WO2021123802A1 - Cellule - Google Patents

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Publication number
WO2021123802A1
WO2021123802A1 PCT/GB2020/053280 GB2020053280W WO2021123802A1 WO 2021123802 A1 WO2021123802 A1 WO 2021123802A1 GB 2020053280 W GB2020053280 W GB 2020053280W WO 2021123802 A1 WO2021123802 A1 WO 2021123802A1
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Prior art keywords
cell
gapdh
cells
isoform
glycolysis
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PCT/GB2020/053280
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English (en)
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Isaac GANNON
Paul Smith
Simon Thomas
Martin PULÉ
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Autolus Limited
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Publication of WO2021123802A1 publication Critical patent/WO2021123802A1/fr

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    • 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
    • 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
    • A61K39/464454—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/71—Oxidoreductases (EC 1.)

Definitions

  • the present invention relates to a cell which expresses a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TOR) and an isoform of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that is resistant to inhibition by one or more glycolysis inhibitors.
  • CAR chimeric antigen receptor
  • TOR transgenic T-cell receptor
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • Fermentation of glucose in cancer cells is the result of a shift from oxidative phosphorylation to glycolysis.
  • the glycolytic metabolism of glucose involves several enzymatic steps ( Figure 1).
  • the rate-limiting step is known to be the conversion of glyceraldehyde 3-phosphate to 1,3- bis-phosphoglycerate by the enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • Chimeric antigen receptors are proteins which graft the specificity of an antibody on to the effector function of a T-cell. Their usual form is that of a type I transmembrane domain protein with an antigen recognizing amino terminus, a spacer, a transmembrane domain all connected to a compound endodomain which transmits T-cell survival and activation signals (see Figure 1a).
  • scFv single-chain variable fragments
  • the present invention provides engineered cells comprising an isoform of glyceraldehyde 3- phosphate dehydrogenase (GAPDH) that is resistant to inhibition by one or more glycolysis inhibitors.
  • GPDH glyceraldehyde 3- phosphate dehydrogenase
  • Such cells are useful in combination therapies with one or more glycolysis inhibitors.
  • the present invention provides a cell which comprises:
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • the cell may be a T cell or a Natural Killer (NK) cell.
  • NK Natural Killer
  • the glycolysis inhibitor may be selected from the group comprising koningic acid (KA), 3- bromopyruvate (3BP), 3-bromopyruvate propyl ester (3-BrOP), pentalenolactone, a- Chlorohydrin, and ornidazole.
  • KA koningic acid
  • BP 3- bromopyruvate
  • 3-BrOP 3-bromopyruvate propyl ester
  • pentalenolactone a- Chlorohydrin
  • ornidazole ornidazole.
  • the glycolysis inhibitor is koningic acid (KA).
  • Isoforms of GAPDH which are resistant to KA may be naturally occurring or may be generated by methods known to those of skill in the art, for example by rational design or directed evolution.
  • the GAPDH isoform may be any GAPDH isoform resistant to inhibition by a glycolysis inhibitor.
  • the GAPDH isoform may be any GAPDH isoform resistant to inhibition by KA, such as those selected from the group comprising: Trichoderma koningii GAPDH 1, Aspergillus oryzae gpdB, Tetragenococcus halophilus GAPDH, and Trichoderma virens GAPDH.
  • the GAPDH isoform may be any GAPDH isoform resistant by pentalenolactone (PL), such as those selected from the group comprising Streptomyces avermitilis GAP1 , Streptomyces arenae GAP1.
  • PL pentalenolactone
  • the GAPDH isoform may have the amino acid sequence of any of SEQ ID NOS: 1, 2, 3, 4, or 5, or an amino acid sequence having at least 50% identity thereto.
  • the GAPDH isoform has the amino acid sequence selected from the group comprising SEQ ID NOS: 1, 2, or 3, or an amino acid sequence having at least 50% identity thereto, which are resistant to inhibition by KA.
  • the GAPDH isoform may have the amino acid sequence selected from the group comprising SEQ ID NOS: 4 or 5, or an amino acid sequence having at least 50% identity thereto, which are resistant to inhibition by PL.
  • the present invention provides a pharmaceutical composition which comprises a plurality of cells according to the first aspect.
  • the present invention provides a cell according to the first aspect, or a pharmaceutical composition according to the second aspect, for use in therapy.
  • the present invention provides a cell according to the first aspect, or a pharmaceutical composition according to the second aspect, for use in a method of treatment of cancer.
  • the method of treatment of cancer may include co-administration of one or more glycolysis inhibitors or pre-treatment with one or more glycolysis inhibitors.
  • the present invention provides a method for treating a disease, which comprises the step of administering a cell according to the first aspect, or a pharmaceutical composition according to the second aspect to a subject in need thereof.
  • the subject may also be administered one or more glycolysis inhibitors or may have been pre-treated with one or more glycolysis inhibitors.
  • the disease may be cancer.
  • the subject may be a human.
  • the present invention provides the use of a cell according to the first aspect, or a pharmaceutical composition according to the second aspect in the manufacture of a medicament for the treatment of cancer.
  • the medicament may be for use in combination with one or more glycolysis inhibitors or may be for use in subjects pre-treated with one or more glycolysis inhibitors.
  • the subject may be a human.
  • a nucleic acid construct which comprises: (i) a first polynucleotide which encodes an isoform of GAPDH that is resistant to inhibition by one or more glycolysis inhibitors; and (ii) a second polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • the first and second polynucleotides may be separated by a co-expression site.
  • the present invention provides a kit of polynucleotides comprising: (i) a first polynucleotide which encodes an isoform of GAPDH that is resistant to inhibition by one or more glycolysis inhibitors; and (ii) a second polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • the present invention provides a vector which comprises a nucleic acid construct according to the seventh aspect of the invention.
  • kits of vectors which comprises: (i) a first vector which comprises a polynucleotide which encodes an isoform of GAPDH that is resistant to inhibition by one or more glycolysis inhibitors; and (ii) a second vector which comprises a polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • a method for making a cell according to the first aspect of the invention which comprises the step of introducing: a nucleic acid construct according to the seventh aspect of the invention, a kit of polynucleotides according to the eighth aspect of the invention, a vector according to the ninth aspect of the invention, or a kit of vectors according to the tenth aspect of the invention into a cell ex vivo.
  • the present inventors provide engineered immune effector cells which are resistant to the effects of one or more glycolysis inhibitors and are useful in combination therapy with one or more glycolysis inhibitors.
  • FIG. 1 Schematic diagram showing an overview of the metabolism of glucose.
  • Glyceraldehyde 3-phophate dehydrogenase is the rate-limiting step in glycolysis.
  • FIG. 2 Schematic diagram showing different generations of chimeric antigen receptors. The basic architecture of a canonical CAR is shown as well as different iterations of the three generations of this form of receptor.
  • Figure 3 Sensitivity of Raji, SupT1 , and Jurkat cancer cell lines to koningic acid: (A) cell count after 48 hours; (B) Percentage of live cells after 48 hours, calculated by SYTOX staining.
  • Figure 4 - KA-sensitivity of native Raji cells and Raji cells engineered to express either GAPDH1 or GAPDH2: (A) cell count after 72 hours; (B) Percentage of live cells after 72 hours, calculated by SYTOX staining.
  • Figure 5 - KA-sensitivity of T cells (A) cell count after 72 hours; (B) Percentage of live cells after 72 hours, calculated by SYTOX staining.
  • Figure 6 Proliferation of T cells, CAR-T cells, and CAR-T cells additionally expressing GAPDH1 in the presence or absence of 15 mM over 48 hours.
  • Figure 7 Proliferation of T cells, CAR-T cells, and CAR-T cells additionally expressing GAPDH1 in the presence or absence of 15 pM over 72 hours.
  • Figure 8 48-hour FACS-based killing assay at 1 :4 E:T ratio.
  • FIG. 11 Cytokine release assay (IFNy): (A) IFNy release 24 hours after activation with anti-idiotype antibody; (B) IFNy normalised to media conditions.
  • FIG. 12 Cytokine release assay (IL-2): (A) IL-2 release 24 hours after activation with anti idiotype antibody; (B) IL-2 normalised to media conditions.
  • IL-2 Cytokine release assay
  • FIG. 13 Alignment of several GAPDH sequences including human GAPDH and various KA-resistant and KA-sensitive forms. Sequences include Glyceraldehyde-3-phosphate dehydrogenase from H. sapiens (Uniprot P04406) (SEQ ID NO: 2); KA sensitive gpdA from Aspergillus oryzae (Uniprot Q9HGY7) (SEQ ID NO: 3); KA sensitive Gpd2 from Trichoderma koningii (Uniprot P17730) (SEQ ID NO: 4); KA resistant gpdB from Aspergillus oryzae (Uniprot Q2U0J7) (SEQ ID NO: 5); and KA resistant Gpd1 from Trichoderma koningii (Uniprot P17729) (SEQ ID NO: 6). A consensus sequence is also provided (SEQ ID NO: 1). DETAILED DESCRIPTION OF THE INVENTION
  • Glycolysis is the metabolic pathway that converts glucose into pyruvate, producing both adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH).
  • ATP adenosine triphosphate
  • NADH nicotinamide adenine dinucleotide
  • the pathway consists of ten enzymatic reactions that act on a number of intermediates, many of which are useful in other metabolic pathways, such as the synthesis nucleotides and amino acids.
  • the intermediate dihydroxyacetone phosphate (DHAP) is a source of glycerol, which is useful in the synthesis of triglycerides.
  • glycolysis After glycolysis is completed pyruvate is transported into mitochondria, where it is the starting point for oxidative phosphorylation. Unlike oxidative phosphorylation, glycolysis is oxygen- independent.
  • T cells also exhibit a glycolytic phenotype.
  • glycolysis is an attractive target for cancer therapy.
  • GAPDH glyceraldehyde-3-phosphate dehydrogenase
  • KA Koningic acid
  • GAPDH a sesquiterpene metabolite from fungus
  • the KA-producing fungi Trichoderma koningii expresses a GAPDH isoform, GAPDH 1 (Uniprot accession number P17729), which is resistant to inhibition by koningic acid.
  • the organism also expresses a KA-sensitive GAPDH isoform, GAPDH2 (Uniprot accession number P 17730).
  • Aspergillus oryzae primarily used in the fermentation of soy sauce, expresses a KA-resistant GAPDH encoded by the gpdB gene (Uniprot accession number Q2U0J7).
  • the A. oryzae gene gpdA encodes a KA-sensitive isoform (Uniprot accession number Q9HGY7).
  • Another organism which plays a role in the fermentation of soy sauce, Tetragenococcus halophilus expresses a KA-resistant GAPDH isoform (Uniprot accession number G4L978).
  • T cells engineered to express a KA- resistant GAPDH are not inhibited by KA.
  • T cells engineered to express a chimeric antigen receptor (CAR) and a KA-resistant GAPDH are capable of killing their target cells in the presence of KA.
  • KA koningic acid
  • KA any glyceraldehyde 3-phosphate dehydrogenase enzyme that is able to convert glyceraldehyde-3-phosphate to 1 ,3-bis-phosphoglycerate in the presence of koningic acid.
  • a GAPDH that is resistant to inhibition by koningic acid will have higher activity than human GAPDH in the presence of KA.
  • a GAPDH that is resistant to inhibition by koningic acid will have an IC50 of at least 10 mM, at least 15 pM, at least 20 pM, at least 25 pM, at least 30 pM, at least 35 pM, at least 40 pM, at least 45 pM, at least 50 pM, at least 55 pM, at least 60 pM, at least 65 pM, at least 70 pM, at least 75 pM, at least 80 pM, at least 85 pM, at least 90 pM, at least 95 pM, 100 pM, at least
  • the sesquiterpenoid antibiotic pentalenolactone also inhibits GAPDH.
  • Streptomyces avermitilis expresses both a PL-resistant GAPDH isoform, GAP1 (Uniprot accession number Q82IZ2), and a PL-sensitive isoform (GAP2, Uniprot accession number Q829W3).
  • Streptomyces arenae expresses both PL-resistant GAP1 (Uniprot accession number P54226) and PL-sensitive GAP2 (Uniprot accession number E3VWI2).
  • GAPDH is also preferentially inhibited by 3-bromopyruvate propyl ester (3-BrOP) (Tang at al.
  • Isoforms of GAPDH that are resistant to inhibition by 3-BrOP are suitable for use in the present invention.
  • a-Chlorohydrin and ornidazole also both inhibit GAPDH.
  • Isoforms of GAPDH that are resistant to inhibition by a-Chlorohydrin and/or ornidazole are suitable for use in the present invention.
  • isoforms of GAPDH resistant to the effect of an inhibitor may be generated by methods known to those of skill in the art, for example by rational design or directed evolution. Rational design of a glycolysis inhibitor resistant isoform of GAPDH might be conducted by comparing the amino acid sequences of naturally occurring glycolysis inhibitor resistant isoforms in order to identity consensus sequences. An alignment of various GAPDH sequences is shown in figure 13. Resistant isoforms may also be generated by directed evolution, which involves one or more rounds of mutagenesis followed by screening for the desired property, i.e. , inhibitor resistance. Mutagenesis may be entirely random or may be focussed on
  • the present invention provides a cell which encodes a GAPDH, wherein the expression of said GAPDH may be “tunable”.
  • tunable means that it is possible to increase, decrease, turn on or turn off the expression of the GAPDH in the engineered immune effector cell.
  • expression of the GAPDH may be controlled or tuned by an inducible promoter.
  • the GAPDH may be regulated by Nuclear factor of activated T cells (NFAT) response element.
  • An NFAT response element may comprise the amino acid sequence set forth in SEQ ID NO: 20 or a variant thereof.
  • Variant sequences of SEQ ID NO: 20 may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 20.
  • the variant sequence is able to function as a NFAT response element.
  • the NFAT response element may comprise repeat units such as 3, 4, 5 or 6 repeat units.
  • the NFAT response element may comprise 3, 4, 5 or 6 repeat units of SEQ ID NO: 20.
  • the NFAT response element may be positioned in front of a promoter (e.g. a CMV promoter).
  • the expression or activity of the GAPDH may be controlled or tuned through interaction with an intracellular retention domain.
  • the GAPDH may be retained within a cellular compartment by interaction with an intracellular retention domain.
  • An agent may be used to disrupt the interaction with the intracellular retention domain, thereby allowing translocation of the GAPDH and expression of the GAPDH in the appropriate cellular localisation of the engineered immune effector cell.
  • the activity of the GAPDH may be controlled or tuned.
  • the present invention relates to a cell which expresses an isoform of GAPDH that is resistant to inhibition by one or more glycolysis inhibitors.
  • the cell may also express a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • the cell may be engineered to express the GAPDH and/or CAR/TCR.
  • an “engineered cell” as used herein means a cell which has been modified to comprise or express a nucleic acid sequence which is not naturally encoded by the cell.
  • Methods for engineering cells are known in the art and include but are not limited to genetic modification of cells e.g. by transduction such as retroviral or lentiviral transduction, transfection (such as transient transfection - DNA or RNA based) including lipofection, polyethylene glycol, calcium phosphate and electroporation. Any suitable method may be used to introduce a nucleic acid sequence into a cell.
  • nucleic acid sequence encoding the GAPDH is not naturally expressed by a corresponding, unmodified cell.
  • An engineered cell is a cell whose genome has been modified e.g. by transduction or by transfection, such as retroviral or lentiviral transduction.
  • the term “introduced” refers to methods for inserting foreign DNA or RNA into a cell.
  • the term introduced includes both transduction and transfection methods. Transfection is the process of introducing nucleic acids into a cell by non-viral methods. Transduction is the process of introducing foreign DNA or RNA into a cell via a viral vector.
  • Engineered cells according to the present invention may be generated by introducing DNA or RNA coding a GAPDH by one of many means including transduction with a viral vector, transfection with DNA or RNA.
  • Cells may be activated and/or expanded prior to the introduction of a nucleic acid sequence encoding the GAPDH, for example by treatment with an anti-CD3 monoclonal antibody or both anti-CD3 and anti-CD28 monoclonal antibodies.
  • activated means that a cell has been stimulated, causing the cell to proliferate, differentiate or initiate an effector function.
  • Methods for measuring cell activation include, for example, measuring the expression of activation markers by flow cytometry, such as the expression of CD69, CD25, CD38 or HLA-DR or measuring intracellular cytokines.
  • expanded means that a cell or population of cells has been induced to proliferate.
  • the expansion of a population of cells may be measured for example by counting the number of cells present in a population.
  • the phenotype of the cells may be determined by methods known in the art such as flow cytometry.
  • an “immune effector cell” as used herein is a cell of the immune system which responds to a stimulus and effects a change.
  • an immune effector cell may a T cell (such as an alpha-beta T cell or a gamma-delta T cell), a B cell (such as a plasma cell), a Natural Killer (NK) cell or a macrophage.
  • T cell such as an alpha-beta T cell or a gamma-delta T cell
  • B cell such as a plasma cell
  • NK Natural Killer
  • Cytolytic immune cell as used herein is a cell which directly kills other cells. Cytolytic cells may kill cancerous cells; virally infected cells or other damaged cells. Cytolytic immune cells include T cells and Natural killer (NK) cells.
  • Cytolytic immune cells can be T cells or T lymphocytes which are a type of lymphocyte that play a central role in cell-mediated immunity.
  • T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of a TCR on their cell surface.
  • Cytolytic T cells (TC cells, or CTLs) destroy virally infected cells and tumour cells, and are also implicated in transplant rejection.
  • CTLs express the CD8 at their surface.
  • CTLs may be known as CD8+ T cells. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of all nucleated cells.
  • MHC class I which is present on the surface of all nucleated cells.
  • IL-10 adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis.
  • the cell of the present invention may be a T-cell, such as an alpha-beta T cell or a gamma- delta T cell.
  • Natural Killer Cells are a type of cytolytic cell which form part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner.
  • NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils and thymus where they then enter into the circulation.
  • LGL large granular lymphocytes
  • the cell of the present invention may be a wild-type killer (NK) cell or a cytokine induced killer cell.
  • the cell may be derived from a patient’s own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party).
  • T or NK cells may be activated and/or expanded prior to being transduced with nucleic acid molecule(s) encoding the polypeptides of the invention, for example by treatment with an anti-CD3 monoclonal antibody.
  • the cell may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T cells.
  • an immortalized T-cell line which retains its lytic function may be used.
  • the cell may be a haematopoietic stem cell (HSC).
  • HSCs can be obtained for transplant from the bone marrow of a suitably matched donor, by leukapheresis of peripheral blood after mobilization by administration of pharmacological doses of cytokines such as G-CSF [peripheral blood stem cells (PBSCs)], or from the umbilical cord blood (UCB) collected from the placenta after delivery.
  • cytokines such as G-CSF [peripheral blood stem cells (PBSCs)]
  • PBSCs peripheral blood stem cells
  • URB umbilical cord blood
  • the marrow, PBSCs, or UCB may be transplanted without processing, or the HSCs may be enriched by immune selection with a monoclonal antibody to the CD34 surface antigen.
  • Classical CARs are chimeric type I trans-membrane proteins which connect an extracellular antigen-recognizing domain (binder) to an intracellular signalling domain (endodomain).
  • the binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-like antigen binding site or on a ligand for the target antigen.
  • mAb monoclonal antibody
  • a spacer domain may be necessary to isolate the binder from the membrane and to allow it a suitable orientation.
  • a common spacer domain used is the Fc of lgG1. More compact spacers can suffice e.g. the stalk from CD8a and even just the lgG1 hinge alone, depending on the antigen.
  • a trans-membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.
  • TNF receptor family endodomains such as the closely related 0X40 and 4-1 BB which transmit survival signals.
  • CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals.
  • CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors.
  • retroviral vectors In this way, a large number of antigen-specific T cells can be generated for adoptive cell transfer.
  • the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on.
  • the CAR directs the specificity and cytotoxicity of the T cell towards cells expressing the targeted antigen.
  • the antigen-binding domain is the portion of a classical CAR which recognizes antigen.
  • Numerous antigen-binding domains are known in the art, including those based on the antigen binding site of an antibody, antibody mimetics, and T-cell receptors.
  • the antigen binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a wild-type ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain binder such as a camelid; an artificial binder single as a Darpin; or a single-chain derived from a T-cell receptor.
  • scFv single-chain variable fragment
  • tumour associated antigens are known, as shown in the following Table 3.
  • the antigen-binding domain used in the present invention may be a domain which is capable of binding a TAA as indicated therein.
  • the transmembrane domain is the sequence of a classical CAR that spans the membrane. It may comprise a hydrophobic alpha helix.
  • the transmembrane domain may be derived from CD28, which gives good receptor stability.
  • the CAR or transgenic TCR for use in the present invention may comprise a signal peptide so that when it is expressed in a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed.
  • the core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix.
  • the signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation.
  • At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase.
  • Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases.
  • the receptor may comprise a spacer sequence to connect the antigen-binding domain with the transmembrane domain.
  • a flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.
  • the spacer sequence may, for example, comprise an lgG1 Fc region, an lgG1 hinge or a human CD8 stalk or the mouse CD8 stalk.
  • the spacer may alternatively comprise an alternative linker sequence which has similar length and/or domain spacing properties as an lgG1 Fc region, an lgG1 hinge or a CD8 stalk.
  • a human lgG1 spacer may be altered to remove Fc binding motifs.
  • the intracellular signalling domain is the signal-transmission portion of a classical CAR.
  • CD3-zeta endodomain which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound.
  • CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signalling may be needed.
  • chimeric CD28 and 0X40 can be used with CD3- Zeta to transmit a proliferative / survival signal, or all three can be used together.
  • the intracellular signalling domain may be or comprise a T cell signalling domain.
  • the intracellular signalling domain may comprise one or more immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAM immunoreceptor tyrosine-based activation motifs
  • An ITAM is a conserved sequence of four amino acids that is repeated twice in the cytoplasmic tails of certain cell surface proteins of the immune system.
  • the motif contains a tyrosine separated from a leucine or isoleucine by any two other amino acids, giving the signature YxxL/l.
  • Two of these signatures are typically separated by between 6 and 8 amino acids in the tail of the molecule (YXXL/IX (6-8) YXXL/I).
  • ITAMs are important for signal transduction in immune cells.
  • the CD3 and z-chains of the T cell receptor complex are found in the tails of important cell signalling molecules such as the CD3 and z-chains of the T cell receptor complex, the CD79 alpha and beta chains of the B cell receptor complex, and certain Fc receptors.
  • the tyrosine residues within these motifs become phosphorylated following interaction of the receptor molecules with their ligands and form docking sites for other proteins involved in the signalling pathways of the cell.
  • the intracellular signalling domain component may comprise, consist essentially of, or consist of the O ⁇ 3-z endodomain, which contains three ITAMs.
  • the O ⁇ 3-z endodomain transmits an activation signal to the T cell after antigen is bound.
  • the intracellular signalling domain may comprise additional co-stimulatory signalling.
  • 4-1 BB also known as CD137
  • CD28 and 0X40 can be used with O ⁇ 3-z to transmit a proliferative / survival signal.
  • the CAR may have the general format: antigen-binding domain-TCR element.
  • TCR element means a domain or portion thereof of a component of the TCR receptor complex.
  • the TCR element may comprise (e.g. have) an extracellular domain and/or a transmembrane domain and/or an intracellular domain e.g. intracellular signalling domain of a TCR element.
  • the TCR element may selected from TCR alpha chain, TCR beta chain, a CD3 epsilon chain, a CD3 gamma chain, a CD3 delta chain, CD3 epsilon chain.
  • T-cell receptor is a molecule found on the surface of T cells which is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the TCR is a heterodimer composed of two different protein chains.
  • the TCR in 95% of T cells the TCR consists of an alpha (a) chain and a beta (b) chain (encoded by TRA and TRB, respectively), whereas in 5% of T cells the TCR consists of gamma and delta (g/d) chains (encoded by TRG and TRD, respectively).
  • antigens recognized by the TCR can include the entire array of potential intracellular proteins, which are processed and delivered to the cell surface as a peptide/MHC complex.
  • heterologous TCR molecules it is possible to engineer cells to express heterologous (i.e. non-native) TCR molecules by artificially introducing the TRA and TRB genes; or TRG and TRD genes into the cell using a vector.
  • the genes for engineered TCRs may be reintroduced into autologous T cells and transferred back into patients for T cell adoptive therapies.
  • Such ‘heterologous’ TCRs may also be referred to herein as ‘transgenic TCRs’.
  • the transgenic TCR for use in the present invention may recognise a tumour associated antigen (TAA) when fragments of the antigen are complexed with major histocompatibility complex (MHC) molecules on the surface of another cell.
  • TAA tumour associated antigen
  • MHC major histocompatibility complex
  • the transgenic TCR for use in the present invention may recognise a TAA listed in Table 3.
  • NUCLEIC ACID CONSTRUCT / KIT OF NUCLEIC ACID SEQUENCES The present invention provides a nucleic acid construct which comprises:
  • the present invention also provides a kit comprising nucleic acid sequences according to the present invention.
  • the kit may comprise
  • polynucleotide As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.
  • the nucleic acid construct may comprise a plurality of nucleic acid sequences which encode a GAPDH; and a CAR or transgenic TCR.
  • the nucleic acid construct may comprise two, three, four or more nucleic acid sequences which encode different components of the invention.
  • the plurality of nucleic acid sequences may be separated by co-expression sites as defined herein. It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code.
  • polynucleotides of the present invention are codon optimised to enable expression in a mammalian cell, in particular an immune effector cell as described herein.
  • Nucleic acids according to the invention may comprise DNA or RNA. They may be single- stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.
  • variant in relation to a nucleotide sequence or amino acid sequence includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid(s) from or to the sequence.
  • a co-expression site is used herein to refer to a nucleic acid sequence enabling co-expression of nucleic acid sequences encoding the GAPDH and a CAR or transgenic TCR according to the present invention.
  • first nucleic acid sequence there may be a co-expression site between the first nucleic acid sequence and the second nucleic acid sequence.
  • the same co-expression site may be used.
  • the co-expression site may be a cleavage site.
  • the cleavage site may be any sequence which enables the two polypeptides to become separated.
  • the cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into individual peptides without the need for any external cleavage activity.
  • cleavage is used herein for convenience, but the cleavage site may cause the peptides to separate into individual entities by a mechanism other than classical cleavage.
  • FMDV Foot-and-Mouth disease virus
  • various models have been proposed for to account for the “cleavage” activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect (Donnelly et al (2001) J. Gen. Virol. 82:1027-1041).
  • the exact mechanism of such “cleavage” is not important for the purposes of the present invention, as long as the cleavage site, when positioned between nucleic acid sequences which encode proteins, causes the proteins to be expressed as separate entities.
  • the cleavage site may be a furin cleavage site.
  • Furin is an enzyme which belongs to the subtilisin-like proprotein convertase family. The members of this family are proprotein convertases that process latent precursor proteins into their biologically active products.
  • Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. Examples of furin substrates include proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta- secretase, membrane type-1 matrix metalloproteinase, beta subunit of pro-nerve growth factor and von Willebrand factor.
  • Furin cleaves proteins just downstream of a basic amino acid target sequence (canonically, Arg-X-(Arg/Lys)-Arg') and is enriched in the Golgi apparatus.
  • the cleavage site may be a Tobacco Etch Virus (TEV) cleavage site.
  • TSV Tobacco Etch Virus
  • TEV protease is a highly sequence-specific cysteine protease which is chymotrypsin-like proteases. It is very specific for its target cleavage site and is therefore frequently used for the controlled cleavage of fusion proteins both in vitro and in vivo.
  • the consensus TEV cleavage site is ENLYFQ ⁇ S (where ‘V denotes the cleaved peptide bond).
  • Mammalian cells such as human cells, do not express TEV protease.
  • the present nucleic acid construct comprises a TEV cleavage site and is expressed in a mammalian cell - exogenous TEV protease must also expressed in the mammalian cell.
  • the cleavage site may encode a self-cleaving peptide.
  • a ‘self-cleaving peptide’ refers to a peptide which functions such that when the polypeptide comprising the proteins and the self- cleaving peptide is produced, it is immediately “cleaved” or separated into distinct and discrete first and second polypeptides without the need for any external cleavage activity.
  • the self-cleaving peptide may be a 2A self-cleaving peptide from an aphtho- or a cardiovirus.
  • the primary 2A/2B cleavage of the aptho- and cardioviruses is mediated by 2A “cleaving” at its own C-terminus.
  • apthoviruses such as foot-and-mouth disease viruses (FMDV) and equine rhinitis A virus
  • the 2A region is a short section of about 18 amino acids, which, together with the N-terminal residue of protein 2B (a conserved proline residue) represents an autonomous element capable of mediating “cleavage” at its own C-terminus (Donelly et al (2001) as above).
  • 2A-like sequences have been found in picornaviruses other than aptho- or cardioviruses, ‘picornavirus-like’ insect viruses, type C rotaviruses and repeated sequences within Trypanosoma spp and a bacterial sequence (Donnelly et al., 2001) as above.
  • the co-expression sequence may be an internal ribosome entry sequence (IRES).
  • the co expressing sequence may be an internal promoter.
  • the present invention also provides a vector, or kit of vectors which comprises one or more nucleic acid sequence(s) or nucleic acid construct(s) of the invention.
  • a vector may be used to introduce the nucleic acid sequence(s) or construct(s) into a host cell so that it expresses a GAPDH as defined herein.
  • the vector may comprise a plurality of nucleic acid sequences which encode different components as provided by the present invention.
  • the vector may comprise two, three, four or more nucleic acid sequences which encode different components, such as the GAPDH and a CAR or transgenic TCR.
  • the plurality of nucleic acid sequences may be separated by co-expression sites as defined herein.
  • the vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon based vector or synthetic mRNA.
  • the vector may be capable of transfecting or transducing a cell.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an engineered immune effector cell according to the present invention or a cell obtainable (e.g. obtained) by a method according to the present invention.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a nucleic acid construct according to the present invention, a first and second polynucleotide as defined herein, or a vector according to the present invention or a first and second vector as defined herein.
  • the invention relates to a pharmaceutical composition containing a cell according to the present invention.
  • the pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
  • Such a formulation may, for example, be in a form suitable for intravenous infusion.
  • the present invention provides a method for treating and/or preventing a disease which comprises the step of administering an engineered immune effector cell according to the invention, or obtainable (e.g. obtained) by a method according to the present invention, or a nucleic acid construct according to the present invention, or a first and second nucleic acid sequence as defined herein; a vector according to the present invention or a first and second vector as described herein (for example in a pharmaceutical composition as described above) to a subject.
  • the present methods for treating and/or preventing a disease may comprise administering an engineered immune effector cell according to the present invention (for example in a pharmaceutical composition as described above) to a subject.
  • the present invention also provides a method for treating and/or preventing a disease in a subject which subject comprises cells of the invention, which method comprises the step of administering one or more glycolysis inhibitors to the subject.
  • this method involves administering one or more glycolysis inhibitors to a subject which already comprises cells of the present invention.
  • the present methods for treating and/or preventing a disease may comprise administering one or more glycolysis inhibitors to a subject to which the engineered immune cell according to the present invention has been administered.
  • the engineered immune cells may be administered to a subject to which one or more glycolysis inhibitors has already been administered.
  • one or more glycolysis inhibitors and the engineered immune cells may be administered simultaneously or substantially simultaneously.
  • a method for treating a disease relates to the therapeutic use of the cells of the present invention.
  • the cells may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
  • the method may involve the steps of:
  • nucleic acid construct according to the present invention introducing the nucleic acid construct according to the present invention, a first and second nucleic acid sequence as defined herein, a vector according to the present invention or a first and second vector as herein to the cell;
  • the engineered immune effector cell may be administered in the form of a pharmaceutical composition.
  • the pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
  • Such a formulation may, for example, be in a form suitable for intravenous infusion.
  • the present invention provides a cell according to the present invention for use in treating a disease.
  • the present invention also relates to the use of a cell according to the present invention for the manufacture of a medicament for the treatment of a disease.
  • the disease to be treated and/or prevented by the method of the present invention may be cancer.
  • the cancer may be a cancer such as neuroblastoma, multiple myeloma, prostate cancer, bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukaemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, and thyroid cancer.
  • the cancer may be neuroblastoma.
  • the cancer may be multiple myeloma.
  • the cancer may be prostate cancer.
  • the cell of the present invention may be capable of killing target cells, such as cancer cells.
  • the target cell may be recognisable by expression of a TAA, for example the expression of a TAA provided above in Table 3.
  • the cancer may be a cancer listed in Table 3.
  • the administration of a cell according to the present invention can be accomplished using any of a variety of routes, such as intraperitoneally, intravenously, subcutaneously, transcutaneously or intramuscularly.
  • the administration of one or more glycolysis inhibitors may also be accomplished using any of a variety of routes, such as intraperitoneally, intravenously, subcutaneously, transcutaneously or intramuscularly.
  • the cell and one or more glycolysis inhibitors may also be administered by intratumoral injection, either simultaneously, separately, or sequentially.
  • Cells of the present invention may be generated by introducing DNA or RNA coding for GAPDH or chimeric antigen receptor , as defined herein, by one of many means including transduction with a viral vector, transfection with DNA or RNA.
  • the cell of the invention may be made by: introducing to a cell (e.g. by transduction or transfection) the polynucleotide according to the present invention, the nucleic acid construct or vector according to the present invention, or a first and second nucleic acid sequence as defined herein, or a first and second vector as defined herein.
  • the cell may be from a sample isolated from a subject.
  • the present invention also provides a method of rendering an immune effector cell less susceptible to koningic acid by introducing a polynucleotide coding for a GAPDH to said immune effector cell.
  • the method may comprise: introducing to a cell (e.g. by transduction or transfection) the polynucleotide according to the present invention, the nucleic acid construct or vector according to the present invention, or a first and second nucleic acid sequence as defined herein, or a first and second vector as defined herein.
  • the method of rendering an immune effector cell less susceptible to koningic acid may comprise maintaining the cell under conditions which allow the expression of the GAPDH.
  • the present invention further relates to the use of a GAPDH to render an immune effector cell less susceptible to koningic acid.
  • Example 1 Effect of Koningic Acid of cancer cells and T cells Cancer Cell line sensitivity to koningic acid
  • Figure 3(A) shows the cell count after 48 hours at each concentration.
  • Figure 3(B) shows the cell count after 48 hours at each concentration.
  • Koningic acid inhibits proliferation from ⁇ 7.5 pM in all cell lines tested. In addition, Raji and Jurkat lines appear to be more susceptible to necrotic cell death than SupT 1.
  • GAPDH1 SFGmR.RQR8-2A-GPD1 -HAtag
  • GAPDH2 S F G m R-RQ R8-2 A-G P D2-H Atag
  • SFGmR is the SFG scaffold attachment region
  • RQR8 is a selection marker/suicide switch as described in WO2013/153391
  • 2A is a self-cleaving peptide sequence
  • GPD1 is GAPDH1 (KA insensitive);
  • GPD2 is GAPDH2 (KA sensitive);
  • HAtag is amino acids 98-106 of human influenza hemagglutinin (HA) surface glycoprotein.
  • Cells were then tested for sensitivity to KA by incubation with 0 mM, 3.125 pM, 6.25 pM, 12.5 pM, 25 pM or 50 pM KA over 72 hours. Cells were counted by FACS at 72 hours and the percentage of live cells was calculated using Sytox staining.
  • Figure 4(A) shows the cell count after 72 hours at each concentration.
  • Figure 4(B) shows the cell count after 72 hours at each concentration.
  • T cells to KA were confirmed by proliferation assay.
  • Isolated PBMCs were activated with anti-CD3 and anti-CD28 antibodies (TransAct) and incubated with 3.125 pM, 6.25 pM, 12.5 pM, 25 pM or 50 pM KA over 72 hours. All cells also received IL-2 at 50 u/ml. Proliferation was monitored by cell count using anti-CD3. Cell viability was also measured.
  • Figure 5(A) shows that proliferation of T cells is reduced in the presence of 6.25 pM KA.
  • Figure 5(B) cell viability was not affected until a concentration of at least 12.5 pM. The effect of KA on T cells is therefore confirmed.
  • Example 2 rescues CAR-T cell proliferation from KA
  • SFGmR is the SFG scaffold attachment region
  • RQR8 is a selection marker/suicide switch as described in WO2013/153391;
  • 2A is a self-cleaving peptide sequence
  • GAPPDH1 is GAPDH1 (KA insensitive); aCD19_HD37_L16H9-CD8STK-TyrpTM-CD28z is an anti-CD19 CAR comprising a CD8 stalk,
  • CD28 co-stimulation domain and CD3zeta signalling domain CD28 co-stimulation domain and CD3zeta signalling domain
  • HAtag is amino acids 98-106 of human influenza hemagglutinin (HA) surface glycoprotein.
  • Proliferation assays were conducted as set out in Example 1 , with the exception that incubations were carried out without additional IL-2. The effect of the presence or absence of 15 mM KA on the proliferation of cells transformed with HD37_28: or HD37_28z_GAPDH1 was investigated.
  • Figure 6 shows that GAPDH1 expression rescues T cell proliferation from the inhibitory effects of KA.
  • CTV Cell Trace Violet
  • target cells either Raji NT cells or Raji cells engineered to express GAPDH1
  • CTV Cell Trace Violet
  • the CTV dye was reconstituted to 5mM in DMSO.
  • the T-cells were resuspended at 2x10 6 cells per ml in PBS, and 1 ul/ml of CTV was added.
  • the T-cells were incubated the CTV for 20 minutes at 37 °C. Subsequently, the cells were quenched by adding 5V of complete media. After a 5 minutes incubation, the T-cells were washed and resuspended in 2ml of complete media. An additional 10-minute incubation at room temperature allows the occurrence of acetate hydrolysis and retention of the dye.
  • Labelled T-cells were co-cultured with target cells (either Raji NT cells or Raji cells engineered to express GAPDH1) for 72 hours.
  • the assay was carried out in a 96-well plate in 0.2 ml total volume using 100,000 Raji cells at an ET ratio of 1:10, in the presence or absence of 15 pM KA.
  • the T-cells were analysed by flow cytometry to measure the dilution of the CTV which occurs as the T-cells divide (Figure 7).
  • Example 3 - GAPDH1 restores CAR T killing in presence of KA FACs-based killing assay (FBK)
  • T cells were co-cultured with the target cells at an ET ratio of 1:4. Both KA-sensitive and KA- insensitive target cells (i.e, those expressing GAPDH2 or GAPDH1 , respectively) were tested.
  • the FBK was carried out after 48h of incubation.
  • IL-2 and IFNy secretion are indicators of T cell activation and was evaluated by using the supernatant of the cytotoxic co-culture assays. Cytokine secretion was analysed for CAR-T cells expression either GAPDH1 or GAPDH2 alongside relevant controls. Production of IL-2 and IFNy was detected by ELISA.

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Abstract

La présente invention concerne une cellule qui exprime un récepteur d'antigène chimère (CAR) ou un récepteur de lymphocyte T transgénique (TCR) et une isoforme de glycéraldéhyde 3-phosphate déshydrogénase (GAPDH) qui est résistante à l'inhibition par un ou plusieurs inhibiteurs de glycolyse.
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Publication number Priority date Publication date Assignee Title
WO2013153391A1 (fr) 2012-04-13 2013-10-17 Ucl Business Plc Polypeptide utile dans la thérapie cellulaire adoptive
WO2016073755A2 (fr) * 2014-11-05 2016-05-12 Board Of Regents, The University Of Texas System Cellules immunitaires effectrices génétiquement modifiées et cellules manipulées pour l'expansion de cellules immunitaires effectrices
WO2017079703A1 (fr) * 2015-11-05 2017-05-11 Juno Therapeutics, Inc. Vecteurs et cellules immunitaires génétiquement modifiées exprimant des modulateurs de voie métabolique et utilisations en thérapie cellulaire adoptive

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WO2013153391A1 (fr) 2012-04-13 2013-10-17 Ucl Business Plc Polypeptide utile dans la thérapie cellulaire adoptive
WO2016073755A2 (fr) * 2014-11-05 2016-05-12 Board Of Regents, The University Of Texas System Cellules immunitaires effectrices génétiquement modifiées et cellules manipulées pour l'expansion de cellules immunitaires effectrices
WO2017079703A1 (fr) * 2015-11-05 2017-05-11 Juno Therapeutics, Inc. Vecteurs et cellules immunitaires génétiquement modifiées exprimant des modulateurs de voie métabolique et utilisations en thérapie cellulaire adoptive

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