the use of a drug.

[0118] Non-limiting examples of autoimmune diseases include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac sprite dermatitis, chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus erythematosus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, tonsillitis, and rheumatoid arthritis. Lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, nephrotic syndrome (including minimal change disease, focal glomerular sclerosis, and membranous nephropathy), pemphigus, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cholangitis, psoriasis, and psoriasis. Examples of autoimmune diseases treatable by the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, systemic sclerosis, Sjögren's syndrome, stiff-man syndrome, systemic lupus erythematosus, ulcerative colitis, uveitis, vasculitis (such as polyarteritis nodosa, Takayasu's arteritis, temporal arteritis / giant cell arteritis, or cutaneous herpetiform vasculitis), vitiligo, and Wegener's granulomatosis. Accordingly, examples of autoimmune diseases treatable by the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, Crohn's disease, ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis.

[0119] VIII. Administration of Therapeutic Compositions In various embodiments, the BTG1-modified cells are administered to an individual in need thereof. Embodiments of the present disclosure relate to methods of using compositions comprising the BTG1-modified cells provided herein to treat or prevent a medical disease or disorder, including cancer, acute or chronic infection, or autoimmune disease. The methods include administering a therapeutically effective amount of the cells to a subject, thereby treating or preventing the disease in the subject, including reducing the risk, severity, and / or delaying the onset of the disease. In certain embodiments, the present disclosure provides for treating cancer or an infection or disease by transferring a composition comprising the cell population.

[0120] Cancers for which embodiments of the present therapeutic methods are useful include any malignant cell type, including those found in solid tumors or hematological tumors. Exemplary solid tumors include, but are not limited to, tumors of organs selected from the pancreas, colon, appendix, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include tumors of the bone marrow, T-cell or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, and the like. Additionally, examples of cancers that may be treated by the methods described herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric cancer or tumors (including gastrointestinal cancer and gastrointestinal stromal tumors), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0121] Cancers for which embodiments of the present methods of treatment are useful may be of the following exemplary histological classifications, including, but not limited to, malignant neoplasms, carcinoma, undifferentiated carcinoma, giant cell and spindle cell carcinoma, small cell carcinoma, papillary carcinoma, squamous cell carcinoma, lymphoepithelial carcinoma, basal cell carcinoma, pilomatrix carcinoma, transitional cell carcinoma, papillary transitional cell carcinoma, adenocarcinoma, malignant gastrinoma, cholangiocarcinoma, hepatocellular carcinoma, mixed hepatocellular and cholangiocarcinoma, trabecular adenocarcinoma, adenoid cystic carcinoma, adenomatous intrapolypoid adenocarcinoma, familial polyposis coli adenocarcinoma, solid carcinoma, malignant carcinoid tumor, bronchioloalveolar adenocarcinoma, papillary adenocarcinoma, chromophobe carcinoma, and pulmonary carcinoma. Acidic carcinoma, oxyphil adenocarcinoma, basophilic carcinoma, clear cell adenocarcinoma, granular cell carcinoma, follicular adenocarcinoma, papillary follicular adenocarcinoma, non-encapsulated sclerosing carcinoma, adrenocortical carcinoma, endometrial carcinoma, skin adnexal carcinoma, apocrine gland carcinoma, sebaceous gland carcinoma, ceruminous gland adenocarcinoma, mucoepidermoid carcinoma, cystadenocarcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, invasive ductal carcinoma, medullary carcinoma, lobular carcinoma, inflammatory carcinoma, Paget's disease of the breast, acinar cell carcinoma, adenosquamous carcinoma, adenocarcinoma with squamous metaplasia, malignant thymoma, malignant ovarian stromal tumor, malignant secoma, malignant granulosa cell tumor, malignant androblastoma, Sertoli cell carcinoma, malignant Leidy's disease Histiocytoma, malignant lipocytoma, malignant paraganglioma, malignant extramammary paraganglioma, pheochromocytoma, glomus angiosarcoma, malignant melanoma, non-melanotic melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentigo melanoma, nodular melanoma, malignant melanoma in giant pigmented nevus, epithelioid cell melanoma, malignant blue nevus, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, malignant mixed tumor, mixed Müllerian tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, malignant mesenchymoma, malignant Brenner tumor, malignant phyllodes tumor, synovial sarcoma, malignant mesothelioma, ovarian dysgerminoma, embryonal carcinoma, malignant teratoma, malignant struma ovari, choriocarcinoma, malignant mesonephroma, angiosarcoma, malignant hemangioendothelioma, Kaposi's sarcoma, malignant hemangiopericytoma, lymphangiosarcoma, osteosarcoma, subcortical osteosarcoma, chondrosarcoma, malignant chondroblastoma, mesenchymal chondrosarcoma, giant cell tumor of bone, Ewing's sarcoma, malignant odontogenic tumor, blastic odontogenic sarcoma, malignant ameloblastoma, blastic fibrosarcoma, malignant pinealoma, chordoma, malignant glioma, ependymoma, astroglioma, protoplasmic astroglioma, fibrous astroglioma, astroblastoma, glioblastoma, oligodendroglioma, oligodendroglioma, undifferentiated neuroectodermal tumor, cerebellar sarcoma,Ganglioneuroblastoma, neuroblastoma, retinoblastoma, olfactory nerve tumor, malignant meningioma, neurofibrosarcoma, malignant neurilemmoma, malignant granular cell tumor, malignant lymphoma, Hodgkin's disease, Hodgkin's tumor, paragranuloma, small lymphocytic lymphoma, diffuse large cell lymphoma, follicular lymphoma, mycosis fungoides, other specified non-Hodgkin's lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small undifferentiated These include cell-type NHL, bulky mass-type NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, malignant histiocytosis, multiple myeloma, mast cell sarcoma, small intestinal immunoproliferative disorders, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myeloid leukemia.

[0122] Therapeutic methods disclosed herein may include the administration of a combination of multiple therapeutic agents, such as a first cancer treatment and a second cancer treatment. These treatments may be administered by any suitable method known to those skilled in the art. For example, the first and second cancer treatments may be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first and second cancer treatments are administered as separate compositions. In some embodiments, the first and second cancer treatments are contained in the same composition. Embodiments of the present disclosure relate to compositions and methods including therapeutic compositions. Different treatments may be administered in one composition or multiple compositions, such as two, three, or four compositions. Combinations of various therapeutic agents may be used. Examples of treatments other than those disclosed herein include surgery, chemotherapy, drug therapy, radiation therapy, hormone therapy, immunotherapy (other than those disclosed herein), or combinations thereof.

[0123] The therapeutic agents of the present disclosure may be administered by the same or different routes of administration. In some embodiments, the cancer treatment is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implant, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implant, by inhalation, intrathecally, intracerebroventricularly, or intranasally. The appropriate dosage can be determined based on the type, severity, and course of the disease being treated, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician.

[0124] The amount administered, both in terms of number of treatments and unit doses, depends on the desired therapeutic effect. An effective dose is understood to mean the amount necessary to achieve a particular effect. A treatment may include various "unit doses." A unit dose is defined as containing a predetermined amount of a therapeutic composition. The amount to be administered, the specific route of administration, and the formulation are within the determinability of those skilled in the art of clinical technology. A unit dose need not be administered as a single dose, but may include continuous infusion over a predetermined period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0125] A therapeutically effective dose of immune cells can be administered by multiple routes of administration, including parenteral administration, such as intravenous, intraperitoneal, intramuscular, intrasternal, or intraarticular injection or infusion.

[0126] An effective dose of immune cells used in adoptive immunotherapy refers to an amount that achieves a desired effect in the treated subject. In embodiments in which the subject has cancer or an acute or chronic infection, the effective dose may be the amount of modified cells necessary to inhibit progression of the disease or ameliorate one or more symptoms, including causing regression of one or more tumors. In embodiments in which the subject has an autoimmune disease, the effective dose may be the amount of modified cells necessary to inhibit progression or cause regression of the autoimmune disease, or an amount capable of alleviating one or more symptoms resulting from the autoimmune disease, such as pain and / or inflammation. It may also be the amount necessary to alleviate symptoms associated with inflammation, such as pain, edema, and elevated body temperature. It may also be the amount necessary to reduce or prevent rejection of a transplanted organ.

[0127] The BTG1-modified cell population can be administered in a treatment regimen appropriate for the disease. For example, it can be administered once or several times over one to several days to ameliorate the disease state, or it can be administered periodically over a long period of time to inhibit disease progression and prevent recurrence. The precise dose employed in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and each subject's circumstances. The number of cells in an effective dose can depend on the subject being treated, the severity and type of the disease, and the method of administration. In some embodiments, a dose that can be used to treat a human subject is at least 1 x 10 4 , at least 1 x 10 5 , at least 1 x 10 6 , at least 1 x 10 7 , at least 1 x 10 8 , at least 1 x 10 9 , or at least 1 × 10 10 cells / m 2 The dose ranges from 1 × 10 4 ~1×10 10 , 1×10 5 ~1×10 10 , 1×10 6 ~1×10 10 , 1×10 7 ~1×1010 , 1×10 8 ~1×10 10 , 1×10 9 ~1×10 10 The exact amount of immune cells can be easily determined by one skilled in the art based on the age, weight, sex, and physiological condition of the subject. Effective doses can be extrapolated based on dose-response curves derived from in vitro or animal model test systems.

[0128] The cells may be administered in combination with one or more other therapeutic agents for the treatment of any disease. Combination therapy will depend on the type of affliction and may include, but is not limited to, one or more antimicrobial agents (e.g., antibiotics, antivirals, and antifungals), antitumor agents (e.g., fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immunodepleting agents (e.g., fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressants (e.g., azathioprine, or glucocorticoids such as dexamethasone or prednisone), anti-inflammatory agents (e.g., glucocorticoids such as hydrocortisone, dexamethasone, or prednisone, or nonsteroidal anti-inflammatory agents such as acetylsalicylic acid, ibuprofen, or naproxen sodium), cytokines (e.g., interleukin-10 or transforming growth factor-β), hormones (e.g., estrogen), or vaccines. Additionally, immunosuppressive or immune tolerance-inducing agents may be administered, such as calcineurin inhibitors (e.g., cyclosporine and tacrolimus), mTOR inhibitors (e.g., rapamycin), mycophenolate mofetil, antibodies (e.g., antibodies recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or B cells), chemotherapeutic agents (e.g., methotrexate, treosulfan, busulfan), radiation, or chemokines, interleukins, or inhibitors thereof (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors). Such additional agents may be administered before, during, or after the administration of immune cells, depending on the desired effect. These cells and agents may be administered via the same route or different routes, and at the same or different sites.

[0129] In certain embodiments, the compositions and methods of the present invention comprise a combination of immune cell populations and at least one additional treatment.The additional treatment may be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the above treatments.The additional treatment may be in the form of adjuvant therapy or neoadjuvant therapy.

[0130] In certain embodiments, any of the cells disclosed herein may be obtained from an appropriate archival source prior to modification. The stored cells may or may not already have modified BTG1 expression and / or activity. The stored cells may or may not already have expression of one or more exogenous genes. In some embodiments, any of the cells disclosed herein are obtained from a subject in need of treatment, modified ex vivo for BTG1 expression and / or activity, and optionally further modified to express one or more exogenous genes, and then readministered to the subject. In such cases, the step of modifying the cells for BTG1 may or may not precede the modification of the cells for one or more exogenous genes.

[0131] [IX. Kit] Certain aspects of the present disclosure also relate to kits containing compositions of the present disclosure or compositions for practicing methods of the present disclosure. In certain embodiments, the kits include intact or cryopreserved cells, which may or may not have been previously activated or expanded. The cells may or may not already express one or more of the components of the elements encompassed herein, including, for example, reagents used to reduce BTG1 expression or activity, reagents used to increase BTG1 expression or activity, cells, vectors, buffers, primers, enzymes, salts, etc. The kits may also include one or more reagents for transfection or transduction of cells, which may include vectors expressing the components, primers for amplifying the components, etc. In some cases, the cells may or may not already express one or more exogenous proteins as defined herein; if not expressing, the kits may include vectors expressing the exogenous proteins, primers for amplifying the exogenous proteins, etc.

[0132] The kits may contain components individually packaged or disposed in tubes, bottles, vials, syringes, or other suitable container means. Individual components may be provided in the kits in concentrated amounts, and in some embodiments, components are provided individually at the same concentration as they would be in solution with the other components. Concentrations of components may be provided, for example, at 1x, 2x, 5x, 10x, or 20x or greater. [Example]

[0133] The following examples are included to demonstrate preferred embodiments of the invention. Those of skill in the art should understand that the techniques disclosed in the examples which follow are techniques discovered by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes for carrying out the invention. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made to the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0134] Example 1: Identification of B-cell translocation gene 1 (BTG1) as a major driver of hyporesponsiveness in exhausted NKT cells and T cells and its application in cancer immunotherapy. T cell exhaustion is an active process characterized by the progressive loss of effector function and proliferative capacity due to persistent antigen stimulation, which occurs in acute or chronic infections and cancer. Therapeutic immune cells, such as T cells or NKT cells, genetically engineered to express tumor-specific chimeric antigen receptors (CARs), also undergo exhaustion, which limits their antitumor activity and is associated with tumor immune escape and disease progression or recurrence. Specific gene expression and epigenetic changes have been implicated in the T cell exhaustion process, but the precise mechanisms underlying the hyporesponsiveness of exhausted cells remain poorly understood. To gain a deeper understanding of the mechanisms of exhaustion in clinically relevant effector cells, single-cell RNA sequencing (scRNAseq) of CAR-NKT cells obtained from 12 neuroblastoma (NB) patients was performed during a phase I clinical trial (NCT03294954). Changes in CAR-NKT gene expression were assessed after patient administration or multiple in vitro tumor cell challenges (repeated tumor challenge assay, or RTC assay). CAR-NKT after five RTCs and peripheral blood CAR-NKT after two weeks of treatment showed similar directional changes in gene expression profiles compared to treated product CAR-NKT, exemplified by the acquisition of terminal effector differentiation and loss of naive / memory programs. These findings support the five RTC (repeated tumor challenge assay) as a model that closely recapitulates the gene expression changes that occur in treated CAR-NKT in patients. Comparison of the gene expression profiles of treated product and five RTC-treated CAR-NKT revealed significant discrepancies between the two groups, identifying 2,904 differentially expressed genes.

[0135] One notable gene that was significantly upregulated in RTC-treated CAR-NKT cells was B-cell translocation gene 1 (BTG1), which promotes mRNA deadenylation and degradation and has recently been reported as a mediator of quiescence in mouse naive T cells. Its role in T cell exhaustion has not previously been described. Consistent with previous reports that BTG1 expression is rapidly downregulated in mouse naive T cells in response to TCR stimulation, BTG1 expression was downregulated during the first 4–6 h after TCR stimulation compared to unstimulated human NKT cells or naive T cells. However, sustained TCR stimulation with a CD3 / CD28-specific monoclonal antibody resulted in sustained upregulation of BTG1 expression in both NKT cells and T cells, as measured at the protein level by Western blot. Overexpression of BTG1 in NKT cells induced a limited number of differentially expressed genes but resulted in an overall decrease in cellular RNA content and reduced proliferative capacity. These differentially expressed genes do not overlap with known regulators of T cell exhaustion (i.e., TOX), suggesting a unique mechanism of hyporesponsiveness mediated by BTG1 in NKT cells and T cells in some embodiments. NKT cells engineered to co-express a tumor-specific CAR and a BTG1-specific shRNA eradicated metastatic NB in ​​mice. Thus, this study uncovered the unexpected role of BTG1 as a critical regulator of NKT cell and T cell function. In certain embodiments, exhausted T cells and NKT cells, like naive T cells, upregulate BTG1 to induce global mRNA degradation, thereby contributing to a quiescent or hyporesponsive state common to at least naive and exhausted T cells or NKT cells. Finally, suppressing BTG1 expression significantly enhances the antitumor activity of CAR-redirected NKT cells, useful, for example, for guiding the rational design of next-generation cancer immunotherapy products.

[0136] Example 2: Role of BTG1 in hyporesponsiveness of exhausted NKT cells and T cells and its application to cancer immunotherapy. This example provides a demonstration of the relevance of BTG1 to exhausted immune cells and its resulting role in adoptive cell therapy.

[0137] Figure 1A shows an example tumor co-culture system in which CAR-NKT cells are re-seeded with fresh CHLA255 neuroblastoma (NB) tumor cells every 5 days, repeatedly repeating this process to induce exhaustion of CAR-NKT cells through chronic antigen exposure. As shown in Figure 1B, the cytotoxic activity of CAR-NKT cells at a given time point during repeated co-culture demonstrates a decline in cytotoxicity over time in many patients. Figure 1C shows uniform manifold approximation and projection (UMAP) projections of scRNA-seq results for the administered product (IP), the administered product co-cultured five times with tumor cells (5RcC), and CAR-NKT cells isolated from peripheral blood (PB) after administration. Figure 1D shows UMAP projections of CAR-NKT gene expression by scRNA-seq obtained from pre-administration and post-5RcC samples. Figure IE shows a volcano plot showing differentially expressed genes (including BTG1) in CAR-NKT after 5RcC compared to pre-treatment. BTG1 expression is elevated in exhausted CAR-NKT.

[0138] Figure 2A shows one embodiment of the design of a retroviral construct encoding BTG1.GFP or GFP alone as a control to evaluate the effect of BTG1 overexpression (OE) on NKTs. The role of BTG1 in regulating the antitumor properties of NKTs was evaluated. BTG1 protein expression at the indicated time points was measured by qPCR and Western blot (Figures 2B and 2C, respectively). Pathway enrichment analysis shows the gene expression program enriched in BTG1 OE NKTs (Figure 2D). The fold proliferation of BTG1 OE NKTs versus control NKTs in six independent donors was assessed (Figure 2E). BTG1 overexpression reduces overall RNA expression and proliferative capacity in NKTs.

[0139] BTG1 protein expression was measured by Western blot, and BTG1 mRNA was measured by qPCR after activation with CD3 / CD28-specific monoclonal antibodies (Figures 3B and 3C).

[0140] Figures 4A and 4B show BTG1 expression in T cells following activation. Peripheral blood T cells were stimulated with plate-immobilized CD3 / CD28 antibodies and cultured in the presence of IL-2. BTG1 expression at the indicated time points was measured by Western blot (Figure 4A). The absolute numbers of BTG1 OE T cells and GFP control T cells after ex vivo culture were measured (Figure 4B).

[0141] BTG1 knockdown in GD2-CAR-NKT is shown in Figure 5. A microRNA targeting BTG1 and a scrambled control were cloned into an MMuLV-based gammaretroviral construct placed downstream of the GD2-CAR (this is just one example of a CAR). Figure 5A shows an example of a retroviral construct design for BTG1 knockdown. The 14g2a scFv for GD2 binding, CD8 hinge and transmembrane domain, CD28 costimulatory domain, and CD3ζ were used in all example constructs. In some cases, IL-15 was used to enhance activity. In some cases, a scrambled microRNA (SCR) was included in the construct, and in other cases, a BTG1 microRNA was used in the construct for knockdown. Figure 5B and Figure 5C show BTG1 transcript levels and protein expression in NKT expressing the given constructs, quantified by qPCR and Western blot, respectively.

[0142] NKT cells were transduced with retroviral vectors encoding CAR and / or IL15, as well as artificial microRNAs (amiRs) specific for BTG1 or a scrambled control. The fold expansion of NKT cells expressing the indicated constructs after transduction is shown in Figure 6A. The frequency of CD62L positivity after transduction in CAR.15 NKT cells with and without BTG1 knockdown is shown in Figure 6B. CD62L expression in CAR.15.amiR-BTG1 NKT cells gated on CAR-positive and CAR-negative populations is shown in Figure 6C. The frequency of PD-1-positive cells in CAR.15 NKT cells with and without BTG1 knockdown is shown in Figure 6D. The cytotoxic activity of CAR.15.amiR.BTG1 and scrambled control NKT cells against the GD2-high and GD2-low CHLA136 neuroblastoma (NB) cell lines was assessed at the indicated coculture time points and is shown in Figure 6E. The frequency of residual tumor cells after 5 days of coculture of CHLA255 cells with designated NKT groups at effector-to-target (E:T) ratios of 1 to 5 is shown in Figure 6F. The fold expansion of CAR-NKT cells after six cycles of coculture with NB cells is shown in Figure 6G. An example experimental design for evaluating the antitumor activity of CAR-NKT in vivo in a high-grade metastatic NB xenograft model is shown in Figure 6H. Bioluminescence images of tumor-bearing mice at designated time points are shown in Figure 6I. The change in tumor burden based on bioluminescence images over time is shown in Figure 6J. Kaplan-Meier survival curves for designated groups of mice are shown in Figure 6K. As described herein, BTG1 knockdown enhances the antitumor activity of GD2-CAR-NKT.

[0143] BTG1 knockdown (KD) enhances the antitumor activity of GD2-CAR T cells (as an example only) after multiple in vitro tumor challenges. The fold expansion of GD2-CAR T cells after three rounds of coculture with CHLA255 neuroblastoma (NB) cells (E:T = 1:1, N = 6, two-tailed paired t-test) is shown in Figure 7A. The change in the percentage of CAR-positive T cells before and after three rounds of coculture with CHLA255 NB cells is shown in Figure 7B.

[0144] BTG1 KD also enhances the antitumor activity of GD2-CAR T cells in a progressive in vivo metastatic NB xenograft model. Figure 8A shows an example of an experimental design for evaluating the antitumor activity of GD2-CAR T cells in this model in vivo. Example designs of BTG1 KD and scrambled miRNA control retroviral constructs (see also Figure 5A). Figure 8C shows bioluminescence images of tumor-bearing mice at weeks 4, 5, 6, and 7. The changes in tumor burden based on Figure 8C are shown graphically in Figure 8D, and Kaplan-Meier survival curves for the mice are also provided (Figure 8E). Quantitative data for human T cells (human CD45 positive) in the total cell population collected from mouse blood on day 10 are shown in Figure 8F.

[0145] Deletion of BTG1 in T cells by CRISPR Cas9-mediated marker (CD34) knock-in (KI) at the BTG1 locus increases the frequency of memory T cells. Figure 9A shows an example of an experimental design using CRISPR technology to knock-in the CD34-Q8 tag at the BTG1 locus. Figure 9B shows a representative flow cytometry analysis of CD34-Q8 tag expression in T cells 5 days after CRISPR KI. Figure 9C shows Western blot analysis of BTG1 protein expression in T cells 7 days after CRISPR KI. Figure 9D shows representative plots and summaries showing the expression of memory markers CD45RA and CCR7 in T cells with the CD34-Q8 tag knocked in at the BTG1 locus and Cas9-only controls.

[0146] All methods disclosed and claimed herein can be performed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that various modifications can be made in the methods described herein and in the steps or sequence of steps thereof without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

Claims

1. A method for enhancing the resistance of cells to exhaustion, comprising the step of reducing the expression and / or activity of B cell translocation gene 1 (BTG1) in the cells.

2. The method according to Claim 1, wherein the cells are not T cells.

3. A method according to claim 1, comprising the case where the cells are one or more immune cells, stem cells, derivatives thereof, or a mixture thereof, and the derivative is an iPSC-derived T cell, NKT cell, or NK cell.

4. A method according to claim 1 or 3, wherein the cells are CD8 T cells, CD4 T cells, natural killer T (NKT) cells, MAIT cells, γ / δ T cells, virus-specific T cells, cytokine-induced killer cells, NK cells, macrophages, or a mixture thereof.

5. A method according to any one of claims 1 to 4, wherein the cells are modified to express one or more foreign genes.

6. The method according to claim 5, wherein the heterogene comprises one or more modified receptors, antibodies, cytokines, suicide genes, costimulators, regulatory factors, or combinations thereof.

7. The method according to claim 6, wherein the modified receptor is a chimeric antigen receptor (CAR) or a T cell receptor.

8. The method according to claim 7, wherein the antigen receptor targets GD2, CD19, GPC3, and / or B7-H3.

9. The method according to any one of claims 6 to 8, wherein the cytokine is IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-33, or a combination thereof.

10. The method according to any one of claims 1 to 9, wherein the step of reducing the amount of a CRISPR drug includes using one or more drugs comprising a CRISPR drug, miRNA, siRNA, shRNA, transposon, one or more small molecule compounds targeting BTG1, or one or more antibodies targeting BTG1.

11. The method according to any one of claims 1 to 10, wherein the step of reducing comprises knock-in, knock-down, or knock-out of the endogenous BTG1 gene in the cell.

12. A plurality of cells produced by the method according to any one of claims 1 to 11.

13. The plurality of cells according to claim 12, wherein the plurality of cells are contained in a pharmaceutically acceptable excipient.

14. Non-cancerous modified cells that have been modified to have reduced BTG1 expression and / or activity, and that express one or more foreign genes.

15. The cell according to claim 14, wherein the foreign gene comprises one or more modified receptors, antibodies, cytokines, suicide genes, costimulators, regulatory factors, or combinations thereof.

16. The cell according to claim 14 or 15, wherein the modified cell is an immune cell or a stem cell.

17. A modified cell according to any one of claims 14 to 16, wherein the cell is a CD8 T cell, a CD4 T cell, a natural killer T (NKT) cell, a MAIT cell, a γ / δ T cell, a virus-specific T cell, a cytokine-induced killer cell, an NK cell, a macrophage, or a mixture thereof.

18. The modified cell according to any one of claims 14 to 17, wherein the reduction is caused by one or more of the CRISPR drug, siRNA, shRNA, transposon, or a mixture thereof.

19. The modified cell according to any one of claims 14 to 18, wherein the decrease in activity is caused by one or more small molecule compounds or one or more antibodies targeting BTG1.

20. The modified cell according to claim 15, wherein the modified receptor is an antigen receptor or a cytokine receptor.

21. The modified cell according to claim 20, wherein the antigen receptor is a CAR or a T cell receptor.

22. The modified cell according to claim 20 or 21, wherein the antigen receptor targets GD2, CD19, GPC3, and / or B7-H3.

23. The modified cell according to any one of claims 15 to 22, wherein the cytokine is IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-33, or a combination thereof.

24. Use of a composition of multiple cells according to claim 12, 13, or 14 in the manufacture of a pharmaceutical for the treatment of cancer and / or acute or chronic infection in an individual, The use wherein the treatment comprises the step of administering a therapeutically effective amount of the plurality of cells to the individual.

25. The use according to claim 24, wherein the infectious disease is human immunodeficiency virus, tuberculosis, herpes, viral hepatitis, or COVID.

26. Use in the manufacture of a pharmacopoeia for the treatment of an autoimmune disease in an individual, comprising cells comprising inducible expression and / or increased activity of BTG1, and / or a drug that increases the expression and / or activity of BTG1, Use comprising the step of administering a therapeutically effective amount of cells and / or the drug to the individual.

27. The use according to claim 26, wherein the cells are one or more immune cells, stem cells, derivatives thereof, or a mixture thereof.

28. The use according to claim 26 or 27, wherein the increase is due to the intracellular expression of BTG1 on the vector.

29. The use according to any one of claims 26 to 28, wherein the increase is due to the introduction of an exogenous promoter into the regulatory region of the endogenous BTG1 gene.

30. The use according to any one of claims 26 to 29, wherein the autoimmune disease is type 1 diabetes, lupus, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, glomerulonephritis, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, juvenile idiopathic arthritis, myasthenia gravis, myocarditis, multiple sclerosis, pemphigus / bullous pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, or vitiligo.

31. Cells expressing endogenous BTG1 or exogenous BTG1 with increased BTG1 expression via a vector, wherein the cells are modified to contain increased BTG1 expression and / or exogenous BTG1.

32. The cell according to claim 31, wherein the increase is due to the introduction of an exogenous promoter into the regulatory region of the endogenous BTG1 gene.

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