EP4580641A2 - Modulation du gène de translocation de lymphocytes b 1 (btg1) destinée à être utilisée dans une thérapie cellulaire adoptive - Google Patents

Modulation du gène de translocation de lymphocytes b 1 (btg1) destinée à être utilisée dans une thérapie cellulaire adoptive

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Publication number
EP4580641A2
EP4580641A2 EP23861278.2A EP23861278A EP4580641A2 EP 4580641 A2 EP4580641 A2 EP 4580641A2 EP 23861278 A EP23861278 A EP 23861278A EP 4580641 A2 EP4580641 A2 EP 4580641A2
Authority
EP
European Patent Office
Prior art keywords
cells
cell
btg1
expression
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23861278.2A
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German (de)
English (en)
Inventor
Leonid S. METELITSA
Andras HECZEY
Xin Xu
Amy N. COURTNEY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baylor College of Medicine
Original Assignee
Baylor College of Medicine
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Baylor College of Medicine filed Critical Baylor College of Medicine
Publication of EP4580641A2 publication Critical patent/EP4580641A2/fr
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4256—Tumor associated carbohydrates
    • A61K40/4258—Gangliosides, e.g. GM2, GD2 or GD3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • BCG1 B-CELL TRANSLOCATION GENE 1
  • Embodiments of the disclosure include methods and compositions for use in therapeutic cells of any kind, including immune effector cells.
  • the therapeutic cells may be tailored and utilized for a particular therapeutic application, such as for cancer treatment, and such cells of this disclosure may be modified to improve one or more activities of the cells.
  • the present disclosure generally concerns modulation of BTG1 in cells either to (1) prevent or reduce undesirable exhaustion of the cells and/or to control excessive activation with associated toxicity of the therapeutic cells in use for individuals (such as those with cancer); or (2) to modulate endogenous cells of an individual to suppress activity of autoimmune cells (such as for those with an autoimmune disorder).
  • a cell for cell therapy is prone to or otherwise has exhaustion, insufficient persistence, insufficient activation, insufficient expansion, insufficient homing, and/or insufficient cytotoxicity.
  • Such cells may be subject to modulation of BTG1 expression and/or activity in order to improve one or more of these characteristics.
  • Some types of immune cells may benefit from modulation of BTG1 expression and/or activity in order to improve one or more of these characteristics.
  • expression and/or activity of endogenous BTG1 is reduced in cells to be used for cell therapy, such as for cancer or chronic infectious disease, and such cells may or may not be particular types of immune cells.
  • T cells of any kind including CD8 T cell, CD4 T cell, 0.0 T cells, y/8 T cells, virus-specific T cells (viral examples including Epstein-Barr virus, cytomegalovirus, BK virus, human herpesvirus, adenovirus, respiratory syncytial virus, Influenza, parainfluenza virus 3, human metapneumovirus, etc.), NKT cells, MAIT cells, cytokine-induced killer cells, NK cells, macrophages, or a mixture thereof, are engineered to have reduced expression and/or activity of endogenous BTG1, and such modulation renders the cells to have reduced or no exhaustion compared to the same type of cells in the absence of such modulation.
  • virus-specific T cells viral examples including Epstein-Barr virus, cytomegalovirus, BK virus, human herpesvirus, adenovirus, respiratory syncytial virus, Influenza, parainfluenza virus 3, human metapneumovirus, etc.
  • NKT cells are engineered
  • Such reduction of expression and/or activity of endogenous BTG1 may render the modulated cells to have reduced exhaustion, enhanced persistence, enhanced activation, enhanced expansion, enhanced homing, and/or enhanced cytotoxicity compared to the same type of cells in the absence of such modulation.
  • expression and/or activity of endogenous BTG1 is increased in cells to be used for cell therapy and/or heterologous BTG1 is introduced in the cells, and such cells may be other particular types of cells.
  • cells may be other particular types of cells.
  • BTG1 upregulation is useful in endogenous autoimmune cells, such as by a drug that facilitates upregulation of BTG1.
  • increased or regulated BTG1 expression can be beneficial for controlling toxicity of therapeutic effector cells in cancer or infections (chronic or acute), such as being used as an “off-switch.”
  • the BTG1 expression may be controlled by an inducible promoter to inhibit or kill therapeutic cells of any kind encompassed herein when needed, such as at the onset of cytokine storm.
  • Embodiments of the disclosure include methods of enhancing cell therapy for an individual, comprising the step of reducing expression and/or activity of B-cell translocation gene 1 (BTG1) in the cells for the cell therapy.
  • BEG1 B-cell translocation gene 1
  • Embodiments of the disclosure include methods of enhancing cell therapy for an individual, comprising the step of reducing expression and/or activity of B-cell translocation gene 1 (BTG1) in the cells, wherein the cells are not T cells.
  • BCG1 B-cell translocation gene 1
  • the cells are immune cells, stem cells, one or more of their derivatives, or a mixture thereof, optionally when the derivatives are iPSC-derived T, NKT or NK cells.
  • the cells may be CD8 T cells, CD4 T cells, natural killer T (NKT) cells, MAIT cells, y/8 T cells, Virus-specific T cells, Cytokine-induced killer cells, NK cells, macrophages, or a mixture thereof.
  • 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.
  • the heterologous gene may comprise one or more engineered receptors, antibodies, cytokines, suicide genes, costimulatory factors regulatory factors, or a combination thereof.
  • the engineered receptor may be an antigen receptor, chemokine receptor, or a cytokine receptor, or the cell may more than one of these types.
  • An antigen receptor may be a chimeric antigen receptor (CAR) or a T cell receptor.
  • a CAR may comprise 1, 2, or more costimulatory domains, such as CD28, 4- IBB, 0X40, CD2, DAP 10, CD40, ICOS, CD27, TLR, MYD88; 2B4, NKG2D, or a combination thereof.
  • the antigen receptor may target GD2, CD19, GPC3, B7-H3, CD20, BCMA, CD30, CD38, CD5, CD7, HER2, PSMA, mesothelin, EGFR, IL13RA2, or a combination thereof, in specific embodiments.
  • the CAR may comprise one or more activating domains, such as CD3( ⁇ , DAP12, 2B4, or a combination thereof.
  • the cytokine is IL-7, IL-12, IL-15, IL- 18, IL-21, IL-23, IL-33, or a combination thereof.
  • the antibody is a monospecific antibody, a bispecific antibody, a tri-specific antibody, or a mixture thereof.
  • the antibody may be a bispecific T cell engager or a tri-specific T cell engager.
  • the reducing step utilizes one or more agents to reduce expression of the endogenous BTG1 gene in the cells.
  • the one or more agents may comprise nucleic acid, peptide, and/or polypeptides.
  • the one or more agents may comprise CRISPR agents, siRNA, shRNA, transposons, or a mixture thereof.
  • the reducing step utilizes one or more agents that reduce activity of BTG1 protein in the cells, and the one or more agents may comprise one or more small molecules or one or more antibodies that target BTG1.
  • the method may further comprise 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 acute or chronic infectious disease.
  • Embodiments of the disclosure may include a plurality of any cells encompassed herein, and the plurality may be comprised in a pharmaceutically acceptable excipient.
  • an engineered non-cancerous cell said cell engineered to comprise a reduction in expression and/or activity of BTG1, wherein the cell expresses one or more heterologous genes.
  • the heterologous gene may comprise one or more engineered receptors, antibodies, cytokines, suicide genes, costimulatory factors regulatory factors, or a combination thereof.
  • the engineered cell may be an immune cell or a stem cell.
  • the cells in specific embodiments are CD8 T cells, CD4 T cells, NKT cells, MAIT cells, y/8 T cells, Virus-specific T cells, Cytokine-induced killer cells, NK cells, macrophages, or a mixture thereof.
  • the reduction in expression was produced by one or more CRISPR agents, siRNA, shRNA, transposons, or a mixture thereof.
  • the reduction in activity may be produced by one or more small molecules.
  • the engineered receptor is an antigen receptor or a cytokine receptor as described elsewhere herein.
  • reduction in activity of BTG1 is by one or more small molecules or one or more antibodies that target BTG1.
  • Embodiments of the disclosure include an engineered CD8 T cell, CD4 T cell, NKT cell, MAIT cell, y/8 T cell, Virus-specific T cell, Cytokine-induced killer cell, NK cell, macrophage, or a mixture thereof, said cell engineered to comprise a reduction in expression and/or activity of BTG1.
  • the cell may express one or more heterologous genes, such as one or more engineered receptors, antibodies, cytokines, suicide genes, costimulatory factors regulatory factors, or a combination thereof.
  • Embodiments of the disclosure include methods of treating cancer and/or acute or chronic infectious disease in an individual, comprising the step of administering to the individual a therapeutically effective amount of any one of the plurality of cells of the disclosure.
  • the plurality of cells comprises NK cells, NK T cells, and/or macrophages comprising a GD2 CAR, a GPC3 CAR, CD 19 CAR, and/or a B7-H3 CAR.
  • the cells may be allogeneic or autologous with respect to the individual.
  • the acute or chronic infectious disease is human immunodeficiency virus, tuberculosis, herpes, viral hepatitis, or CO VID.
  • Embodiments of the disclosure include methods of treating an autoimmune disease in an individual, comprising the step of administering to the individual a therapeutically effective amount of cells comprising an increase in 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 expression and/or activity of BTG1 in endogenous cells of the individual.
  • the cells are immune cells, stem cells, one or more of their derivatives, or a mixture thereof.
  • the increase in expression may be from expression of BTG1 on a vector in the cells, and the vector may be an extrachromosomal vector or an integrating vector.
  • the increase in expression is from introduction of a heterologous promoter in a regulatory region of the endogenous BTG1 gene in the cell.
  • the increase in activity of BTG1 may be from introduction of a small molecule into the cell.
  • the cell may or may not express one or more heterologous genes.
  • the autoimmune disease is Type 1 diabetes, Lupus, Alopecia areata, Autoimmune hemolytic anemia, Autoimmune hepatitis, Dermatomyositis, Glomerulonephritis, Granulomatosis with polyangiitis, Graves’ disease Guillain-Barre 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, Sjogren’s syndrome, Systemic lupus erythematosus, thyroiditis, uveitis, or Vitiligo.
  • the cells may be allogeneic or autologous with respect to the individual.
  • Embodiments of the disclosure include an engineered cell, wherein the cell is engineered to have an increase in expression of endogenous BTG1 and/or that comprises a vector that expresses heterologous BTG1.
  • the vector may be an extrachromosomal vector or an integrating vector.
  • the increase in expression may be from introduction of a heterologous promoter in a regulatory region of the endogenous BTG1 gene in the cell.
  • the increase in activity of BTG1 may be from introduction of a small molecule into the cell, and the cell may express one or more heterologous genes.
  • Embodiments include methods of controlling activity and/or toxicity of a cell therapy, comprising the step of increasing expression and/or activity of BTG1 in the cells of the cell therapy.
  • the increase in expression of BTG1 may be by an inducible promoter.
  • Embodiments may include methods of reducing expression of the endogenous BTG1 gene in the cells, such as by using one or more agents comprise CRISPR agents, miRNA, siRNA, shRNA, transposons, or a mixture thereof, for example.
  • FIGS. 3A-3C Evaluating the role of BTG1 in regulating the antitumor properties of NKTs.
  • FIG. 3A BTG1 protein expression as determined by western blot at indicated timepoints in repeat tumor challenge assay (RTC).
  • FIG. 3B Protein expression of BTG1 measured by western blot after activation with CD3/CD28 specific monoclonal antibodies
  • FIG. 3C BTG1 mRNA expression as determined by qPCR at indicated timepoints.
  • FIGS. 4A-4B BTG1 expression in T cells upon activation: Peripheral blood T cells were stimulated with plate bound CD3/CD28 antibody and cultured in the presence of IL2.
  • FIG. 4A BTG1 protein expression measured by Western blot at indicated timepoints.
  • FIG. 4B Absolute number of BTG1 OE vs GFP control T cells after ex vivo culture.
  • FIGS. 5A-5C BTG1 KD in GD2-CAR NKTs. MicroRNAs targeting BTG1 and scrambled control were cloned into MMuLV-based gamma-retroviral constructs downstream from the GD2-CAR.
  • FIG. 5A Retroviral construct designs for BTG1 knockdown.
  • FIGS. 5B, 5C BTG1 transcript and protein expression in NKTs expressing indicated constructs quantified by qPCR and Western blot, respectively.
  • FIGS. 7A and 7B BTG1 KD enhances the antitumor activity of GD2-CAR T cells.
  • x, y, and/or z can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.
  • an "autoimmune disease” refers to a disease in which the immune system produces an immune response (for example, a B cell or a T cell response) against an antigen that is part of the normal host (that is, an autoantigen), with consequent injury to tissues.
  • An autoantigen may be derived from a host cell, or may be derived from a commensal organism such as the micro-organisms (known as commensal organisms) that normally colonize mucosal surfaces.
  • a “reduction of expression” or “disruption” or “alteration” of a gene refers to the total or partial elimination or decrease of expression of one or more gene products encoded by the subject gene in a cell, compared to the level of expression of the gene product in the absence of the alteration.
  • Exemplary gene products include mRNA and protein products encoded by the gene. Alteration in some cases is transient or reversible and in other cases is permanent. Alteration in some cases is of a functional or full length protein or mRNA, despite the fact that a truncated or non-functional product may be produced.
  • gene activity or function, as opposed to expression is disrupted.
  • Gene alteration is generally induced by artificial methods, i.e., by addition or introduction of a compound, molecule, complex, or composition, and/or by alteration of nucleic acid of or associated with the gene, such as 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 technology, such as RNAi, siRNA, shRNA, and/or ribozymes, which generally result in transient reduction of expression, as well as gene editing techniques which result in targeted gene inactivation or alteration, e.g., by induction of breaks and/or homologous recombination. Examples include insertions, mutations, and deletions.
  • the alterations typically result in the repression and/or complete absence of expression of a normal or "wild type" product encoded by the gene.
  • exemplary of such gene alterations are insertions, frameshift and missense mutations, deletions, knock-in (including knock-in with a marker, such as a CD34-Q8 tag, GFP, or another selection marker), and knock-out of the gene or part of the gene, including deletions 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, functional product, or any product, such as by insertion of a stop codon.
  • Such alterations may also occur by alterations in the promoter or enhancer or other region affecting activation of transcription, so as to prevent transcription of the gene.
  • Gene alterations include gene targeting, including targeted gene inactivation by homologous recombination.
  • engineered refers to an entity that is generated by the hand of man, including a cell, nucleic acid, polypeptide, vector, and so forth.
  • an engineered entity is synthetic and comprises elements that are not naturally present or configured in the manner in which it is utilized in the disclosure.
  • a vector is engineered through recombinant nucleic acid technologies, and a cell is engineered through transfection or transduction of an engineered vector.
  • Cells may be engineered to express heterologous proteins that are not naturally expressed by the cells, either because the heterologous proteins are recombinant or synthetic or because the cells do not naturally express the proteins.
  • An engineered entity is not found in nature.
  • exhaustted refers to immune cells, including T cells, in which the cells become dysfunctional by exhibiting poor effector function, sustained expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion can occur during chronic infection, autoimmune disease, or cancer, and it can prevent optimal control of infection and tumors, respectively.
  • exogenous when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means; or in relation to a cell, the term refers to a cell that was isolated and subsequently introduced to other cells or to an organism by artificial or natural means.
  • An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid that occurs naturally within the organism or cell.
  • An exogenous cell may be from a different organism, or it may be from the same organism.
  • an exogenous nucleic acid is one that is in a chromosomal location different from where it would be in natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature.
  • NKT cells may refer to a subset of innate-like T lymphocytes that recognize glycolipids presented by the monomorphic MHC-like molecule CD Id. Unlike T cells, NKTs do not recognize HLA class I or class II molecules. Type I or invariant NKTs express invariant T cell receptor (TCR) a-chain Va24-Jal8, which in specific embodiments is paired with vpi l. This subset of NKTs can be identified using monoclonal antibody clone 6B 11 or by reactivity to a synthetic glycolipid alpha-galactosylceramide. In specific embodiments, the NKT cells express one or more engineered antigen receptors, such as one or more CARs.
  • TCR T cell receptor
  • phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate.
  • the preparation of a pharmaceutical composition comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure.
  • animal (e.g., human) administration it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.
  • “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, efc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be
  • aqueous solvents
  • the term “subject,” as used herein, generally refers to an individual that has or is suspected of having cancer, or in other cases that has or is suspected of having acute or chronic infectious disease and/or autoimmune disease.
  • the subject can be any organism or animal subject that is an object of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (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., have or be suspected of having a disease (that may be referred to as a medical condition), such as benign or malignant neoplasias, or cancer.
  • a disease that may be referred to as a medical condition
  • the subject may being undergoing or having undergone treatment.
  • the subject may be asymptomatic.
  • the subject may be healthy individuals but that are desirous of prevention of cancer, infectious disease, or autoimmune disease.
  • the term “individual” may be used interchangeably, in at least some cases.
  • the “subject” or “individual”, as used herein, may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility.
  • the individual may be receiving one or more medical compositions via the internet.
  • An individual may comprise any age of a human or non-human animal and therefore includes both adult and juveniles (i.e., children) and infants and includes in utero individuals. It is not intended that the term connote a need for medical treatment, therefore, an individual may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies. The subject may be participating in a clinical trial.
  • treatment includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g., cancer, infectious disease, or autoimmune disease.
  • Treatment can involve optionally either the reduction or amelioration of one or more symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. Treating may mean alleviation of at least one symptom of the disease or condition.
  • treatment of cancer refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of one or more signs or symptoms of a disease.
  • treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis.
  • treatment of cancer may also refer to prolonging survival and/or increasing quality of life of a subject with cancer.
  • cell therapies for individuals in need thereof are prepared such that the cells have modulated expression and/or activity of endogenous BTG1 or in other cases have heterologous BTG1 introduced into the cells.
  • the disclosure encompasses embodiments wherein cellular therapies for a disease are generated based on the disease being treated.
  • an individual has a medical condition for which cells for therapy for the condition are needed to have persistence and to avoid exhaustion.
  • cancer or chronic viral infectious disease such as HIV, chronic viral hepatitis
  • BTG1 plays a role in the exhaustion of NKT and T cells, for example, and its knockdown or knockout or reduced activity is beneficial to the persistence of the cells.
  • Such cells are effective as therapy for cancer or chronic viral infectious disease.
  • 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 extent of exhaustion.
  • Embodiments of the disclosure include methods of enhancing cell therapies for an individual, comprising the step of reducing expression and/or activity of BTG1 in the cells for the cell therapy or increasing expression and/or activity of BTG1 in the cells for the cell therapy.
  • the cells may or may not be T cells.
  • Embodiments of the disclosure include methods of producing cells for cell therapy for an individual, comprising the step of reducing expression and/or activity of BTG1 in the cells, optionally wherein the cells are not T cells.
  • methods of producing cells for cell therapy for an individual comprising the step of increasing expression and/or activity of BTG1 in the cells or in endogenous cells of the individual.
  • the cells for cancer therapy or acute or chronic infectious disease therapy have modulation of expression of BTG1
  • the cells for cancer therapy or acute or chronic infectious disease therapy have modulation of activity of BTG1, or both.
  • the modulation of expression includes disruption or reduction of expression of the endogenous BTG1 gene to an extent that is greater than in the absence of the modulation.
  • the modulation of activity includes reduction of activity of endogenous BTG1 protein to an extent that is greater than in the absence of the modulation.
  • expression of the endogenous BTG1 gene is reduced in cells for cancer therapy or therapy for acute or chronic infectious disease.
  • the expression may be engineered to be reduced by any suitable manner.
  • reduction in BTG1 gene expression is carried out by effecting a disruption in the gene, such as a knock-out, insertion, missense or frameshift mutation, such as biallelic frameshift mutation, deletion of all or part of the gene, e.g., one or more exons or portions therefore, and/or knock-in.
  • a disruption in the gene such as a knock-out, insertion, missense or frameshift mutation, such as biallelic frameshift mutation, deletion of all or part of the gene, e.g., one or more exons or portions therefore, and/or knock-in.
  • the altered BTG1 gene expression can be effected by sequence-specific or targeted nucleases, including DNA-binding targeted nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases such as a CRISPR-associated nuclease (Cas), specifically designed to be targeted to the sequence of the BTG1 gene or a portion thereof.
  • ZFN zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • RNA-guided nucleases such as a CRISPR-associated nuclease (Cas), specifically designed to be targeted to the sequence of the BTG1 gene or a portion thereof.
  • gene alteration is carried out by induction of one or more double-stranded breaks and/or one or more single-stranded breaks in the BTG1 gene, typically in a targeted manner.
  • the double-stranded or single- stranded breaks are made by a nuclease, e.g. an endonuclease, such as a gene-targeted nuclease.
  • the breaks are induced in the coding region of the gene, e.g. in an exon.
  • 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.
  • NHEJ non-homologous end-joining
  • HDR homology-directed repair
  • the repair process is error-prone and results in disruption of the gene, such as a frameshift mutation, e.g., biallelic frameshift mutation, which can result in complete knockout of the gene.
  • the disruption comprises inducing a deletion, mutation, and/or insertion.
  • the disruption results in the presence of an early stop codon.
  • the presence of an insertion, deletion, translocation, frameshift mutation, and/or a premature stop codon results in disruption of the expression, activity, and/or function of the gene.
  • the DNA-targeting molecule includes a BTG1 -binding protein such as one or more zinc finger protein (ZFP) or transcription activator-like protein (TAL), fused to an effector protein such as an endonuclease.
  • ZFP zinc finger protein
  • TAL transcription activator-like protein
  • an effector protein such as an endonuclease. Examples include ZFNs, TALEs, and TALENs.
  • the BTG1 -targeting molecule comprises one or more zinc- finger proteins (ZFPs) or domains thereof that bind to the BTG1 gene in a sequence-specific manner.
  • ZFP or domain thereof is a protein or domain within a larger 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 through coordination of a zinc ion.
  • the term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.
  • the ZFPs are artificial ZFP domains targeting specific DNA sequences, typically 9-18 nucleotides long, generated by assembly of individual fingers.
  • ZFPs include those in which a single finger domain is approximately 30 amino acids in length and contains an alpha helix containing two invariant histidine residues coordinated through zinc with two cysteines of a single beta turn, and having two, three, four, five, or six fingers.
  • sequence-specificity of a ZFP may be altered by making amino acid substitutions at the four helix positions (-1, 2, 3 and 6) on a zinc finger recognition helix.
  • the ZFP or ZFP-containing molecule is non-naturally occurring, e.g., is engineered to bind to a target site of choice.
  • the B TGI -targeting molecule is or comprises a zinc-finger DNA binding domain fused to a DNA cleavage domain to form a zinc-finger nuclease (ZFN).
  • fusion proteins comprise the cleavage domain (or cleavage halfdomain) from at least one Type liS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered.
  • the cleavage domain is from the Type liS restriction endonuclease Fok I. Fok I generally catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other.
  • the BTG1 -targeting molecule comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as in a transcription activator-like protein effector (TALE) protein, See, e.g., U.S. Patent Publication No. 2011/0301073, incorporated by reference in its entirety herein.
  • TAL transcription activator-like protein
  • TALE transcription activator-like protein effector
  • a TALE DNA binding domain or TALE is a polypeptide comprising one or more TALE repeat domains/units.
  • the repeat domains are involved in binding of the TALE to its cognate target DNA sequence.
  • a single “repeat unit” (also referred to as a “repeat") is typically 33-35 amino acids in length 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 making up the Repeat Variable Diresidue (RVD), typically at positions 12 and/or 13 of the repeat.
  • RVD Repeat Variable Diresidue
  • TALEs The natural (canonical) code for DNA recognition of these TALEs has been determined such that an HD sequence at positions 12 and 13 leads to a binding to cytosine (C), NG binds to T, NI to A, NN binds to G or A, and NO binds to T and non- canonical (atypical) RVDs are also known.
  • C cytosine
  • NG binds to T
  • NI to A binds to G or A
  • NO binds to T and non- canonical (atypical) RVDs are also known.
  • TALEs may be targeted to any gene by design of TAL arrays with specificity to the target DNA sequence.
  • the target sequence generally begins with a thymidine.
  • the molecule is a DNA binding endonuclease, such as a TALE nuclease (TALEN).
  • TALEN is a fusion protein comprising a DNA- binding domain derived from a TALE and a nuclease catalytic domain to cleave a nucleic acid target sequence.
  • the TALEN recognizes and cleaves the target sequence in the BTG1 gene.
  • cleavage of the DNA results in double-stranded breaks.
  • the breaks stimulate the rate of homologous recombination or non-homologous end joining (NHEJ).
  • NHEJ non-homologous end joining
  • repair mechanisms involve rejoining of what remains of the two DNA ends through direct re-ligation or via the so-called microhomology -mediated end joining.
  • repair via NHEJ results in small insertions or deletions and can be used to disrupt and thereby repress the gene.
  • the modification may be a substitution, deletion, or addition of at least one nucleotide.
  • cells in which a cleavage-induced mutagenesis event, i.e. a mutagenesis event consecutive to an NHEJ event, has occurred can be identified and/or selected by well-known methods in the art.
  • TALE repeats are assembled to specifically target a gene.
  • a library of TALENs targeting 18,740 human protein-coding genes has been constructed (Kim et al., 2013).
  • Custom-designed TALE arrays are commercially available through Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, Ky., USA), and Life Technologies (Grand Island, N.Y., USA).
  • TALENs that target CD38 are commercially available (See Gencopoeia, catalog numbers HTN222870-1, HTN222870-2, and HTN222870-3).
  • Exemplary molecules are described, e.g., in U.S. Patent Publication Nos. US 2014/0120622, and 2013/0315884.
  • the TALEN s are introduced as trans genes encoded by one or more plasmid vectors.
  • the plasmid vector can contain a selection marker which provides for identification and/or selection of cells which received said vector.
  • the alteration is carried out using one or more DNA-binding nucleic acids, such as alteration via an RNA-guided endonuclease (RGEN).
  • RGEN RNA-guided endonuclease
  • the alteration can be carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins.
  • CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g.
  • tracrRNA or an active partial tracrRNA encompassing a "direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system
  • atracr-mate sequence encompassing a "direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system
  • guide sequence also referred to as a "spacer” in the context of an endogenous CRISPR system
  • the CRISPR/Cas nuclease or CRISPR/Cas nuclease system can include a noncoding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains).
  • a CRISPR system can derive from a type I, type II, or type III CRISPR system, e.g., derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.
  • target sequence generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex.
  • Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.
  • the CRISPR system can induce double stranded breaks (DSBs) at the target site, followed by disruptions or alterations as discussed herein.
  • Cas9 variants deemed “nickases,” are 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 different gRNAs targeting sequences such that upon introduction of the nicks simultaneously, a 5' overhang is introduced.
  • catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor or activator, to affect gene expression.
  • the target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • the target sequence may be located in the nucleus or cytoplasm of the cell, such as within an organelle of the cell.
  • a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an "editing template” or "editing polynucleotide” or “editing sequence”.
  • an exogenous template polynucleotide may be referred to as an editing template.
  • the recombination is homologous recombination.
  • the CRISPR complex (comprising the guide sequence hybridized to the target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.
  • the tracr sequence which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g.
  • tracr sequence has sufficient complementarity to a 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% of sequence complementarity along the length of the tracr mate sequence when optimally aligned.
  • One or more vectors driving expression of one or more elements of the CRISPR system can be introduced into the cell such that expression of the elements of the CRISPR system direct formation of the CRISPR complex at one or more target sites.
  • Components can also be delivered to cells as proteins and/or RNA.
  • a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors.
  • two or more of the elements expressed from the same or different regulatory elements may 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 may comprise one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site").
  • a restriction endonuclease recognition sequence also referred to as a "cloning site”
  • one or more insertion sites are located upstream and/or downstream of one or more sequence elements of one or more vectors.
  • the CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia).
  • the CRISPR enzyme can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence.
  • the vector can encode a CRISPR enzyme that is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
  • an aspartate-to-alanine substitution D10A in the RuvC I catalytic domain of Cas9 from S.
  • Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • the predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence.
  • the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • the CRISPR enzyme may be part of a fusion protein comprising one or more heterologous protein domains.
  • a CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains.
  • protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
  • Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluore scent proteins including blue fluorescent protein (BFP).
  • GST glutathione-5-transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • beta galactosidase beta-glucuronidas
  • a CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US 20110059502, incorporated herein by reference.
  • the activity of the endogenous BTG1 protein is reduced in the cells for the cancer therapy or acute or chronic infectious disease therapy, and this may be in addition to or alternative to reducing expression of the endogenous BTG1 gene.
  • the protein may be reduced in activity by any suitable manner, such as with one or more small molecules, one or more antibodies, or a combination thereof. Small molecules may be selected for use with the cells for the cancer therapy from a library and based on their ability to reduce activity in vitro, for example.
  • one or more antibodies that bind BTG1 may inhibit its activity at least in part or in some cases in full.
  • the antibodies may be of any kind, including monoclonal, polyclonal, etc. BTG1 antibodies are commercially available, or they may be generated by standard means.
  • the cells of a cell therapy benefit from the increase in expression and/or activity of BTG1 or endogenous cells of a recipient individual have an increase in expression and/or activity of BTG1.
  • such cells are those for use in therapy for any one or more autoimmune diseases, as examples.
  • the cells may have an increase in expression and/or activity by any suitable means.
  • the cells have an increase in expression of the BTG1 gene, the cells have an increase in activity of the endogenous BTG1 protein, or both, and in particular embodiments the expression may be inducible to avoid potential toxicity.
  • the cells may be from expression of a heterologous BTG1 gene, such as on a vector.
  • the increase in expression of the BTG1 gene comes from an increase in expression of the endogenous BTG1 gene in the cells, such as upon introduction of a promoter into a regulatory or other region in a position to be able to regulate the endogenous BTG1 gene.
  • a constitutive promoter in a regulatory region of the endogenous BTG1 gene, such as the SV40, CMV, UBC, EFl A, PGK promoters.
  • Vectors include but are not limited to, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g. derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV etc), lentiviral vectors (e.g.
  • retroviral vectors e.g. derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV etc
  • lentiviral vectors e.g.
  • adenoviral vectors including replication competent, replication deficient and gutless forms thereof, adeno-associated viral (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, polio virus vectors, vesicular stomatitis virus vectors, maraba virus vectors and so forth.
  • Ad adenoviral vectors including replication competent, replication deficient and gutless forms thereof, adeno-associated viral (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine ma
  • the vector is a multi ci str onic vector, and in such cases, a single vector may encode the BTG1 gene and one or more heterologous proteins, such as one or more CARs and/or TCRs, a suicide gene, one or more cytokines, etc.
  • Viral vectors encoding one or more gene products encompassed herein may be provided in certain aspects of the present disclosure.
  • non-essential genes are typically replaced with a gene or coding sequence for a heterologous (or non-native) protein.
  • a viral vector is a kind of expression construct that utilizes viral sequences to introduce nucleic acid and possibly proteins into a cell. The ability of certain viruses to infect cells or enter cells via receptor mediated-endocytosis, and to integrate into host cell genomes and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells).
  • Nonlimiting examples of virus vectors that may be used to deliver a nucleic acid of certain aspects of the present invention include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors.
  • any cells of the disclosure are modified to express one or more heterologous proteins.
  • the heterologous proteins may facilitate activity of the cells in any manner, including at least their activation, persistence, expansion, homing, and/or cytotoxicity, in some embodiments.
  • the modulation of the cells to include one or more heterologous proteins may occur before, during, or after the modulation of expression and/or activity of BTG1.
  • the one or more heterologous proteins may be introduced on the same or a different vector as one or more agents that result in decreased expression or activity of BTG1 or one or more agents that result in increased expression or activity of BTG1.
  • the cells are modified to express one or more bispecific or multi-specific antibodies, although in other cases the cells do not express the antibodies but the antibodies are utilized in conjunction with the cells.
  • the antibodies may be engagers that bridge a particular immune effector cell with a particular target cell for destruction of the target cell.
  • the engineered cells are used with standard T-cell engagers (BiTEs) because in specific embodiments they have been modified to express CD3 that in many cases is the T cell antigen to which the BiTE engager binds.
  • the BiTE may also target a cancer antigen or viral antigen that may be tailored to the medical condition of an intended recipient individual.
  • the BiTE may be tailored to bind a cancer antigen that is characteristic of the cancer cells of a cancer of the individual.
  • the cells may be NK cells modified to express (or not to express but instead used in conjunction with) one or more bispecific NK engagers (BiKEs).
  • the BiKE may comprise an antibody that binds a surface protein on an NK cell, including a naturally expressed surface protein on NK cells, and also comprises an antibody that binds a desired target antigen.
  • the BiKE may target the NK cells through an antibody for a NK surface protein, such as CD 16, CS1, CD56, NKG2D, NKG2C, DNAM, 2B4, CD2, an NCR, or KIR, for example.
  • the BiKE used in the disclosure may also target a cancer antigen or viral antigen that may be tailored to the medical condition of an intended recipient individual.
  • the BiKE may be tailored to bind a cancer antigen that is characteristic of the cancer cells of a cancer of the individual.
  • the engineered antigen receptor is a chimeric antigen receptor (CAR).
  • the cells may be modified to encode at least one CAR, and the CAR may be first generation, second generation, or third or a subsequent generation, for example.
  • the CAR may or may not be bispecific for two or more different antigens.
  • the CAR may comprise one or more costimulatory domains.
  • Each costimulatory domain may comprise the costimulatory domain of any one or more of, for example, members of the TNFR superfamily, CD28, CD 137 (4-1BB), CD134 (0X40), DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1 (CDl la/CD18), Lek, TNFR-I, TNFR-II, Fas, CD30, CD27, NKG2D, 2B4M, CD40, ICOS, TLR, MYD88; 2B4, or combinations thereof, for example.
  • the CAR lacks one or more specific costimulatory domains; for example, the CAR may lack 4- IBB and/or lack CD28.
  • the CAR polypeptide in the cells comprises an extracellular spacer domain that links the antigen binding domain and the transmembrane domain, and this may be referred to as a hinge.
  • Extracellular spacer domains may include, but are not limited to, Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions antibodies, artificial spacer sequences or combinations thereof.
  • Examples of extracellular spacer domains include but are not limited to CD8-alpha hinge, CD28, artificial spacers made of polypeptides such as Gly3, or CHI, CH3 domains of IgGs (such as human IgGl or IgG4).
  • the extracellular spacer domain may comprise (i) a hinge, CH2 and CH3 regions of IgG4, (ii) a hinge region of IgG4, (iii) a hinge and CH2 of IgG4, (iv) a hinge region of CD8-alpha or CD4, (v) a hinge, CH2 and CH3 regions of IgGl, (vi) a hinge region of IgGl or (vii) a hinge and CH2 of IgGl, (viii) a hinge region of CD28, or a combination thereof.
  • the hinge is from IgGl and in certain aspects the CAR polypeptide comprises a particular IgGl hinge amino acid sequence or is encoded by a particular IgGl hinge nucleic acid sequence.
  • the transmembrane domain in the CAR may be derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein.
  • Transmembrane regions include those derived from (/. ⁇ ., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154, ICOS/CD278, GITR/CD357, NKG2D, and DAP molecules, such as DAP 10 or DAP12.
  • the transmembrane domain in some embodiments is synthetic.
  • the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine may be found at each end of a synthetic transmembrane domain.
  • the CAR comprises one or more activating domains, such as CD3( ⁇ , DAP 12, 2B4, or a combination thereof.
  • the engineered receptors utilize one or more homing receptors (that can home to a target not necessarily because of a signal release, such as in the event that they utilize adhesion molecules) and/or one or more chemokine receptors.
  • chemokine receptors include CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors and XC chemokine receptors.
  • the chemokine receptor is a receptor for CCR2, CCR3, CCR5, CCR8, CCR7, CXCR3, L-selectin (CD62L) CXCR1, CXCR2, or CX3CR1.
  • TCRs T Cell Receptors
  • the engineered antigen receptors include recombinant TCRs and/or TCRs cloned from naturally occurring T cells.
  • a "T cell receptor” or “TCR” refers to a molecule that contains a variable alpha and beta chains (also known as TCRalpha and TCRbeta, respectively) or a variable gamma and delta chains (also known as TCRgamma and TCRdelta, respectively) and that is capable of specifically binding to an antigen peptide bound to a MHC receptor.
  • the TCR is in the alpha/beta form.
  • TCRs that exist in alpha/beta and gamma/delta forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions.
  • a TCR can be found on the surface of a cell or in soluble form.
  • a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • a TCR also can contain a constant domain, a transmembrane domain and/or a short cytoplasmic tail.
  • each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end.
  • a TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction.
  • the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full- length TCRs, including TCRs in the alpha/beta form or gamma/delta form.
  • TCR includes any TCR or functional fragment, such as an antigen-binding portion of a TCR that binds to a specific antigenic peptide bound in an MHC molecule, i.e. MHC-peptide complex.
  • An "antigen-binding portion" or antigen-binding fragment" of a TCR which can be used interchangeably, refers to a molecule that contains a portion of the structural domains of a TCR, but that binds the antigen (e.g. MHC- peptide complex) to which the full TCR binds.
  • an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable .beta, chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex, such as generally where each chain contains three complementarity determining regions.
  • variable domains of the TCR chains associate to form loops, or complementarity determining regions (CDRs) analogous to immunoglobulins, which confer antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule and determine peptide specificity.
  • CDRs are separated by framework regions (FRs).
  • FRs framework regions
  • CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the beta chain interacts with the C-terminal part of the peptide.
  • CDR2 is thought to recognize the MHC molecule.
  • the variable region of the beta-chain can contain a further hypervariability (HV4) region.
  • the TCR chains contain a constant domain.
  • the extracellular portion of TCR chains e.g., alpha-chain, beta-chain
  • 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 domain of the TCR domain contains short connecting sequences in which a cysteine residue forms a disulfide bond, making a link between the two chains.
  • a TCR may have an additional cysteine residue in each of the alpha and beta chains such that the TCR contains two disulfide bonds in the constant domains.
  • the TCR chains can contain a transmembrane domain.
  • the transmembrane domain is positively charged.
  • the TCR chains contains a cytoplasmic tail.
  • the structure allows the TCR to associate with other molecules like CD3.
  • a TCR containing constant domains with a transmembrane region can anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex.
  • CD3 is a multi-protein complex that can possess three distinct chains (gamma, delta, and epsilon) in mammals and the zeta-chain.
  • the complex can contain a CD3gamma chain, a CD3delta chain, two CD3epsilon. chains, and a homodimer of CD3zeta chains.
  • the CD3gamma, CD3delta, and CD3epsilon chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain.
  • the transmembrane regions of the CD3gamma, CD3delta, and CD3 epsilon 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 CD3gamma, CD3delta, and CD3epsilon chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or IT AM, whereas each CD3zeta chain has three.
  • ITAMs are involved in the signaling capacity of the TCR complex.
  • These accessory molecules have negatively charged transmembrane regions and play a role in propagating the signal from the TCR into the cell.
  • the CD3- and .zeta. -chains, together with the TCR, form what is known as the T cell receptor complex.
  • the TCR may be a heterodimer of two chains alpha and beta (or optionally gamma and delta) or it may be a single chain TCR construct.
  • the TCR is a heterodimer containing two separate chains (alpha and beta chains or gamma and delta chains) that are linked, such as by a disulfide bond or disulfide bonds.
  • a TCR for a target antigen e.g., a cancer antigen
  • 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.
  • the TCR is obtained from a biological source, such as from cells such as from a T cell (e.g. cytotoxic T cell), T cell hybridomas or other publicly available source.
  • the T cells can be obtained from in vivo isolated cells.
  • a high-affinity T cell clone can be isolated from a patient, and the TCR isolated.
  • the T cells can be a cultured T cell hybridoma or clone.
  • the TCR clone for a target antigen has been generated in transgenic mice engineered with human immune system genes (e.g., the human leukocyte antigen system, or HLA).
  • phage display is used to isolate TCRs against a target antigen (see, e.g., Varela-Rohena et al., 2008 and Li, 2005).
  • the TCR or antigen-binding portion thereof can be synthetically generated from knowledge of the sequence of the TCR.
  • cytokine release syndrome which may also be referred to as cytokine storm
  • the individual may have elevated inflammatory cytokine(s) (merely as examples: interferon-gamma, granulocyte macrophage colony-stimulating factor, IL- 10, IL-6 and TNF-alpha); fever; fatigue; hypotension; hypoxia, tachycardia; nausea; capillary leak; cardiac/renal/hepatic dysfunction; or a combination thereof, for example.
  • the individual may have confusion, delirium, aplasia, and/or seizures.
  • the individual is tested for a marker associated with onset and/or severity of cytokine release syndrome, such as C-reactive protein, IL-6, TNF-alpha, and/or ferritin.
  • the cells may be administered in combination with one or more other therapeutic agents for the treatment of any affliction.
  • Combination therapies would depend on the affliction and can include, but are not limited to, one or more anti-microbial agents (for example, antibiotics, anti-viral agents and anti-fungal agents), anti-tumor agents (for example, fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immune-depleting agents (for example, fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressive agents (for example, azathioprine, or glucocorticoids, such as dexamethasone or prednisone), anti-inflammatory agents (for example, glucocorticoids such as hydrocortisone, dexamethasone or prednisone, or non-steroidal anti-inflammatory agents such as acetylsalicy
  • immunosuppressive or tolerogenic agents including but not limited to calcineurin inhibitors (e.g., cyclosporin and tacrolimus); mTOR inhibitors (e.g., Rapamycin); mycophenolate mofetil, antibodies (e.g., recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., Methotrexate, Treosulfan, Busulfan); irradiation; or chemokines, interleukins or their inhibitors (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors) can be administered.
  • additional pharmaceutical agents can be administered before, during, or after administration of the immune cells, depending on the desired effect. This administration of the cells and the agent can be by the same route or by different routes, and either at the same site or at a different site.
  • compositions and methods of the present embodiments involve an immune cell population in combination with at least one additional therapy.
  • the additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing.
  • the additional therapy may be in the form of adjuvant or neoadjuvant therapy.
  • any of the cells of the disclosure may be obtained from suitable storage prior to modulation.
  • the cells in storage may or may not already have modulation of BTG1 expression and/or activity.
  • the cells in storage may or may not already have expression of any one or more heterologous genes.
  • any of the cells of the disclosure are obtained from the individual in need of the therapy, are modulated ex vivo with respect to BTG1 expression and/or activity, are optionally modulated to express one or more heterologous genes, and are administered back to the individual.
  • a step of modulating the cells for BTG1 may or may not precede modulating the cells for the one or more heterologous genes.
  • the cells may or may not also express one or more heterologous proteins as defined herein, and when they do not, the kit may comprise vectors that express the heterologous protein(s), primers for amplification of the heterologous protein(s), and so forth.
  • Kits may comprise components that may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means. Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more, as examples.
  • BCG1 B-CELL TRANSLOCATION GENE 1
  • T cell exhaustion is an active process characterized by a progressive loss of effector function and proliferative capacity because of 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 tumor-specific chimeric antigen receptor (CAR) (as one example) also undergo exhaustion that limits their antitumor potency and is associated with tumor escape and disease progression or recurrence.
  • CAR tumor-specific chimeric antigen receptor
  • scRNAseq single cell RNA sequencing
  • CAR-NKTs CAR NKT cells from 12 patients with neuroblastoma (NB) was performed in the course of a phase I clinical trial (NCT03294954). Changes were assessed in CAR-NKT gene expression following their infusion to patients or multiple rounds of tumor cell challenge in vitro (repeat tumor challenge assay (RTC) assay).
  • RTC tumor challenge assay
  • CAR-NKTs after 5 RTC rounds and peripheral blood CAR-NKTs 2 weeks post-infusion had similar directional changes in their gene expression profiles compared to infusion product CAR-NKTs, exemplified by the acquisition of terminal effector differentiation and loss of the naive/memory program.
  • BTG1 B-cell translocation gene 1
  • BTG1 as a critical regulator of NKT and T cell function.
  • exhausted T cells and NKT cells upregulate BTG1 to induce global mRNA degradation, contributing to the state of quiescence or hypo-responsiveness shared at least by naive T and exhausted T cells or NKT cells.
  • BTG1 downregulation strongly enhances antitumor activity of CAR-redirected NKTs, which is useful to guide the rational design of nextgeneration cancer immunotherapy products, for example.
  • the present example provides demonstration of BTG1 association with exhausted immune cells and their consequential role in adoptive cell therapy.
  • FIG. 1 A shows one embodiment of a tumor co-culture system in which CAR-NKTs are re-plated with fresh CHLA255 NB tumor cells every five days for multiple cycles, thereby inducing exhaustion in CAR-NKTs via chronic antigen exposure.
  • the cytotoxic activity of CAR-NKTs during repeated co-culturing at indicated timepoints shows that with time, for many patients the cytotoxicity becomes reduced (FIG. IB).
  • FIG. IE shows a Volcano plot demonstrating differentially expressed genes in CAR-NKTs post-5RcC versus pre-infusion products, including BTG1. BTG1 expression is elevated in exhausted CAR-NKTs.
  • FIG. 2A provides one embodiment of a design of retroviral constructs encoding BTG1.GFP or GFP only as a control to evaluate the impact of BTG1 overexpression (OE) in NKTs.
  • OE BTG1 overexpression
  • FIG. 5A illustrates example of retroviral construct designs for BTG1 knockdown.
  • the 14g2a scFv for GD2 binding was utilized in all examples of constructs, as was CD8 hinge and transmembrane domain, CD28 costimulatory domain, and CD3zeta.
  • IL-15 was utilized for enhancement of activity.
  • NKTs were transduced with retroviral vectors encoding the CAR +/- IL 15 and/or artificial microRNAs (amiR) specific for BTG1 or a scrambled control.
  • Fold expansion of NKTs expressing indicated constructs post-transduction is provided in FIG. 6A.
  • CD62L frequency post-transduction in CAR.15 NKTs with or without BTG1 KD is demonstrated in FIG. 6B.
  • CD62L expression in CAR.15.amiR-BTGl NKTs gated on CAR+ and CAR- populations is provided in FIG. 6C.
  • FIG. 6D shows the frequency of PD-1+ CAR.15 NKTs with and without BTG1 KD.
  • FIG. 61 Bioluminescence images of tumor-bearing mice at specified timepoints are demonstrated (FIG. 61). Tumor burden changes based on bioluminescence images over time (FIG. 6J). Kaplan- Meier survival curves for mice in indicated groups, as shown in FIG. 6K. As provided herein, BTG1 knockdown enhances the antitumor activity of GD2-CARNKTs/
  • BTG1 knockdown enhances the antitumor activity of GD2-CAR T cells (as examples) after multiple rounds of in vitro tumor challenge.
  • the CAR % change of T cells is provided in FIG. 7B before or after three rounds of co-culture with CHLA255 NB cells.

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Abstract

Des modes de réalisation de la divulgation concernent des méthodes et des compositions associés à des cellules modifiées et leurs utilisations, les cellules étant modifiées par rapport à l'expression du gène de translocation de lymphocytes B 1 (BTG1). Dans des modes de réalisation spécifiques, les cellules modifiées sont utilisées pour une thérapie cellulaire adoptive contre des états pathologiques particuliers. Dans certains modes de réalisation, les cellules modifiées sont des cellules immunitaires, telles que des lymphocytes T, ayant une expression réduite de BTG1 et sont utilisées pour améliorer une thérapie contre le cancer et/ou une maladie infectieuse. Dans certains modes de réalisation, les cellules modifiées sont des cellules ayant une expression inductible accrue de BTG1 et sont utilisées pour une thérapie contre une maladie auto-immune. Dans certains modes de réalisation, l'expression de BTG1 est augmentée dans des cellules endogènes chez un individu atteint d'une maladie auto-immune.
EP23861278.2A 2022-09-02 2023-08-31 Modulation du gène de translocation de lymphocytes b 1 (btg1) destinée à être utilisée dans une thérapie cellulaire adoptive Pending EP4580641A2 (fr)

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