WO2023070080A1 - Inactivation de regnase-1 et de roquin-1 pour améliorer l'activité de cellule car-t - Google Patents

Inactivation de regnase-1 et de roquin-1 pour améliorer l'activité de cellule car-t Download PDF

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WO2023070080A1
WO2023070080A1 PCT/US2022/078496 US2022078496W WO2023070080A1 WO 2023070080 A1 WO2023070080 A1 WO 2023070080A1 US 2022078496 W US2022078496 W US 2022078496W WO 2023070080 A1 WO2023070080 A1 WO 2023070080A1
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cells
cell
tcr
car
antigen
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Neil Sheppard
David Mai
Carl H. June
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The Trustees Of The University Of Pennsylvania
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/464468—Mesothelin [MSLN]
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • chimeric antigen receptor (CAR) T cell therapy has demonstrated clinical success in several hematological malignancies, it has limited efficacy against solid tumors. This is partially due to both tumor intrinsic and T cell intrinsic mechanisms that limit cytotoxic effector and memory T cell responses. Efforts to modulate T cell intrinsic functional regulators have focused largely on surface receptors, such as the co-inhibitory receptors PD1 and CTLA4, or transcription factors such as c-Jun.
  • An underexplored category of immune regulators falls in the class of RNA binding proteins (RBPs). Specifically, Regnase-1 and Roquin-1 are RBPs that act as negative immune regulators that serve to coordinate fine tuning and restriction of inflammatory gene expression.
  • Regnase-1 knockout enhances the function of mouse OT-1 cells, Pmel TCR-T cells, and CD19-CAR CD8+ T cells (Wei et al. (2019) Nature. 576(7787): 471- 476).
  • Knocking out Regnase-1, Roquin-1 & -2 or all three genes increased IL-2 and IFN-g mRNA expression in human Jurkat T cells stimulated by the non-physiological stimulus of PMA and ionomycin, which bypasses the TCR complex (Cui et al. (2017) The Journal of Immunology, 199:4066-4077).
  • the disclosure provides a modified immune cell or precursor cell thereof, wherein endomenous Roquin-1 has been disrupted and/or knocked out.
  • the disclosure provides a modified immune cell or precursor cell thereof, wherein endogenous Roquin-1 and endogenous Regnase-1 have been disrupted and/or knocked out.
  • the disclosure provides a method of treating cancer in a subject in need thereof.
  • the method comprises administering to the subject a composition comprising any of the modified immune cells or precursor cells thereof contemplated herein.
  • the cell comprises a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.
  • the antigen binding domain binds a tumor associated antigen (TAA).
  • TAA tumor associated antigen
  • the tumor TAA is expressed by cells on a solid tumor.
  • the TAA is mesothelin.
  • the CAR comprises a nucleotide sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17.
  • the cell comprises an exogenous T cell receptor (TCR).
  • TCR exogenous T cell receptor
  • the exogenous TCR binds a TAA.
  • the TAA is NY-ESO-1.
  • the exogenous TCR comprises a nucleotide sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18.
  • the exogenous TRAC been disrupted and/or knocked out.
  • the cell is a primary cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a T cell.
  • the endomenous Regnase-1 has been disrupted and/or knocked out via a CRISPR/Cas9 system.
  • the CRISPR/Cas9 system comprises an sgRNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1-3.
  • the endomenous Roquin-1 has been disrupted and/or knocked out via a CRISPR/Cas9 system.
  • the CRISPR/Cas9 system comprises an sgRNA comprising the nucleotide sequence of SEQ ID NO: 12.
  • Fig. 1 Scheme of generation of Regl/Roql single and double KO human CAR-T cells.
  • Fig. 2 Expansion kinetics and transduction efficiencies of Regl/Roql KO human CAR-T cells.
  • Fig. 3 Western blot validation of protein-level Regl/Roql KO.
  • Fig. 4 Sequencing based validation of genetic Regl/Roql KO.
  • Fig. 5 Phenotype of Regl/Roql KO CAR-T cells thawed after primary expansion.
  • Fig. 6 CD4/CD8 distribution of Regl/Roql KO CAR-T cells thawed after primary expansion.
  • Fig. 7 Basal ICOS levels of Regl/Roql KO CAR-T cells.
  • Fig. 8 CD25/CD69 expression of Regl/Roql KO CAR-T cells 18hr post-stimulation against antigen-negative cells.
  • Fig. 9 Antigen-independent cytokine release of Regl/Roql KO CAR-T cells.
  • Fig. 10 Volcano plots of differentially regulated genes in Regl/Roql KO T cells at baseline.
  • Fig. 11 Expression of various inflammatory-associated genes in Regl/Roql KO T cells at baseline.
  • Fig. 12 PCA clustering analysis of Regl/Roql KO cells from 3 independent donors and gene set enrichment analysis of Regl-KO and DKO cells.
  • Fig. 13 Cytotoxicity over different E:T ratios and killing over time at a 1 : 10 E:T ratio of Regl/Roql KO CAR-T cells.
  • Fig. 14 Proliferation of Regl/Roql KO CD4 and CD8 CAR-T cells after antigen exposure.
  • Fig. 15 CD25/CD69 expression on Regl/Roql KO CAR-T cells with antigen exposure.
  • Fig. 16 Fold change in cytokine secretion compared to mock CAR-T cells of Regl/Roql KO CAR-T cells with antigen exposure.
  • Fig. 17 Schematic demonstrating Regl/Roql degradation upon T cell activation and experimental scheme for a chronic antigen exposure (CAE) assay.
  • Fig. 18 Regl/Roql KO CAR-T cell proliferation under CAE. Left panel - mean and SD of 2-3 donors, right panel - results of individual donors.
  • Fig. 19 CAR expression over time of Regl/Roql KO CAR-T cells under CAE.
  • Fig. 20 CD4/CD8 distribution over time of Regl/Roql KO CAR-T cells under CAE.
  • Fig. 21 ICOS expression over time of Regl/Roql KO CAR-T cells under CAE.
  • Fig. 22 Co-inhibitory marker expression over time of Regl/Roql KO CAR-T cells under CAE.
  • Fig. 23 Mean Fluorescence Intensity (MFI) of co-inhibitory makers PD1, TIM3, and LAG3, and phenotype of Regl/Roql KO CAR-T cells after 28 days of CAE.
  • MFI Mean Fluorescence Intensity
  • Fig. 24 Experimental scheme for an in vitro restimulation assay and cytotoxicity of Regl/Roql KO CAR-T cells after 4 rounds of restimulation.
  • Fig. 25 Cytokine and effector molecule production by intracellular cytokine staining of CAR-T cells after 4 rounds of restimulation.
  • Fig. 26 Experimental scheme to investigate in vivo tumor burden control and tumor growth kinetics over time of CAR-T cell treated mice.
  • Fig. 27 Tumor volumes of CAR-T cell treated mice at 4 weeks after treatment.
  • Fig. 28 Enumeration of T cells in the peripheral blood from CAR-T cell treated mice and CD8 percentage of those T cells at 21 days after treatment.
  • Fig. 29 Kaplan-Meyer curve plotting survival of CAR-T cell treated mice over time.
  • Fig. 30 In vivo experimental scheme for analysis of TILs and T cells in the spleen and associated tumor growth curve up to 14 days before organ harvesting.
  • Fig. 31 Enumeration of CD4+ and CD8+ TILs in CAR-T cell treated mice after 14 days.
  • Fig. 32 Enumeration of CD4+ and CD8+ T cells in the spleen in CAR-T cell treated mice after 14 days.
  • Fig. 33 Flow characterization of CD4 and CD8 TILs from CAR-T cell treated mice after 14 days.
  • Fig. 34 T cell subset characterization of CD4 and CD8 TILs from CAR-T cell treated mice after 14 days.
  • Fig. 35 Cytokine levels in the serum of mice treated with Regl/Roql CAR-T cells after 14 days or treatment.
  • Fig. 36 In vivo experimental scheme for rechallenging tumor-cleared mice, associated tumor growth curve from cleared mice rechallenged with tumor cells on the opposite flank, and tumor volumes of rechallenged mice after 21 days of rechallenge.
  • Fig. 37 CD4 and CD8 T cells in the blood of rechallenged mice after 21 days of rechallenge.
  • Fig. 38 Comparison of T cell counts in the blood 21 days after initial tumor challenge and 21 days after tumor rechallenge with at least a month between initial tumor clearance and rechallenge.
  • Fig. 39 Comparison of T cell counts in the blood 21 days after initial tumor challenge and without tumor rechallenge in Regl-KO and Roql-KO CAR-T cell treated mice.
  • Fig. 40 TCR-T cell expansion kinetics over time and transduction efficiencies after primary expansion.
  • Fig. 41 Western blot validation of Regl and/or Roql KO in KO TCR-T cells.
  • Fig. 42 Sequencing based validation of Regl and/or Roql KO in KO TCR-T cells.
  • Fig. 43 CD4/CD8 distribution of Regl/Roql KO TCR-T cells thawed after primary expansion.
  • Fig. 44 Basal ICOS levels of Regl/Roql KO TCR-T cells.
  • Fig. 45 CD25/CD69 expression of Regl/Roql KO TCR-T cells 18hr post-stimulation against antigen-negative cells.
  • Fig. 46 Antigen-independent cytokine release of Regl/Roql KO TCR-T cells.
  • Fig. 47 Phenotype of Regl/Roql KO TCR-T cells thawed after primary expansion.
  • Fig. 48 Cytotoxicity over different E:T ratios and killing over time at a 1 :3 E:T ratio of Regl/Roql KO TCR-T cells.
  • Fig. 49 Proliferation of Regl/Roql KO CD4 and CD8 TCR-T cells after antigen exposure.
  • Fig. 50 Fold change in cytokine secretion compared to mock TCR-T cells of Regl/Roql KO TCR-T cells with antigen exposure.
  • Fig. 51 CD25/CD69 expression on Regl/Roql KO TCR-T cells with antigen exposure.
  • Fig. 52 Experimental scheme to investigate in vivo tumor burden control and tumor growth kinetics over time of TCR-T cell treated mice.
  • Fig. 53 Tumor volumes of TCR-T cell treated mice at 4 weeks after treatment.
  • Fig. 54 Enumeration of T cells in the peripheral blood from TCR-T cell treated mice at 21 days after treatment.
  • the present invention is based on the unexpected finding that genetic disruption of Regnase-1 and/or Roquin-1 enhances inflammatory gene signatures long-term and facilitates greater function and persistence of human CAR-T cells against cancer.
  • RNA binding proteins that regulate the inflammatory response.
  • Regnase and Roquin target immune response associated transcripts, such as transcription factors and cytokines. Both bind secondary structures called stem loops located at the 3’UTR of mRNAs.
  • Regnase localizes to the ribosome and ER to target translationally active mRNA.
  • Roquin localizes to stress granules and processing bodies to target translationally inactive mRNA. Mutants or deletions of these proteins in mice and humans cause autoimmunity.
  • Regnase is a ribonuclease that controls mRNA stability (Uehata, T. & Takeuchi, O., Journal of Interferon & Cytokine Research 37, 220-229 (2017)). From a family of 4 paralogues Regnase-1 most well-studied. Activity and regulation seems to be context dependent. It has deubiquitinase activity (TRAF2, TRAF3, TRAF6, NEMO). Recent literature suggests Regnase-1 deficiency generates longer-lived, better performing T cells (Reina-Campos, M. & Goldrath, A. W., Nature 2021 576:7787 576, 392-393 (2019); Wenting, Z. et a!., Blood 138, 122-135 (2021)).
  • Roquin is an atypical E3 ubiquitin ligase (Uehata, T. & Takeuchi, O., Journal of Interferon & Cytokine Research 37, 220-229 (2017)). It is part of a family of 2 paralogues. It has the RING-domain characteristic of E3 ubiquitin ligases but there has been no reported function of that nature to date. It has a role in regulating miRNAs (miR146a, miR24) involved in immune regulation. It is a multi-functional regulator of immune homeostasis that remains to be fully characterized.
  • compositions and methods comprising modified immune cells or precursor cells thereof (e.g. T cells, e.g. CAR T cells) wherein endogenous Roquin-1 has been discrupted and/or knocked out. Also provided are compositions and methods comprising modified immune cells or precursor cells thereof (e.g. T cells, e.g. CAR T cells) wherein endogenous Roquin-1 and Regnase-1 have been disrupted and/or knocked out. Methods of treatment are also provided.
  • modified immune cells or precursor cells thereof e.g. T cells, e.g. CAR T cells
  • an element means one element or more than one element.
  • “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • Activation refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions.
  • the term “activated T cells” refers to, among other things, T cells that are undergoing cell division.
  • to “alleviate” a disease means reducing the severity of one or more symptoms of the disease.
  • antigen as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • antibody production or the activation of specific immunologically-competent cells, or both.
  • any macromolecule including virtually all proteins or peptides, can serve as an antigen.
  • antigens can be derived from recombinant or genomic DNA.
  • any DNA which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response.
  • an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
  • autologous is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.
  • a “co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation.
  • Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor.
  • a “co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to T cell proliferation and/or upregulation or downregulation of key molecules.
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
  • a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
  • downstreamregulation refers to the decrease or elimination of gene expression of one or more genes.
  • Effective amount or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to an amount that when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the invention. The immune response can be readily assessed by a plethora of art-recognized methods.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • ex vivo refers to cells that have been removed from a living organism, e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).
  • expression is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
  • “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • Identity refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage.
  • the identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.
  • immune response is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and/or activate lymphocytes to remove the antigen.
  • immunosuppressive is used herein to refer to reducing overall immune response.
  • “Insertion/deletion”, commonly abbreviated “indel,” is a type of genetic polymorphism in which a specific nucleotide sequence is present (insertion) or absent (deletion) in a genome.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • a “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.
  • modified is meant a changed state or structure of a molecule or cell of the invention.
  • Molecules may be modified in many ways, including chemically, structurally, and functionally.
  • Cells may be modified through the introduction of nucleic acids.
  • modulating is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject.
  • the term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.
  • A refers to adenosine
  • C refers to cytosine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • oligonucleotide typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which “U” replaces “T.”
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.
  • nucleotide as used herein is defined as a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and polynucleotides as used herein are interchangeable.
  • nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
  • recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample.
  • an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific.
  • an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
  • the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
  • a particular structure e.g., an antigenic determinant or epitope
  • stimulation is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex.
  • a stimulatory molecule e.g., a TCR/CD3 complex
  • Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and/or reorganization of cytoskeletal structures, and the like.
  • a “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.
  • a “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like.
  • Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.
  • subject is intended to include living organisms in which an immune response can be elicited e.g., mammals).
  • a “subject” or “patient,” as used therein, may be a human or non-human mammal.
  • Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals.
  • the subject is human.
  • a “target site” or “target sequence” refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.
  • a target sequence refers to a genomic nueleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.
  • T cell receptor refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen.
  • the TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules.
  • TCR is composed of a heterodimer of an alpha (a) and beta (P) chain, although in some cells the TCR consists of gamma and delta (y/8) chains.
  • TCRs may exist in alpha/beta and gamma/delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain.
  • the TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell.
  • a helper T cell including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell.
  • terapéutica as used herein means a treatment and/or prophylaxis.
  • a therapeutic effect is obtained by suppression, remission, or eradication of a disease state.
  • transfected or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • the term “variant” when used in conjunction to an amino acid sequence refers to a sequence that is at least, or about, 85%, 90%, 91%, 92%, 93%solv 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence.
  • the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions.
  • the substitution is a conservative substitution.
  • a “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • the present invention provides modified immune cells or precursors thereof e.g., T cells) for use in immunotherapy (e.g. CAR T cells).
  • the invention provides a modified immune cell or precursor cell thereof (e.g., T cell) wherein endogenous Roquin-1 has been disrupted and/or knocked out.
  • the invention provides a modified immune cell or precursor cell thereof (e.g., T cell) wherein endogenous Roquin-1 and endogenous Regnase-1 have been disrupted and/or knocked out.
  • the invention provides a modified immune cell or precursor cell thereof (e.g., T cell) comprising a chimeric antigen receptor (CAR), wherein Roquin-1 has been disrupted and/or knocked out.
  • the invention provides a modified immune cell or precursor cell thereof (e.g., T cell) comprising a chimeric antigen receptor (CAR), wherein endogenous Roquin-1 and endogenous Regnase-1 have been disrupted and/or knocked out.
  • a subject cell can comprise any CAR known in the art as well as those described in detail elsewhere herein.
  • a subject CAR comprises affinity for an antigen on a target cell. Accordingly, such modified cells possess the specificity directed by the CAR that is expressed therein.
  • a modified cell of the present disclosure comprising a mesothelin CAR possesses specificity for mesothelin on a target cell.
  • the invention provides a modified immune cell or precursor cell thereof (e.g., T cell) comprising an exogenous T Cell Receptor (TCR), wherein Roquin-1 has been disrupted and/or knocked out.
  • TCR T Cell Receptor
  • the invention provides a modified immune cell or precursor cell thereof (e.g., T cell) comprising an exogenous T Cell Receptor (TCR), wherein endogenous Roquin-1 and endogenous Regnase-1 have been disrupted and/or knocked out.
  • a modified cell e.g., a modified cell comprising an exogenous CAR and/or TCR
  • the gene-edited immune cells e.g., T cells
  • Gene editing technologies include, without limitation, homing endonucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector (TALE) nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) (e.g. CRISPR/Cas9).
  • ZFNs zinc-finger nucleases
  • TALE transcription activator-like effector
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Homing endonucleases generally cleave their DNA substrates as dimers, and do not have distinct binding and cleavage domains.
  • ZFNs recognize target sites that consist of two zinc-finger binding sites that flank a 5- to 7-base pair (bp) spacer sequence recognized by the FokI cleavage domain.
  • TALENs recognize target sites that consist of two TALE DNA-binding sites that flank a 12- to 20-bp spacer sequence recognized by the FokI cleavage domain.
  • the Cas9 nuclease is targeted to DNA sequences complementary to the targeting sequence within the single guide RNA (gRNA) located immediately upstream of a compatible protospacer adjacent motif (PAM). Accordingly, one of skill in the art would be able to select the appropriate gene editing technology for the present invention.
  • the present invention provides a modified immune cell or precursor cell thereof (e.g., a T cell) comprising a CRISPR-mediated modification in an endogenous gene locus encoding Roquin-1 that is capable of downregulating gene expression of endogenous Roquin-1, and an exogenous CAR and/or TCR as described herein.
  • a modified immune cell or precursor cell thereof e.g., a T cell
  • a CRISPR-mediated modification in an endogenous gene locus encoding Roquin-1 that is capable of downregulating gene expression of endogenous Roquin-1, and an exogenous CAR and/or TCR as described herein.
  • the present invention provides a modified immune cell or precursor cell thereof (e.g., a T cell) comprising a CRISPR-mediated modification in an endogenous gene locus encoding Roquin-1 and a CRISPR-mediated modification in an endogenous gene locus encoding Regnase-1 that are capable of downregulating gene expression of endogenous Roquin-1 and Regnase-1; and an exogenous CAR and/or TCR as described herein.
  • the CRISPR-mediated modification is introduced via a CRISPR/Cas9 system, comprising a Cas9 enzyme and at least one guide RNA (gRNA).
  • the guide RNA comprises a guide sequence that is sufficiently complementary with a target sequence in the endogenous gene locus encoding Roquin-1 and/or Regnase-1.
  • the guide RNA comprises or consists of the nucleic acid sequence set forth in any one of SEQ ID NOs. 1-16.
  • Non-limiting types of CRISPR-mediated modifications include a substitution, an insertion, a deletion, and an insertion/deletion (INDEL).
  • the modification can be located in any part of the endogenous gene locus encoding Roquin-1 and/or Regnase-1, including but not limited to an exon, a splice donor, or a splice acceptor.
  • the disruption is carried out by gene editing using an RNA-guided nuclease such as a CRISPR-Cas system, such as CRISPR-Cas9 system, specific for the gene (e.g., Roquin-1 and/or Regnase-1) being disrupted.
  • an agent containing a Cas9 and a guide RNA (gRNA) containing a targeting domain, which targets a region of the genetic locus is introduced into the cell.
  • the agent is or comprises a ribonucleoprotein (RNP) complex of a Cas9 polypeptide and a gRNA (Cas9/gRNA RNP).
  • the introduction includes contacting the agent or portion thereof with the cells in vitro, which can include cultivating or incubating the cell and agent for up to 24, 36 or 48 hours or 3, 4, 5, 6, 7, or 8 days.
  • the introduction further can include effecting delivery of the agent into the cells.
  • the methods, compositions and cells according to the present disclosure utilize direct delivery of ribonucleoprotein (RNP) complexes of Cas9 and gRNA to cells, for example by electroporation.
  • the RNP complexes include a gRNA that has been modified to include a 3' poly- A tail and a 5' Anti-Reverse Cap Analog (ARCA) cap.
  • RNP ribonucleoprotein
  • the CRISPR/Cas9 system is a facile and efficient system for inducing targeted genetic alterations.
  • Target recognition by the Cas9 protein requires a ‘seed’ sequence within the guide RNA (gRNA) and a conserved di-nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region.
  • the CRISPR/Cas9 system can thereby be engineered to cleave virtually any DNA sequence by redesigning the gRNA in cell lines (such as 293T cells), primary cells, and TCR T cells.
  • the CRISPR/Cas9 system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, making this system suited for multiple gene editing or synergistic activation of target genes.
  • the Cas9 protein and guide RNA form a complex that identifies and cleaves target sequences.
  • Cas9 is comprised of six domains: REC I, REC II, Bridge Helix, PAM interacting, HNH, and RuvC.
  • the REC I domain binds the guide RNA, while the Bridge helix binds to target DNA.
  • the HNH and RuvC domains are nuclease domains.
  • Guide RNA is engineered to have a 5’ end that is complementary to the target DNA sequence. Upon binding of the guide RNA to the Cas9 protein, a conformational change occurs activating the protein. Once activated, Cas9 searches for target DNA by binding to sequences that match its protospacer adjacent motif (PAM) sequence.
  • PAM protospacer adjacent motif
  • a PAM is a two or three nucleotide base sequence within one nucleotide downstream of the region complementary to the guide RNA.
  • the PAM sequence is 5’-NGG-3’.
  • CRISPRi induces permanent gene disruption that utilizes the RNA-guided Cas9 endonuclease to introduce DNA double stranded breaks which trigger error-prone repair pathways to result in frame shift mutations.
  • a catalytically dead Cas9 lacks endonuclease activity.
  • a DNA recognition complex is generated that specifically interferes with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently represses expression of targeted genes.
  • the CRISPR/Cas gene disruption occurs when a guide nucleic acid sequence specific for a target gene and a Cas endonuclease are introduced into a cell and form a complex that enables the Cas endonuclease to introduce a double strand break at the target gene.
  • the CRISPR/Cas system comprises an expression vector, such as, but not limited to, a pAd5F35-CRISPR vector.
  • the Cas expression vector induces expression of Cas9 endonuclease.
  • endonucleases may also be used, including but not limited to, Casl2a (Cpfl), T7, Cas3, Cas8a, Cas8b, CaslOd, Csel, Csyl, Csn2, Cas4, CaslO, Csm2, Cmr5, Fokl, other nucleases known in the art, and any combinations thereof.
  • inducing the Cas expression vector comprises exposing the cell to an agent that activates an inducible promoter in the Cas expression vector.
  • the Cas expression vector includes an inducible promoter, such as one that is inducible by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, for example doxycycline).
  • an antibiotic e.g., by tetracycline or a derivative of tetracycline, for example doxycycline.
  • Other inducible promoters known by those of skill in the art can also be used.
  • the inducing agent can be a selective condition (e.g., exposure to an agent, for example an antibiotic) that results in induction of the inducible promoter. This results in expression of the Cas expression vector.
  • guide RNA refers to any nucleic acid that promotes the specific association (or “targeting”) of an RNA-guided nuclease such as a Cas9 to a target sequence (e.g., a genomic or episomal sequence) in a cell.
  • target sequence e.g., a genomic or episomal sequence
  • a “modular” or “dual RNA” guide comprises more than one, and typically two, separate RNA molecules, such as a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), which are usually associated with one another, for example by duplexing.
  • crRNA CRISPR RNA
  • tracrRNA trans-activating crRNA
  • a “unimolecular gRNA,” “chimeric gRNA,” or “single guide RNA (sgRNA)” comprises a single RNA molecule.
  • the sgRNA may be a crRNA and tracrRNA linked together.
  • the 3’ end of the crRNA may be linked to the 5’ end of the tracrRNA.
  • a crRNA and a tracrRNA may be joined into a single unimolecular or chimeric gRNA, for example, by means of a four nucleotide (e.g., GAAA) “tetraloop” or “linker” sequence bridging complementary regions of the crRNA (at its 3' end) and the tracrRNA (at its 5' end).
  • GAAA four nucleotide
  • a “repeat” sequence or region is a nucleotide sequence at or near the 3’ end of the crRNA which is complementary to an anti-repeat sequence of a tracrRNA.
  • an “anti-repeat” sequence or region is a nucleotide sequence at or near the 5’ end of the tracrRNA which is complementary to the repeat sequence of a crRNA.
  • gRNA / Cas9 complex for genome editing may be found in, at least, Mali et al. Science, 339(6121), 823- 826 (2013); Jiang et al. Nat. Biotechnol. 31(3). 233-239 (2013); and Jinek et al. Science, 337(6096), 816-821 (2012); which are incorporated by reference herein.
  • a “guide sequence” or “targeting sequence” refers to the nucleotide sequence of a gRNA, whether unimolecular or modular, that is fully or partially complementary to a target domain or target polynucleotide within a DNA sequence in the genome of a cell where editing is desired.
  • Guide sequences are typically 10-30 nucleotides in length, preferably 16-24 nucleotides in length (for example, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length), and are at or near the 5' terminus of a Cas9 gRNA.
  • a “target domain” or “target polynucleotide sequence” or “target sequence” is the DNA sequence in a genome of a cell that is complementary to the guide sequence of the gRNA.
  • target sequence refers to a sequence to which a guide sequence is designed to have some complementarity, where hybridization between a 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.
  • a target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • a target sequence is located in the nucleus or cytoplasm of a cell. In other embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or nucleus.
  • a CRISPR complex comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins
  • formation of a CRISPR complex results in cleavage of one or both strands in or near (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) the target sequence.
  • complete complementarity is not needed, provided this is sufficient to be functional.
  • one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell, such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites.
  • a Cas nuclease, a crRNA, and a tracrRNA 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.
  • CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5’ with respect to (“upstream” of) or 3’ with respect to (“downstream” of) a second element.
  • the coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction.
  • a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron).
  • the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme).
  • 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.
  • a tagged CRISPR enzyme is used to identify the location of a target sequence.
  • Non-viral vector delivery systems include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell (Anderson, 1992, Science 256:808-813; and Yu, et al., 1994, Gene Therapy 1 : 13-26).
  • the CRISPR/Cas is derived from a type II CRISPR/Cas system.
  • the CRISPR/Cas sytem is derived from a Cas9 nuclease.
  • Exemplary Cas9 nucleases that may be used in the present invention include, but are not limited to, S. pyogenes Cas9 (SpCas9), S. aureus Cas9 (SaCas9), S. thermophilus Cas9 (StCas9), N meningitidis Cas9 (NmCas9), C. jejuni Cas9 (CjCas9), and Geobacillus Cas9 (GeoCas9).
  • Cas proteins comprise at least one RNA recognition and/or RNA binding domain. RNA recognition and/or RNA binding domains interact with the guiding RNA. Cas proteins can also comprise nuclease domains (ie., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains.
  • the Cas proteins can be modified to increase nucleic acid binding affinity and/or specificity, alter an enzymatic activity, and/or change another property of the protein.
  • the Cas-like protein of the fusion protein can be derived from a wild type Cas9 protein or fragment thereof.
  • the Cas can be derived from modified Cas9 protein.
  • the amino acid sequence of the Cas9 protein can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, and so forth) of the protein.
  • domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein.
  • a Cas9 protein comprises at least two nuclease (z.e., DNase) domains.
  • a Cas9 protein can comprise a RuvC-like nuclease domain and a HNH- like nuclease domain.
  • the Cas9-derived protein can be modified to contain only one functional nuclease domain (either a RuvC-like or a HNH-like nuclease domain).
  • the Cas9-derived protein can be modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., the nuclease activity is absent).
  • the Cas9-derived protein is able to introduce a nick into a double-stranded nucleic acid (such protein is termed a “nickase”), but not cleave the doublestranded DNA.
  • nickase a double-stranded nucleic acid
  • any or all of the nuclease domains can be inactivated by one or more deletion mutations, insertion mutations, and/or substitution mutations using well-known methods, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art.
  • a vector drives the expression of the CRISPR system.
  • the art is replete with suitable vectors that are useful in the present invention.
  • the vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells.
  • Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • the vectors of the present invention may also be used for nucleic acid standard gene delivery protocols. Methods for gene delivery are known in the art (U.S. Patent Nos. 5,399,346, 5,580,859 & 5,589,466, incorporated by reference herein in their entireties).
  • the vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (4th Edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012), and in other virology and molecular biology manuals.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sindbis virus, gammaretrovirus and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01/96584; WO 01/29058; and U.S. Patent No. 6,326,193).
  • guide RNA(s) and Cas9 can be delivered to a cell as a ribonucleoprotein (RNP) complex (e.g., a Cas9/RNA-protein complex).
  • RNP ribonucleoprotein
  • RNPs are comprised of purified Cas9 protein complexed with gRNA and are well known in the art to be efficiently delivered to multiple types of cells, including but not limited to stem cells and immune cells (Addgene, Cambridge, MA, Minis Bio LLC, Madison, WI).
  • the Cas9/RNA-protein complex is delivered into a cell by electroporation.
  • a gene edited modified cell of the present disclosure is edited using CRISPR/Cas9 to disrupt an endogenous gene locus encoding Roquin-1 and/or Regnase-1.
  • Suitable gRNAs for use in disrupting Roquin-1 and/or Regnase-1 are set forth in SEQ ID NOs: 1-16). It will be understood to those of skill in the art that guide RNA sequences may be recited with a thymidine (T) or a uridine (U) nucleotide.
  • a modified immune cell or precursor cell thereof comprising a CRISPR-mediated modification in an endogenous gene locus encoding Roquin-1 and/or Regnase-1, wherein the modification is capable of downregulating gene expression of endogenous Roquin-1 and/or Regnase-1; and an exogenous CAR and/or TCR comprising affinity for an antigen on a target cell.
  • the nucleic acid encoding an exogenous TCR and/or CAR is inserted into an exon of the endogenous gene locus encoding Roquin-1 and/or Regnase-1. In some embodiments, the nucleic acid encoding an exogenous TCR and/or CAR is inserted into a splice donor of the endogenous gene locus encoding Roquin-1 and/or Regnase-1. In some embodiments, the nucleic acid encoding an exogenous TCR and/or CAR is inserted into a splice acceptor of the endogenous gene locus encoding Roquin-1 and/or Regnase-1. The nucleic acid encoding an exogenous TCR and/or CAR may be inserted into any target site (e.g., genomic sequence) that results in the simultaneous disruption of the expression of endogenous Roquin-1 and/or Regnase-1.
  • any target site e.g., genomic sequence
  • the modified cell is an autologous cell. In certain embodiments, the modified cell is a cell isolated from a human subject. In certain embodiments, the modified cell is a modified immune cell. In certain embodiments, the modified cell is a modified T cell. In certain embodiments, the modified cell is a modified T cell resistant to T cell exhaustion.
  • compositions and methods include those in which at least or greater than about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of immune cells in a composition of immune cells contain the desired genetic modification.
  • about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of immune cells in a composition of cells into which an agent (e.g. gRNA/Cas9) for knockout or genetic disruption of endogenous gene (e.g., Roquin-1 and/or Regnase-1) was introduced contain the genetic disruption; do not express the targeted endogenous polypeptide, do not contain a contiguous and/or functional copy of the targeted gene.
  • an agent e.g. gRNA/Cas9
  • endogenous gene e.g., Roquin-1 and/or Regnase-1
  • the methods, compositions and cells according to the present disclosure include those in which at least or greater than about 50%, 60%, 65%, 70%. 75%, 80%, 85%, 90% or 95% of cells in a composition of cells into which an agent (e.g. gRNA/Cas9) for knockout or genetic disruption of a targeted gene was introduced do not express the targeted polypeptide, such as on the surface of the immune cells.
  • at least or greater than about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of cells in a composition of cells into which an agent (e.g. gRNA/Cas9) for knockout or genetic disruption of the targeted gene was introduced are knocked out in both alleles, i.e. comprise a biallelic deletion, in such percentage of cells.
  • compositions and methods in which the Cas9- mediated cleavage efficiency (% indel) in or near the targeted gene is at least or greater than about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% in cells of a composition of cells into which an agent (e.g. gRNA/Cas9) for knockout or genetic disruption of a targeted gene has been introduced.
  • an agent e.g. gRNA/Cas9
  • the provided cells, compositions and methods results in a reduction or disruption of signals delivered via the endogenous in at least or greater than about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of cells in a composition of cells into which an agent (e.g. gRNA/Cas9) for knockout or genetic disruption of a targeted gene was introduced.
  • an agent e.g. gRNA/Cas9 for knockout or genetic disruption of a targeted gene was introduced.
  • the modified cell of the present disclosure is genetically edited to disrupt the expression of Roquin-1 and/or Regnase-1.
  • disruption of Roquin-1 and/or Regnase-1 is shown by the present disclosure to enhance immune cell (e.g., T cell) function.
  • disrupting the expression of Roquin-1 and/or Regnase-1 may result in reduced expression of genes involved in negatively regulating immune cell function (e.g., T cell survival) and/or enhanced expression of genes involved in positively regulating immune cell function (e.g., pro-survival factors), thus increasing efficacy of the gene edited immune cells.
  • disrupting the expression of Roquin-1 and/or Regnase-1 enhances inflammatory gene signatures long-term and facilitates greater function and persistence of human CAR-T cells against cancer.
  • the modified cell of the present disclosure is genetically edited to disrupt the expression of an additional endogeneous gene.
  • the cell may be further edited to disrupt an endogenous PDCD1 gene product (e.g. Programmed Death 1 receptor; PD- 1). Disrupting the expression of endogenous PD-1 may create “checkpoint” resistant modified cells, resulting in increased tumor control.
  • PDCD1 Endogenous PDCD1 gene product
  • Checkpoint resistant modified cells may also be created by disrupting the expression of, for example, without limitation, the Adenosine A2A receptor (A2AR), B7-H3 (CD276), B7-H4 (VTCN1), the B and T Lymphocyte Attenuator protein (BTLA/CD272), CD96, the Cytotoxic T-Lymphocyte Associated protein 4 (CTLA- 4/CD152), Indoleamine 2,3 -dioxygenase (IDO), the Killer-cell Immunoglobulin-like Receptor (KIR), the Lymphocyte Activation Gene-3 (LAG3), the T cell immunoreceptor with Ig and ITIM domains (TIGIT), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3), the CD200 Receptor (CD200R), or the V-domain Ig suppressor of T cell activation (VISTA).
  • A2AR Adenosine A2A receptor
  • B7-H3 CD276
  • B7-H4 B
  • the cell may be further edited to disrupt other endogeneous genes, including but not limited to, the T Cell Receptor Alpha Chain (TRAC), T Cell Receptor Beta Chain (TRBC), Programmed Death- 1 (PD- 1) Ligand 1 (PDL1), and Transforming Growth Gactor P (TGF-P) Receptor (TGFpR).
  • T Cell Receptor Alpha Chain T Cell Receptor Beta Chain
  • TRBC T Cell Receptor Beta Chain
  • PD- 1 PD- 1
  • PDL1 Transforming Growth Gactor P
  • TGF-P Transforming Growth Gactor P Receptor Receptor
  • compositions according to the provided disclosure that comprise cells engineered with a recombinant receptor and comprise the reduction, deletion, elimination, knockout or disruption in expression of an endogenous gene (e.g. genetic disruption of Roquin-1 and/or Regnase-1) retain the functional property or activities of the receptor compared to the receptor expressed in engineered cells of a corresponding or reference composition comprising the receptor but do not comprise the genetic disruption of a gene or express the polypeptide when assessed under the same conditions.
  • an endogenous gene e.g. genetic disruption of Roquin-1 and/or Regnase-1
  • the engineered cells of the provided compositions retain a functional property or activity compared to a corresponding or reference composition comprising engineered cells in which such are engineered with the recombinant receptor but do not comprise the genetic disruption or express the targeted polypeptide when assessed under the same conditions.
  • the cells retain cytotoxicity, proliferation, survival or cytokine secretion compared to such a corresponding or reference composition.
  • the immune cells in the composition retain a phenotype of the immune cell or cells compared to the phenotype of cells in a corresponding or reference composition when assessed under the same conditions.
  • cells in the composition include naive cells, effector memory cells, central memory cells, stem central memory cells, effector memory cells, and long-lived effector memory cells.
  • the percentage of T cells, or T cells expressing the recombinant receptor e.g.
  • TCR and/or CAR TCR and/or CAR
  • a targeted gene e.g., Roquin-1 and/or Regnase-1
  • activity or phenotype can be measured in an in vitro assay, such as by incubation of the cells in the presence of an antigen targeted by the TCR and/or CAR, a cell expressing the antigen and/or an antigen-receptor activating substance.
  • any of the assessed activities, properties or phenotypes can be assessed at various days following electroporation or other introduction of the agent, such as after or up to 3, 4, 5, 6, 7 days.
  • such activity, property or phenotype is retained by at least 80%, 85%, 90%, 95% or 100% of the cells in the composition compared to the activity of a corresponding composition containing cells engineered with the recombinant receptor but not comprising the genetic disruption of the targeted gene when assessed under the same conditions.
  • a "corresponding composition” or a “corresponding population of immune cells” refers to immune cells (e.g., T cells) obtained, isolated, generated, produced and/or incubated under the same or substantially the same conditions, except that the immune cells or population of immune cells were not introduced with the agent.
  • immune cells e.g., T cells
  • such immune cells are treated identically or substantially identically as immune cells that have been introduced with the agent, such that any one or more conditions that can influence the activity or properties of the cell, including the upregulation or expression of the inhibitory molecule, is not varied or not substantially varied between the cells other than the introduction of the agent.
  • T cell markers Methods and techniques for assessing the expression and/or levels of T cell markers are known in the art. Antibodies and reagents for detection of such markers are well known in the art, and readily available. Assays and methods for detecting such markers include, but are not limited to, flow cytometry, including intracellular flow cytometry, ELISA, ELISPOT, cytometric bead array or other multiplex methods, Western Blot and other immunoaffinity-based methods. In some embodiments, antigen receptor (e.g. TCR and/or CAR)-expressing cells can be detected by flow cytometry or other immunoaffinity based method for expression of a marker unique to such cells, and then such cells can be co-stained for another T cell surface marker or markers.
  • flow cytometry including intracellular flow cytometry, ELISA, ELISPOT, cytometric bead array or other multiplex methods, Western Blot and other immunoaffinity-based methods.
  • antigen receptor e.g. TCR and/or CAR
  • the cells, compositions and methods provide for the deletion, knockout, disruption, or reduction in expression of the target gene in immune cells (e.g. T cells) to be adoptively transferred (such as cells engineered to express an exogenous TCR and/or CAR).
  • the methods are performed ex vivo on primary cells, such as primary immune cells (e.g. T cells) from a subject.
  • methods of producing or generating such genetically engineered T cells include introducing into a population of cells containing immune cells (e.g. T cells) one or more nucleic acid encoding a recombinant receptor (e.g.
  • exogenous TCR and/or CAR and an agent or agents that is capable of disrupting, a gene that encode the endogenous receptor to be targeted.
  • introducing encompasses a variety of methods of introducing DNA into a cell, either in vitro or in vivo, such methods including transformation, transduction, transfection (e.g. electroporation), and infection.
  • Vectors are useful for introducing DNA encoding molecules into cells. Possible vectors include plasmid vectors and viral vectors. Viral vectors include retroviral vectors, lentiviral vectors, or other vectors such as adenoviral vectors or adeno-associated vectors.
  • the population of cells containing T cells can be cells that have been obtained from a subject, such as obtained from a peripheral blood mononuclear cells (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product.
  • T cells can be separated or selected to enrich T cells in the population using positive or negative selection and enrichment methods.
  • the population contains CD4+, CD8+ or CD4+ and CD8+ T cells.
  • the step of introducing the nucleic acid encoding a genetically engineered antigen receptor and the step of introducing the agent e.g.
  • Cas9/gRNA RNP can occur simultaneously or sequentially in any order.
  • the cells subsequent to introduction of the exogenous receptor and one or more gene editing agents (e.g. Cas9/gRNA RNP), the cells are cultured or incubated under conditions to stimulate expansion and/or proliferation of cells.
  • gene editing agents e.g. Cas9/gRNA RNP
  • the genetically engineered cells exhibit increased expansion and/or persistence when administered in vivo to a subject, as compared to certain available methods.
  • the provided immune cells exhibit increased persistence when administered in vivo to a subject.
  • the persistence of genetically engineered immune cells, in the subject upon administration is greater as compared to that which would be achieved by alternative methods, such as those involving administration of cells genetically engineered by methods in which T cells were not introduced with an agent that reduces expression of or disrupts a gene encoding an endogenous receptor.
  • the persistence is increased at least or about at least 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 60- fold, 70-fold, 80-fold, 90-fold, 100-fold or more.
  • the immune cell or precursor cell thereof is a T cell.
  • the T cell is a human T cell.
  • the cell is an autologous cell (e.g. an autologous T cell).
  • the modified cells can comprise any chimeric antigen receptor (CAR) disclosed herein.
  • CAR chimeric antigen receptor
  • cells, compositions and methods that enhance immune cell, such as T cell, function in adoptive cell therapy including those offering improved efficacy, such as by increasing activity and potency of administered genetically engineered cells, while maintaining persistence or exposure to the transferred cells over time.
  • the degree or extent of persistence of administered cells can be detected or quantified after administration to a subject.
  • quantitative PCR qPCR
  • persistence is quantified as copies of DNA or plasmid encoding the exogenous receptor per microgram of DNA, or as the number of receptor-expressing cells per microliter of the sample, e.g., of blood or serum, or per total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells per microliter of the sample.
  • PBMCs peripheral blood mononuclear cells
  • flow cytometric assays detecting cells expressing the receptor generally using antibodies specific for the receptors also can be performed.
  • Cell-based assays may also be used to detect the number or percentage of functional cells, such as cells capable of binding to and/or neutralizing and/or inducing responses, e.g., cytotoxic responses, against cells of the disease or condition or expressing the antigen recognized by the receptor.
  • the extent or level of expression of another marker associated with the modified cell can be used to distinguish the administered cells from endogenous cells in a subject.
  • Disruption or reduction of Regnase-1 and/or Roquin-1 can carried out by any means known in the art including but not limited to miRNA, siRNA, a drug, an antibody, an inhibitor, and the like.
  • the present invention provides compositions and methods for modified immune cells or precursors thereof, e.g., modified T cells, comprising a chimeric antigen receptor (CAR).
  • modified T cells comprising a chimeric antigen receptor (CAR).
  • CARs of the present invention comprise an antigen binding domain, a transmembrane domain, and an intracellular domain.
  • the antigen binding domain of a CAR is an extracellular region of the CAR for binding to a specific target antigen including proteins, carbohydrates, and glycolipids.
  • the CAR comprises affinity to a target antigen on a target cell.
  • the target antigen may include any type of protein, or epitope thereof, associated with the target cell.
  • the CAR may comprise affinity to a target antigen on a target cell that indicates a particular disease state of the target cell.
  • the target cell antigen is a tumor associated antigen (TAA).
  • TAAs tumor associated antigens
  • TAAs include but are not limited to, differentiation antigens such as MART-l/MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EB VA and the human papillomavirus (HPV) antigens E6 and
  • the antigen binding domain of the CAR targets an antigen that includes but is not limited to CD 19, CD20, CD22, BCMA, ROR1, Mesothelin, CD33/IL3Ra, c-Met, PSMA, PSCA, Glycolipid F77, EGFRvIII, GD-2, TnMUCl, NY-ESO- 1 TCR, MAGE A3 TCR, and the like.
  • the CAR of the invention can be engineered to include the appropriate antigen binding domain that is specific to the desired antigen target.
  • an antibody for CD 19 can be used as the antigen bind moiety for incorporation into the CAR of the invention.
  • the target cell antigen is mesothelin.
  • a CAR of the present disclosure has affinity for mesothelin on a target cell.
  • the target cell antigen is CD 19.
  • a CAR of the present disclosure has affinity for CD19 on a target cell. This should not be construed as limiting in any way, as a CAR having affinity for any target antigen is suitable for use in a composition or method of the present invention.
  • a CAR of the present disclosure having affinity for a specific target antigen on a target cell may comprise a target-specific binding domain.
  • the target-specific binding domain is a murine target-specific binding domain, e.g., the targetspecific binding domain is of murine origin.
  • the target-specific binding domain is a human target-specific binding domain, e.g., the target-specific binding domain is of human origin.
  • a CAR of the present disclosure having affinity for CD 19 on a target cell may comprise a CD 19 binding domain.
  • a CAR of the present disclosure may have affinity for one or more target antigens on one or more target cells.
  • a CAR may have affinity for one or more target antigens on a target cell.
  • the CAR is a bispecific CAR, or a multispecific CAR.
  • the CAR comprises one or more target-specific binding domains that confer affinity for one or more target antigens.
  • the CAR comprises one or more target-specific binding domains that confer affinity for the same target antigen.
  • a CAR comprising one or more target-specific binding domains having affinity for the same target antigen could bind distinct epitopes of the target antigen.
  • the binding domains may be arranged in tandem and may be separated by linker peptides.
  • the binding domains are connected to each other covalently on a single polypeptide chain, through an oligo- or polypeptide linker, an Fc hinge region, or a membrane hinge region.
  • the antigen binding domain is selected from the group consisting of an antibody, an antigen binding fragment (Fab), and a single-chain variable fragment (scFv).
  • a CD 19 binding domain of the present invention is selected from the group consisting of a CD19-specific antibody, a CD19-specific Fab, and a CD19-specific scFv.
  • a CD 19 binding domain is a CD19-specific antibody.
  • a CD19 binding domain is a CD19-specific Fab.
  • a CD19 binding domain is a CD19-specific scFv.
  • a mesothelin binding domain of the present invention is selected from the group consisting of a mesothelin-specific antibody, a mesothelin- specific Fab, and a mesothelin-specific scFv.
  • a mesothelin binding domain is a mesothelin-specific antibody.
  • a mesothelin binding domain is an a mesothelin-specific Fab.
  • an a mesothelin binding domain is an a mesothelin- specific scFv.
  • the antigen binding domain can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, and any fragment thereof.
  • the antigen binding domain portion comprises a mammalian antibody or a fragment thereof. The choice of antigen binding domain may depend upon the type and number of antigens that are present on the surface of a target cell.
  • single-chain variable fragment is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH::VL heterodimer.
  • the heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoding linker, which connects the N- terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N- terminus of the VL.
  • the antigen binding domain (e.g., PSCA binding domain) comprises an scFv having the configuration from N-terminus to C-terminus, VH - linker - VL. In some embodiments, the antigen binding domain comprises an scFv having the configuration from N-terminus to C-terminus, VL - linker - VH. Those of skill in the art would be able to select the appropriate configuration for use in the present invention.
  • the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility.
  • the linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain.
  • Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014/087010, the contents of which are hereby incorporated by reference in their entireties.
  • GS linker sequences include, without limitation, glycine serine (GS) linkers such as (GS)n, (GSGGS)n (SEQ ID NO: 19), (GGGS)n (SEQ ID NO: 20), and (GGGGS)n (SEQ ID NO: 21), where n represents an integer of at least 1.
  • GS glycine serine
  • Exemplary linker sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO: 22), GGSGG (SEQ ID NO:23), GSGSG (SEQ ID NO: 24), GSGGG (SEQ ID NO: 25), GGGSG (SEQ ID NO: 26), GSSSG (SEQ ID NO: 27), GGGGS (SEQ ID NO: 28), GGGGSGGGGSGGGGS (SEQ ID NO: 29) and the like.
  • GGSG SEQ ID NO: 22
  • GGSGG SEQ ID NO:23
  • GSGSG SEQ ID NO: 24
  • GSGGG SEQ ID NO: 25
  • GGGSG SEQ ID NO: 26
  • GSSSG SEQ ID NO: 27
  • GGGGS SEQ ID NO: 28
  • GGGGSGGGGSGGGGS SEQ ID NO: 29
  • an antigen binding domain of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL is separated by the linker sequence having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 29), which may be encoded by the nucleic acid sequence GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT (SEQ ID NO: 30).
  • Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754.
  • Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3): 173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40).
  • Fab refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).
  • an antibody digested by the enzyme papain yields two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).
  • F(ab')2 refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab') (bivalent) regions, wherein each (ab') region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S — S bond for binding an antigen and where the remaining H chain portions are linked together.
  • a “F(ab')2” fragment can be split into two individual Fab' fragments.
  • the antigen binding domain may be derived from the same species in which the CAR will ultimately be used.
  • the antigen binding domain of the CAR may comprise a human antibody or a fragment thereof.
  • the antigen binding domain may be derived from a different species in which the CAR will ultimately be used.
  • the antigen binding domain of the CAR may comprise a murine antibody or a fragment thereof.
  • the antigen binding domain may be operably linked to another domain of the CAR, such as the transmembrane domain or the intracellular domain, both described elsewhere herein, for expression in the cell.
  • a first nucleic acid sequence encoding the antigen binding domain is operably linked to a second nucleic acid encoding a transmembrane domain, and further operably linked to a third a nucleic acid sequence encoding an intracellular domain.
  • the antigen binding domains described herein can be combined with any of the transmembrane domains described herein, any of the intracellular domains or cytoplasmic domains described herein, or any of the other domains described herein that may be included in a CAR of the present invention.
  • a subject CAR of the present invention may also include a hinge domain as described herein.
  • a subject CAR of the present invention may also include a spacer domain as described herein.
  • each of the antigen binding domain, transmembrane domain, and intracellular domain is separated by a linker.
  • CARs of the present invention may comprise a transmembrane domain that connects the antigen binding domain of the CAR to the intracellular domain of the CAR.
  • the transmembrane domain of a subject CAR is a region that is capable of spanning the plasma membrane of a cell (e.g., an immune cell or precursor thereof).
  • the transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane.
  • the transmembrane domain is interposed between the antigen binding domain and the intracellular domain of a CAR.
  • the transmembrane domain is naturally associated with one or more of the domains in the CAR.
  • the transmembrane domain can be selected or modified by one or more amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, to minimize interactions with other members of the receptor complex.
  • the transmembrane domain may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein, e.g., a Type I transmembrane protein. Where the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates insertion of the CAR into a cell membrane, e.g., an artificial hydrophobic sequence. Examples of the transmembrane domain of particular use in this invention include, without limitation, transmembrane domains derived from (i.e.
  • the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that may be included in a subject CAR.
  • the transmembrane domain further comprises a hinge region.
  • a subject CAR of the present invention may also include a hinge region.
  • the hinge region of the CAR is a hydrophilic region which is located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR.
  • the hinge region is an optional component for the CAR.
  • the hinge region may include a domain selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof.
  • hinge regions include, without limitation, a CD8a hinge, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), as well as CHI and CH3 domains of IgGs (such as human IgG4).
  • a subject CAR of the present disclosure includes a hinge region that connects the antigen binding domain with the transmembrane domain, which, in turn, connects to the intracellular domain.
  • the hinge region is preferably capable of supporting the antigen binding domain to recognize and bind to the target antigen on the target cells (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135).
  • the hinge region is a flexible domain, thus allowing the antigen binding domain to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as tumor cell (Hudecek et al., supra). The flexibility of the hinge region permits the hinge region to adopt many different conformations.
  • the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).
  • the hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa.
  • the hinge region can have a length of greater than 5 aa, greater than 10 aa, greater than 15 aa, greater than 20 aa, greater than 25 aa, greater than 30 aa, greater than 35 aa, greater than 40 aa, greater than 45 aa, greater than 50 aa, greater than 55 aa, or more.
  • Suitable hinge regions can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g, Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.
  • Suitable hinge regions can have a length of greater than 20 amino acids (e.g., 30, 40, 50, 60 or more amino acids).
  • hinge regions include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 19) and (GGGS)n (SEQ ID NO:20), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.
  • Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components.
  • Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142).
  • Exemplary hinge regions can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO:22), GGSGG (SEQ ID NO: 23), GSGSG (SEQ ID NO: 24), GSGGG (SEQ ID NO: 25), GGGSG (SEQ ID NO: 26), GSSSG (SEQ ID NO: 27), and the like.
  • the hinge region is an immunoglobulin heavy chain hinge region.
  • Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al., Proc. Natl. Acad. Sci. USA (1990) 87(1): 162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789.
  • an immunoglobulin hinge region can include one of the following amino acid sequences: DKTHT (SEQ ID NO: 31); CPPC (SEQ ID NO: 32); CPEPKSCDTPPPCPR (SEQ ID NO: 33) (see, e.g, Glaser et al, J. Biol. Chem.
  • ELKTPLGDTTHT SEQ ID NO: 34
  • KSCDKTHTCP SEQ ID NO: 35
  • KCCVDCP SEQ ID NO: 36
  • KYGPPCP SEQ ID NO: 37
  • EPKSCDKTHTCPPCP SEQ ID NO: 38
  • ELKTPLGDTTHTCPRCP SEQ ID NO: 40
  • SPNMVPHAHHAQ SEQ ID NO: 41
  • the hinge region can comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region.
  • the hinge region can include one or more amino acid substitutions and/or insertions and/or deletions compared to a wild-type (naturally-occurring) hinge region.
  • His229 of human IgGl hinge can be substituted with Tyr, so that the hinge region comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 41); see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897.
  • the hinge region can comprise an amino acid sequence derived from human CD8, or a variant thereof.
  • the CAR comprises a CD8a hinge domain and/or a CD8a transmembrane domain.
  • a subject CAR of the present invention also includes an intracellular signaling domain.
  • the terms “intracellular signaling domain” and “intracellular domain” are used interchangeably herein.
  • the intracellular signaling domain of the CAR is responsible for activation of at least one of the effector functions of the cell in which the CAR is expressed (e.g., immune cell).
  • the intracellular signaling domain transduces the effector function signal and directs the cell (e.g., immune cell) to perform its specialized function, e.g., harming and/or destroying a target cell.
  • an intracellular domain for use in the invention examples include, but are not limited to, the cytoplasmic portion of a surface receptor, co-stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in the T cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability.
  • intracellular signaling domain examples include, without limitation, the C, chain of the T cell receptor complex or any of its homologs, e.g., q chain, FcsRIy and P chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (A, 6 and a), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28.
  • the intracellular signaling domain may be human CD3 zeta chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (IT AM) bearing cytoplasmic receptors, and combinations thereof.
  • the intracellular signaling domain of the CAR includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD2, CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof.
  • intracellular domain examples include a fragment or domain from one or more molecules or receptors including, but not limited to, TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, 0X40, CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4,
  • intracellular domains include, without limitation, intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, intracellular signaling domains may include signaling domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol.
  • NKp30 B7-H6
  • DAP 12 see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212
  • NKG2D NKp44
  • NKp46 NKp46
  • DAP10 CD3z
  • Intracellular signaling domains suitable for use in a subject CAR of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation of the CAR (z.e., activated by antigen and dimerizing agent).
  • the intracellular signaling domain includes at least one (e.g., one, two, three, four, five, six, etc.) IT AM motifs as described below.
  • the intracellular signaling domain includes DAP10/CD28 type signaling chains.
  • the intracellular signaling domain is not covalently attached to the membrane bound CAR, but is instead diffused in the cytoplasm.
  • Intracellular signaling domains suitable for use in a subject CAR of the present invention include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides.
  • ITAM immunoreceptor tyrosine-based activation motif
  • an IT AM motif is repeated twice in an intracellular signaling domain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids.
  • the intracellular signaling domain of a subject CAR comprises 3 ITAM motifs.
  • intracellular signaling domains includes the signaling domains of human immunoglobulin receptors that contain immunoreceptor tyrosine based activation motifs (IT AMs) such as, but not limited to, FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).
  • IT AMs immunoreceptor tyrosine based activation motifs
  • a suitable intracellular signaling domain can be an ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif.
  • a suitable intracellular signaling domain can be an ITAM motif-containing domain from any ITAM motif-containing protein.
  • a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived.
  • ITAM motif-containing polypeptides include, but are not limited to: DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).
  • the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX- activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor associated protein; killer-activating receptor-associated protein; etc.).
  • DAP12 also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX- activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor associated protein; killer-activating receptor-associated protein; etc.
  • the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon Rl-gamma; fcRgamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.).
  • FCER1G also known as FCRG
  • Fc epsilon receptor I gamma chain Fc receptor gamma-chain
  • fcRgamma fcRgamma
  • fceRl gamma high affinity immunoglobulin epsilon receptor subunit gamma
  • immunoglobulin E receptor high affinity, gamma chain; etc.
  • the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T-cell receptor T3 delta chain; T-cell surface glycoprotein CD3 delta chain; etc.).
  • T-cell surface glycoprotein CD3 delta chain also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T-cell receptor T3 delta chain; T-cell surface glycoprotein CD3 delta chain; etc.
  • the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T- cell surface antigen T3/Leu-4 epsilon chain, T-cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.).
  • the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 gamma chain (also known as CD3G, T-cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.).
  • the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T-cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.).
  • the intracellular signaling domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig- alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.).
  • an intracellular signaling domain suitable for use in a CAR of the present disclosure includes a DAP10/CD28 type signaling chain. In one embodiment, an intracellular signaling domain suitable for use in a CAR of the present disclosure includes a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain includes a cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR includes a cytoplasmic signaling domain of human CD3 zeta.
  • intracellular signaling domain While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.
  • the intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • the intracellular signaling domains described herein can be combined with any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that may be included in the CAR.
  • the present invention provides compositions and methods for modified immune cells or precursors thereof (e.g., modified T cells) comprising an exogenous T cell receptor (TCR).
  • modified T cells comprising an exogenous T cell receptor (TCR).
  • the cell has been altered to contain specific T cell receptor (TCR) genes (e.g., a nucleic acid encoding an alpha/beta TCR).
  • TCRs or antigen-binding portions thereof include those that recognize a peptide epitope or T cell epitope of a target polypeptide, such as an antigen of a tumor, viral or autoimmune protein.
  • the TCR has binding specificity for a tumor associated antigen, e.g., human NY-ESO-1.
  • the immune cell or precursor thereof comprises a modification in the endogenous gene locus encoding Roquin-1 that is capable of downregulating gene expression of the endogenous Roquin-1, and an exogeneous TCR comprising affinity for an antigen on a target cell.
  • the immune cell or precursor thereof comprises a modification in the endogenous gene locus encoding Regnase-1 that is capable of downregulating gene expression of the endogenous Regnase-1, and an exogeneous TCR comprising affinity for an antigen on a target cell.
  • the immune cell or precursor thereof comprises a modification in the endogenous gene locus encoding Roquin-1 and Regnase-1 that is capable of downregulating gene expression of the endogenous Roquin-1 and Regnase-1, and an exogeneous TCR comprising affinity for an antigen on a target cell.
  • the invention provides a modified immune cell or precursor cell thereof, wherein the endogenous Roquin-1 has been reduced or knocked out, and an exogeneous TCR comprising affinity for an antigen on a target cell.
  • the modified immune cell or precursor thereof comprises an exogeneous TCR comprising affinity for an antigen on a target cell, and the endogenous Regnase-1 has been reduced or knocked out.
  • the modified immune cell or precursor thereof comprises an exogeneous TCR comprising affinity for an antigen on a target cell, and the endogenous Roquin-1 and Regnase-1 have been reduced or knocked out.
  • a TCR is a disulfide-linked heterodimeric protein comprised of six different membrane bound chains that participate in the activation of T cells in response to an antigen.
  • alpha/beta TCRs and gamma/delta TCRs There exists alpha/beta TCRs and gamma/delta TCRs.
  • An alpha/beta TCR comprises a TCR alpha chain and a TCR beta chain.
  • T cells expressing a TCR comprising a TCR alpha chain and a TCR beta chain are commonly referred to as alpha/beta T cells.
  • Gamma/delta TCRs comprise a TCR gamma chain and a TCR delta chain.
  • T cells expressing a TCR comprising a TCR gamma chain and a TCR delta chain are commonly referred to as gamma/delta T cells.
  • a TCR of the present disclosure is a TCR comprising a TCR alpha chain and a TCR beta chain.
  • the TCR alpha chain and the TCR beta chain are each comprised of two extracellular domains, a variable region and a constant region.
  • the TCR alpha chain variable region and the TCR beta chain variable region are required for the affinity of a TCR to a target antigen.
  • Each variable region comprises three hypervariable or complementarity-determining regions (CDRs) which provide for binding to a target antigen.
  • CDRs hypervariable or complementarity-determining regions
  • the constant region of the TCR alpha chain and the constant region of the TCR beta chain are proximal to the cell membrane.
  • a TCR further comprises a transmembrane region and a short cytoplasmic tail. CD3 molecules are assembled together with the TCR heterodimer.
  • CD3 molecules comprise a characteristic sequence motif for tyrosine phosphorylation, known as immunoreceptor tyrosine-based activation motifs (IT AMs). Proximal signaling events are mediated through the CD3 molecules, and accordingly, TCR-CD3 complex interaction plays an important role in mediating cell recognition events.
  • IT AMs immunoreceptor tyrosine-based activation motifs
  • TCR Stimulation of TCR is triggered by major histocompatibility complex molecules (MHCs) on antigen presenting cells that present antigen peptides to T cells and interact with TCRs to induce a series of intracellular signaling cascades. Engagement of the TCR initiates both positive and negative signaling cascades that result in cellular proliferation, cytokine production, and/or activation-induced cell death.
  • MHCs major histocompatibility complex molecules
  • a TCR of the present invention can be a wild-type TCR, a high affinity TCR, and/or a chimeric TCR.
  • a high affinity TCR may be the result of modifications to a wild-type TCR that confers a higher affinity for a target antigen compared to the wild-type TCR.
  • a high affinity TCR may be an affinity-matured TCR. Methods for modifying TCRs and/or the affinitymaturation of TCRs are known to those of skill in the art.
  • TCR heterodimers which include the native disulphide bridge which connects the respective subunits (Garboczi, et al., (1996), Nature 384(6605): 134-41; Garboczi, et al., (1996), J Immunol 157(12): 5403-10; Chang et al., (1994), PNAS USA 91 : 11408-11412; Davodeau et al., (1993), J. Biol. Chem. 268(21): 15455-15460; Golden et al., (1997), J. Imm. Meth. 206: 163-169; U.S. Pat. No. 6,080,840).
  • the exogenous TCR is a full TCR or an antigen-binding portion or antigen-binding fragment thereof.
  • the TCR is an intact or full-length TCR, including TCRs in the aP form or y6 form.
  • the TCR is an antigenbinding portion that is less than a full-length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC-peptide complex.
  • an antigen-binding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as 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 P chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex.
  • the variable chains of a TCR contain complementarity determining regions (CDRs) involved in recognition of the peptide, MHC and/or MHC-peptide complex.
  • variable domains of the TCR contain hypervariable loops, or CDRs, which generally are the primary contributors to antigen recognition and binding capabilities and specificity.
  • CDRs hypervariable loops
  • a CDR of a TCR or combination thereof forms all or substantially all of the antigen-binding site of a given TCR molecule.
  • the various CDRs within a variable region of a TCR chain generally are separated by framework regions (FRs), which generally display less variability among TCR molecules as compared to the CDRs (see, e.g., lores et al, Proc. Nat'l Acad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J.
  • CDR3 is the main CDR responsible for antigen binding or specificity, or is the most important among the three CDRs on a given TCR variable region for antigen recognition, and/or for interaction with the processed peptide portion of the peptide-MHC complex.
  • the CDR1 of the alpha chain can interact with the N-terminal part of certain antigenic peptides.
  • CDR1 of the beta chain can interact with the C-terminal part of the peptide.
  • CDR2 contributes most strongly to or is the primary CDR responsible for the interaction with or recognition of the MHC portion of the MHC-peptide complex.
  • the variable region of the P-chain can contain a further hypervariable region (CDR4 or HVR4), which generally is involved in superantigen binding and not antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).
  • a TCR contains a variable alpha domain (Va) and/or a variable beta domain (Vp) or antigen-binding fragments thereof.
  • the a-chain and/or P-chain of a TCR also can contain a constant domain, a transmembrane domain and/or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3 Ed., Current Biology Publications, p. 4:33, 1997).
  • the a chain constant domain is encoded by the TRAC gene (IMGT nomenclature) or is a variant thereof.
  • the P chain constant region is encoded by TRBC1 or TRBC2 genes (IMGT nomenclature) or is a variant thereof.
  • the constant domain is adjacent to the cell membrane.
  • the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains, and two membrane-distal variable domains, which variable domains each contain CDRs.
  • IMGT International Immunogenetics Information System
  • the CDR1 sequences within a TCR Va chain and/or VP chain correspond to the amino acids present between residue numbers 27-38, inclusive
  • the CDR2 sequences within a TCR Va chain and/or VP chain correspond to the amino acids present between residue numbers 56-65, inclusive
  • the CDR3 sequences within a TCR Va chain and/or VP chain correspond to the amino acids present between residue numbers 105-117, inclusive.
  • the IMGT numbering system should not be construed as limiting in any way, as there are other numbering systems known to those of skill in the art, and it is within the level of the skilled artisan to use any of the numbering systems available to identify the various domains or regions of a TCR.
  • the TCR may be a heterodimer of two chains a and P (or optionally y and 6) that are linked, such as by a disulfide bond or disulfide bonds.
  • the constant domain of the TCR may contain short connecting sequences in which a cysteine residue forms a disulfide bond, thereby linking the two chains of the TCR.
  • a TCR may have an additional cysteine residue in each of the a and P chains, such that the TCR contains two disulfide bonds in the constant domains.
  • each of the constant and variable domains contain disulfide bonds formed by cysteine residues.
  • the TCR for engineering cells as described is one generated from a known TCR sequence(s), such as sequences of Va,P chains, for which a substantially full- length coding sequence is readily available. Methods for obtaining full-length TCR sequences, including V chain sequences, from cell sources are well known.
  • nucleic acids encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of TCR-encoding nucleic acids within or isolated from a given cell or cells, or synthesis 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.
  • the T cells can be obtained from in vivo isolated cells.
  • the T- cells can be a cultured T cell hybridoma or clone.
  • the TCR or antigen-binding portion thereof can be synthetically generated from knowledge of the sequence of the TCR.
  • a high-affinity T cell clone for a target antigen e.g., a cancer antigen is identified, isolated from a patient, and introduced into the cells.
  • 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). See, e.g., tumor antigens (see, e.g., Parkhurst et al. (2009) Clin Cancer Res. 15: 169-180 and Cohen et al. (2005) J Immunol. 175:5799-5808).
  • human immune system genes e.g., the human leukocyte antigen system, or HLA
  • tumor antigens see, e.g., Parkhurst et al. (2009) Clin Cancer Res. 15: 169-180 and Cohen et al. (2005) J Immunol. 175:5799-5808.
  • phage display is used to isolate TCRs against a target antigen (see, e.g., Varela-Rohena et al. (2008) Nat Med. 14: 1390-1395 and Li (2005) Nat Biotechnol. 23:3
  • the TCR or antigen-binding portion thereof is one that has been modified or engineered.
  • directed evolution methods are used to generate TCRs with altered properties, such as with higher affinity for a specific MHC-peptide complex.
  • directed evolution is achieved by display methods including, but not limited to, yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci U S A, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), or T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84).
  • display approaches involve engineering, or modifying, a known, parent or reference TCR.
  • a wild-type TCR can be used as a template for producing mutagenized TCRs in which in one or more residues of the CDRs are mutated, and mutants with an desired altered property, such as higher affinity for a desired target antigen, are selected.
  • the TCR can contain an introduced disulfide bond or bonds.
  • the native disulfide bonds are not present.
  • the one or more of the native cysteines (e.g. in the constant domain of the a chain and P chain) that form a native interchain disulfide bond are substituted with another residue, such as with a serine or alanine.
  • an introduced disulfide bond can be formed by mutating non-cysteine residues on the alpha and beta chains, such as in the constant domain of the a chain and P chain, to cysteine. Exemplary non-native disulfide bonds of a TCR are described in published International PCT No.
  • cysteines can be introduced at residue Thr48 of the a chain and Ser57 of the P chain, at residue Thr45 of the a chain and Ser77 of the P chain, at residue TyrlO of the a chain and Serl7 of the P chain, at residue Thr45 of the a chain and Asp59 of the P chain and/or at residue Serl5 of the a chain and Glul5 of the P chain.
  • the presence of non-native cysteine residues e.g.
  • resulting in one or more non-native disulfide bonds) in a recombinant TCR can favor production of the desired recombinant TCR in a cell in which it is introduced over expression of a mismatched TCR pair containing a native TCR chain.
  • the TCR chains contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some aspects, each chain (e.g. alpha or beta) 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. In some embodiments, a TCR, for example via the cytoplasmic tail, is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. In some cases, the structure allows the TCR to associate with other molecules like CD3 and subunits thereof.
  • a TCR containing constant domains with a transmembrane region may anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex.
  • the intracellular tails of CD3 signaling subunits e.g. CD3y, CD35, CD3s and CD3( ⁇ chains) contain one or more immunoreceptor tyrosine-based activation motif or IT AM that are involved in the signaling capacity of the TCR complex.
  • the TCR is a full-length TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR (sc-TCR). A TCR may be cell-bound or in soluble form. In some embodiments, for purposes of the provided methods, the TCR is in cell-bound form expressed on the surface of a cell.
  • a dTCR contains a first polypeptide wherein a sequence corresponding to a TCR a chain variable region sequence is fused to the N terminus of a sequence corresponding to a TCR a chain constant region extracellular sequence, and a second polypeptide wherein a sequence corresponding to a TCR P chain variable region sequence is fused to the N terminus a sequence corresponding to a TCR P chain constant region extracellular sequence, the first and second polypeptides being linked by a disulfide bond.
  • the bond can correspond to the native interchain disulfide bond present in native dimeric aP TCRs. In some embodiments, the interchain disulfide bonds are not present in a native TCR.
  • one or more cysteines can be incorporated into the constant region extracellular sequences of dTCR polypeptide pair.
  • both a native and a non-native disulfide bond may be desirable.
  • the TCR contains a transmembrane sequence to anchor to the membrane.
  • a dTCR contains a TCR a chain containing a variable a domain, a constant a domain and a first dimerization motif attached to the C-terminus of the constant a domain, and a TCR P chain comprising a variable P domain, a constant P domain and a first dimerization motif attached to the C-terminus of the constant P domain, wherein the first and second dimerization motifs easily interact to form a covalent bond between an amino acid in the first dimerization motif and an amino acid in the second dimerization motif linking the TCR a chain and TCR P chain together.
  • the TCR is a scTCR, which is a single amino acid strand containing an a chain and a P chain that is able to bind to MHC -peptide complexes.
  • a scTCR can be generated using methods known to those of skill in the art, See e.g., International published PCT Nos. WO 96/13593, WO 96/18105, W099/18129, WO04/033685, W02006/037960, WO2011/044186; U.S. Patent No. 7,569,664; and Schlueter, C. J. et al. J. Mol. Biol. 256, 859 (1996).
  • a scTCR contains a first segment constituted by an amino acid sequence corresponding to a TCR a chain variable region, a second segment constituted by an amino acid sequence corresponding to a TCR P chain variable region sequence fused to the N terminus of an amino acid sequence corresponding to a TCR P chain constant domain extracellular sequence, and a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
  • a scTCR contains a first segment constituted by an amino acid sequence corresponding to a TCR P chain variable region, a second segment constituted by an amino acid sequence corresponding to a TCR a chain variable region sequence fused to the N terminus of an amino acid sequence corresponding to a TCR a chain constant domain extracellular sequence, and a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
  • a scTCR contains a first segment constituted by an a chain variable region sequence fused to the N terminus of an a chain extracellular constant domain sequence, and a second segment constituted by a P chain variable region sequence fused to the N terminus of a sequence P chain extracellular constant and transmembrane sequence, and, optionally, a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
  • a scTCR contains a first segment constituted by a TCR P chain variable region sequence fused to the N terminus of a P chain extracellular constant domain sequence, and a second segment constituted by an a chain variable region sequence fused to the N terminus of a sequence comprising an a chain extracellular constant domain sequence and transmembrane sequence, and, optionally, a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
  • the a and P chains must be paired so that the variable region sequences thereof are orientated for such binding.
  • a linker sequence is included that links the a and P chains to form the single polypeptide strand.
  • the linker should have sufficient length to span the distance between the C terminus of the a chain and the N terminus of the P chain, or vice versa, while also ensuring that the linker length is not so long so that it blocks or reduces bonding of the scTCR to the target peptide-MHC complex.
  • the linker of a scTCRs that links the first and second TCR segments can be any linker capable of forming a single polypeptide strand, while retaining TCR binding specificity.
  • the linker sequence may, for example, have the formula -P-AA-P-, wherein P is proline and AA represents an amino acid sequence wherein the amino acids are glycine and serine.
  • the first and second segments are paired so that the variable region sequences thereof are orientated for such binding.
  • the linker has a sufficient length to span the distance between the C terminus of the first segment and the N terminus of the second segment, or vice versa, but is not too long to block or reduces bonding of the scTCR to the target ligand.
  • the linker can contain from or from about 10 to 45 amino acids, such as 10 to 30 amino acids or 26 to 41 amino acids residues, for example 29, 30, 31 or 32 amino acids.
  • a scTCR contains a disulfide bond between residues of the single amino acid strand, which, in some cases, can promote stability of the pairing between the a and P regions of the single chain molecule (see e.g. U.S. Patent No. 7,569,664).
  • the scTCR contains a covalent disulfide bond linking a residue of the immunoglobulin region of the constant domain of the a chain to a residue of the immunoglobulin region of the constant domain of the P chain of the single chain molecule.
  • the disulfide bond corresponds to the native disulfide bond present in a native dTCR. In some embodiments, the disulfide bond in a native TCR is not present. In some embodiments, the disulfide bond is an introduced non-native disulfide bond, for example, by incorporating one or more cysteines into the constant region extracellular sequences of the first and second chain regions of the scTCR polypeptide. Exemplary cysteine mutations include any as described above. In some cases, both a native and a non-native disulfide bond may be present.
  • any of the TCRs can be linked to signaling domains that yield an active TCR on the surface of a T cell.
  • the TCR is expressed on the surface of cells.
  • the TCR does contain a sequence corresponding to a transmembrane sequence.
  • the transmembrane domain can be a Ca or CP transmembrane domain.
  • the transmembrane domain can be from a non-TCR origin, for example, a transmembrane region from CD3z, CD28 or B7.1.
  • the TCR does contain a sequence corresponding to cytoplasmic sequences.
  • the TCR contains a CD3z signaling domain. In some embodiments, the TCR is capable of forming a TCR complex with CD3. In some embodiments, the TCR or antigen binding portion thereof may be a recombinantly produced natural protein or mutated form thereof in which one or more property, such as binding characteristic, has been altered. In some embodiments, a TCR may be derived from one of various animal species, such as human, mouse, rat, or other mammal.
  • the TCR comprises affinity to a target antigen on a target cell.
  • the target antigen may include any type of protein, or epitope thereof, associated with the target cell.
  • the TCR may comprise affinity to a target antigen on a target cell that indicates a particular disease state of the target cell.
  • the target antigen is processed and presented by MHCs.
  • the target cell antigen is a New York esophageal-1 (NY-ESO-1) peptide.
  • NY-ESO-1 belongs to the cancer-testis (CT) antigen group of proteins.
  • CT cancer-testis
  • NY-ESO-1 is a highly immunogenic antigen in vitro and is presented to T cells via the MHC.
  • a high affinity TCR recognizing the NY-ESO157-165 epitope may recognize HLA-A2-positive, NY-ESO-1 positive cell lines (but not to cells that lack either HLA- A2 or NY-ESO). Accordingly, a TCR of the present disclosure may be a HLA-A2-restricted NY- ESO-1 (SLLMWITQC; SEQ ID NO: 95)-specific TCR. In one embodiment, an NY-ESO-1 TCR of the present disclosure is a wild-type NY-ESO-1 TCR.
  • a wild-type NY-ESO-1 TCR may include, without limitation, the 8F NY-ESO-1 TCR (also referred to herein as “8F” or “8F TCR”), and the 1G4 NY-ESO-1 TCR (also referred to herein as “1G4” or “1G4 TCR”).
  • an NY-ESO-1 TCR of the present disclosure is an affinity enhanced 1G4 TCR, also called Ly95.
  • 1G4 TCR and affinity enhanced 1G4 TCR is described in U.S. Patent No. 8,143,376. This should not be construed as limiting in any way, as a TCR having affinity for any target antigen is suitable for use in a composition or method of the present invention.
  • the modified cells e.g., cells wherein endogenous Roquin-1 and endogenous Regnase-1 have been disrupted and/or knocked out; e.g., CAR T cells
  • the composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier.
  • a therapeutically effective amount of the pharmaceutical composition comprising the modified cells may be administered.
  • the invention includes a method for adoptive cell transfer therapy comprising administering to a subject in need thereof a modified immune cell or precursor cell thereof e.g. T cell) of the present invention.
  • the invention includes a method of treating a disease or condition in a subject comprising administering to a subject in need thereof a population of modified immune cells or precursor cells thereof (e.g. T cells).
  • the cell therapy e.g., adoptive T cell therapy is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject.
  • the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
  • the cell therapy e.g., adoptive T cell therapy
  • the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject.
  • the cells then are administered to a different subject, e.g., a second subject, of the same species.
  • the first and second subjects are genetically identical.
  • the first and second subjects are genetically similar.
  • the second subject expresses the same HLA class or supertype as the first subject.
  • the subject has been treated with a therapeutic agent targeting the disease or condition, e.g. the tumor, prior to administration of the cells or composition containing the cells.
  • the subject is refractory or non-responsive to the other therapeutic agent.
  • the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT.
  • the administration effectively treats the subject despite the subject having become resistant to another therapy.
  • the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden.
  • the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.
  • the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT.
  • HSCT hematopoietic stem cell transplantation
  • the administration effectively treats the subject despite the subject having become resistant to another therapy.
  • the modified immune cells of the present invention can be administered to an animal, preferably a mammal, even more preferably a human, to treat a cancer.
  • the cells of the present invention can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell(s), where it is desirable to treat or alleviate the disease.
  • the types of cancers to be treated with the modified cells or pharmaceutical compositions of the invention include, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas.
  • cancers include but are not limited breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and the like.
  • the cancers may be non-solid tumors (such as hematological tumors) or solid tumors.
  • Adult tumors/cancers and pediatric tumor s/cancers are also included.
  • the cancer is a solid tumor or a hematological tumor.
  • the cancer is a carcinoma.
  • the cancer is a sarcoma.
  • the cancer is a leukemia.
  • the cancer is a solid tumor.
  • Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas se
  • Carcinomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular
  • Sarcomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.
  • the modified immune cells of the invention are used to treat a myeloma, or a condition related to myeloma.
  • myeloma or conditions related thereto include, without limitation, light chain myeloma, non-secretory myeloma, monoclonal gamopathy of undertermined significance (MGUS), plasmacytoma (e.g., solitary, multiple solitary, extramedullary plasmacytoma), amyloidosis, and multiple myeloma.
  • a method of the present disclosure is used to treat multiple myeloma.
  • a method of the present disclosure is used to treat refractory myeloma.
  • a method of the present disclosure is used to treat relapsed myeloma.
  • the modified immune cells of the invention are used to treat a melanoma, or a condition related to melanoma.
  • melanoma or conditions related thereto include, without limitation, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, amelanotic melanoma, or melanoma of the skin (e.g., cutaneous, eye, vulva, vagina, rectum melanoma).
  • a method of the present disclosure is used to treat cutaneous melanoma.
  • a method of the present disclosure is used to treat refractory melanoma.
  • a method of the present disclosure is used to treat relapsed melanoma.
  • the modified immune cells of the invention are used to treat a sarcoma, or a condition related to sarcoma.
  • sarcoma or conditions related thereto include, without limitation, angiosarcoma, chondrosarcoma, Ewing’s sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, and synovial sarcoma.
  • a method of the present disclosure is used to treat synovial sarcoma.
  • a method of the present disclosure is used to treat liposarcoma such as myxoid/round cell liposarcoma, differentiated/dedifferentiated liposarcoma, and pleomorphic liposarcoma.
  • a method of the present disclosure is used to treat myxoid/round cell liposarcoma.
  • a method of the present disclosure is used to treat a refractory sarcoma.
  • a method of the present disclosure is used to treat a relapsed sarcoma.
  • the cells of the invention to be administered may be autologous, with respect to the subject undergoing therapy.
  • the administration of the cells of the invention may be carried out in any convenient manner known to those of skill in the art.
  • the cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
  • the compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the cells of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, alymph node, an organ, a tumor, and the like.
  • the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type(s) and/or a desired ratio of cell types.
  • the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio.
  • the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types.
  • the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.
  • the populations or sub-types of cells are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells.
  • the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg.
  • the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight.
  • the individual populations or sub-types are present at or near a desired output ratio (such as CD4 + to CD8 + ratio), e.g., within a certain tolerated difference or error of such a ratio.
  • a desired output ratio such as CD4 + to CD8 + ratio
  • the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired dose of CD4+ cells and/or a desired dose of CD8+ cells.
  • the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg.
  • the desired dose is at or above a minimum number of cells of the population or subtype, or minimum number of cells of the population or sub-type per unit of body weight.
  • the dosage is based on a desired fixed dose of total cells and a desired ratio, and/or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or subpopulations.
  • the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4 + to CD8 + cells, and/or is based on a desired fixed or minimum dose of CD4 + and/or CD8 + cells.
  • the cells, or individual populations of sub-types of cells are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million
  • the dose of total cells and/or dose of individual sub-populations of cells is within a range of between at or about IxlO 5 cells/kg to about IxlO 11 cells/kg 10 4 and at or about 10 11 cells/kilograms (kg) body weight, such as between 10 5 and 10 6 cells / kg body weight, for example, at or about 1 x 10 5 cells/kg, 1.5 x 10 5 cells/kg, 2 x 10 5 cells/kg, or 1 x 10 6 cells/kg body weight.
  • the cells are administered at, or within a certain range of error of, between at or about 10 4 and at or about 10 9 T cells/kilograms (kg) body weight, such as between 10 5 and 10 6 T cells / kg body weight, for example, at or about 1 x 10 5 T cells/kg, 1.5 x 10 5 T cells/kg, 2 x 10 5 T cells/kg, or 1 x 10 6 T cells/kg body weight.
  • a suitable dosage range of modified cells for use in a method of the present disclosure includes, without limitation, from about IxlO 5 cells/kg to about IxlO 6 cells/kg, from about IxlO 6 cells/kg to about IxlO 7 cells/kg, from about IxlO 7 cells/kg about IxlO 8 cells/kg, from about IxlO 8 cells/kg about IxlO 9 cells/kg, from about IxlO 9 cells/kg about IxlO 10 cells/kg, from about IxlO 10 cells/kg about IxlO 11 cells/kg.
  • a suitable dosage for use in a method of the present disclosure is about IxlO 8 cells/kg.
  • a suitable dosage for use in a method of the present disclosure is about IxlO 7 cells/kg. In other embodiments, a suitable dosage is from about IxlO 7 total cells to about 5xl0 7 total cells. In some embodiments, a suitable dosage is from about IxlO 8 total cells to about 5xl0 8 total cells. In some embodiments, a suitable dosage is from about 1.4xl0 7 total cells to about l.lxlO 9 total cells. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 7xl0 9 total cells.
  • the cells are administered at or within a certain range of error of between at or about 10 4 and at or about 10 9 CD4 + and/or CD8 + cells/kilograms (kg) body weight, such as between 10 5 and 10 6 CD4 + and/or CD8 + cells / kg body weight, for example, at or about 1 x 10 5 CD4 + and/or CD8 + cells/kg, 1.5 x 10 5 CD4 + and/or CD8 + cells/kg, 2 x 10 5 CD4 + and/or CD8 + cells/kg, or 1 x 10 6 CD4 + and/or CD8 + cells/kg body weight.
  • the cells are administered at or within a certain range of error of, greater than, and/or at least about 1 x 10 6 , about 2.5 x 10 6 , about 5 x 10 6 , about 7.5 x 10 6 , or about 9 x 10 6 CD4 + cells, and/or at least about 1 x 10 6 , about 2.5 x 10 6 , about 5 x 10 6 , about 7.5 x 10 6 , or about 9 x 10 6 CD8+ cells, and/or at least about 1 x 10 6 , about 2.5 x 10 6 , about 5 x 10 6 , about 7.5 x 10 6 , or about 9 x 10 6 T cells.
  • the cells are administered at or within a certain range of error of between about 10 8 and 10 12 or between about 10 10 and 10 11 T cells, between about 10 8 and 10 12 or between about 10 10 and 10 11 CD4 + cells, and/or between about 10 8 and 10 12 or between about 10 10 and 10 11 CD8 + cells.
  • the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4+ and CD8+ cells or sub-types.
  • the desired ratio can be a specific ratio or can be a range of ratios, for example, in some embodiments, the desired ratio (e.g., ratio of CD4 + to CD8 + cells) is between at or about 5: 1 and at or about 5: 1 (or greater than about 1 :5 and less than about 5: 1), or between at or about 1 :3 and at or about 3 : 1 (or greater than about 1 :3 and less than about 3: 1), such as between at or about 2: 1 and at or about 1 :5 (or greater than about 1 :5 and less than about 2: 1, such as at or about 5: 1, 4.5: 1, 4: 1, 3.5: 1, 3: 1, 2.5: 1, 2: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1,
  • the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.
  • a dose of modified cells is administered to a subject in need thereof, in a single dose or multiple doses. In some embodiments, a dose of modified cells is administered in multiple doses, e.g., once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion.
  • the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician.
  • the compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.
  • the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
  • the cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order.
  • the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa.
  • the cells are administered prior to the one or more additional therapeutic agents.
  • the cells are administered after the one or more additional therapeutic agents.
  • the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence.
  • the methods comprise administration of a chemotherapeutic agent.
  • the modified cells of the invention may be administered to a subject in combination with an immune checkpoint antibody e.g., an anti-PDl, anti-CTLA-4, or anti-PDLl antibody).
  • an immune checkpoint antibody e.g., an anti-PDl, anti-CTLA-4, or anti-PDLl antibody.
  • the modified cell may be administered in combination with an antibody or antibody fragment targeting, for example, PD-1 (programmed death 1 protein).
  • PD-1 programmeed death 1 protein
  • anti-PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA®, formerly lambrolizumab, also known as MK- 3475), and nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®) or an antigenbinding fragment thereof.
  • the modified cell may be administered in combination with an anti-PD-Ll antibody or antigen-binding fragment thereof.
  • anti-PD-Ll antibodies include, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®, Atezolizumab), and MEDI4736 (Durvalumab, Imfinzi).
  • the modified cell may be administered in combination with an anti-CTLA-4 antibody or antigen-binding fragment thereof.
  • An example of an anti- CTLA-4 antibody includes, but is not limited to, Ipilimumab (trade name Yervoy).
  • Other types of immune checkpoint modulators may also be used including, but not limited to, small molecules, siRNA, miRNA, and CRISPR systems. Immune checkpoint modulators may be administered before, after, or concurrently with the modified cell comprising the CAR.
  • combination treatment comprising an immune checkpoint modulator may increase the therapeutic efficacy of a therapy comprising a modified cell of the present invention.
  • the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods.
  • Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry.
  • the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004).
  • the biological activity of the cells is measured by assaying expression and/or secretion of one or more cytokines, such as CD 107a, IFNy, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
  • cytokines such as CD 107a, IFNy, IL-2, and TNF.
  • the subject is provided a secondary treatment. Secondary treatments include but are not limited to chemotherapy, radiation, surgery, and medications.
  • the subject can be administered a conditioning therapy prior to CAR T cell therapy.
  • the conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject.
  • the conditioning therapy comprises administering an effective amount of fludarabine to the subject.
  • the conditioning therapy comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject.
  • Administration of a conditioning therapy prior to CAR T cell therapy may increase the efficacy of the CAR T cell therapy.
  • a specific dosage regimen of the present disclosure includes a lymphodepletion step prior to the administration of the modified T cells.
  • the lymphodepletion step includes administration of cyclophosphamide and/or fludarabine.
  • a specific dosage regimen of the present disclosure does not include a lymphodepletion step prior to the administration of the modified T cells.
  • the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg/m 2 /day and about 2000 mg/m 2 /day (e.g., 200 mg/m 2 /day, 300 mg/m 2 /day, or 500 mg/m 2 /day).
  • the dose of cyclophosphamide is about 300 mg/m 2 /day.
  • the lymphodepletion step includes administration of fludarabine at a dose of between about 20 mg/m 2 /day and about 900 mg/m 2 /day (e.g., 20 mg/m 2 /day, 25 mg/m 2 /day, 30 mg/m 2 /day, or 60 mg/m 2 /day).
  • the dose of fludarabine is about 30 mg/m 2 /day.
  • the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg/m 2 /day and about 2000 mg/m 2 /day (e.g., 200 mg/m 2 /day, 300 mg/m 2 /day, or 500 mg/m 2 /day), and fludarabine at a dose of between about 20 mg/m 2 /day and about 900 mg/m 2 /day (e.g., 20 mg/m 2 /day, 25 mg/m 2 /day, 30 mg/m 2 /day, or 60 mg/m 2 /day).
  • the lymphodepletion step includes administration of cyclophosphamide at a dose of about 300 mg/m 2 /day, and fludarabine at a dose of about 30 mg/m 2 /day.
  • the dosing of cyclophosphamide is 300 mg/m 2 /day over three days
  • the dosing of fludarabine is 30 mg/m 2 /day over three days.
  • Dosing of lymphodepletion chemotherapy may be scheduled on Days -6 to -4 (with a -1 day window, i.e., dosing on Days -7 to -5) relative to T cell (e.g., CAR-T, TCR-T, a modified T cell, etc.) infusion on Day 0.
  • T cell e.g., CAR-T, TCR-T, a modified T cell, etc.
  • the subject receives lymphodepleting chemotherapy including 300 mg/m 2 of cyclophosphamide by intravenous infusion 3 days prior to administration of the modified T cells. In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg/m 2 of cyclophosphamide by intravenous infusion for 3 days prior to administration of the modified T cells.
  • the subject receives lymphodepleting chemotherapy including fludarabine at a dose of between about 20 mg/m 2 /day and about 900 mg/m 2 /day (e.g., 20 mg/m 2 /day, 25 mg/m 2 /day, 30 mg/m 2 /day, or 60 mg/m 2 /day).
  • the subject receives lymphodepleting chemotherapy including fludarabine at a dose of 30 mg/m 2 for 3 days.
  • the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of between about 200 mg/m 2 /day and about 2000 mg/m 2 /day (e.g., 200 mg/m 2 /day, 300 mg/m 2 /day, or 500 mg/m 2 /day), and fludarabine at a dose of between about 20 mg/m 2 /day and about 900 mg/m 2 /day (e.g., 20 mg/m 2 /day, 25 mg/m 2 /day, 30 mg/m 2 /day, or 60 mg/m 2 /day).
  • lymphodepleting chemotherapy including cyclophosphamide at a dose of between about 200 mg/m 2 /day and about 2000 mg/m 2 /day (e.g., 200 mg/m 2 /day, 300 mg/m 2 /day, or 500 mg/m 2 /day)
  • fludarabine at a dose of between about 20 mg/m 2 /day and about 900 mg
  • the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of about 300 mg/m 2 /day, and fludarabine at a dose of 30 mg/m 2 for 3 days.
  • lymphodepleting chemotherapy including cyclophosphamide at a dose of about 300 mg/m 2 /day, and fludarabine at a dose of 30 mg/m 2 for 3 days.
  • Cells of the invention can be administered in dosages and routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Cell compositions may be administered multiple times at dosages within these ranges. Administration of the cells of the invention may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art.
  • CRS cytokine release syndrome
  • Clinical features include: high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia/hypotension, capillary leak, cardiac dysfunction, renal impairment, hepatic failure, and disseminated intravascular coagulation.
  • Dramatic elevations of cytokines including interferon-gamma, granulocyte macrophage colony-stimulating factor, IL- 10, and IL-6 have been shown following CAR T-cell infusion.
  • One CRS signature is elevation of cytokines including IL-6 (severe elevation), IFN-gamma, TNF-alpha (moderate), and IL-2 (mild).
  • CRS C-reactive protein
  • the invention provides for, following the diagnosis of CRS, appropriate CRS management strategies to mitigate the physiological symptoms of uncontrolled inflammation without dampening the antitumor efficacy of the engineered cells (e.g., CAR T cells).
  • CRS management strategies are known in the art.
  • systemic corticosteroids may be administered to rapidly reverse symptoms of sCRS (e.g., grade 3 CRS) without compromising initial antitumor response.
  • an anti-IL-6R antibody may be administered.
  • An example of an anti-IL-6R antibody is the Food and Drug Administration-approved monoclonal antibody tocilizumab, also known as atlizumab (marketed as Actemra, or RoActemra).
  • Tocilizumab is a humanized monoclonal antibody against the interleukin-6 receptor (IL-6R).
  • IL-6R interleukin-6 receptor
  • CRS is generally managed based on the severity of the observed syndrome and interventions are tailored as such. CRS management decisions may be based upon clinical signs and symptoms and response to interventions, not solely on laboratory values alone.
  • the first-line management of CRS may be tocilizumab, in some embodiments, at the labeled dose of 8 mg/kg IV over 60 minutes (not to exceed 800 mg/dose); tocilizumab can be repeated Q8 hours. If suboptimal response to the first dose of tocilizumab, additional doses of tocilizumab may be considered.
  • Tocilizumab can be administered alone or in combination with corticosteroid therapy.
  • CRS management guidance may be based on published standards (Lee et al. (2019) Biol Blood Marrow Transplant, doi.org/10.1016/j.bbmt.2018.12.758; Neelapu et al. (2016) Nat Rev Clin Oncology, 15:47; Teachey et al. (2016) Cancer Discov, 6(6):664-679).
  • MAS Macrophage Activation Syndrome
  • HHLH Hemophagocytic lymphohistiocytosis
  • MAS appears to be a reaction to immune activation that occurs from the CRS, and should therefore be considered a manifestation of CRS.
  • MAS is similar to HLH (also a reaction to immune stimulation).
  • the clinical syndrome of MAS is characterized by high grade non-remitting fever, cytopenias affecting at least two of three lineages, and hepatosplenomegaly. It is associated with high serum ferritin, soluble interleukin-2 receptor, and triglycerides, and a decrease of circulating natural killer (NK) activity.
  • NK circulating natural killer
  • the modified immune cells of the present invention when used in a method of treatment as described herein, enhances the ability of the modified immune cells in carrying out their function. Accordingly, the present invention provides a method for enhancing a function of a modified immune cell for use in a method of treatment as described herein.
  • the invention includes a method of treating cancer in a subject in need thereof, comprising administering to the subject any one of the modified immune or precursor cells disclosed herein.
  • Yet another aspect of the invention includes a method of treating cancer in a subject in need thereof, comprising administering to the subject a modified immune or precursor cell generated by any one of the methods disclosed herein.
  • the present disclosure provides methods for producing or generating a modified immune cell or precursor thereof (e.g., a T cell) of the invention for tumor immunotherapy, e.g., adoptive immunotherapy.
  • the cells generally are engineered by introducing one or more genetically engineered nucleic acids encoding the exogenous receptors (e.g., a TCR and/or CAR).
  • the cells also are introduced, either simultaneously or sequentially with the nucleic acid encoding the exogenous receptor, with an agent (e.g. Cas9/gRNA RNP) that is capable of disrupting a targeted gene (e.g., a gene encoding endogenous Roquin-1 and/or Regnase-1).
  • an agent e.g. Cas9/gRNA RNP
  • the invention provides a method for generating a modified immune cell or precursor cell thereof, comprising introducing into an immune or precursor cell a CRISPR system comprising one or more polypeptides and/or nucleic acids capable of downregulating gene expression of endogenous Roquin-1 and/or Regnase-1; and introducing into the immune or precursor cell a nucleic acid encoding an exogenous TCR and/or CAR, wherein the exogenous TCR and/or CAR comprises affinity for an antigen on a target cell.
  • the invention provides a method for generating a modified immune cell or precursor cell thereof, comprising introducing into an immune or precursor cell one or more polypeptides and/or nucleic acids capable of downregulating gene expression of endogenous Roquin-1 and/or Regnase-1; and introducing into the immune or precursor cell a nucleic acid encoding an exogenous TCR and/or CAR, wherein the nucleic acid encoding an exogenous TCR and/or CAR is inserted into an endogenous gene locus encoding Roquin-1 and/or Regnase-1, and wherein the exogenous TCR and/or CAR comprises affinity for an antigen on a target cell.
  • the immune cell or precursor cell thereof is a T cell.
  • the T cell is human T cell.
  • T cell is an autologous T cell.
  • the exogenous receptor e.g., TCR and/or CAR
  • the exogenous receptor is introduced into a cell by an expression vector.
  • Expression vectors comprising a nucleic acid sequence encoding a TCR and/or CAR of the present invention are provided herein.
  • Suitable expression vectors include lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno associated virus (AAV) vectors, adenovirus vectors, engineered hybrid viruses, naked DNA, including but not limited to transposon mediated vectors, such as Sleeping Beauty, Piggybak, and Integrases such as Phi31.
  • Some other suitable expression vectors include Herpes simplex virus (HS V) and retrovirus expression vectors.
  • the nucleic acid encoding an exogenous TCR and/or CAR is introduced into the cell via viral transduction.
  • the viral transduction comprises contacting the immune or precursor cell with a viral vector comprising the nucleic acid encoding an exogenous TCR and/or CAR.
  • the viral vector is an adeno-associated viral (AAV) vector.
  • the AAV vector comprises a 5’ ITR and a 3’ITR derived from AAV6.
  • the AAV vector comprises a Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE).
  • WPRE Woodchuck Hepatitis Virus post-transcriptional regulatory element
  • the AAV vector comprises a polyadenylation (poly A) sequence.
  • the polyA sequence is a bovine growth hormone (BGH) polyA sequence.
  • Adenovirus expression vectors are based on adenoviruses, which have a low capacity for integration into genomic DNA but a high efficiency for transfecting host cells.
  • Adenovirus expression vectors contain adenovirus sequences sufficient to: (a) support packaging of the expression vector and (b) to ultimately express the CAR in the host cell.
  • the adenovirus genome is a 36 kb, linear, double stranded DNA, where a foreign DNA sequence (e.g., a nucleic acid encoding an exogenous CAR) may be inserted to substitute large pieces of adenoviral DNA in order to make the expression vector of the present invention (see, e.g., Danthinne and Imperiale, Gene Therapy (2000) 7(20): 1707-1714).
  • a foreign DNA sequence e.g., a nucleic acid encoding an exogenous CAR
  • AAV adeno associated virus
  • Another expression vector is based on an adeno associated virus (AAV), which takes advantage of the adenovirus coupled systems.
  • AAV expression vector has a high frequency of integration into the host genome. It can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue cultures or in vivo.
  • the AAV vector has a broad host range for infectivity. Details concerning the generation and use of AAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368.
  • Retrovirus expression vectors are capable of integrating into the host genome, delivering a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and being packaged in special cell lines.
  • the retroviral vector is constructed by inserting a nucleic acid (e.g., a nucleic acid encoding an exogenous TCR and/or CAR) into the viral genome at certain locations to produce a virus that is replication defective.
  • a nucleic acid e.g., a nucleic acid encoding an exogenous TCR and/or CAR
  • the retroviral vectors are able to infect a broad variety of cell types, integration and stable expression of the TCR and/or CAR requires the division of host cells.
  • Lentiviral vectors are derived from lentiviruses, which are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function (see, e.g., U.S. Patent Nos. 6,013,516 and 5,994, 136).
  • Some examples of lentiviruses include the Human Immunodeficiency Viruses (HIV-1, HIV-2) and the Simian Immunodeficiency Virus (SIV).
  • Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.
  • Lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression, e.g., of a nucleic acid encoding a CAR (see, e.g., U.S. Patent No. 5,994,136).
  • Expression vectors including a nucleic acid of the present disclosure can be introduced into a host cell by any means known to persons skilled in the art.
  • the expression vectors may include viral sequences for transfection, if desired.
  • the expression vectors may be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like.
  • the host cell may be grown and expanded in culture before introduction of the expression vectors, followed by the appropriate treatment for introduction and integration of the vectors.
  • the host cells are then expanded and may be screened by virtue of a marker present in the vectors.
  • markers that may be used are known in the art, and may include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.
  • the terms "cell,” “cell line,” and “cell culture” may be used interchangeably.
  • the host cell an immune cell or precursor thereof, e.g., a T cell, an NK cell, or an NKT cell.
  • the present invention also provides genetically engineered cells which include and stably express a TCR and/or CAR of the present disclosure.
  • the genetically engineered cells are genetically engineered T-lymphocytes (T cells), naive T cells (TN), memory T cells (for example, central memory T cells (TCM), effector memory cells (TEM)), natural killer cells (NK cells), and macrophages capable of giving rise to therapeutically relevant progeny.
  • the genetically engineered cells are autologous cells.
  • Modified cells may be produced by stably transfecting host cells with an expression vector including a nucleic acid of the present disclosure. Additional methods for generating a modified cell of the present disclosure include, without limitation, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and/or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer and/or hydrodynamic delivery) and/or particle-based methods (e.g., impalefection, using a gene gun and/or magnetofection). Transfected cells expressing a TCR and/or CAR of the present disclosure may be expanded ex vivo.
  • chemical transformation methods e.g., using calcium phosphate, dendrimers, liposomes and/or cationic polymers
  • non-chemical transformation methods e.g., electroporation, optical transformation, gene electrotransfer and/or hydrodynamic delivery
  • particle-based methods e.g., impalefection, using a
  • Physical methods for introducing an expression vector into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells including vectors and/or exogenous nucleic acids are well- known in the art. See, e.g., Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Chemical methods for introducing an expression vector into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Lipids suitable for use can be obtained from commercial sources.
  • DMPC dimyristyl phosphatidylcholine
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform may be used as the only solvent since it is more readily evaporated than methanol.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10).
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine-nucleic acid complexes are also contemplated.
  • assays include, for example, molecular biology assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemistry assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • molecular biology assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemistry assays such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • the nucleic acids introduced into the host cell are RNA.
  • the RNA is mRNA that comprises in vitro transcribed RNA or synthetic RNA.
  • the RNA may be produced by in vitro transcription using a polymerase chain reaction (PCR)- generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase.
  • the source of the DNA may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.
  • PCR may be used to generate a template for in vitro transcription of mRNA which is then introduced into cells.
  • Methods for performing PCR are well known in the art.
  • Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR.
  • “Substantially complementary,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR.
  • the primers can be designed to be substantially complementary to any portion of the DNA template.
  • the primers can be designed to amplify the portion of a gene that is normally transcribed in cells (the open reading frame), including 5' and 3' UTRs.
  • the primers may also be designed to amplify a portion of a gene that encodes a particular domain of interest.
  • the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5' and 3' UTRs.
  • Primers useful for PCR are generated by synthetic methods that are well known in the art.
  • “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified.
  • Upstream is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand.
  • reverse primers are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified.
  • Downstream is used herein to refer to a location 3' to the DNA sequence to be amplified relative to the coding strand.
  • the RNA preferably has 5' and 3' UTRs.
  • the 5' UTR is between zero and 3000 nucleotides in length.
  • the length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.
  • the 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest.
  • UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
  • the use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
  • the 5' UTR can contain the Kozak sequence of the endogenous gene.
  • a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence.
  • Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art.
  • the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells.
  • various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the mRNA.
  • a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed.
  • the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
  • the promoter is a T7 polymerase promoter, as described elsewhere herein.
  • Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
  • the mRNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
  • RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells.
  • the transcription of plasmid DNA linearized at the end of the 3' UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.
  • phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).
  • the polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100T tail (size can be 50-5000 T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination.
  • Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.
  • Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly (A) polymerase, such as E. coli poly A polymerase (E-PAP).
  • E-PAP E. coli poly A polymerase
  • increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA.
  • the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds.
  • ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
  • RNAs produced by the methods disclosed herein include a 5' cap.
  • the 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7: 1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun, 330:958-966 (2005)).
  • the RNA is electroporated into the cells, such as in vitro transcribed RNA.
  • Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
  • a nucleic acid encoding a TCR and/or CAR of the present disclosure will be RNA, e.g., in vitro synthesized RNA.
  • Methods for in vitro synthesis of RNA are known in the art; any known method can be used to synthesize RNA comprising a sequence encoding a TCR and/or CAR.
  • Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053.
  • Introducing RNA comprising a nucleotide sequence encoding a TCR and/or CAR into a host cell can be carried out in vitro, ex vivo or in vivo.
  • a host cell e.g., an NK cell, a cytotoxic T lymphocyte, etc.
  • RNA comprising a nucleotide sequence encoding a TCR and/or CAR.
  • the disclosed methods can be applied to the modulation of T cell activity in basic research and therapy, in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including the assessment of the ability of the genetically modified T cell to kill a target cancer cell.
  • the methods also provide the ability to control the level of expression over a wide range by changing, for example, the promoter or the amount of input RNA, making it possible to individually regulate the expression level. Furthermore, the PCR-based technique of mRNA production greatly facilitates the design of the mRNAs with different structures and combination of their domains.
  • RNA transfection is essentially transient and a vector-free.
  • An RNA transgene can be delivered to a lymphocyte and expressed therein following a brief in vitro cell activation, as a minimal expressing cassette without the need for any additional viral sequences. Under these conditions, integration of the transgene into the host cell genome is unlikely. Cloning of cells is not necessary because of the efficiency of transfection of the RNA and its ability to uniformly modify the entire lymphocyte population.
  • IVT-RNA Genetic modification of T cells with in I’z/zvz-transcribed RNA (IVT-RNA) makes use of two different strategies both of which have been successively tested in various animal models.
  • Cells are transfected with in vz/ro-transcribed RNA by means of lipofection or electroporation. It is desirable to stabilize IVT-RNA using various modifications in order to achieve prolonged expression of transferred IVT-RNA.
  • IVT vectors are known in the literature which are utilized in a standardized manner as template for in vitro transcription and which have been genetically modified in such a way that stabilized RNA transcripts are produced.
  • protocols used in the art are based on a plasmid vector with the following structure: a 5' RNA polymerase promoter enabling RNA transcription, followed by a gene of interest which is flanked either 3' and/or 5' by untranslated regions (UTR), and a 3' polyadenyl cassette containing 50-70 A nucleotides.
  • UTR untranslated regions
  • the circular plasmid Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenyl cassette by type II restriction enzymes (recognition sequence corresponds to cleavage site).
  • the polyadenyl cassette thus corresponds to the later poly(A) sequence in the transcript.
  • some nucleotides remain as part of the enzyme cleavage site after linearization and extend or mask the poly(A) sequence at the 3' end. It is not clear, whether this nonphy si ologi cal overhang affects the amount of protein produced intracellularly from such a construct.
  • the RNA construct is delivered into the cells by electroporation.
  • electroporation See, e.g., the formulations and methodology of electroporation of nucleic acid constructs into mammalian cells as taught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841 Al, US 2004/0059285A1, US 2004/0092907A1.
  • the various parameters including electric field strength required for electroporation of any known cell type are generally known in the relevant research literature as well as numerous patents and applications in the field. See e.g., U.S. Pat. No. 6,678,556, U.S. Pat. No. 7,171,264, and U.S. Pat. No. 7,173,116.
  • Apparatus for therapeutic application of electroporation are available commercially, e.g., the MedPulserTM DNA Electroporation Therapy System (Inovio/Genetronics, San Diego, Calif.), and are described in patents such as U.S. Pat. No. 6,567,694; U.S. Pat. No. 6,516,223, U.S. Pat. No. 5,993,434, U.S. Pat. No. 6,181,964, U.S. Pat. No. 6,241,701, and U.S. Pat. No. 6,233,482; electroporation may also be used for transfection of cells in vitro as described e.g. in US20070128708A1.
  • Electroporation may also be utilized to deliver nucleic acids into cells in vitro. Accordingly, electroporation-mediated administration into cells of nucleic acids including expression constructs utilizing any of the many available devices and electroporation systems known to those of skill in the art presents an exciting new means for delivering an RNA of interest to a target cell.
  • the immune cells can be incubated or cultivated prior to, during and/or subsequent to introducing the nucleic acid molecule encoding the exogenous receptor (e.g., the TCR and/or CAR) and the gene editing agent (e.g. Cas9/gRNA RNP).
  • the cells e.g. T cells
  • the cells can be incubated or cultivated prior to, during or subsequent to the introduction of the nucleic acid molecule encoding the exogenous receptor, such as prior to, during or subsequent to the transduction of the cells with a viral vector (e.g. lentiviral vector) encoding the exogenous receptor.
  • the cells e.g.
  • T cells can be incubated or cultivated prior to, during or subsequent to the introduction of the gene editing agent (e.g. Cas9/gRNA RNP), such as prior to, during or subsequent to contacting the cells with the agent or prior to, during or subsequent to delivering the agent into the cells, e.g. via electroporation.
  • the incubation can be both in the context of introducing the nucleic acid molecule encoding the exogenous receptor and introducing the gene editing agent, e.g. Cas9/gRNA RNP.
  • the method includes activating or stimulating cells with a stimulating or activating agent (e.g. anti-CD3/anti-CD28 antibodies) prior to introducing the nucleic acid molecule encoding the exogenous receptor and the gene editing agent, e.g. Cas9/gRNA RNP.
  • a stimulating or activating agent e.g. anti-CD3/anti-CD28 antibodies
  • introducing the gene editing agent e.g. Cas9/gRNA RNP
  • introducing the gene editing agent is done after introducing the nucleic acid molecule encoding the exogenous receptor.
  • the cells prior to the introducing of the agent, the cells are allowed to rest, e.g. by removal of any stimulating or activating agent.
  • the stimulating or activating agent and/or cytokines are not removed. Those of skill in the art will be able to determine the order in which each of the one or more nucleic acid sequences are introduced into the host cell.
  • a source of immune cells is obtained from a subject (e.g. for ex vivo manipulation).
  • Sources of cells manipulation may also include, e.g., autologous or heterologous donor blood, cord blood, or bone marrow.
  • the source of immune cells may be from the subject to be treated with the modified immune cells of the invention, e.g., the subject's blood, the subject's cord blood, or the subject's bone marrow.
  • subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • the subject is a human.
  • Immune cells can be obtained from a number of sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs.
  • Immune cells are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells.
  • Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs).
  • the cells are human cells. With reference to the subject to be treated, the cells may be allogeneic and/or autologous.
  • the cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen.
  • the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cells), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell a hematopoietic stem cell, a natural killer cell (NK cell) or a dendritic cell.
  • a CD8+ T cell e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell
  • a CD4+ T cell e.g., a CD4+ T cell, a natural killer T cell (NKT cells), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell a hematopoietic stem cell, a natural killer cell (NK cell) or
  • the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
  • the cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell generated from a subject, manipulated to alter (e.g., induce a mutation in) or manipulate the expression of one or more target genes, and differentiated into, e.g., a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell or a hematopoietic stem cell.
  • iPS induced pluripotent stem
  • the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.
  • T cells or other cell types such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.
  • TN cells naive T cells
  • TEFF effector T cells
  • memory T cells and sub-types thereof such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells
  • TIL tumor-infiltrating lymphocytes
  • immature T cells mature T cells
  • helper T cells cytotoxic T cells
  • mucosa- associated invariant T (MAIT) cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells
  • follicular helper T cells alpha/beta T cells, and delta/gamma T cells.
  • any number of T cell lines available in the art may be used.
  • the methods include isolating immune cells from the subject, preparing, processing, culturing, and/or engineering/modifying them.
  • preparation of the engineered cells includes one or more culture and/or preparation steps.
  • the cells for engineering/modifying as described may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
  • the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered.
  • the subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
  • the cells in some embodiments are primary cells, e.g., primary human cells.
  • the samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed.
  • Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
  • the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product.
  • exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom.
  • Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
  • the cells are derived from cell lines, e.g., T cell lines.
  • the cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, nonhuman primate, and pig.
  • isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps.
  • cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents.
  • cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
  • cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis.
  • the samples contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
  • the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions.
  • the cells are resuspended in a variety of biocompatible buffers after washing.
  • components of a blood cell sample are removed and the cells directly resuspended in culture media.
  • the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
  • immune are obtained cells from the circulating blood of an individual are obtained by apheresis or leukapheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps.
  • PBS phosphate buffered saline
  • wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps.
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS.
  • a variety of biocompatible buffers such as, for example, Ca-free, Mg-free PBS.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation.
  • the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
  • Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use.
  • negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population. The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker.
  • positive selection of or enrichment for cells of a particular type refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker.
  • negative selection, removal, or depletion of cells of a particular type refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
  • multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.
  • a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection.
  • multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
  • one or more of the T cell populations is enriched for or depleted of cells that are positive for (marker+) or express high levels (marker 111811 ) of one or more particular markers, such as surface markers, or that are negative for (marker -) or express relatively low levels (marker low ) of one or more markers.
  • specific subpopulations of T cells such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.
  • such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells).
  • the cells such as the CD8+ cells or the T cells, e.g., CD3+ cells
  • the cells are enriched for (i.e., positively selected for) cells that are positive or expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD 127, and/or CD62L and/or depleted of (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA.
  • cells are enriched for or depleted of cells positive or expressing high surface levels of CD 122, CD95, CD25, CD27, and/or IL7-Ra (CD 127).
  • CD8+ T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and for CD62L.
  • CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
  • T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14.
  • a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells.
  • Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
  • CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation.
  • enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve longterm survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations.
  • combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.
  • memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes.
  • PBMC can be enriched for or depleted of CD62L-CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.
  • a CD4+ T cell population and a CD8+ T cell sub-population e.g., a subpopulation enriched for central memory (TCM) cells.
  • the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L.
  • enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L.
  • Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order.
  • the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4- based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
  • CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.
  • CD4+ lymphocytes can be obtained by standard methods.
  • naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells.
  • central memory CD4+ cells are CD62L+ and CD45RO+.
  • effector CD4+ cells are CD62L- and CD45RO.
  • a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD1 lb, CD 16, HLA-DR, and CD8.
  • the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection.
  • the cells are incubated and/or cultured prior to or in connection with genetic engineering/modification.
  • the incubation steps can include culture, cultivation, stimulation, activation, and/or propagation.
  • the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
  • the conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
  • the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex.
  • the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell.
  • Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and/or one or more cytokines.
  • the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml).
  • the stimulating agents include IL-2 and/or IL- 15, for example, an IL-2 concentration of at least about 10 units/mL.
  • the modified cells are expanded without any stimulating agents.
  • the modified cells are expanded in vivo.
  • T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient.
  • T cells can be isolated from an umbilical cord.
  • a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.
  • the cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody.
  • Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • a preferred method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8.
  • the concentration of cells and surface can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/ml is used. In a further embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.
  • T cells can also be frozen after the washing step, which does not require the monocyteremoval step. While not wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
  • the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank.
  • the population of T cells is comprised within cells such as peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line.
  • peripheral blood mononuclear cells comprise the population of T cells.
  • purified T cells comprise the population of T cells.
  • T regulatory cells can be isolated from a sample.
  • the sample can include, but is not limited to, umbilical cord blood or peripheral blood.
  • the Tregs are isolated by flow-cytometry sorting.
  • the sample can be enriched for Tregs prior to isolation by any means known in the art.
  • the isolated Tregs can be cryopreserved, and/or expanded prior to use. Methods for isolating Tregs are described in U.S. Patent Numbers: 7,754,482, 8,722,400, and 9,555,105, and U.S. Patent Application No. 13/639,927, contents of which are incorporated herein in their entirety.
  • the cells can be activated and expanded in number using methods as described, for example, in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681 ; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Publication No. 20060121005.
  • the T cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells.
  • T cell populations may be stimulated by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
  • a ligand that binds the accessory molecule is used for co-stimulation of an accessory molecule on the surface of the T cells.
  • T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
  • an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and these can be used in the invention, as can other methods and reagents known in the art (see, e.g., ten Berge et al., Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2): 53-63).
  • Expanding T cells by the methods disclosed herein can be multiplied by about 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater, and any and all whole or partial integers therebetween.
  • the T cells expand in the range of about 20 fold to about 50 fold.
  • the T cells can be incubated in cell medium in a culture apparatus for a period of time or until the cells reach confluency or high cell density for optimal passage before passing the cells to another culture apparatus.
  • the culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro.
  • the level of confluence is 70% or greater before passing the cells to another culture apparatus. More preferably, the level of confluence is 90% or greater.
  • a period of time can be any time suitable for the culture of cells in vitro.
  • the T cell medium may be replaced during the culture of the T cells at any time. Preferably, the T cell medium is replaced about every 2 to 3 days.
  • the T cells are then harvested from the culture apparatus whereupon the T cells can be used immediately or cryopreserved to be stored for use at a later time.
  • the invention includes cry opreserving the expanded T cells.
  • the cryopreserved T cells are thawed prior to introducing nucleic acids into the T cell.
  • the method comprises isolating T cells and expanding the T cells.
  • the invention further comprises cryopreserving the T cells prior to expansion.
  • the cryopreserved T cells are thawed for electroporation with the RNA encoding the chimeric membrane protein.
  • ex vivo culture and expansion of T cells comprises the addition to the cellular growth factors, such as those described in U.S. Pat. No. 5,199,942, or other factors, such as flt3-L, IL-1, IL-3 and c-kit ligand.
  • expanding the T cells comprises culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3 and c-kit ligand.
  • the culturing step as described herein can be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours.
  • the culturing step as described further herein can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.
  • Cell culture refers generally to cells taken from a living organism and grown under controlled condition.
  • a primary cell culture is a culture of cells, tissues or organs taken directly from an organism and before the first subculture.
  • Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and/or division, resulting in a larger population of the cells.
  • the rate of cell proliferation is typically measured by the amount of time required for the cells to double in number, otherwise known as the doubling time.
  • Each round of subculturing is referred to as a passage.
  • cells When cells are subcultured, they are referred to as having been passaged.
  • a specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times it has been passaged.
  • a cultured cell population that has been passaged ten times may be referred to as a P10 culture.
  • the primary culture i.e., the first culture following the isolation of cells from tissue, is designated P0.
  • the cells are described as a secondary culture (Pl or passage 1).
  • the cells become a tertiary culture (P2 or passage 2), and so on.
  • the number of population doublings of a culture is greater than the passage number.
  • the expansion of cells (i.e., the number of population doublings) during the period between passaging depends on many factors, including but is not limited to the seeding density, substrate, medium, and time between passaging.
  • the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between.
  • Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-a or any other additives for the growth of cells known to the skilled artisan.
  • serum e.g., fetal bovine or human serum
  • IL-2 interleukin-2
  • insulin IFN-gamma
  • IL-4 interleukin-7
  • GM-CSF GM-CSF
  • IL-10 interleukin-12
  • IL-15 IL-15
  • additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.
  • Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells.
  • Antibiotics e.g., penicillin and streptomycin
  • the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).
  • the medium used to culture the T cells may include an agent that can co-stimulate the T cells.
  • an agent that can stimulate CD3 is an antibody to CD3
  • an agent that can stimulate CD28 is an antibody to CD28.
  • a cell isolated by the methods disclosed herein can be expanded approximately 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater.
  • the T cells expand in the range of about 20 fold to about 50 fold, or more.
  • human T regulatory cells are expanded via anti-CD3 antibody coated KT64.86 artificial antigen presenting cells (aAPCs).
  • aAPCs antigen presenting cells
  • the method of expanding the T cells can further comprise isolating the expanded T cells for further applications.
  • the method of expanding can further comprise a subsequent electroporation of the expanded T cells followed by culturing.
  • the subsequent electroporation may include introducing a nucleic acid encoding an agent, such as a transducing the expanded T cells, transfecting the expanded T cells, or electroporating the expanded T cells with a nucleic acid, into the expanded population of T cells, wherein the agent further stimulates the T cell.
  • the agent may stimulate the T cells, such as by stimulating further expansion, effector function, or another T cell function.
  • compositions containing such cells and/or enriched for such cells are also provided.
  • compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy.
  • therapeutic methods for administering the cells and compositions to subjects e.g., patients.
  • compositions including the cells for administration including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof.
  • the pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient.
  • the composition includes at least one additional therapeutic agent.
  • pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and/or by the method of administration. Accordingly, there are a variety of suitable formulations.
  • the pharmaceutical composition can contain preservatives.
  • Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arg
  • Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
  • the formulations can include aqueous solutions.
  • the formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
  • chemotherapeutic agents e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
  • the pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount.
  • Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
  • the cell populations are administered parenterally.
  • parenteral includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration.
  • the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
  • compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH.
  • sterile liquid preparations e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH.
  • Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues.
  • Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyoi (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
  • carriers can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyoi (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
  • Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.
  • a suitable carrier such as a suitable carrier, diluent, or excipient
  • the compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
  • compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
  • antimicrobial preservatives for example, parabens, chlorobutanol, phenol, and sorbic acid.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • CD4+ and CD8+ primary human T cells were combined in a 1 : 1 ratio. Cells were washed three times in cold reduced serum media (Opti-MEM) and then resuspended in supplemented P3 Primary Cell Nucleofector Solution (Lonza) at up to 10e 6 cells per lOOpL volume, which was the volume for each electroporation. Washed and resuspended cells were kept on ice. 20pg of Cas9 protein was combined with lOpg of sgRNA and incubated at room temperature for 10 minutes to form RNP complexes. For DKO, RNP complexes were prepared separately for each knockout.
  • Opti-MEM cold reduced serum media
  • P3 Primary Cell Nucleofector Solution Loxza
  • IOOUL of cells were added and mixed to RNP complexes and then transferred to a Nucleocuvette vessel (Lonza).
  • a Nucleocuvette vessel Lionza
  • cells were mixed with one set of RNP complexes and then subsequently mixed with the other set of RNP complexes.
  • the vessel was placed into a 4D-Nucleofector Core Unit electroporator (Lonza) and electroporated using the setting code ‘EH111’ for high-fidelity, unstimulated human T cells. Electroporated cells were transferred into culture media at a density of 5e 6 cells/mL and placed in a 30°C incubator for 48 hours.
  • cells were activated by adding CD3/CD28-targeting magnetic beads (CTS Dynabeads CD3/CD28) at a 3: 1 ratio of beads to cells and diluted to a culture density of le 6 cells/mL.
  • CTS Dynabeads CD3/CD28 CD3/CD28-targeting magnetic beads
  • cells were transduced with lentivirus encoding a CAR at an MOI of 3 transducing units per cell.
  • beads were removed from the cells.
  • Cells were diluted to 0.6e 6 cells/mL every other day until the average cell volume reached ⁇ 300fL, then sampled for determination of knockout efficiency by sequencing and western blot, and then cryopreserved.
  • SEQ ID NO: 1 AAGGAGGTCTTCTCCTGCCG
  • SEQ ID NO: 2 CAGTGTTTGTGCCATCCTGG
  • SEQ ID NO: 3 CAGCTCCCTCTAGTCCCGCG
  • PCR primer sequences for PCR amplification of Regnase-1 knockout locus SEQ ID NO: 4: Forward Set # 1 (5’ ⁇ 3’): GGAACTGGCACTGGGAATGGA SEQ ID NO: 5: Reverse Set # 1 (5’ ⁇ 3’): AATGACCACCATTCAGAGCAGG SEQ ID NO: 6: Forward Set # 2 (5’ ⁇ 3’): GTGTGTAGTGCCACCAATCC SEQ ID NO: 7: Reverse Set # 2 (5’ ⁇ 3’): TTACCAGTAGCGCGACCCT
  • SEQ ID NO: 8 Forward Set # 1 (5’ ⁇ 3 ’): TCCAAAGGGGAAGGGACTGC SEQ ID NO: 9: Reverse Set # 1 (5’ ⁇ 3’): TCTCTCTTGGGGAGAACCAGG SEQ ID NO: 10: Forward Set # 2 (5’ ⁇ 3 ’): GCGCTATTCACCGTCCCTAA SEQ ID NO: 11 : Reverse Set # 2 (5’ ⁇ 3’): CCTCCCTCTAGACCAGTCCC sgRNA sequences that facilitate efficient Roquin-1 knockout (5’— >3’):
  • SEQ ID NO: 12 TGAAGACACAAAGCATTATG
  • SEQ ID NO: 13 Forward (5’ ⁇ 3’): CAAAACAAAGGAATGCAAAGATACA
  • SEQ ID NO: 14 Reverse (5’ ⁇ 3’): GTGCAGTAATGGCAACTTCCG
  • SEQ ID NO: 15 Forward (5’ ⁇ 3’): TGGGCGTTTTGATGTTACCAG
  • SEQ ID NO: 16 Reverse (5’ ⁇ 3’): TGATGGAAAGCACTGCTGAAG
  • Example 1 CAR-T cell function
  • Knockout (KO) CAR-T cells were generated using the scheme depicted in Fig 1. Briefly, CD4+ and CD8+ T cells were combined in a 1 : 1 ratio and electroporated with RNP complexes with sgRNAs targeting Regnase-1 (Regl) and/or Roquin-1 (Roql). Cells were then placed in 30°C at 5e6 cells/mL for 48 hours, before stimulation with CD3/CD28 coated beads at a 3: 1 ratio of beads to T cells. After 24 hours, cells were transduced with virus encoding the mesothelin- targeting M5 CAR at a MOI of 3.
  • KO of Regnase-1 and/or Roquin-1 did not affect the number of population doublings or the cell volume during initial expansion (Fig 2).
  • KO cells were validated on both the protein and DNA level using western blotting against respective targets and sequencing at respective genetic KO loci and showed high efficiency KO (Fig. 3, Fig. 4).
  • KO CAR-T cells displayed similar transduction efficiencies, indicating that KO of Regnase-1 and/or Roquin-1 does not affect transduction efficiency (Fig. 2).
  • Regnase-1 KO and double knock-out (DKO) CAR-T cells retained a greater percentage of central memory cells compared to Roquin-1 KO and mock CAR-T cells (Fig. 5).
  • All KO CAR-T cell groups displayed enhanced CD4 expression on CD8 T cells and ICOS expression compared to mock CAR-T cells (Fig. 6, Fig. 7).
  • CAR-T cells were assayed for their activation potential via staining for CD25 and CD69 after coculture with antigen-negative cells.
  • KO CAR-T cells displayed a greater activation profile compared to mock CAR-T cells against antigen-negative cells, with Regnase-1 and DKO CAR-T cells showing enhanced CD69 expression and altered CD25 expression compared to mock CAR-T cells (Fig. 8).
  • CAR-T cells were assayed for their baseline cytokine activity using a Luminex assay. Briefly, CAR-T cells were cocultured overnight with antigen-negative cells at a E:T ratio of 1 :2 and supernatants were isolated for analysis. Against antigen-negative cells, Regl-KO and DKO CAR-T cells displayed substantial increased production of IL2, TNFa, IFNg, IL6 and GM-CSF (Fig. 9).
  • RNA sequencing The baseline transcriptional profiles of Regnase-1 and/or Roquin-1 KO engineered T cells were probed using bulk RNA sequencing. Briefly, cells were thawed and rested overnight in IL7 and IL 15 before RNA isolation, library preparation, and sequencing. Differential expression analysis using DESeq revealed upregulation of various inflammatory genes in Reg-1 KO and DKO T cells at baseline as depicted using a volcano plot (Fig. 10). Focused analysis of a set of inflammatory genes including various cytokines, costimulatory receptors, and inhibitory receptors revealed general upregulation of these transcripts in Regl-KO and DKO T cells (Fig. 11).
  • RNAseq data from three different donors revealed that while the largest variation was due to biological differences between donors, among each donor, Regl-KO and DKO groups clustered more closely together while Roql-KO and Mock groups clustered more closely together (Fig. 12).
  • Gene set enrichment analysis revealed upregulation of inflammatory gene sets in Regl-KO and DKO groups but not the Roql-KO group (Fig. 12).
  • CAR-T cells were assayed for cytotoxicity using a luciferase-based assay. Briefly, CAR- T cells were seeded at E:T ratios 10: 1, 3: 1, 1 : 1, 0.3: 1, 0.1 :1, 0.03: 1, and tumor only against antigen-positive cells transduced with GFP and CBG. After 24 hours, luciferin substrate was added to each well and read for luminescence signal. All CAR-T cells displayed similar cytotoxicity across all E:T ratios in this experiment against antigen-positive cells (Fig. 13). CAR- T cells were assayed for killing ability over time using an impedance assay.
  • CAR-T cells were seeded at E:T ratios 1 : 1, 1 :3, and 1 : 10 against adherent antigen-positive cells and the cell index, a measure of cell impedance indicative of live cells adhered to the bottom of the well, was measured over time. All CAR-T cells were able to clear tumor similarly across all E:T ratios in this experiment (Fig. 13).
  • CAR-T cells were assayed for their proliferation via CellTraceViolet staining after coculture for 7 days with antigen-positive cells at a 1 :5 E:T ratio. All CAR-T cell groups demonstrated similar proliferation via CTV staining in the CD8 compartment, with evidence of a modest increase in CD4 CAR-T cell proliferation for Reg 1 -KO and especially for DKO (Fig. 14).
  • CAR-T cells were assayed for their activation profile via flow cytometry for CD25 and CD69 after coculture with antigen-positive cells.
  • KO CAR-T cells displayed slightly greater percentages of CD25+CD69+ cells after stimulation with antigen-positive cells compared to mock CAR-T cells (Fig. 15).
  • CAR-T cells were assayed for their cytokine secretion using a Luminex assay. Briefly, CAR-T cells were cocultured overnight with antigen-positive cells at a E:T ratio of 1 :2 and supernatants were isolated for analysis. Against antigen-positive cells, Regl-KO and DKO CAR- T cells displayed enhanced production of several cytokines, such as IFNg and IL6 (Fig. 16).
  • CAR-T cells were assayed for their long-term activity and phenotype with a chronic antigen exposure (CAE) assay (Fig. 17). Briefly, CAR-T cells were counted and reseeded with additional tumor cells twice a week for 4 weeks. Regl-KO and DKO CAR-T cells demonstrated increased cumulative proliferation over the course of CAE compared to mock (Fig. 18). All KO cells also demonstrated greater retention of CAR expression over time compared to mock (Fig. 19). All groups displayed progressive loss of CD4+ cells under CAE (Fig. 20). Roql-KO and DKO CAR-T cells retained greater ICOS expression under CAE (Fig. 21).
  • CAE chronic antigen exposure
  • KO cells generally displayed greater expression of exhaustion markers PD1, TIM3, and LAG3 under CAE, with Roql-KO and DKO CAR-T cells displaying the greatest percentage of co-expressed exhaustion markers at the end of CAE (Fig. 22). After 28 days of CAE, Roql-KO and DKO CAR-T cells expressed more PD1 and TIM3 (Fig. 23). KO cells generally displayed greater retention of a central memory population subset compared to mock, indicative of less differentiation (Fig. 23).
  • CAR-T cells were assayed for their long-term activity and function using a restimulation assay (Fig. 24). Briefly, CAR-T cells were stimulated with irradiated antigen-positive cells twice a week for a total of 4 stimulations before subjecting them to functional assays. After 4 stimulations, DKO CAR-T cells had the greatest cytotoxicity as determined by a luciferase-based assay described elsewhere herein (Fig. 24). DKO CAR-T cells also displayed the greatest cytokine activity and retention of effector molecules as determined by intracellular cytokine staining (Fig. 25).
  • CAR-T cells were assayed for their in vivo activity with a xenograft NSG mouse model (Fig. 26). Briefly, NSG mice were injected with 2e6 AsPCl cells subcutaneously and measured weekly until tumor sizes reached -250 cubic mm. 0.5e6 CAR-T cells were then injected and tumors were measured weekly via caliper measurements and BLI imaging. 21 days after CAR-T cell administration, blood was sampled to measure the number of CD3+ or CD45+ cells in circulation. In this experiment, DKO CAR-T cell treated mice were the only group to complete clear tumor burden, while treatment with Regnase-1 or Roquin-1 KO CAR-T cells resulted in better tumor control compared to mock CAR-T cells (Fig. 26, Fig. 27). DKO CAR-T cell treated mice retained more T cells per volume blood than mock CAR-T cells (Fig. 28). In this experiment, Roql-KO and DKO CAR-T cells extended survival longer compared to mock (Fig. 29).
  • CAR-T cells were assayed for their intratumoral activity using a xenograph NSG mouse model (Fig. 30).
  • the experimental scheme is the same as described above, except that 2 weeks after CAR-T cell administration the tumors and spleens were harvested from the mice and analyzed. In this experiment, the tumor growth kinetics for the 2 weeks before tumor harvest were comparable across all groups (Fig. 30).
  • Regl-KO and DKO CAR-T cells displayed a greater number of TILs as well as a greater number of CD45+ cells in the spleen per mass of tissue compared to mock (Fig. 31, Fig. 32).
  • Cytokine levels of CAR-T cell treated mice were measured using a Luminex assay. Briefly, peripheral blood was isolated from mice 14 days after treatment and spun down to isolate the plasma or serum. Many cytokines were not detectable, though DKO CAR-T cell treated mice showed elevated IFNg, TNFa, IL8, and GM-CSF in the blood (Fig. 35).
  • CAR-T cells were assayed for their persistence using a tumor rechallenge experiment. Briefly, mice previously treated with CAR-T cells that demonstrated complete tumor clearance were rechallenged with 2e6 AsPCl cells by a subcutaneous injection on the opposite (left) flank and monitored over time. Mice previously treated with KO CAR-T cells demonstrated sustained tumor burden suppression compared to the Mock CAR-T cell treated mouse (Fig. 36). KO CAR- T cell treated mice also demonstrated enhanced retention of CD4 and CD8 T cells in the blood at 21 days after rechallenge, which was especially notable for DKO CAR-T cells (Fig. 37).
  • KO TCR-T cells were generated largely using the scheme depicted in Fig. 1 except with the addition of TRAC knockout and elimination of the 2 day 30°C recovery period. Briefly, CD4+ and CD8+ T cells were combined in a 1 : 1 ratio and electroporated with RNP complexes with sgRNAs targeting TRAC and Regnase-1 and/or Roquin-1 and immediately stimulated with CD3/CD28 coated beads at a 3: 1 ratio of beads to T cells. After 24 hours, cells were transduced with virus encoding the NY-ESO-1 -targeting 8F TCR at a MOI of 3.
  • KO of Regnase-1 and/or Roquin-1 did not affect the number of population doublings or the cell volume during initial expansion (Fig 40). KO cells were validated on both the protein and DNA level using western blotting against respective targets and sequencing at respective genetic KO loci and showed high efficiency KO (Fig. 41, Fig. 42).
  • KO TCR-T cells displayed similar transduction efficiencies, indicating that KO of Regnase-1 and/or Roquin-1 does not affect transduction efficiency (Fig. 40).
  • Regnase-1 KO and DKO TCR-T cells retained a greater percentage of central memory cells compared to Roquin-1 KO and mock CAR-T cells (Fig. 47).
  • All KO CAR-T cell groups displayed enhanced CD4 expression on CD8 T cells and ICOS expression compared to mock CAR-T cells (Fig. 43, Fig. 44).
  • TCR-T cells were assayed for their activation potential via staining for CD25 and CD69 after coculture with antigen-negative cells.
  • KO TCR-T cells displayed a slightly greater but largely similar activation profile compared to mock CAR-T cells against antigen-negative cells (Fig. 45).
  • TCR-T cells were assayed for their baseline cytokine activity using a Luminex assay. Briefly, TCR-T cells were cocultured overnight with antigen-negative cells at a E:T ratio of 1 :2 and supernatants were isolated for analysis. Against antigen-negative cells, DKO TCR-T cells displayed increased production of GM-CSF (Fig. 46).
  • TCR-T cells were assayed for cytotoxicity using a luciferase-based assay. Briefly, TCR-T cells were seeded at E:T ratios 10: 1, 3: 1, 1 : 1, 0.3: 1, 0.1 : 1, 0.03: 1, and tumor only against antigen-positive cells transduced with GFP and CBG. After 24 hours, luciferin substrate was added to each well and read for luminescence signal. All TCR-T cells displayed similar cytotoxicity across all E:T ratios in this experiment against antigen-positive cells (Fig. 48). TCR- T cells were assayed for killing ability over time using an imaging-based assay.
  • TCR-T cells were seeded at E:T ratios 1 :3 against adherent antigen-positive cells and the GFP mean, a measure of live tumor cells remaining, was measured over time. All TCR-T cells demonstrated similar killing over time in this experiment (Fig. 48).
  • TCR-T cells were assayed for their proliferation via CellTrace Violet staining after coculture for 7 days with antigen-positive cells at a 1 :3 E:T ratio. All KO TCR-T cell groups demonstrated emnhanced proliferation via CTV staining in the CD8 compartment compared to mock TCR-T, with the DKO group also demonstrating enhanced proliferation in the CD4 compartment (Fig. 49). TCR-T cells were assayed for their cytokine secretion using a Luminex assay. Briefly, TCR-T cells were cocultured overnight with antigen-positive cells at a E:T ratio of 1 :2 and supernatants were isolated for analysis.
  • Regl-KO and DKO CAR- T cells displayed enhanced production of several cytokines, such as IFNg and IL6 (Fig. 50).
  • TCR-T cells were assayed for their activation profile via flow cytometry for CD25 and CD69 after coculture with antigen-positive cells.
  • KO TCR-T cells displayed slightly greater percentages of CD25+CD69+ cells after stimulation with antigen-positive cells compared to mock TCR-T cells (Fig. 51).
  • TCR-T cells were assayed for their in vivo activity with a xenograft NSG mouse model (Fig. 52). Briefly, NSG mice were injected with 4e6 A549-ESO-HLA-II cells subcutaneously and measured weekly until tumor sizes reached -150 cubic mm. 3e6 TCR-T cells were then injected and tumors were measured weekly via caliper measurements and BLI imaging. 21 days after CAR-T cell administration, blood was sampled to measure the number of CD3+ or CD45+ cells in circulation. In this experiment, Regl-KO and DKO TCR-T cell treated mice demonstrated enhanced tumor burden control (Fig. 52, Fig. 53). DKO TCR-T cell treated mice retained more T cells per volume blood than mock TCR-T cells (Fig. 54).
  • Embodiment 1 provides a modified immune cell or precursor cell thereof, wherein endomenous Roquin-1 has been disrupted and/or knocked out.
  • Embodiment 2 provides a modified immune cell or precursor cell thereof, wherein endogenous Roquin-1 and endogenous Regnase-1 have been disrupted and/or knocked out.
  • Embodiment 3 provides the modified immune cell or precursor cell thereof of any preceding embodiment, wherein the cell comprises a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.
  • CAR chimeric antigen receptor
  • Embodiment 4 provides the modified immune cell or precursor cell thereof of embodiment 3, wherein the antigen binding domain binds a tumor associated antigen (TAA).
  • Embodiment 5 provides the modified immune cell or precursor cell thereof of embodiment 4, wherein the tumor associated antigen (TAA) is expressed by cells on a solid tumor.
  • TAA tumor associated antigen
  • Embodiment 6 provides the modified immune cell or precursor cell thereof of embodiment 4 or 5, wherein the TAA is mesothelin.
  • Embodiment 7 provides the modified immune cell or precursor cell thereof of embodiment 6, wherein the CAR comprises a nucleotide sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17.
  • Embodiment 8 provides the modified immune cell or precursor cell thereof of any of the preceding embodiments, wherein the cell comprises an exogenous T cell receptor (TCR).
  • TCR T cell receptor
  • Embodiment 9 provides the modified immune cell or precursor cell thereof of embodiment 8, wherein the exogenous TCR binds a TAA.
  • Embodiment 10 provides the modified immune cell or precursor cell thereof of embodiment 9, wherein the TAA is NY-ESO-1.
  • Embodiment 11 provides the modified immune cell or precursor cell thereof of embodiment 10, wherein the exogenous TCR comprises a nucleotide sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18.
  • Embodiment 12 provides the modified immune cell or precursor cell thereof of any of embodiments 8-11, wherein the exogenous TRAC been disrupted and/or knocked out.
  • Embodiment 13 provides the modified immune cell or precursor cell thereof of any of the preceding embodiments, wherein the cell is a primary cell.
  • Embodiment 14 provides the modified immune cell or precursor cell thereof of any of the preceding embodiments, wherein the cell is a human cell.
  • Embodiment 15 provides the modified immune cell or precursor cell thereof of any of the preceding embodiments, wherein the cell is a T cell.
  • Embodiment 16 provides the modified immune cell or precursor cell thereof of any of the preceding embodiments, wherein the endomenous Regnase-1 has been disrupted and/or knocked out via a CRISPR/Cas9 system.
  • Embodiment 17 provides the modified immune cell or precursor cell thereof of embodiment 16, wherein the CRISPR/Cas9 system comprises an sgRNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1-3.
  • Embodiment 18 provides the modified immune cell or precursor cell thereof of any of the preceding embodiments, wherein the endomenous Roquin-1 has been disrupted and/or knocked out via a CRISPR/Cas9 system.
  • Embodiment 18 provides the modified immune cell or precursor cell thereof of embodimentl8, wherein the CRISPR/Cas9 system comprises an sgRNA comprising the nucleotide sequence of SEQ ID NO: 12.
  • Embodiment 20 provides a method of treating cancer in a subject in need thereof.
  • the method comprises administering to the subject a composition comprising the modified immune cell or precursor cell thereof of any of the preceding embodiments.

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Abstract

La présente invention concerne une cellule immunitaire modifiée ou une cellule précurseur de celle-ci comprenant des récepteurs d'antigène chimériques (CAR) et/ou des récepteurs de lymphocytes T exogènes (TCR), le Roquin-1 endogène et/ou le Regnase-1 endogène ayant été dissociés et/ou inactivés pour le traitement du cancer chez un sujet en ayant besoin. L'invention concerne par ailleurs des compositions et des méthodes de traitement.
PCT/US2022/078496 2021-10-22 2022-10-21 Inactivation de regnase-1 et de roquin-1 pour améliorer l'activité de cellule car-t WO2023070080A1 (fr)

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WO2020219682A2 (fr) * 2019-04-24 2020-10-29 St. Jude Children's Research Hospital, Inc. Knock-out de gènes pour améliorer la fonction des lymphocytes t

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