Modulation of b-cell translocation gene 1 (BTG1) for use in adoptive cell therapy

Abstract

Embodiments of the present disclosure include methods and compositions related to modified cells, and uses thereof, wherein the cells have been engineered with respect to expression of B-cell translocation gene 1 (BTG1). In particular embodiments, the engineered cells are used for adoptive cell therapy for special disease states. In some embodiments, the engineered cells are immune cells, such as T cells, having reduced expression of BTG1, and are useful for enhancing therapy for cancer and / or infectious diseases. In some embodiments, the engineered cells are cells having increased inducible expression of BTG1, and are useful in therapy for autoimmune diseases. In some embodiments, the expression of BTG1 is increased in endogenous cells in an individual having an autoimmune disease.

Description

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

[0002] I. TECHNICAL FIELD

[0003] The present disclosure relates at least to the fields of immunology, cell biology, molecular biology, and medicine (including at least cancer medicine).

[0004] II. BACKGROUND OF THE INVENTION

[0005] T cell exhaustion is an active process characterized by the progressive loss of effector function and proliferative capacity due to prolonged antigen stimulation that occurs in chronic infections and cancer. Therapeutic immune cells such as T cells or NKT cells engineered to express tumor-specific chimeric antigen receptors (CARs) also undergo exhaustion, which limits their anti-tumor efficacy and is associated with tumor escape and disease progression or recurrence. Although certain gene expression and epigenetic changes have been implicated in the T cell exhaustion process, the exact mechanisms responsible for the hypo-responsiveness of exhausted cells remain uncertain.

[0006] The present disclosure provides a solution to a long-standing need in the field of improving cell therapies. SUMMARY OF THE INVENTION

[0008] Embodiments of the present disclosure include methods and compositions for use in any type of therapeutic cell, including immune effector cells. The therapeutic cells can be adapted and used for specific therapeutic applications, such as for cancer treatment, and such cells of the present disclosure can be modified to improve one or more activities of the cells. In various embodiments, the present disclosure generally relates to the regulation of BTG1 in cells to (1) prevent or reduce unwanted exhaustion of the cells and / or control overactivation associated with toxicity of the therapeutic cells in the use for an individual (e.g., those having cancer); or (2) regulate endogenous cells of an individual to inhibit the activity of autoreactive cells (e.g., for those having an autoimmune disorder).

[0009] In some embodiments, the expression and / or activity of BTG1 is modulated in cells for adoptive cell therapy for any purpose, and the type of modulation is associated with the intended therapeutic use of the cells that have been modified accordingly. In various embodiments, the modulation can be downregulation of the expression of the endogenous BTG1 gene in the cells to be used for therapy and / or reduction of the activity of the BTG1 protein in the cells. In other embodiments, the modulation can be upregulation of the expression of the endogenous BTG1 gene in the cells, and / or introduction of heterologous BTG1 into the cells (including by overexpression), and / or increase of the activity of the BTG1 protein in the cells.

[0010] In particular embodiments, the cells for cell therapy tend to or otherwise have exhaustion, insufficient persistence, insufficient activation, insufficient expansion, insufficient homing, and / or insufficient cytotoxicity. Such cells can undergo modulation of BTG1 expression and / or activity to improve one or more of these characteristics. Some types of immune cells can benefit from modulation of BTG1 expression and / or activity to improve one or more of these characteristics. In particular embodiments, the expression and / or activity of endogenous BTG1 is reduced in cells to be used for cell therapy (e.g., for cancer or chronic infectious diseases), and such cells can be or can not be a particular type of immune cell. In various embodiments, any type of T cell (including CD8 T cells, CD4 T cells, αβ T cells, γ / δ T cells, virus-specific T cells (virus examples include Epstein-Barr virus, cytomegalovirus, BK virus, human herpesvirus, adenovirus, respiratory syncytial virus, influenza virus, parainfluenza virus 3, human metapneumovirus, etc.), NKT cells, MAIT cells, cytokine-induced killer cells, NK cells, macrophages, or mixtures thereof) can be engineered to have reduced expression and / or activity of endogenous BTG1, and such modulation renders the cells having reduced exhaustion or no exhaustion compared to the same type of cells without such modulation. Such reduction of the expression and / or activity of endogenous BTG1 can render the modulated cells having reduced exhaustion, enhanced persistence, enhanced activation, enhanced expansion, enhanced homing, and / or enhanced cytotoxicity compared to the same type of cells without such modulation.

[0011] In particular embodiments, the expression and / or activity of endogenous BTG1 is increased in cells to be used for cell therapy, and / or heterologous BTG1 is introduced into the cells, and such cells can be other particular types of cells. In particular embodiments, for cells to be used for cell therapy, it is desired that they tend to or otherwise have exhaustion, have insufficient persistence, have insufficient activation, have insufficient expansion, have insufficient homing, and / or have insufficient cytotoxicity.

[0012] In certain embodiments, upregulation of BTG1 is useful in endogenous autoreactive cells, such as by a drug that promotes BTG1 upregulation.

[0013] In some embodiments, increased or regulated BTG1 expression can be beneficial for controlling the toxicity of therapeutic effector cells in cancer or infection (chronic or acute), such as serving as an "off-switch". In some embodiments, BTG1 expression can be controlled by an inducible promoter to inhibit or kill any type of therapeutic cell covered herein when needed, such as at the onset of a cytokine storm.

[0014] Embodiments of the present disclosure include methods of enhancing cell therapy for an individual, which include the step of reducing the expression and / or activity of B-cell translocation gene 1 (BTG1) in cells used for the cell therapy.

[0015] Embodiments of the present disclosure include methods of enhancing cell therapy for an individual, which include the step of reducing the expression and / or activity of B-cell translocation gene 1 (BTG1) in cells, wherein the cells are not T cells.

[0016] In particular embodiments, the cells are immune cells, stem cells, one or more of their derivatives, or mixtures thereof, optionally when the derivatives are iPSC-derived T, NKT, or NK cells. The cells can be 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 mixtures thereof. In particular embodiments, the cells are modified to express one or more heterologous genes, although in other cases, the cells are not modified to express one or more heterologous genes. In particular embodiments, the heterologous genes can include one or more engineered receptors, antibodies, cytokines, suicide genes, costimulatory factors, regulatory factors, or combinations thereof. The engineered receptors can be antigen receptors, chemokine receptors, or cytokine receptors, or the cells can be more than one of these types. The antigen receptor can be a chimeric antigen receptor (CAR) or a T cell receptor. The CAR can comprise 1, 2, or more costimulatory domains, such as CD28, 4-1BB, OX40, CD2, DAP10, CD40, ICOS, CD27, TLR, MYD88, 2B4, NKG2D, or combinations thereof. In particular embodiments, the antigen receptor can target GD2, CD19, GPC3, B7-H3, CD20, BCMA, CD30, CD38, CD5, CD7, HER2, PSMA, mesothelin, EGFR, IL13RA2, or combinations thereof. The CAR can comprise one or more activation domains, such as CD3ζ, DAP12, 2B4, or combinations thereof. In particular cases, the cytokines are IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-33, or combinations thereof. In particular embodiments, the antibodies are monospecific antibodies, bispecific antibodies, trispecific antibodies, or mixtures thereof. The antibodies can be bispecific T cell engagers or trispecific T cell engagers. In some embodiments, the reducing step utilizes one or more reagents to reduce the expression of the endogenous BTG1 gene in the cells. The one or more reagents can include nucleic acids, peptides, and / or polypeptides. The one or more reagents can include CRISPR reagents, siRNA, shRNA, transposons, or mixtures thereof. In some cases, the reducing step utilizes one or more reagents that reduce the activity of the BTG1 protein in the cells, and the one or more reagents can include one or more small molecules or one or more antibodies that target BTG1. The method further comprises the step of 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 infectious disease.

[0017] Embodiments of the present disclosure may include a plurality of any cells covered herein, and the plurality of cells may be included in a pharmaceutically acceptable excipient.

[0018] In some embodiments, engineered non-cancerous cells are provided, the cells being engineered to comprise a decrease in the expression and / or activity of BTG1, wherein the cells express one or more heterologous genes. The heterologous genes may include one or more engineered receptors, antibodies, cytokines, suicide genes, co-stimulatory factors, regulatory factors, or combinations thereof. The engineered cells may be immune cells or stem cells. In particular embodiments, the cells are CD8 T cells, CD4 T cells, NKT cells, MAIT cells, γ / δ T cells, virus-specific T cells, cytokine-induced killer cells, NK cells, macrophages, or mixtures thereof. In some embodiments, the decrease in expression is produced by one or more CRISPR reagents, siRNA, shRNA, transposons, or mixtures thereof. The decrease in activity may be produced by one or more small molecules. In some embodiments, the engineered receptor is an antigen receptor or cytokine receptor described elsewhere herein. In some embodiments, the decrease in the activity of BTG1 is effected by one or more small molecules or one or more antibodies targeting BTG1.

[0019] Embodiments of the present disclosure include engineered CD8 T cells, CD4 T cells, NKT cells, MAIT cells, γ / δ T cells, virus-specific T cells, cytokine-induced killer cells, NK cells, macrophages, or mixtures thereof, the cells being engineered to comprise a decrease in the expression and / or activity of BTG1. The cells may express one or more heterologous genes, such as one or more engineered receptors, antibodies, cytokines, suicide genes, co-stimulatory factors, regulatory factors, or combinations thereof.

[0020] Embodiments of the present disclosure include methods of treating cancer and / or acute or chronic infectious diseases in an individual, which include the step of administering to the individual a therapeutically effective amount of any one of the plurality of cells of the present disclosure. In some embodiments, the plurality of cells include NK cells, NK T cells, and / or macrophages, which comprise GD2 CAR, GPC3 CAR, CD19 CAR, and / or B7-H3 CAR. The cells may be allogeneic or autologous relative to the individual. In particular embodiments, the acute or chronic infectious disease is human immunodeficiency virus, tuberculosis, herpes, viral hepatitis, or COVID.

[0021] Embodiments of the present disclosure include methods of treating an autoimmune disease in an individual, which include the step of administering to the individual a therapeutically effective amount of cells comprising an increase in the inducible expression and / or activity of BTG1, and / or which include the step of administering to the individual a therapeutically effective amount of a drug that increases the expression and / or activity of BTG1 in the endogenous cells of the individual. In some embodiments, the cells are immune cells, stem cells, one or more derivatives thereof, or a mixture thereof. The increased expression can result from the expression of BTG1 on a vector in the cells, and the vector can be an episomal vector or an integrating vector. In some embodiments, the increased expression results from the introduction of a heterologous promoter into the regulatory region of the endogenous BTG1 gene in the cells. The increase in the activity of BTG1 can result from the introduction of a small molecule into the cells. The cells may or may not express one or more heterologous genes. In particular cases, 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 / 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. The cells can be allogeneic or autologous relative to the individual.

[0022] Embodiments of the present disclosure include modified cells, wherein the cells are modified to have an increase in the expression of endogenous BTG1 and / or to comprise a vector expressing heterologous BTG1. The vector can be an episomal vector or an integrating vector. The increased expression can result from the introduction of a heterologous promoter into the regulatory region of the endogenous BTG1 gene in the cells. The increase in the activity of BTG1 can result from the introduction of a small molecule into the cells, and the cells can express one or more heterologous genes.

[0023] Embodiments include methods of controlling the activity and / or toxicity of a cell therapy, which include the step of increasing the expression and / or activity of BTG1 in the cells of the cell therapy. The increase in the expression of BTG1 can be effected by an inducible promoter. Embodiments can include methods of reducing the expression of the endogenous BTG1 gene in the cells, for example by using one or more reagents, including for example CRISPR reagents, miRNA, siRNA, shRNA, transposons, or a mixture thereof.

[0024] 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 the 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present invention. The disclosure can be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0027] Figure 1A-1E : BTG1 expression is elevated in exhausted chimeric antigen receptor (CAR)-natural killer T (NKT) cells. FIG. 1A. Experimental design for a repeated tumor co-culture system to induce exhaustion in CAR-NKTs via chronic antigen exposure. The prepared CAR-NKTs were re-plated with fresh CHLA255 NB tumor cells every five days for multiple cycles. FIG. 1B. Cytotoxic activity of CAR-NKTs during repeated co-culture at the indicated time points. FIG. 1C. Uniform Manifold Approximation and Projection (UMAP) projection of single-cell RNA sequencing (scRNAseq) results from: the prepared CAR-NKT infusion product (IP), CAR-NKTs after five cycles of repeated co-culture with tumor cells (5RcC), and CAR-NKTs isolated from peripheral blood (PB) after infusion. FIG. 1D. UMAP projection of CAR-NKT gene expression by scRNAseq from pre-infusion and 5RcC samples. FIG. 1E. Volcano plot showing differentially expressed genes in CAR-NKTs after 5RcC compared to the pre-infusion product.

[0028] Figure 2A-2E: Overexpression of BTG1 reduces global RNA expression and proliferative capacity in NKT. Figure 2A. Design of retroviral constructs encoding BTG1-green fluorescent protein fusion (BTG1.GFP) or GFP and firefly luciferase (GFP control) controls for evaluation of the effect of BTG1 overexpression (OE) in NKT. Figure 2B. BTG1 expression in NKT expressing the GFP.BTG1 construct vs. wild-type (WT) NKT by qPCR. Figure 2C. BTG1 expression in NKT expressing the GFP.BTG1 construct vs. wild-type (WT) NKT by Western blot. Figure 2D. Pathway enrichment analysis indicating gene expression programs enriched in BTG1 OE NKT. Figure 2E. Fold expansion of BTG1 OE vs. GP control NKT; six independent donors were evaluated.

[0029] Figure 3A-3C . Evaluate the role of BTG1 in regulating the anti-tumor properties of NKT. Figure 3A. BTG1 protein expression measured by Western blot at the indicated time points in the repeated tumor challenge assay (RTC). Figure 3B. Protein expression of BTG1 measured by Western blot after activation with CD3 / CD28-specific monoclonal antibodies. Figure 3C. BTG1 mRNA expression measured by qPCR at the indicated time points.

[0030] Figure 4A-4B . BTG1 expression in T cells after activation. 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 blot at the indicated time points. Figure 4B. Absolute number of BTG1 OE vs. GFP control T cells after ex vivo culture.

[0031] Figure 5A-5C . BTG1 KD in GD2-CAR NKT. MicroRNAs targeting BTG1 and scrambled controls were cloned into an MMuLV-based γ-retroviral construct downstream of GD2-CAR. Figure 5A. Design of retroviral construct for BTG1 knockdown. Figures 5B and 5C. BTG1 transcript and protein expression in NKT expressing the indicated constructs quantified by qPCR and Western blot, respectively.

[0032] Figure 6A-6K. Knockdown (KD) of BTG1 enhances the anti-tumor activity of GD2-CAR NKT. NKT cells were transduced with retroviral vectors encoding CAR+ / - interleukin-15 (IL15) and / or artificial microRNA (amiR) specific to BTG1 or scrambled control. Figure 6A. Fold expansion of NKT cells expressing the indicated constructs after transduction. Figure 6B. CD62L frequency after transduction in CAR.15 NKT with or without BTG1 KD. Figure 6C. CD62L expression in CAR.15.amiR-BTG1 NKT gated for CAR+ and CAR- populations. Figure 6D. Frequency of PD-1+ CAR.15 NKT with and without BTG1 KD. Figure 6E. Cytolytic activity of CAR.15.amiR.BTG1 vs. scrambled control NKT against GD2-high CHLA255 and GD2-low CHLA136NB cell lines evaluated at the indicated co-culture time points. Figure 6F. Residual tumor cell frequency after five-day co-culture of the indicated NKT groups and CHLA255 cells at an E:T ratio of 1:5. Figure 6G. Fold expansion of CAR-NKT cells after six cycles of co-culture with NB cells. 6H. Experimental design for in vivo evaluation of CAR-NKT anti-tumor activity in an aggressive metastatic NB xenograft model. Figure 6I. Bioluminescence images of tumor-bearing mice at the specifically designated time points. Figure 6J. Change in tumor burden based on bioluminescence images over time. Figure 6K. Kaplan-Meier survival curves for mice in the indicated groups. 10 mice / treatment group, survival comparison was performed by Gehan-Breslow-Wilcoxon test.

[0033] Figures 7A and 7B. BTG1 KD enhances the anti-tumor activity of GD2-CAR T cells. Figure 7A) Fold expansion 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) Percentage change in CAR of T cells before or after three rounds of co-culture with CHLA255 NB cells (N = 6, two-tailed paired t-test).

[0034] Figure 8A-8F. BTG1 KD enhances the anti-tumor activity of GD2-CAR T cells in an in vivo metastatic NB xenograft model. Figure 8A) Experimental design for in vivo evaluation of the anti-tumor activity of GD2-CAR T cells in an aggressive metastatic NB 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 the specified time points. Figure 8D) Tumor burden changes over time based on the bioluminescence images in C). Figure 8E) Kaplan-Meier survival curves for mice in the indicated groups, 10 mice / group, with survival comparisons performed by Gehan-Breslow-Wilcoxon test. Figure 8F) Quantification of human T cells (human CD45+ in the total cell population collected from mouse blood on day 10).

[0035] Figure 9A-9D . BTG1 deletion in T cells increases the frequency of memory T cells. Figure 9A) Experimental design for KI CD34-Q8 tag at the BTG1 locus using the CRISPR method. Figure 9B) Representative flow cytometry of CD34-Q8 tag expression in T cells five days after CRISPR KI. Figure 9C) BTG1 protein expression in T cells by Western blot seven days after CRISPR KI. Figure 9D) Representative plots and summary showing the expression of memory markers CD45RA and CCR7 in T cells with CD34-Q8 tag KI at the BTG1 locus and Cas9 control only (N = 4). DETAILED DESCRIPTION OF THE INVENTION

[0037] I. DEFINITION EXAMPLES

[0038] Consistent with long-standing patent law practice, when used in this specification (including the claims) in conjunction with the word "comprising (including)", the words "a" and "an" 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. It is contemplated that any method or composition described herein may be practiced in relation to any other method or composition described herein, and different embodiments may be combined.

[0039] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprising" and "include" will be understood to imply the inclusion of the stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are essential or mandatory and that no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified for the listed elements in the present disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are essential or mandatory, but that other elements are optional and may or may not be present, depending on whether they affect the activity or action of the listed elements.

[0040] Throughout this specification, reference to "an embodiment", "(an) embodiment", "(a) particular embodiment", "(a) related embodiment", "a certain embodiment", "(an) additional embodiment" or "(a) further embodiment" or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Further, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.

[0041] As used herein, the terms "or" and "and / or" are used to describe multiple components that are combined or mutually exclusive. For example, "x, y and / or z" can mean "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 embodiment.

[0042] Throughout this application, the term "about" is used in its plain and ordinary sense in the field of cell and molecular biology to indicate that a value includes the standard deviation with respect to the device or method used to measure the value.

[0043] "Autoimmune disease" refers to a disease in which the immune system mounts an immune response (e.g., a B cell or T cell response) against an antigen that is part of the normal host (i.e., a self-antigen), resulting in damage to tissue. Self-antigens can be derived from host cells or can be derived from commensal organisms such as microorganisms that normally colonize mucosal surfaces (referred to as commensals).

[0044] As used herein, "reduced", "disrupted", or "altered" expression of a gene refers to the complete or partial elimination or decrease in the expression of one or more gene products encoded by the gene of interest in a cell, compared to the level of expression of the gene product in the absence of such alteration. Exemplary gene products include mRNA and protein products encoded by a gene. The alteration can be transient or reversible in some cases, and persistent in other cases. The alteration can be in the functional or full-length protein or mRNA in some cases, despite the fact that truncated or non-functional products may be produced. In some embodiments herein, gene activity or function, rather than expression, is disrupted. Gene alterations are typically induced by artificial means, i.e., by the addition or introduction of a compound, molecule, complex, or composition, and / or by alteration of the gene or nucleic acid associated with the gene, e.g., at the DNA level. Exemplary methods for gene alteration include gene silencing, knockdown, knockout, and / or gene alteration techniques, such as gene editing. Examples include antisense techniques, such as RNAi, siRNA, shRNA, and / or ribozymes, which typically result in transient reduction in expression, and gene editing techniques, which result in targeted gene inactivation or alteration, e.g., by introducing breaks and / or homologous recombination. Examples include insertions, mutations, and deletions. The alteration typically results in repression and / or complete absence of expression of the normal or "wild-type" product encoded by the gene. Examples of such gene alterations are insertions, frameshift and missense mutations, deletions, knock-ins (including knock-ins with markers such as CD34-Q8 tag, GFP, or another selectable marker), and knockout of a gene or a portion of a gene, including deletion of the entire gene. Such alterations can occur in the coding region, e.g., in one or more exons, resulting in the inability to produce a full-length product, a functional product, or any product, e.g., by introducing a stop codon. Such alterations can also occur by alterations in the promoter or enhancer or other regions affecting transcriptional activation, so as to prevent transcription of the gene. Gene alteration includes gene targeting, including targeted gene inactivation by homologous recombination.

[0045] The term "engineered" as used herein refers to an entity generated by the hand of man, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, the engineered entity is synthetic and contains elements that do not occur or are not configured in nature in the manner in which they are used in the present disclosure. In particular embodiments, vectors are engineered by recombinant nucleic acid techniques, and cells are engineered by transfection or transduction with the engineered vector. Cells can be engineered to express a heterologous protein not naturally expressed by the cell, either because the heterologous protein is recombinant or synthetic, or because the cell does not naturally express the protein. Engineered entities are not found in nature.

[0046] As used herein, the term "exhausted" or "exhaustion" refers to immune cells, including T cells, that become dysfunctional by exhibiting poor effector function, persistent expression of inhibitory receptors, and a transcriptional state distinct from functional effector or memory T cells. Exhaustion can occur during chronic infection, autoimmune disease, or cancer, and it can respectively prevent optimal control of infection and tumors.

[0047] When used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism, the term "exogenous" refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means; or when used in relation to a cell, the term refers to a cell that has been isolated and subsequently introduced into other cells or organisms by artificial or natural means. Exogenous nucleic acids can be from a different organism or cell, or it can be one or more additional copies of a nucleic acid that naturally occurs within the organism or cell. Exogenous cells can be from a different organism, or it can be from the same organism. As a non-limiting example, an exogenous nucleic acid is a nucleic acid that is at a chromosomal location different from where it is found in a natural cell, or is flanked by nucleic acid sequences different from those found in nature.

[0048] As used herein, "natural killer T (NKT) cells" can refer to a subset of innate-like T lymphocytes that recognize glycolipids presented by the monomorphic MHC-like molecule CD1d. Unlike T cells, NKT do not recognize class I or class II HLA molecules. Type I or invariant NKT express an invariant T cell receptor (TCR) α-chain Vα24-Jα18, which pairs with Vβ11 in particular embodiments. This NKT subset can be identified by using the monoclonal antibody clone 6B11 or by reactivity to the synthetic glycolipid α-galactosylceramide. In particular embodiments, the NKT cells express one or more engineered antigen receptors, such as one or more CARs.

[0049] The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, such as a human (where appropriate). Given the present disclosure, the preparation of pharmaceutical compositions containing antibodies or other active ingredients will be known to those of skill in the art. In addition, for administration to an animal (e.g., a human), it will be understood that the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA's Bureau of Biologics Standards.

[0050] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose solution, etc.), non-aqueous solvents (e.g., 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, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrating agents, lubricants, sweetening agents, flavoring agents, dyes, and nutrient supplements, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0051] As used herein, the term "subject" generally refers to an individual having or suspected of having cancer or otherwise having or suspected of having an acute or chronic infectious disease and / or an autoimmune disease. The subject can be any biological or animal subject that is the target of a method or material, including mammals such as humans, laboratory animals (e.g., primates, rats, mice, rabbits), domestic animals (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. The subject can be a patient, e.g., having or suspected of having a disease (which can be referred to as a medical condition), such as benign or malignant neoplasia or cancer. The subject can be undergoing or have undergone treatment. The subject can be asymptomatic. The subject can be a healthy individual desiring to prevent cancer, infectious disease, or autoimmune disease. In at least some instances, the term "individual" can be used interchangeably. As used herein, a "subject" or "individual" can or can not live in a medical institution and can be treated as an outpatient of a medical institution. The individual can be receiving one or more pharmaceutical compositions via the Internet. The individual can include humans or non-human animals of any age and thus includes adults and juveniles (i.e., children) and infants and includes individuals in utero. It is not intended that the term implies a need for medical treatment, and thus the individual can voluntarily or involuntarily be part of an experiment (whether clinical or in support of basic scientific research). The subject can be participating in a clinical trial.

[0052] As used herein, "treatment" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and can include even a minimal reduction in one or more measurable markers of the disease or condition being treated (e.g., cancer, infectious disease or autoimmune disease). Treatment can optionally involve alleviation or improvement of one or more symptoms of the disease or condition, or delay in the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition or its associated symptoms. Treatment can mean remission of at least one symptom of the disease or condition.

[0053] As used throughout this application, the term "therapeutically effective" means anything that promotes or enhances the well-being of a subject with respect to the medical management of the condition. This includes, but is not limited to, a decrease in the frequency or severity of one or more signs or symptoms of a disease. For example, treatment of cancer can involve, for example, a decrease in tumor size, a decrease in tumor invasiveness, a decrease in cancer growth rate, or prevention of metastasis. For example, treatment of cancer can also mean prolonging the survival of a subject with cancer and / or improving the quality of life of a subject with cancer.

[0054] II. General Embodiments

[0055] In various embodiments, a cell therapy is prepared for an individual in need thereof such that the cells have a regulated expression and / or activity of endogenous BTG1, or in other cases, a heterologous BTG1 introduced into the cells.

[0056] The present disclosure encompasses embodiments in which a cell therapy for a disease is generated based on the disease being treated. For some embodiments, the individual has a medical condition for which the cells for therapy of the condition need to have persistence and avoid exhaustion. In particular embodiments (by way of example only) in which cancer or a chronic viral infectious disease (e.g., HIV, chronic viral hepatitis) is being treated, it is beneficial for the cells of the cell therapy to avoid exhaustion or have a reduced degree of exhaustion. As provided elsewhere herein, BTG1 plays a role in the exhaustion of, for example, NKT and T cells, and its knockdown or knockout or reduced activity is beneficial for the persistence of such cells. Such cells are effective as a therapy for cancer or a chronic viral infectious disease.

[0057] However, in some embodiments in which, for example, an autoimmune disease is being treated, it is beneficial for the cells of the cell therapy to have exhaustion or have an increased or accelerated degree of exhaustion.

[0058] Embodiments of the present disclosure include methods of enhancing cell therapy for an individual, which include reducing the expression and / or activity of BTG1 in the cells used for the cell therapy, or increasing the expression and / or activity of BTG1 in the cells used for the cell therapy. In some cases, the cells may or may not be T cells.

[0059] Embodiments of the present disclosure include methods of generating cells for cell therapy for an individual, which include reducing the expression and / or activity of BTG1 in the cells, optionally wherein the cells are not T cells. In particular embodiments, provided herein are methods of generating cells for cell therapy for an individual, which include increasing the expression and / or activity of BTG1 in the cells or in the endogenous cells of the individual.

[0060] In particular embodiments, the present disclosure provides methods including the step of reducing the expression and / or activity of BTG1 in the cells, optionally wherein the cells are not T cells. In some embodiments, provided herein are methods including the step of increasing the expression and / or activity of BTG1 in the cells or in the endogenous cells of the individual.

[0061] III. Reduction of the expression and / or activity of BTG1

[0062] In particular embodiments, cells for use in cancer therapy or therapy of acute or chronic infectious diseases are required to have reduced exhaustion or no exhaustion compared to, for example, natural corresponding cells in vivo or cells lacking modification of BTG1 (e.g., by human hand). In particular cases, such cells may be immune cells, including at least any kind of T cells (including CD8 T cells, CD4 T cells, αβ T cells, γ / δ T cells, virus-specific T cells, NKT cells, MAIT cells, cytokine-induced killer cells, NK cells, macrophages, or mixtures thereof). In particular embodiments, the cells are any kind of T cells, or NKT cells. In various embodiments, the cells for cancer therapy or therapy of acute or chronic infectious diseases have regulation of the expression of BTG1, the cells for cancer therapy or therapy of acute or chronic infectious diseases have regulation of the activity of BTG1, or both. In particular embodiments, the regulation of the expression includes disruption or reduction of the expression of the endogenous BTG1 gene to a greater extent than in the absence of the regulation. In particular embodiments, the regulation of the activity includes reduction of the activity of the endogenous BTG1 protein to a greater extent than in the absence of the regulation.

[0063] In particular embodiments, the expression of the endogenous BTG1 gene is reduced in cells for cancer therapy or for therapy of acute or chronic infectious diseases. The expression can be engineered to be reduced by any suitable means.

[0064] In some embodiments, the reduction of BTG1 gene expression is effected by introducing disruptions in the gene, such as knockouts, insertions, missense or frameshift mutations, such as biallelic frameshift mutations, deletions of all or part of the gene (e.g., one or more exons or portions thereof), and / or knock-ins. For example, the altered BTG1 gene expression can be effected by sequence-specific or targeted nucleases, including DNA-binding targeted nucleases (e.g., zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)) and RNA-guided nucleases (e.g., CRISPR-associated nucleases (Cas)), which are specifically designed to be targeted to the sequence of the BTG1 gene or a portion thereof.

[0065] In some embodiments, the alteration of the expression, activity, and / or function of the BTG1 gene is effected by disrupting the gene. In some aspects, the gene is modified such that its expression is reduced by at least about or approximately 20, 30, or 40%, typically at least about or approximately 50, 60, 70, 80, 90, or 95%, compared to the expression in the absence of the gene modification or in the absence of the component introduced to effect the modification.

[0066] In some embodiments, the alteration is transient or reversible such that the expression of the gene is restored at a later time. In other embodiments, the alteration is not reversible or transient, e.g., is permanent.

[0067] In some embodiments, the gene alteration is effected by introducing one or more double-strand breaks and / or one or more single-strand breaks (typically, in a targeted manner) in the BTG1 gene. In some embodiments, the double-strand or single-strand breaks are created by a nuclease (e.g., an endonuclease, such as a gene-targeted nuclease). In some aspects, the break is induced in the coding region of the gene, e.g., in an exon. For example, in some embodiments, the induction occurs near the N-terminal portion of the coding region, e.g., in the first exon, in the second exon, or in a subsequent exon.

[0068] In some aspects, the double-stranded or single-stranded breaks undergo repair via cellular repair processes such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). In some aspects, the repair process is error-prone and results in disruption of the gene, such as a frameshift mutation, such as a bi-allelic frameshift mutation, which can result in a complete knockout of the gene. For example, in some aspects, the disruption includes inducing deletions, mutations, and / or insertions. In some embodiments, the disruption results in the presence of an early termination codon. In some aspects, the presence of an insertion, deletion, translocation, frameshift mutation, and / or premature termination codon results in disruption of the expression, activity, and / or function of the gene.

[0069] In some embodiments, gene alteration is achieved using antisense techniques, such as by RNA interference (RNAi), using short interfering RNAs (siRNAs), short hairpin (shRNAs), and / or ribozymes to selectively inhibit or repress the expression of the BTG1 gene. siRNA technology is RNAi using double-stranded RNA molecules having a sequence homologous to and complementary to the nucleotide sequence of the mRNA transcribed from the gene. siRNAs typically are homologous / complementary to a region of the mRNA transcribed from the gene or can be siRNAs comprising multiple RNA molecules homologous / complementary to different regions. In some aspects, siRNAs are included in a polycistronic construct, such as included together with an expression construct that produces a heterologous gene product.

[0070] A. 1 ZFP and ZFN

[0071] In some embodiments, the molecule targeting DNA includes a BTG1 binding protein, such as one or more zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs), which are fused to an effector protein such as an endonuclease. Examples include ZFNs, TALEs, and TALENs.

[0072] In some embodiments, the molecule targeting BTG1 comprises one or more zinc finger proteins (ZFPs) or domains thereof that bind to the BTG1 gene in a sequence-specific manner. A ZFP or domain thereof is a protein or a domain within a larger such protein that binds DNA in a sequence-specific manner through one or more zinc fingers (regions of amino acid sequence within the binding domain whose structure is stabilized by coordination of zinc ions). The term "zinc finger DNA binding protein" is often abbreviated to zinc finger protein or ZFP. Included among ZFPs are artificial ZFP domains that target specific DNA sequences (typically, 9 - 18 nucleotides in length), which are generated by the assembly of individual fingers.

[0073] ZFPs include those ZFPs in which the length of a single zinc finger domain is approximately 30 amino acids and contains an α-helix that contains two invariant histidine residues coordinated by zinc to two cysteines of a single β-turn, and has 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 molecule comprising a ZFP is non-naturally occurring, e.g., engineered to bind a selected target site.

[0074] In some embodiments, the molecule targeting BTG1 is or comprises a zinc finger DNA binding domain that is fused to a DNA cleavage domain to form a zinc finger nuclease (ZFN). In some embodiments, the fusion protein comprises a cleavage domain (or cleavage half-domain) from at least one IIS-type restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. In some embodiments, the cleavage domain is from the IIS-type restriction endonuclease Fok I. Fok I typically catalyzes double-stranded cleavage of DNA nine nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand.

[0075] Many gene-specific engineered zinc fingers are commercially available. For example, Sangamo Biosciences (Richmond, Calif., USA) has collaborated with Sigma-Aldrich (St. Louis, Mo., USA) to develop a platform (CompoZr) for zinc finger construction, which allows researchers to completely bypass zinc finger construction and validation and provides specific targeting 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 catalog numbers CSTZFND, CSTZFN, CTil-1KT, and PZD0020).

[0076] B. TAL, TALE, and TALEN

[0077] In some embodiments, the molecule targeting BTG1 comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as in a transcription activator-like effector (TALE) protein. See, e.g., U.S. Patent Publication No. 2011 / 0301073, which is incorporated herein by reference in its entirety.

[0078] The TALE DNA binding domain or TALE is a polypeptide comprising one or more TALE repeat domains / units. The repeat domain is involved in the binding of the TALE to its associated target DNA sequence. A single "repeat unit" (also referred to as a "repeat sequence") typically has a length of 33-35 amino acids and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. Each TALE repeat unit includes 1 or 2 DNA binding residues, which constitute the Repeat Variable Diresidue (RVD), typically at positions 12 and / or 13 of the repeat sequence. The natural (canonical) code for DNA recognition by these TALEs has been determined, such that the HD sequence at positions 12 and 13 results in binding to cytosine (C), NG binds to T, NI binds to A, NN binds to G or A, and NO binds to T, and non-canonical (atypical) RVDs are also known. In some embodiments, a TALE can be targeted to any gene by designing a TAL array with specificity for the target DNA sequence. The target sequence typically begins with thymidine.

[0079] In some embodiments, the molecule is a DNA-binding endonuclease, such as a TALE nuclease (TALEN). In some aspects, the TALEN is a fusion protein that comprises a DNA-binding domain derived from a TALE and a nuclease catalytic domain for cleaving a nucleic acid target sequence.

[0080] In some embodiments, the TALEN recognizes and cleaves a target sequence in the BTG1 gene. In some aspects, the cleavage of DNA results in a double-strand break. In some aspects, the break stimulates the rate of homologous recombination or non-homologous end joining (NHEJ). Typically, NHEJ is an imperfect repair process that often results in changes to the DNA sequence at the cleavage site. In some aspects, the repair mechanism involves the rejoining of the remaining portions of the two DNA ends by direct re-ligation or via so-called microhomology-mediated end joining. In some embodiments, repair via NHEJ results in small insertions or deletions and can be used to disrupt and thereby inhibit the gene. In some embodiments, the modification can be a substitution, deletion, or addition of at least one nucleotide. In some aspects, cells in which a cleavage-induced mutagenesis event (i.e., a mutagenesis event in coherence with an NHEJ event) has occurred can be identified and / or selected by methods well known in the art.

[0081] In some embodiments, TALE repeats are assembled to specifically target 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 arrays are commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, Ky., USA), and Life Technologies (Grand Island, N.Y., USA). In particular, TALENs targeting CD38 are commercially available (see Gencopoeia, catalog numbers HTN222870-1, HTN222870-2, and HTN222870-3). Exemplary molecules are described, for example, in U.S. Patent Publication Nos. US2014 / 0120622 and 2013 / 0315884.

[0082] In some embodiments, the TALENs are introduced as transgenes encoded by one or more plasmid vectors. In some aspects, the plasmid vector may contain a selectable marker that provides for the identification and / or selection of cells that have received the vector.

[0083] C.RGEN (CRISPR / Cas system)

[0084] In some embodiments, the alteration is performed using one or more DNA-binding nucleic acids, such as an alteration via an RNA-guided endonuclease (RGEN). For example, the alteration can be performed using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. Generally, the "CRISPR system" collectively refers to transcripts and other elements involved in the expression or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or an active portion of tracrRNA), tracr-mate sequences (covering "direct repeats" and the portion of direct repeats processed by tracrRNA, in the context of an endogenous CRISPR system), guide sequences (also referred to as "spacers", in the context of an endogenous CRISPR system), and / or other sequences and transcripts from the CRISPR locus.

[0085] The 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) having nuclease functionality (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, e.g., from a particular organism containing an endogenous CRISPR system, such as Streptococcus pyogenes.

[0086] In some aspects, a Cas nuclease and a gRNA (including a fusion of a crRNA specific for a target sequence and a fixed tracrRNA) are introduced into a cell. Typically, the Cas nuclease is targeted to the target site, e.g., a gene, at the target site at the 5' end of the gRNA by using complementary base pairing. The target site can be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence (e.g., typically NGG or NAG). In this regard, the gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Typically, the CRISPR system is characterized by elements that facilitate the formation of the CRISPR complex at the site of the target sequence. Typically, a "target sequence" generally refers to a sequence for which a guide sequence is designed to have complementarity, where hybridization between the target sequence and the guide sequence facilitates the formation of the CRISPR complex. Complete complementarity is not necessarily required, provided that there is sufficient complementarity to cause hybridization and facilitate the formation of the CRISPR complex.

[0087] The CRISPR system can induce a double-strand break (DSB) at the target site, followed by breakage or alteration, as discussed herein. In other embodiments, a Cas9 variant that is considered a "nickase" is used to nick a single strand at the target site. Paired nickases can be used, e.g., to improve specificity, each directed by a pair of gRNAs targeting different sequences, thereby introducing 5' overhangs simultaneously upon introduction of the nicks. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor or activator to affect gene expression.

[0088] The target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide. The target sequence can be located in the nucleus or cytoplasm of a cell, such as within an organelle of the cell. Generally, a sequence or template that can be used for recombination into the targeted locus containing the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In some aspects, an exogenous template polynucleotide can be referred to as an editing template. In some aspects, the recombination is homologous recombination.

[0089] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (which comprises a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more bases). The tracr sequence (which can comprise all or part of a wild-type tracr sequence (e.g., about or greater than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides) of the wild-type tracr sequence or consist thereof) can also form part of the CRISPR complex, such as by hybridization along at least a portion of the tracr sequence with all or part of a tracr mate sequence operably linked to the guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in formation of the CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned.

[0090] One or more vectors that drive the expression of one or more components of the CRISPR system can be introduced into a cell such that the expression of the components of the CRISPR system directs the formation of CRISPR complexes at one or more target sites. The components can also be delivered to the cell 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 of the components expressed from the same or different regulatory elements can be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. The 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, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences within a cell.

[0091] The vector can contain a regulatory element operably linked to an enzyme-encoding sequence that encodes a CRISPR enzyme (e.g., a Cas protein). Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also referred to as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified forms thereof. These enzymes are known; for example, the amino acid sequence of Streptococcus pyogenes Cas9 protein can be found under accession number Q99ZW2 in the SwissProt database.

[0092] The CRISPR enzyme can be Cas9 (e.g., from Streptococcus pyogenes or Streptococcus pneumoniae (S. pneumoniae)). The CRISPR enzyme can direct cleavage of one or both strands at the location of the target sequence, e.g., within the target sequence and / or within the complement of the target sequence. The vector can encode a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartic acid to alanine substitution (D10A) in the RuvCI catalytic domain of Cas9 from S. pyogenes converts Cas9 from a double-strand cleaving nuclease to a nickase (cleaving single strand). In some embodiments, Cas9 nickase can be used in combination with guide sequences, e.g., two guide sequences that respectively target the sense and antisense strands of a DNA target. This combination allows nicks to be made in both strands and is used to induce NHEJ or HDR.

[0093] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell (e.g., a eukaryotic cell). The eukaryotic cell can be those of a particular organism or derived from a particular organism, the particular organism being, for example, a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. Generally, codon optimization refers to the process of modifying a nucleic acid sequence for enhanced expression in a host cell of interest 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 that host cell while maintaining the native amino acid sequence. Various species exhibit a particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) is often related to the translational efficiency of messenger RNA (mRNA), which is thought to depend particularly on the properties of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of the tRNAs selected in a cell is usually a reflection of the codons that are most frequently used in peptide synthesis. Thus, genes can be adapted for optimal gene expression in a given organism based on codon optimization.

[0094] Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0095] Optimal alignments can be determined by using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., Burrows Wheeler Aligner), ClustalW, 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).

[0096] The CRISPR enzyme can be part of a fusion protein that includes one or more heterologous protein domains. The CRISPR enzyme fusion protein can include any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include: histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to: glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins (including blue fluorescent protein (BFP)). The CRISPR enzyme can be fused to a gene sequence encoding a protein or a fragment of a protein that binds to a DNA molecule or other cellular molecule, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding protein (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can form part of a fusion protein containing the CRISPR enzyme are described in US20110059502 (which is incorporated herein by reference).

[0097] *****

[0098] In other embodiments, the activity of endogenous BTG1 protein is reduced in cells for use in the cancer therapy or the therapy of acute or chronic infectious diseases, and this can be additional or alternative to reducing the expression of the endogenous BTG1 gene. The protein can be reduced in activity by any suitable means, such as with one or more small molecules, one or more antibodies, or a combination thereof. For example, small molecules can be selected from a library and based on their ability to reduce activity in vitro for use with cells for the cancer therapy. In some embodiments, one or more antibodies that bind BTG1 can at least partially or in some cases completely inhibit its activity. The antibodies can be of any kind, including monoclonal antibodies, polyclonal antibodies, etc. BTG1 antibodies are commercially available, or they can be generated by standard means.

[0099] IV. Increase in the expression and / or activity of BTG1

[0100] In certain embodiments, cells for cell therapy benefit from an increase in the expression and / or activity of BTG1, or the recipient individual's endogenous cells have an increase in the expression and / or activity of BTG1. In particular embodiments, by way of example, such cells are those for use in the therapy of any one or more autoimmune diseases. The cells can have an increase in expression and / or activity by any suitable means. In particular embodiments, the cells have an increase in the expression of the BTG1 gene, the cells have an increase in the activity of the endogenous BTG1 protein, or both, and in particular embodiments, the expression can be inducible to avoid potential toxicity. In embodiments in which the cells have an increase in the expression of the protein, it can be from the expression of a heterologous BTG1 gene (e.g., on a vector). In some embodiments, the increase in the expression of the BTG1 gene comes from an increase in the expression of the endogenous BTG1 gene in the cells, such as after introducing a promoter into a regulatory region or other region capable of regulating the endogenous BTG1 gene. For example, a constitutive promoter can be capable of being introduced into the regulatory region of the endogenous BTG1 gene, such as the SV40, CMV, UBC, EF1A, PGK promoters.

[0101] In the case of introducing heterologous BTG1 into cells on a vector, those skilled in the art will be able to construct the vector by standard recombinant techniques. Vectors include, but are not limited to: plasmids, cosmids, viruses (phages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., derived from Moloney murine leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenovirus (Ad) vectors (including their replication-competent, replication-deficient, and empty forms), adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus vectors, herpesvirus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, maraba virus vectors, etc.

[0102] In particular embodiments, the vector is a polycistronic vector, and in such cases, a single vector can encode the BTG1 gene and one or more heterologous proteins, such as one or more CARs and / or TCRs, suicide genes, one or more cytokines, etc.

[0103] In certain aspects of the present disclosure, viral vectors encoding one or more gene products encompassed herein can be provided. In generating recombinant viral vectors, non-essential genes are typically replaced with genes or coding sequences for heterologous (or non-native) proteins. Viral vectors are a class of expression constructs that utilize viral sequences to introduce nucleic acids and possibly proteins into cells. The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis and integrate into the host cell genome and stably and efficiently express viral genes has made them attractive candidates for transferring foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver nucleic acids for certain aspects of the present invention include retroviral vectors, lentiviral vectors, adenovirus vectors, and adeno-associated virus vectors.

[0104] V. Heterologous Proteins

[0105] In particular embodiments, any cell of the present disclosure is modified to express one or more heterologous proteins. In some embodiments, the heterologous protein(s) can promote the activity of the cell in any way, including at least its activation, persistence, expansion, homing, and / or cytotoxicity. Modulating the cell to include one or more heterologous proteins can occur before, during, or after modulation of the expression and / or activity of BTG1. In some cases, the one or more heterologous proteins can be introduced on the same or different vectors as one or more reagents that result in decreased BTG1 expression or activity or one or more reagents that result in increased BTG1 expression or activity.

[0106] A. Bispecific or multispecific antibodies

[0107] In some embodiments, the cell is modified to express one or more bispecific or multispecific antibodies, while in other cases, the cell does not express the antibodies but instead the antibodies are used in conjunction with the cell.

[0108] In cases where the cell is modified to express the antibody, the antibody can be an adaptor that bridges a specific immune effector cell and a specific target cell for the destruction of the target cell. In certain embodiments, the engineered cells are used in conjunction with a standard T-cell engager (BiTE) because in particular embodiments they have been modified to express CD3, which in many cases is the T-cell antigen to which the BiTE engager binds. In such cases, the BiTE can also target a cancer antigen or a viral antigen, which can be tailored to the medical condition of the intended recipient individual. For example, the BiTE can be tailored to bind a cancer antigen that is characteristic of the cancer cells of the individual.

[0109] In some cases, the cell can be an NK cell that is modified to express one or more bispecific NK engagers (BiKEs) (or does not express one or more bispecific NK engagers but is used in conjunction with them). The BiKE can comprise an antibody that binds to a surface protein on the NK cell (including a surface protein that is naturally expressed on the NK cell), and also comprises an antibody that binds to a desired target antigen. The BiKE can target the NK cell via an antibody to an NK surface protein (e.g., CD16, CS1, CD56, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR, or KIR). In such cases, the BiKEs used in the present disclosure can also target a cancer antigen or a viral antigen, which can be tailored to the medical condition of the intended recipient individual. For example, the BiKE can be tailored to bind a cancer antigen that is characteristic of the cancer cells of the individual.

[0110] B. Modified receptor

[0111] In particular embodiments, the cells are modified to express one or more modified receptors. In some cases, the modified receptor can be a modified antigen receptor targeting any type of cancer or viral antigen. The receptor can be adapted to target the desired antigen based on cells associated with the medical condition of the intended recipient individual.

[0112] 1. Chimeric antigen receptor

[0113] In some embodiments, the modified antigen receptor is a chimeric antigen receptor (CAR). The cells can be modified to encode at least one CAR, and the CAR can be, for example, a first-generation, second-generation, or third-generation or subsequent generation. The CAR can be or can not be bispecific for two or more different antigens. The CAR can comprise one or more co-stimulatory domains. Each co-stimulatory domain can comprise a co-stimulatory domain of any one or more of the following, for example: members 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 co-stimulatory domains; for example, the CAR can lack 4-1BB and / or lack CD28.

[0114] In particular embodiments, the CAR polypeptide in the cell comprises an extracellular spacer domain that links the antigen-binding domain and the transmembrane domain, and this may be referred to as a hinge. The extracellular spacer domain may include, but is not limited to: the Fc fragment of an antibody or its fragment or derivative, the hinge region of an antibody or its fragment or derivative, the CH2 region of an antibody, the CH3 region of an antibody, an artificial spacer sequence, or a combination thereof. Examples of the extracellular spacer domain include, but are not limited to: the CD8-α hinge, CD28, an artificial spacer made of a polypeptide such as Gly3, or the CH1 and CH3 domains of IgG (e.g., human IgG1 or IgG4). In particular cases, the extracellular spacer domain may 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-α or CD4, (v) the hinge, CH2, and CH3 regions of IgG1, (vi) the hinge region of IgG1, (vii) the hinge and CH2 of IgG1, (viii) the hinge region of CD28, or a combination thereof. In particular embodiments, the hinge is from IgG1, and in some aspects, the CAR polypeptide comprises a specific IgG1 hinge amino acid sequence or is encoded by a specific IgG1 hinge nucleic acid sequence.

[0115] The transmembrane domain in the CAR may be derived from a natural or synthetic source. In cases where the source is natural, in some aspects, the domain is derived from any membrane-binding protein or transmembrane protein. Transmembrane regions include those derived from (i.e., transmembrane regions that at least comprise) the α, β, or ζ chains 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 such as DAP10 or DAP12. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain mainly comprises hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan, and valine may be found at each end of the synthetic transmembrane domain.

[0116] In particular embodiments, the CAR comprises one or more activation domains such as CD3ζ, DAP12, 2B4, or a combination thereof.

[0117] In some embodiments, the engineered receptor utilizes one or more homing receptors (which can home to a target, not necessarily because of signal release, such as in the case where they utilize 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 particular cases, the chemokine receptor is a receptor for CCR2, CCR3, CCR5, CCR8, CCR7, CXCR3, L-selectin (CD62L), CXCR1, CXCR2, or CX3CR1.

[0118] 2. T cell receptor (TCR)

[0119] In some embodiments, the engineered antigen receptor includes 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 α and β chains (also referred to as TCRα and TCRβ, respectively) or variable γ and δ chains (also referred to as TCRγ and TCRδ, respectively) and is capable of specifically binding to an antigen peptide bound to an MHC receptor. In some embodiments, the TCR is in the α / β form.

[0120] Typically, TCRs that exist in the α / β and γ / δ forms are generally structurally similar, but the T cells that express them can have different anatomical localizations or functions. TCRs can be found on the surface of a cell or in a soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes), where it is typically responsible for recognizing an antigen bound to a major histocompatibility complex (MHC) molecule. In some embodiments, the TCR can also comprise a constant domain, a transmembrane domain, and / or a short cytoplasmic tail region. For example, in some aspects, each chain of the TCR can have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail region at the C-terminus. In some embodiments, the TCR associates with invariant proteins of the CD3 complex involved in mediating signal transduction. Unless otherwise specified, the term "TCR" should be understood to encompass its functional TCR fragments. The term also encompasses full-length or complete TCRs, including TCRs in the α / β form or γ / δ form.

[0121] Thus, for the purposes herein, reference to a TCR includes any TCR or functional fragment, such as the antigen-binding portion of a TCR, which binds to a specific antigenic peptide (i.e., MHC-peptide complex) bound in an MHC molecule. The "antigen-binding portion" or "antigen-binding fragment" of a TCR (which may be used interchangeably) refers to a molecule that includes a portion of the structural domains of a TCR, but binds the antigen (e.g., MHC-peptide complex) to which the intact TCR binds. In some cases, the antigen-binding portion includes the variable domains of a TCR, such as the variable alpha and variable beta chains of a TCR, which are sufficient to form a binding site for binding to a specific MHC-peptide complex, such as typically where each chain includes three complementarity determining regions.

[0122] In some embodiments, the variable domains of the TCR chains associate to form loops or complementarity determining regions (CDRs) similar to immunoglobulins, which confer antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule. Typically, like immunoglobulins, the CDRs are separated by framework regions (FRs). In some embodiments, CDR3 is the major CDR responsible for recognizing the processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of the antigenic peptide, and CDR1 of the beta chain has been shown to interact with the C-terminal portion of the peptide. CDR2 is thought to recognize the MHC molecule. In some embodiments, the variable region of the beta chain may include a further hypervariable (HV4) region.

[0123] In some embodiments, the TCR chains include constant domains. For example, like immunoglobulins, the extracellular portion of a TCR chain (e.g., alpha-chain, beta-chain) may include two immunoglobulin domains, a variable domain at the N-terminus (e.g., V a or Vp; typically, amino acids 1 to 116 based on Kabat numbering, Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.), and a constant domain adjacent to the cell membrane (e.g., alpha-chain constant domain or C a, typically, based on Kabat amino acids 117 to 259; the β-chain constant domain or Cp, typically, based on Kabat amino acids 117 to 295). For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains and two membrane-distal variable domains containing CDRs. The constant domains of the TCR domains contain short linker sequences in which cysteine residues form disulfide bonds, thereby constituting the connection between the two chains. In some embodiments, the TCR can have additional cysteine residues in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant domain.

[0124] In some embodiments, the TCR chain can contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail region. In some cases, this structure allows the TCR to associate with other molecules such as CD3. For example, a TCR containing a constant domain with a transmembrane region can be anchored to a protein in the cell membrane and associate with an invariant subunit of the CD3 signal transducer or complex.

[0125] Typically, CD3 is a multi-protein complex that can have three different chains (γ, δ, and ε) (in mammals) and the ζ-chain. For example, in mammals, the complex can contain one CD3γ chain, one CD3δ chain, two CD3ε chains, and a homodimer of the CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif (called the immunoreceptor tyrosine-based activation motif or ITAM), while each CD3ζ chain has three. Typically, ITAM is involved in the signaling capacity of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in transmitting signals from the TCR into the cell. The CD3- chains and the ζ-chain together with the TCR form the so-called T cell receptor complex.

[0126] In some embodiments, the TCR can be a heterodimer of two chains, α and β (or optionally γ and δ), or it can be a single-chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two separate chains (α and β chains or γ and δ chains) that are linked (e.g., by a disulfide bond). In some embodiments, a TCR specific for a target antigen (e.g., a cancer antigen) is identified and introduced into a cell. In some embodiments, the nucleic acid encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, such as a cell, e.g., a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available sources. In some embodiments, the T cells can be obtained from cells isolated in vivo. In some embodiments, high-affinity T cell clones can be isolated from a patient, and the TCRs isolated. In some embodiments, the T cells can be cultured T cell hybridomas or clones. In some embodiments, TCR clones specific for a target antigen are generated in transgenic mice engineered with human immune system genes (e.g., the human leukocyte antigen system or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate TCRs specific for a target antigen (see, e.g., Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, the TCR or an antigen-binding portion thereof can be generated synthetically from knowledge of the sequence of the TCR.

[0127] C. Cytokines

[0128] In some embodiments, the cells are engineered to express one or more heterologous cytokines and / or are engineered to upregulate the normal expression of one or more heterologous cytokines. The cells may or may not be transduced or transfected for one or more cytokines on the same vector as other heterologous genes (e.g., CAR).

[0129] One or more cytokines can be co-expressed from the vector, including polypeptides separate from any component of the heterologous gene product. For example, interleukin-15 (IL-15) is tissue-restricted and is only observable in serum or systemically at any level under pathological conditions. IL-15 has several properties desirable for adoptive therapy. IL-15 is a homeostatic cytokine that induces the development and cell proliferation of natural killer cells, promotes the eradication of established tumors by alleviating the functional inhibition of tumor-resident cells, and inhibits activation-induced cell death (AICD). In addition to IL-15, other cytokines are contemplated. These include, but are not limited to: cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells for human applications. Cells expressing IL-15 are capable of sustained supportive cytokine signaling, which is useful for their survival after infusion.

[0130] In particular embodiments, the cells express one or more exogenously provided cytokines. As an example, the cytokine is IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, GMCSF, or a combination thereof. The cytokine can be provided exogenously to the NK cells as it is expressed from an expression vector within the cells. In an alternative case, endogenous cytokines in the cells are upregulated after manipulating the expression regulation of the endogenous cytokines (e.g., genetic recombination at the promoter site of the cytokine). In the case where the cytokine is provided to the cells on an expression construct, the cytokine can be encoded from the same vector as one or more components of the CD3 complex with or without the TCR complex.

[0131] D. Antigen

[0132] The engineered antigen receptors and antibodies encompassed by the present disclosure can target one or more specific antigens. Among the antigens targeted by the antibodies and / or engineered antigen receptors are those expressed in the context of a disease, condition, or cell type to be targeted via adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematological cancers, cancers of the immune system, such as lymphoma, leukemia, and / or myeloma, such as B, T, and myeloid leukemia, lymphoma, and multiple myeloma. In some embodiments, the antigen is selectively expressed or overexpressed on the cells of the disease or condition (e.g., tumor or pathogenic cells) as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on the engineered cells.

[0133] Any suitable antigen can be targeted in the method of the present invention. In some cases, the antigen may be associated with certain cancer cells and not with non-cancerous cells. Exemplary antigens include, but are not limited to, antigenic molecules from infectious agents, auto / self-antigens, tumor / cancer-associated antigens, and tumor neoantigens. In particular embodiments, the antigen targeted by the engineered receptor is GD2, CD19, GPC3, and / or B7-H3.

[0134] In particular aspects, the antigens include: 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, CD33, 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, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, DAM-10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-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, phosphoinositide 3-kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, beta-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 signal transducer 1 (TACSTD1), TACSTD2, receptor tyrosine kinases (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), signal transducer and activator of transcription STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor-κB (NF-κB), Notch receptors (e.g., Notch1-4), NY-ESO 1, c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK), and its regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-α, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoint, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, 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, XAGE 1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, and LRRN1.,

[0135] Tumor-associated antigens can be derived from prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, kidney cancer, mesothelioma, ovarian cancer, liver cancer, brain cancer, bone cancer, gastric cancer, spleen cancer, testicular cancer, cervical cancer, anal cancer, gallbladder cancer, thyroid cancer, or melanoma. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE 1, MAGE 3, and MAGE 4 (or other MAGE antigens such as those disclosed in International Patent Publication No. WO 99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY-ESO-1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types (e.g., melanoma, lung cancer, sarcoma, and bladder cancer). See, for example, U.S. Patent No. 6,544,518. Prostate cancer tumor-associated antigens include, for example, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostate acid phosphatase, NKX3.1, and prostate six-transmembrane epithelial antigen (STEAP).

[0136] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. Additionally, tumor antigens can be autologous peptide hormones, such as full-length gonadotropin-releasing hormone (GnRH) (a short 10-amino acid peptide), which is useful in the treatment of many cancers.

[0137] Antigens can include epitope regions or epitope peptides derived from genes mutated in tumor cells or from genes transcribed at different levels in tumor cells compared to normal cells, such as telomerase, survivin, mesothelin, mutant ras, bcr / abl rearrangement, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and aberrantly expressed intronic sequences such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myeloma and B-cell lymphoma; tumor antigens containing epitope regions or epitope peptides derived from tumor virus processes, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; non-mutated carcinoembryonic proteins with tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[0138] E. Suicide genes

[0139] In particular embodiments, a suicide gene is used in conjunction with the cell therapy to control its use and allow termination of the cell therapy at a desired event and / or time. A suicide gene is employed in the transduced cells to induce death of the transduced cells when needed. Cells of the present disclosure that have been modified to possess one or more vectors (e.g.,) covered by the present disclosure can contain one or more suicide genes. In some embodiments, the term "suicide gene" as used herein is defined as a gene that effects a conversion of a gene product to a compound that kills its host cell upon administration of a prodrug or other reagent. In other embodiments, the suicide gene encodes a gene product that is targeted by a reagent (e.g., an antibody) that targets the suicide gene product when desired.

[0140] In some cases, the cell therapy can be subjected to the use of one or more suicide genes of any kind when an individual who is receiving and / or has received the cell therapy exhibits one or more symptoms of one or more adverse events (e.g., cytokine release syndrome, neurotoxicity, allergic / anaphylactic reactions, and / or on-target / off tumor toxicity (by way of example)) or is considered at risk (including emergently) of having one or more of such symptoms. The use of the suicide gene can be part of a planned protocol of the therapy or can be used only when it is recognized that it is needed. In some cases, the cell therapy is terminated by using a reagent that targets the suicide gene or the gene product therefrom because the therapy is no longer needed.

[0141] The utilization of the suicide gene can be initiated at the onset of at least one adverse event for the individual, and that adverse event can be identified in any manner, including according to regular monitoring that can or can not be continuous since the start of the cell therapy. The adverse event can be detected after examination and / or testing. In the case where the individual has cytokine release syndrome (which can also be referred to as cytokine storm), the individual can, for example, have elevated inflammatory cytokines (by way of example only: interferon-γ, granulocyte-macrophage colony-stimulating factor, IL-10, IL-6, and TNF-α); fever; fatigue; hypotension; hypoxia; tachycardia; nausea; capillary leak; heart / kidney / liver dysfunction; or a combination thereof. In the case where the individual has neurotoxicity, the individual can have confusion, delirium, hypoplasia, and / or seizures. In some cases, the individual is tested 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.

[0142] Examples of suicide gene / prodrug combinations that can be used are 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 cytarabine. The Escherichia coli purine nucleoside phosphorylase can be utilized, which is a so - called suicide gene that converts the prodrug 6 - methylpurine deoxyribonucleoside to the toxic purine 6 - methylpurine. Other suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzyme (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), xanthine guanine ribosyltransferase (XGRTP), glycosidase, methionine - α,γ - lyase (MET), EGFRv3, and thymidine phosphorylase (TP), by way of example.

[0143] VI. Infectious Diseases

[0144] In some embodiments, the cells for cell therapy are engineered to have reduced expression of BTG1, and these cells are then used in the therapy of one or more chronic or acute infectious diseases. The types of infectious diseases can be caused by the following: 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 of infectious origin 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.

[0145] In particular embodiments, a therapeutically effective amount of cells, such as certain immune cells, are administered to an individual having an acute or chronic infectious disease, wherein BTG1 is inducible and / or its activity is increased by the administration of a drug. In some embodiments, the cells are also engineered to express one or more heterologous proteins, such as engineered antigen receptors.

[0146] VII. Autoimmune Diseases

[0147] In some embodiments, an individual is in need of a therapy for an autoimmune disease, such as an autoimmune disease that would benefit from cells in which BTG1 is increased in expression and / or activity. In some cases, a therapeutically effective amount of the engineered cells in which BTG1 is increased in expression and / or activity (above levels of cells not engineered in the same manner) is administered to an individual having an autoimmune disease (including one or more symptoms of an autoimmune disease). In particular embodiments, the expression of BTG1 is inducible, for example to prevent toxicity. In other cases, the expression and / or activity of BTG1 is increased in an individual having an autoimmune disease, for example with a drug.

[0148] Non-limiting examples of autoimmune diseases: 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, dermatitis herpetiformis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, 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, lichen planus, lupus erythematosus, Ménière's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes, myasthenia gravis, nephrotic syndrome (e.g., minimal change disease, focal glomerulosclerosis or membranous nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-person syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitis (e.g., polyarteritis nodosa, Takayasu arteritis, temporal arteritis / giant cell arteritis, or dermatitis herpetiformis, vasculitis), vitiligo, and Wegener's granulomatosis. Thus, some examples of autoimmune diseases that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Crohn's disease, ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis.

[0149] VIII. Administration of the Therapeutic Composition

[0150] In various embodiments, BTG1-modulated cells are administered to an individual in need thereof. Embodiments of the present disclosure relate to methods for treating or preventing medical diseases or disorders (including cancer, acute or chronic infectious diseases, or autoimmune diseases) using compositions comprising the BTG1-modulated cells provided herein. The methods include administering to the subject a therapeutically effective amount of the cells, thereby treating or preventing the disease in the subject, including reducing the risk of the disease, reducing the severity of the disease, and / or delaying the onset of the disease. In certain embodiments of the present disclosure, cancer or an infection or disease is treated by transferring a composition comprising the cell population.

[0151] Cancers for which embodiments of the present treatment methods are useful include any malignant cell type, such as those found in solid tumors or hematological malignancies. Exemplary solid tumors can include, but are not limited to, tumors of organs selected from the group consisting of: pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological malignancies include tumors of the bone marrow, T or B cell malignancies, leukemia, lymphoma, blastoma, myeloma, and the like. Further examples of cancers that can be treated using the methods provided 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 or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0152] The cancer can particularly be of the following histological types, although not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelioma; basal cell carcinoma; trichoblastoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; oxyphilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; ceruminous gland carcinoma; 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, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipoid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomus sarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo maligna melanoma; acral lentiginous melanoma; nodular melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; müllerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor; phyllodes tumor, malignant; synovial sarcoma; mesothelioma; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; hemangioendothelioma, malignant; kaposi sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligoastrocytoma; primary neuroectodermal tumor; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma;Retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granulocytic sarcoma, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin; granulomatosis with polyangiitis; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin lymphoma; B-cell lymphoma; low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate / follicular NHL; intermediate diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenström macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; 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 myelogenous leukemia.

[0153] The therapies provided herein can include administering a combination of therapeutic agents, such as a first cancer therapy and a second cancer therapy. The therapies can be administered in any suitable manner known in the art. For example, the first and second cancer treatments can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first and second cancer treatments are administered in separate compositions. In some embodiments, the first and second cancer treatments are in the same composition. Embodiments of the present disclosure relate to compositions and methods that comprise a therapeutic composition. Different therapies can be administered in one composition or in more than one composition (e.g., 2 compositions, 3 compositions, or 4 compositions). Various combinations of the agents can be employed. Examples of other therapies besides those of the present disclosure include surgery, chemotherapy, pharmaceutical therapy, radiation, hormone therapy, immunotherapy (other than that of the present disclosure), or combinations thereof.

[0154] The therapeutic agents of the present disclosure can be administered by the same route of administration or by different routes of administration. In some embodiments, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly or intranasally. Suitable dosages can be determined based on the type of disease to be treated, the severity and course of the disease, the clinical condition of the individual, the clinical history of the individual and the response to treatment, and the judgment of the attending physician.

[0155] The amount to be administered (depending on the number of treatments and the unit dose) depends on the desired therapeutic effect. An effective dose is understood to mean the amount necessary to achieve a particular effect. The treatment can include various "unit doses". A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount to be administered and the particular route and formulation are within the decision-making skills of a person skilled in the clinical art. The unit dose does not need to be administered as a single injection, but can include a continuous infusion over a set period of time. In some embodiments, the unit dose contains a single administrable dose.

[0156] A therapeutically effective amount of immune cells can be administered by a number of routes, including parenteral administration, such as intravenous, intraperitoneal, intramuscular, intrasternal or intra-articular injection or infusion.

[0157] A therapeutically effective amount of immune cells for use in adoptive cell therapy is the amount that achieves the desired effect in the subject being treated. In embodiments where the subject being treated has cancer or an acute or chronic infectious disease, this can be the amount of such modified cells that is necessary to improve one or more symptoms, including to inhibit the progression of the disease or cause the regression of one or more tumors. In embodiments where the subject being treated has an autoimmune disease, this can be the amount of such modified cells that is necessary to inhibit the progression or cause the regression of the autoimmune disease, or that is capable of alleviating one or more symptoms caused by the autoimmune disease, such as pain and / or inflammation. It can be the amount necessary to alleviate symptoms associated with inflammation, such as pain, edema and elevated body temperature. It can also be the amount necessary to reduce or prevent rejection of a transplanted organ.

[0158] The BTG1-modulated cell population can be administered in a treatment regime consistent with the disease, for example, in one to several doses over a period of one to several days to improve the disease state, or in regular doses over an extended period of time to inhibit disease progression and prevent disease recurrence. The precise dose to be employed in the formulation may 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 attending physician and the circumstances of each patient. The therapeutically effective amount of cells can depend on the subject being treated, the severity and type of the affliction, and the mode of administration. In some embodiments, the doses that can be used in the treatment of human subjects can vary from at least 1×10 4 to at least 1×10 5 to at least 1×10 6 to at least 1×10 7 to at least 1×10 8 to at least 1×10 9 or at least 1×10 10 cells / m 2 . The doses can vary from 1×10 4 to 1×10 10 , including from 1×10 5 to 1×10 10 , or from 1×10 6 to 1×10 10 , or from 1×10 7 to 1×10 10 , or from 1×10 8 to 1×10 10 , or from 1×10 9 to 1×10 10 etc. The exact amount of immune cells can be readily determined by those skilled in the art based on the age, weight, sex, and physiological condition of the subject. The effective dose can be extrapolated from a dose-response curve derived from in vitro or animal model test systems.

[0159] The cells can be administered in combination with one or more other therapeutic agents for treating any disorder. The combination therapy will depend on the disorder and can include, but is not limited to: one or more antimicrobial agents (e.g., antibiotics, antiviral agents, and antifungal agents), anti-tumor agents (e.g., fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressive agents (e.g., fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressive agents (e.g., azathioprine, or glucocorticoids such as dexamethasone or prednisone), anti-inflammatory agents (e.g., glucocorticoids such as hydrocortisone, dexamethasone, or prednisone, or non-steroidal 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 tolerogenic agents can be administered, including but not limited to: calcium-dependent phosphatase inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors (e.g., rapamycin); mycophenolate mofetil, antibodies (e.g., those that recognize CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., methotrexate, treosulfan, busulfan); radiation; or chemokines, interleukins, or their inhibitors (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors). Such additional pharmaceutical agents can be administered before, during, or after the administration of the immune cells, depending on the desired effect. The administration of the cells and the agents can be by the same route or by different routes, and at the same site or at different sites.

[0160] In certain embodiments, the compositions and methods of the present embodiments relate to populations of immune cells in combination with at least one additional therapy. The additional therapy can be radiotherapy, surgery (e.g., mastectomy and lumpectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the above. The additional therapy can be in the form of adjuvant or neoadjuvant therapy.

[0161] In particular embodiments, any of the cells of the present disclosure can be obtained from a suitable stock prior to modulation. The cells in the stock may or may not already have modulation of BTG1 expression and / or activity. The cells in the stock may or may not already have expression of any one or more heterologous genes. In some embodiments, any of the cells of the present disclosure are obtained from an individual in need of the therapy, modulated ex vivo with respect to BTG1 expression and / or activity, optionally modulated to express one or more heterologous genes, and administered back to the individual. In such cases, the step of modulating the cells with respect to BTG1 may or may not precede the step of modulating the cells with respect to the one or more heterologous genes.

[0162] IX. Kits

[0163] Certain aspects of the present disclosure also relate to kits that include a composition of the present disclosure or a composition for practicing a method of the present disclosure. In particular embodiments, the kit includes cells that are fresh or frozen and may or may not already be pre-activated or expanded. The cells may or may not already express one or more components of the elements encompassed herein, such as reagents, cells, vectors, buffers, primers, enzymes, salts, etc. used in reducing or increasing BTG1 expression or activity. The kit may include one or more reagents for transfecting or transducing cells, including reagents such as vectors expressing the components, primers for amplifying the components, etc. In some cases, the cells may or may not also express one or more heterologous proteins as defined herein, and when they do not, the kit may include vectors expressing the heterologous proteins, primers for amplifying the heterologous proteins, etc.

[0164] The kit may include components that can be individually packaged or placed in a container (e.g., a tube, bottle, vial, syringe, or other suitable container means). The individual components may also be provided in concentrated amounts in the kit; in some embodiments, the components may be provided individually at the same concentration as they would be in a solution with other components. The concentration of the components may be provided, for example, as 1x, 2x, 5x, 10x, or 20x or higher.

[0165] X. Examples

[0166] The following examples are included to demonstrate preferred embodiments of the present invention. Those skilled in the art will recognize that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present invention and, thus, can be considered to constitute preferred modes for the practice of the present invention. However, from the present disclosure, those skilled in the art will recognize that many changes can be made in the particular embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention.

[0167] Example 1

[0168] Identification of B-cell translocation gene 1 (BTG1) as a key driver of hyporesponsiveness in exhausted NKT and T cells and its use for cancer immunotherapy

[0169] T cell exhaustion is an active process characterized by the progressive loss of effector function and proliferative capacity due to prolonged antigen stimulation that occurs in acute or chronic infections and cancer. Therapeutic immune cells such as T cells or NKT cells engineered to express a tumor-specific chimeric antigen receptor (CAR) (as an example) also undergo exhaustion, which limits their anti-tumor efficacy and is associated with tumor escape and disease progression or recurrence. Although certain gene expression and epigenetic changes have been implicated in the T cell exhaustion process, the exact mechanisms responsible for the hyporesponsiveness of exhausted cells remain uncertain. To gain insight into the exhaustion mechanisms in clinically relevant effector cells, single-cell RNA sequencing (scRNAseq) of CAR NKT cells from 12 patients with neuroblastoma (NB) was performed during a Phase I clinical trial (NCT03294954). Changes in CAR-NKT gene expression were evaluated after their infusion into patients or multiple rounds of in vitro tumor cell attack (repeated tumor attack (RTC) assay). Compared to the infused product CAR-NKT, CAR-NKT after 5 RTC rounds and peripheral blood CAR-NKT 2 weeks after infusion had similar directional changes in their gene expression profiles, exemplified by the acquisition of terminal effector differentiation and the loss of naive / memory programs. These findings support the five-cycle RTC (repeated tumor attack assay) as a model that closely recapitulates the gene expression changes that occur in CAR-NKT in patients after infusion. Comparison of the gene expression profiles of the infused product with five-cycle RTC CAR-NKT revealed significant divergence between the two groups, with 2904 differentially expressed genes identified.

[0170] A notable gene significantly upregulated in RTC CAR-NKT is B-cell translocation gene 1 (BTG1), which promotes mRNA deadenylation and degradation and has recently been described as a mediator of quiescence in naive murine T cells. The role of this gene in T cell exhaustion has not been described. Consistent with the previously reported rapid downregulation of BTG1 in murine naive T cells in response to TCR stimulation, BTG1 expression was downregulated relative to unstimulated human NKT or naive T cells during the first four to six hours after TCR stimulation. However, continuous TCR stimulation with CD3 / CD28-specific monoclonal antibodies resulted in the sustained upregulation of BTG1 in both NKT and T cells, as measured at the protein level by Western blot. Overexpression of BTG1 in NKT induced a limited number of differentially expressed genes but resulted in a global decrease in RNA content in the cells and reduced proliferative capacity. The differentially expressed genes did not overlap with previously identified regulators of T exhaustion (i.e., TOX), which in some embodiments indicates a unique mechanism of BTG1-mediated hyporesponsiveness in NKT and T cells. NKT engineered to co-express a tumor-specific CAR and BTG1-specific shRNA eradicated metastatic NB in mice. Thus, this study reveals an unexpected role of BTG1 as a key regulator of NKT and T cell function. In particular embodiments, like naive T cells, exhausted T cells and NKT cells upregulate BTG1 to induce global mRNA degradation, thus contributing to a quiescent or hyporesponsive state shared at least by naive T cells and exhausted T cells or NKT cells. Finally, BTG1 downregulation strongly enhances the anti-tumor activity of CARredirected NKT, which is useful for guiding the rational design of, for example, next-generation cancer immunotherapy products.

[0171] Example 2

[0172] Role of BTG1 in hyporesponsiveness in exhausted NKT and T cells and its use in cancer immunotherapy

[0173] This example provides proof of BTG1 associated with exhausted immune cells and its consequent role in adoptive cell therapy.

[0174] Figure 1A shows an embodiment of a tumor co-culture system in which CAR-NKT is re-plated with fresh CHLA255 NB tumor cells every five days for multiple cycles, thereby inducing exhaustion in CAR-NKT via chronic antigen exposure. The cytotoxic activity of CAR-NKT during repeated co-culture at the indicated time points shows that, over time, cytotoxicity becomes reduced for many patients (Figure 1B). Uniform Manifold Approximation and Projection (UMAP) projections of scRNAseq results from the following are shown in Figure 1C: the infusion product (IP), co-culture of the repeated infusion product with tumor cells (5RcC) for five cycles, and CAR-NKT isolated from peripheral blood (PB) after infusion; and UMAP projections of CAR-NKT gene expression by scRNAseq from pre-infusion and 5RcC samples are shown in Figure 1D. Figure 1E shows a volcano plot that reveals differentially expressed genes in CAR-NKT compared to the pre-product after 5RcC, including BTG1. BTG1 expression is elevated in exhausted CAR-NKT.

[0175] Figure 2A provides an embodiment of the design of a retroviral construct encoding BTG1.GFP or GFP only (as a control) for evaluating the effect of BTG1 overexpression (OE) in NKT. The role of BTG1 in regulating the anti-tumor properties of NKT was evaluated. BTG1 protein expression by qPCR and Western blot at the indicated time points (Figure 2B and 2C, respectively). Pathway enrichment analysis that indicates gene expression programs enriched in BTG1 OE NKT (Figure 2D). The fold expansion of BTG1 OE vs. control NKT was evaluated in six independent donors (Figure 2E). Overexpression of BTG1 reduces global RNA expression and proliferative capacity in NKT.

[0176] After activation with CD3 / CD28-specific monoclonal antibodies, BTG1 protein expression was measured by Western blot and BTG1 mRNA was measured by qPCR (Figure 3B and 3C).

[0177] Figure 4A-4B BTG1 expression in T cells after activation was revealed. Peripheral blood T cells were stimulated with plate-bound CD3 / CD28 antibodies and cultured in the presence of IL2. BTG1 expression was measured by Western blot at the indicated time points (Figure 4A). The absolute numbers of BTG1 OE vs. GFP control T cells were determined after ex vivo culture (Figure 4B).

[0178] Knockdown of BTG1 in GD2-CAR NKT is shown in Figure 5. MicroRNAs targeting BTG1 and scrambled controls were cloned into an MMuLV-based γ-retroviral construct, downstream of GD2-CAR (as just an example of a CAR). Figure 5A illustrates an example of the retroviral construct design for BTG1 knockdown. The 14g2a scFv for GD2 binding was used in all construct examples, as were the CD8 hinge and transmembrane domains, the CD28 co-stimulatory domain, and CD3ζ. In some cases, IL-15 was used for enhanced activity. In some cases, a control scrambled miRNA (SCR) was included in the construct, while in other cases, the BTG1 miRNA for knockdown was used in the construct. Figures 5B and 5C show the BTG1 transcript levels and protein expression in NKT expressing the indicated constructs, quantified by qPCR and Western blot, respectively.

[0179] NKT were transduced with retroviral vectors encoding CAR + / - IL15 and / or artificial microRNA (amiR) specific to BTG1 or scrambled controls. The fold expansion of NKT expressing the indicated constructs after transduction is provided in Figure 6A. The CD62L frequency after transduction in CAR.15 NKT with or without BTG1 KD is shown in Figure 6B. CD62L expression in CAR.15.amiR-BTG1 NKT gated for CAR+ and CAR- populations is provided in Figure 6C. Figure 6D shows the frequency of PD-1+ CAR.15 NKT with and without BTG1 KD. Figure 6E provides the cytotoxic activity of CAR.15.amiR.BTG1 vs. scrambled control NKT against GD2-high CHLA255 and GD2-low CHLA136 NB cell lines, evaluated at the indicated co-culture time points. The frequency of residual tumor cells after a five-day co-culture of the indicated NKT groups and CHLA255 cells at an E:T ratio of 1 to 5 is shown (Figure 6F). The fold expansion of CAR-NKT cells after six cycles of co-culture with NB cells is provided in Figure 6G. An exemplary experimental design for in vivo evaluation of CAR-NKT anti-tumor activity in an aggressive metastatic NB xenograft model is provided in Figure 6H. Bioluminescence images of tumor-bearing mice at the specifically designated time points are shown (Figure 6I). The change in tumor burden over time based on bioluminescence images (Figure 6J). Kaplan-Meier survival curves for the mice in the indicated groups, as shown in Figure 6K. As provided herein, BTG1 knockdown enhances the anti-tumor activity of GD2-CAR NKT.

[0180] BTG1 knockdown (KD) enhances the anti-tumor activity of GD2-CAR T cells (as an example) after multiple rounds of in vitro tumor challenge. The fold expansion of GD2-CAR T cells after three rounds of co-culture with CHLA255 neuroblastoma (NB) cells is provided in Figure 7A (E:T = 1:1, N = 6, two-tailed paired t-test). The change in CAR% of T cells before or after three rounds of co-culture with CHLA255 NB cells is provided in Figure 7B.

[0181] BTG1 KD also enhances the anti-tumor activity of GD2-CAR T cells in an aggressive in vivo metastatic NB xenograft model. Figure 8A shows an example of the experimental design for in vivo evaluation of GD2-CAR T cell anti-tumor activity in the model. An example of a schematic of retroviral constructs for BTG1 KD and scrambled miRNA control (see also Figure 5A). Figure 8C shows bioluminescence images of tumor-bearing mice at weeks 4, 5, 6, and 7. The change in tumor burden based on the bioluminescence images in Figure 8C is shown graphically in Figure 8D, and a Kaplan-Meier survival curve for the mice is also provided (Figure 8E). Quantification of human T cells (human CD45+ in the total cell population collected from mouse blood on day 10) is provided (Figure 8F).

[0182] CRISPR Cas9-mediated BTG1 deletion with a marker (CD34) knock-in (KI) at the BTG1 locus in T cells increases the frequency of memory T cells. Figure 9A shows an example of the experimental design for using the CRISPR method to KI the CD34-Q8 tag at the BTG1 locus. Representative flow cytometry of CD34-Q8 tag expression in T cells five days after CRISPR KI is shown. Figure 9C shows BTG1 protein expression in T cells by Western blot seven days after CRISPR KI. Representative plots and summaries are provided in Figure 9D, which show the expression of the memory markers CD45RA and CCR7 in T cells with CD34-Q8 tag KI at the BTG1 locus and with only Cas9 control.

[0183] ***

[0184] All methods disclosed and claimed herein can be made and executed in accordance with the present disclosure without undue experimentation. While the compositions and methods of the invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made to the methods described herein and to the steps or the order of the steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically related reagents can be substituted for the reagents described herein and that the same or similar results will be achieved. All such similar substitutes and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims.

Claims

1. A method of enhancing cell therapy for an individual, comprising the step of reducing the expression and / or activity of B-cell translocation gene 1 (BTG1) in cells for said cell therapy.

2. A method of enhancing cell therapy for an individual, comprising the step of reducing the expression and / or activity of B-cell translocation gene 1 (BTG1) in cells, wherein said cells are not T cells.

3. The method according to claim 1, wherein said cells are immune cells, stem cells, one or more of their derivatives, or a mixture thereof, optionally when said derivatives are iPSC-derived T, NKT or NK cells.

4. The method according to claim 1 or 3, wherein said 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. The method according to any one of claims 1-4, wherein said cells are modified to express one or more heterologous genes.

6. The method according to claim 5, wherein said heterologous genes comprise one or more engineered receptors, antibodies, cytokines, suicide genes, co-stimulatory factors, regulatory factors or combinations thereof.

7. The method according to claim 6, wherein said engineered receptor is an antigen receptor or a cytokine receptor.

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

9. The method according to claim 8, wherein said CAR comprises 1, 2 or more co-stimulatory domains.

10. The method according to claim 9, wherein said co-stimulatory domains comprise CD28, 4-1BB, OX40, CD2, DAP10, CD40, ICOS, CD27, TLR, MYD88, 2B4, NKG2D or combinations thereof.

11. The method according to any one of claims 7-10, wherein said antigen receptor targets GD2, CD19, GPC3 and / or B7-H3.

12. The method according to any one of claims 8-11, wherein said CAR comprises one or more activation domains.

13. The method according to claim 12, wherein said one or more activation domains comprise CD3ζ, DAP12, 2B4 or combinations thereof.

14. The method according to any one of claims 6-13, wherein said cytokine is IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-33 or combinations thereof.

15. The method according to any one of claims 6-14, wherein said antibody is a monospecific antibody, bispecific antibody, trispecific antibody or a mixture thereof.

16. The method according to any one of claims 6-15, wherein said antibody is a bispecific T cell engager or a trispecific T cell engager.

17. The method according to any one of claims 1-16, wherein the reducing step utilizes one or more reagents to reduce the expression of the endogenous BTG1 gene in the cell.

18. The method according to claim 17, wherein the one or more reagents comprise nucleic acids, peptides, and / or polypeptides.

19. The method according to claim 17 or 18, wherein the one or more reagents comprise CRISPR reagents, miRNA, siRNA, shRNA, transposons, or a mixture thereof.

20. The method according to any one of claims 1-19, wherein the reducing step utilizes one or more reagents that reduce the activity of BTG1 protein in the cell.

21. The method according to claim 20, wherein the one or more reagents comprise one or more small molecules targeting BTG1 or one or more antibodies.

22. The method according to any one of claims 1-21, further comprising the step of administering a therapeutically effective amount of the cells to an individual in need thereof.

23. The method according to claim 22, wherein the individual has cancer.

24. The method according to any one of claims 1-17, wherein the reducing step comprises knock-in or knockdown or knockout of the endogenous BTG1 gene in the cell.

25. The method according to claim 24, wherein the knock-in comprises knock-in of a detectable marker.

26. A plurality of cells produced by the method according to any one of claims 1-25.

27. The plurality of cells according to claim 26, wherein the plurality of cells are contained in a pharmaceutically acceptable excipient.

28. Engineered non-cancerous cells, the cells being engineered to comprise a reduction in the expression and / or activity of BTG1, wherein the cells express one or more heterologous genes.

29. The cells according to claim 28, wherein the heterologous genes comprise one or more engineered receptors, antibodies, cytokines, suicide genes, co-stimulatory factors, regulatory factors, or combinations thereof.

30. The cells according to claim 28 or 29, wherein the engineered cells are immune cells or stem cells.

31. The cells according to any one of claims 28-30, wherein the cells are CD8 T cells, CD4 T cells, natural killer T (NKT) cells, NKT cells, MAIT cells, γ / δ T cells, virus-specific T cells, cytokine-induced killer cells, NK cells, macrophages, or a mixture thereof.

32. The cells according to any one of claims 28-31, wherein the reduction in expression is produced by one or more CRISPR reagents, siRNA, shRNA, transposons, or a mixture thereof.

33. The cells according to any one of claims 28-32, wherein the reduction in activity is produced by one or more small molecules.

34. The cells according to claim 29, wherein the engineered receptor is an antigen receptor or a cytokine receptor.

35. The cell according to claim 34, wherein the antigen receptor is a CAR or a T cell receptor.

36. The cell according to claim 35, wherein the CAR comprises 1, 2 or more co-stimulatory domains.

37. The cell according to claim 36, wherein the co-stimulatory domain comprises CD28, 4-1BB, OX40, CD2, DAP10, CD40, ICOS, CD27, TLR, MYD88, 2B4, NKG2D or a combination thereof.

38. The cell according to any one of claims 34-37, wherein the antigen receptor targets GD2, CD19, GPC3 and / or B7-H3.

39. The cell according to any one of claims 35-38, wherein the CAR comprises one or more activation domains.

40. The cell according to claim 39, wherein the one or more activation domains comprise CD3ζ, DAP12, 2B4 or a combination thereof.

41. The cell according to any one of claims 28-40, wherein the cytokine is IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-33 or a combination thereof.

42. The cell according to any one of claims 29-41, wherein the antibody is a monospecific antibody, a bispecific antibody or a trispecific antibody.

43. The cell according to any one of claims 29-42, wherein the antibody is a bispecific T cell engager or a trispecific T cell engager.

44. The cell according to any one of claims 28-43, wherein the reduction of the activity of BTG1 is effected by one or more small molecules or one or more antibodies targeting BTG1.

45. A plurality of cells according to any one of claims 28-44.

46. The plurality of cells according to claim 45, wherein the plurality of cells are comprised in a pharmaceutically acceptable excipient.

47. An engineered CD8 T cell, CD4 T cell, NKT cell, MAIT cell, γ / δ T cell, virus-specific T cell, cytokine-induced killer cell, NK cell, macrophage or a mixture thereof, the cell being engineered to comprise a reduction in the expression and / or activity of BTG1.

48. The cell according to claim 47, wherein the cell expresses one or more heterologous genes.

49. The cell according to claim 48, wherein the heterologous gene comprises one or more engineered receptors, antibodies, cytokines, suicide genes, co-stimulatory factors, regulatory factors or a combination thereof.

50. A plurality of cells according to any one of claims 47-49.

51. The plurality of cells according to claim 50, wherein the plurality of cells are comprised in a pharmaceutically acceptable excipient.

52. A method for treating cancer and / or acute or chronic infectious diseases in an individual, comprising the step of administering to the individual a therapeutically effective amount of any one of the plurality of cells according to claim 26, 27, 45, 46, 50 or 51.

53. The method according to claim 52, wherein the plurality of cells comprise NK cells, NK T cells and / or macrophages, which comprise GD2 CAR, GPC3 CAR and / or B7-H3 CAR.

54. The method according to claim 52 or 53, wherein the cells are autologous with respect to the individual.

55. The method according to claim 52 or 53, wherein the cells are allogeneic with respect to the individual.

56. The method according to any one of claims 52-55, wherein the infectious disease is human immunodeficiency virus, tuberculosis, herpes, viral hepatitis or COVID.

57. A method for treating an autoimmune disease in an individual, comprising the step of administering to the individual a therapeutically effective amount of cells comprising an increased inducible expression and / or activity of BTG1, and / or comprising the step of administering to the individual a therapeutically effective amount of a drug that increases the expression and / or activity of BTG1 in the endogenous cells of the individual.

58. The method according to claim 57, wherein the cells are immune cells, stem cells, one or more of their derivatives, or a mixture thereof.

59. The method according to claim 57 or 58, wherein the increased expression is from the expression of BTG1 on a vector in the cells.

60. The method according to claim 59, wherein the vector is an episomal vector.

61. The method according to claim 59, wherein the vector is an integrative vector.

62. The method according to any one of claims 57-61, wherein the increased expression is from the introduction of a heterologous promoter into the regulatory region of the endogenous BTG1 gene in the cells.

63. The method according to any one of claims 57-62, wherein the increased activity of BTG1 is from introducing a small molecule into the cells.

64. The method according to any one of claims 57-63, wherein the cells express one or more heterologous genes.

65. The method according to any one of claims 57-64, 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 / 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.

66. The method according to any one of claims 57 - 65, wherein the cells are autologous to the individual.

67. The method according to any one of claims 57 - 65, wherein the cells are allogeneic to the individual.

68. A modified cell, wherein the cell is modified to have an increased expression of endogenous BTG1 and / or comprises a vector expressing heterologous BTG1.

69. The cell according to claim 68, wherein the vector is an episomal vector.

70. The cell according to claim 68, wherein the vector is an integrative vector.

71. The cell according to any one of claims 68 - 70, wherein the increased expression results from introducing a heterologous promoter into the regulatory region of the endogenous BTG1 gene in the cell.

72. The cell according to any one of claims 68 - 71, wherein the increased activity of BTG1 results from introducing a small molecule into the cell.

73. The cell according to any one of claims 68 - 72, wherein the cell expresses one or more heterologous genes.

74. A plurality of cells according to any one of claims 68 - 73.

75. The plurality of cells according to claim 74, wherein the plurality of cells are contained in a pharmaceutically acceptable excipient.

76. A method of controlling the activity and / or toxicity of a cell therapy, which comprises the step of increasing the expression and / or activity of BTG1 in the cells of the cell therapy.

77. The method according to claim 76, wherein the increase in the expression of BTG1 is effected by an inducible promoter.

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