WO2024033544A1 - Deglycosylation of native glycoproteins expressed on a tumor cell surface - Google Patents

Deglycosylation of native glycoproteins expressed on a tumor cell surface Download PDF

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Publication number
WO2024033544A1
WO2024033544A1 PCT/EP2023/072345 EP2023072345W WO2024033544A1 WO 2024033544 A1 WO2024033544 A1 WO 2024033544A1 EP 2023072345 W EP2023072345 W EP 2023072345W WO 2024033544 A1 WO2024033544 A1 WO 2024033544A1
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Prior art keywords
sequence
glycosidase
cell
seq
car
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PCT/EP2023/072345
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French (fr)
Inventor
Monica CASUCCI
Beatrice Greco
Maria Chiara Bonini
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Ospedale San Raffaele S.R.L.
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Publication of WO2024033544A1 publication Critical patent/WO2024033544A1/en

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    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464428—CD44 not IgG
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01052—Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (3.5.1.52), i.e. glycopeptidase
    • 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
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/15—Non-antibody based
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag

Definitions

  • the present invention relates to deglycosylating enzymes and modified versions thereof.
  • the invention also provides combinations of deglycosylating enzymes and binding molecules, such as CARs, for example for improving the therapeutic activity of CAR-containing cells.
  • Methods and uses involving the deglycosylating enzymes of the invention are also provided.
  • BACKGROUND TO THE INVENTION Chimeric antigen receptors (CARs) are synthetic biology molecules commonly constructed by fusing an antigen-binding moiety, often derived from a tumor-reactive monoclonal antibody, with intracellular signaling domains derived from T lymphocytes.
  • CAR-T cell therapies Clinical testing of CAR-T cell therapies has increased rapidly in recent years, leading to marketing authorizations for different CAR-T cell products targeting either CD19 or BCMA in relapsed/refractory B-cell lymphomas, B-cell acute lymphoblastic leukemia of children and young adults and multiple myeloma.
  • CAR-T therapies objective response rates in patients with solid tumors are far less frequent and improving therapeutic efficacy against these malignancies represents one of the biggest challenges in the field.
  • the relative resistance of solid tumors to CAR-T cells highlights the need for improved understanding of different determinants of CAR-T cell activity and therapeutic efficacy.
  • N-glycans may protect tumors from CAR-T cells by interfering with immunological synapse formation and by promoting the interaction between co-inhibitory molecules, such as PD-1 and PD-L1. Besides fostering mechanisms of resistance by tumor cells, N-glycans might also support the inhibitory function exerted by the tumor microenvironment (TME). Moreover, it has been observed that blocking N-glycan synthesis in tumor cells with glycosylation inhibitors, such as the glucose/mannose analogue 2-deoxi-2-glucose (2DG), resulted in higher CAR-T cell activation, improved tumor cell lysis, and superior antitumor activity in different xenograft mouse models (Greco et al.
  • glycosylation inhibitors such as the glucose/mannose analogue 2-deoxi-2-glucose (2DG)
  • CAR-T therapies may still have limited efficacy in certain therapeutic scenarios, for example, when treating solid tumors.
  • CAR-T therapies may still have limited efficacy in certain therapeutic scenarios, for example, when treating solid tumors.
  • the present inventors have developed deglycosylating enzymes (glycosidases) and that may be used in combination with tumor specific CAR-T cells to create single CAR T cell agents that are endowed de-glycosylating activity.
  • glycanases being unsuitable lysosomal enzymes that function at an acidic pH
  • the inventors have surprisingly developed and validated a modified glycosidase (a peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase; PNGase) that is functional in the extracellular space and may deglycosylate proteins exposed on the surface of tumor cells.
  • PNGase peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase
  • the inventors have demonstrated in model studies that CAR T cells engineered to express the glycosidase display improved killing of tumor cells, such as pancreatic adenocarcinoma cells and lung adenocarcinoma cells.
  • a polynucleotide comprising (a) a nucleotide sequence encoding a glycosidase; and (b) a nucleotide sequence encoding a chimeric antigen receptor (CAR).
  • a product comprising (a) a first polynucleotide comprising a nucleotide sequence encoding a glycosidase; and (b) a second polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR).
  • the product may be, for example, a kit or a composition.
  • kits comprising (a) a first polynucleotide comprising a nucleotide sequence encoding a glycosidase; and (b) a second polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR).
  • a composition comprising (a) a first polynucleotide comprising a nucleotide sequence encoding a glycosidase; and (b) a second polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR).
  • the first polynucleotide is comprised in a first vector and the second polynucleotide is comprised in a second vector.
  • the glycosidase is a secreted glycosidase.
  • the glycosidase is secreted from a cell.
  • the cell is a CAR cell, i.e., it displays a chimeric antigen receptor.
  • the glycosidase is secreted by a CAR cell.
  • Glycosidases may comprise a signal peptide, to facilitate their secretion.
  • the glycosidase comprises a signal peptide.
  • the signal peptide may be cleaved from the glycosidase during its export from a cell.
  • the nucleotide sequence encoding a glycosidase further encodes a signal peptide operably linked to the glycosidase.
  • the signal peptide is selected from the group consisting of: a CD8 signal peptide, an IgG variable region heavy chain signal peptide, an Ig kappa chain V-III region VG signal peptide, a GM-CFS/CSF signal peptide, and a CSFR2A signal peptide.
  • the signal peptide is a CD8 signal peptide.
  • the signal peptide is an IgG variable region heavy chain signal peptide.
  • the signal peptide comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence as set forth in the group consisting of SEQ ID NOs: 24 – 28 or a fragment thereof.
  • the glycosidase is functional in the extracellular environment.
  • the glycosidase hydrolyses cell-surface displayed glycoproteins or glycopeptides.
  • the glycosidase has a substrate that is an N-linked glycan.
  • the glycosidase is an N-glycanase. In one embodiment, the glycosidase hydrolyses a bond (e.g. a b-aspartylglucosaminyl bond) between an N-linked glycan and an asparagine (Asn) residue. In one embodiment, the glycosidase hydrolyses a bond (e.g. a b-aspartylglucosaminyl bond) between a core- chitobiose region of an N-linked glycan and an asparagine (Asn) residue.
  • a bond e.g. a b-aspartylglucosaminyl bond
  • the glycosidase is peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), or a variant thereof.
  • the glycosidase is human peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), or a variant thereof.
  • the PNGase variant is a truncated PNGase.
  • the PNGase lacks a PUB domain.
  • the PNGase lacks a PAW domain.
  • the PNGase lacks both a PUB and a PAW domain.
  • the glycosidase comprises or consists of: (a) a sequence that has at least 70% sequence identity to SEQ ID NO: 1, or a fragment thereof; (b) a sequence that has at least 70% sequence identity to SEQ ID NO: 2, or a fragment thereof; (c) a sequence that has at least 70% sequence identity to SEQ ID NO: 3, or a fragment thereof; or (d) a sequence that has at least 70% sequence identity to SEQ ID NO: 34, or a fragment thereof.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, or a fragment thereof.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, or a fragment thereof.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3, or a fragment thereof.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34, or a fragment thereof.
  • the CAR is a 44v6.28z CAR.
  • the CAR comprises or consists of a sequence with at least 70% sequence identity to SEQ ID NO: 22.
  • the CAR is encoded by a polynucleotide sequence that comprises or consists of a sequence with at least 70% sequence identity to SEQ ID NO: 23.
  • the CAR comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22, or a fragment thereof.
  • the CAR is encoded by a polynucleotide sequence that comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23, or a fragment thereof.
  • the nucleotide sequences encoding the glycosidase and the CAR are operably linked to one or more promoter(s).
  • the nucleotide sequences encoding the glycosidase and the CAR are operably linked to the same promoter(s).
  • nucleotide sequences encoding the glycosidase and the CAR are independently operably linked to one or more promoter(s). In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR are operably linked to separate promoter(s). In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR are in opposing directions. In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR are in opposing directions and are independently operably linked to separate promoters. In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR in the same direction.
  • the promoter is selected from the group consisting of: a cytomegalovirus promoter (CMV), a human phosphoglycerate kinase promoter (PGK), an EF-1 ⁇ promoter and an inducible NFAT promoter.
  • the promoter is a cytomegalovirus (CMV) promoter.
  • the promoter is a minimal cytomegalovirus (mCMV) promoter.
  • a vector comprising the polynucleotide or product according to the invention.
  • the vector is a viral vector.
  • the vector is a lentiviral vector.
  • the lentiviral vector is a bidirectional lentiviral vector.
  • the vector is an adeno-associated viral (AAV) vector.
  • AAV adeno-associated viral
  • a glycosidase wherein the glycosidase is a peptide-N(4)- (N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), and wherein the PNGase: (a) comprises a signal peptide; and/or (b) lacks a PUB domain, a PAW domain, or both.
  • a glycosidase wherein the glycosidase is a peptide-N(4)- (N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), and wherein the PNGase: (a) lacks a signal peptide; and/or (b) lacks a PUB domain, a PAW domain, or both.
  • the glycosidase comprises a signal peptide.
  • the glycosidase lacks a signal peptide.
  • the glycosidase is secreted from a cell.
  • the glycosidase lacks a PUB domain.
  • the glycosidase lacks a PAW domain. In one embodiment, the glycosidase lacks both a PUB domain and a PAW domain. In one embodiment, the glycosidase comprises a signal peptide and lacks a PUB domain. In one embodiment, the glycosidase comprises a signal peptide and lacks a PAW domain. In a preferred embodiment, the glycosidase comprises a signal peptide and lacks a PUB domain and a PAW domain. In one embodiment, the glycosidase lacks a signal peptide and lacks a PUB domain. In one embodiment, the glycosidase lacks a signal peptide and lacks a PAW domain.
  • the glycosidase lacks a signal peptide and lacks a PUB domain and a PAW domain.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity to SEQ ID NO: 6, or a fragment thereof.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6, or a fragment thereof.
  • the glycosidase comprises a signal peptide and lacks a PUB domain.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity to SEQ ID NO: 7, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7, or a fragment thereof. In one embodiment, the glycosidase comprises a signal peptide and lacks a PAW domain. In one embodiment, the glycosidase comprises a signal peptide and lacks a PUB domain and a PAW domain.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity to SEQ ID NO: 8, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8, or a fragment thereof.
  • the glycosidase comprises or consists of: (a) a sequence that has at least 70% sequence identity to SEQ ID NO: 1, 4, 6, or 9, or a fragment thereof; (b) a sequence that has at least 70% sequence identity to SEQ ID NO: 2, 7, or 10, or a fragment thereof; (c) a sequence that has at least 70% sequence identity to SEQ ID NO: 3, 8, or 11 or a fragment thereof; or (d) a sequence that has at least 70% sequence identity to SEQ ID NO: 34 or a fragment thereof.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, 4, 6, or 9, or a fragment thereof.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, 7, or 10, or a fragment thereof.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3, 8, or 11, or a fragment thereof.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34, or a fragment thereof.
  • a polynucleotide comprising a nucleotide sequence encoding the glycosidase of the invention.
  • the glycosidase is encoded by a polynucleotide that comprises or consists of: (a) a sequence that has at least 70% sequence identity to SEQ ID NO: 5 or 12, or a fragment thereof; (b) a sequence that has at least 70% sequence identity to SEQ ID NO: 13, or a fragment thereof; or (c) a sequence that has at least 70% sequence identity to SEQ ID NO: 14 or a fragment thereof.
  • the glycosidase is encoded by a polynucleotide that comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5 or 12, or a fragment thereof.
  • the glycosidase is encoded by a polynucleotide that comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13, or a fragment thereof.
  • the glycosidase is encoded by a polynucleotide that comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14 or a fragment thereof.
  • a vector comprising the polynucleotide according to the invention.
  • the vector is a non-viral vector.
  • the vector is a nanoparticle.
  • the vector is a viral vector.
  • the vector is a lentiviral vector.
  • the lentiviral vector is a bidirectional lentiviral vector.
  • the vector is an adeno-associated viral (AAV) vector.
  • the polynucleotide or vector comprises a promoter that is operably linked to the nucleotide sequence encoding the glycosidase.
  • the promoter is a PGK promoter.
  • a cell comprising the polynucleotide or product, the vector, or the glycosidase according to the invention.
  • the cell is a eukaryotic cell, such as a mammalian cell.
  • the cell is selected from the group consisting of a rodent cell, such as a mouse or rat cell, a feline cell, a canine cell, and a human cell.
  • the cell is a human cell.
  • the cell is an immune cell.
  • the cell is a T cell.
  • the polynucleotide or product, the vector, the glycosidase, or the cell according to the invention for use in therapy.
  • the polynucleotide or product, the vector, the glycosidase, or the cell according to the invention for use in the treatment of cancer.
  • the cancer is a solid tumor.
  • the polynucleotide or product, the vector, the glycosidase, or the cell according to the invention for improving CAR cell activity.
  • a method of producing a CAR cell comprising introducing the polynucleotide or product, the vector, or the glycosidase according to the invention into a cell.
  • the therapeutic activity of the CAR cell or CAR-T cell is improved.
  • the therapeutic activity is target cell killing.
  • a method of treatment comprising producing a CAR cell according to the method of the invention, and administering the CAR cell to a subject in need thereof.
  • the subject is a human subject.
  • the subject has cancer.
  • the glycosidase of the invention may be used in combination with T cell receptors (TCRs), thus additional aspects of the invention relate to the use of the glycosidase with a TCR instead of the CAR as disclosed herein.
  • TCRs T cell receptors
  • a polynucleotide comprising (a) a nucleotide sequence encoding a glycosidase; and (b) a nucleotide sequence encoding a chimeric antigen receptor (CAR).
  • a product comprising (a) a first polynucleotide comprising a nucleotide sequence encoding a glycosidase; and (b) a second polynucleotide comprising a nucleotide sequence encoding a T cell receptor (TCR).
  • the product may be, for example, a kit or a composition.
  • a kit comprising (a) a first polynucleotide comprising a nucleotide sequence encoding a glycosidase; and (b) a second polynucleotide comprising a nucleotide sequence encoding a T cell receptor (TCR).
  • composition comprising (a) a first polynucleotide comprising a nucleotide sequence encoding a glycosidase; and (b) a second polynucleotide comprising a nucleotide sequence encoding a T cell receptor (TCR).
  • TCR T cell receptor
  • the cell of the invention may be a tumour-infiltrating lymphocyte (TIL).
  • TIL tumour-infiltrating lymphocyte
  • FIG. 1 Schematic representation of lentiviral vectors cloned to generate T3M-4 model cell lines expressing (a) wild-type PNGase (wtPNGase) or (b) secreted PNGase (sPNGase). (c) Flow- cytometry profile of PHA-L binding to T3M-4 transduced to express wtPNGase compared to control untransduced cells. (d) Flow-cytometry profile of PHA-L binding to T3M-4 transduced to express sPNGase compared to control untransduced cells.
  • FIGURE 2 T cells co-expressing 44v6.28z CAR and secreted PNGase reduce binding of PHA-L lectin to tumor cells.
  • FIGURE 3 Expansion and phenotype of 44v6.28z_sPNGase CAR-T cells.
  • FIGURE 444v6.28z_sPNGase CAR-T cells improve killing of T3M-4 pancreatic adenocarcinoma cells in co-culture.
  • (b) Killing of PC9 cells at different E:T ratios (n 1 donor and 3 technical replicates). Killing is expressed as Elimination Index with respect to control UT.
  • Glycosidases are enzymes that catalyse the hydrolysis of glycosidic bonds in sugars.
  • Glycosidases that act upon carbohydrates that are bound to proteins, e.g., as N- or O-linked glycans, may also be referred to herein as deglycosylating enzymes as they catalyse removal of glycans or sugar moieties.
  • Glycosidases are abundant and diverse and similarly have diverse functionality. For example, glycosidases may vary in size, structure, target specificity, catalytic activity, and the conditions under which they are catalytically active.
  • glycosidases may catalyse the removal of individual sugars at the termini of glycan structures, others catalyse the removal of entire glycans by the targeted hydrolysis of the inner-most sugar-protein bond. In either scenario, the enzymatic removal of sugars may be considered to be deglycosylation.
  • Glycosidases function endogenously in diverse biological processes, and in different cellular compartments, under different conditions.
  • the glycosidase of the invention is functional, i.e., enzymatically active, in the extracellular space.
  • such an enzyme will be able to deglycosylate proteins in the extracellular environment, such as those exposed on the surface of cells, e.g., tumor cells.
  • enzymes may function over a range of conditions, e.g., pH, with varying activity.
  • An enzyme according to the invention may be used under non-optimal conditions and yet still retain functionality, i.e., an enzyme does not need to be optimally functioning to still be functional and retain the activity of deglycosylation.
  • the glycosidase is functional in the extracellular environment. Functionality in the extracellular environment may be a natural property of the enzyme, or it may be introduced e.g., by mutation, to an enzyme, for example constituting a variant.
  • Glycosidases that are functional in the extracellular environment may catalyze the removal of glycans (or “act upon” glycans) from diverse substrates.
  • the glycosidase deglycosylates glycoproteins or glycopeptides.
  • the glycosidase deglycosylates cell-surface displayed glycoproteins or glycopeptides.
  • the glycosidase has a substrate that is an N-linked glycan.
  • the glycosidase deglycosylates N-linked glycans.
  • the glycosidase is an N-glycanase.
  • the glycosidase has a substrate that is an O-linked glycan. In one embodiment, the glycosidase deglycosylates O-linked glycans. In one embodiment, the glycosidase is an O-glycanase. In one embodiment, the glycosidase catalyzes the removal of one or more sugar moieties. In one embodiment, the glycosidase catalyzes the removal of an entire glycan chain. As used herein, “a glycan” may refer to an entire glycan structure, or fragments of said structure, such as individual sugars (mono-, di-, poly-saccharides).
  • Exemplary glycosidases An exemplary glycosidase is peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), such as human PNGase. Further, modified versions of human PNGase described herein may also be considered exemplary and may be used in the present invention.
  • the glycosidase is PNGase. In one embodiment, the glycosidase is human PNGase.
  • Exemplary human PNGase [Uniprot (Q96IV0-1)] (SEQ ID NO: 1): AAAALGSSSGSASPAVAELCQNTPETFLEASKLLLTYADNILRNPNDEKYRSIRIGNT AFSTRLLPVRGAVECLFEMGFEEGETHLIFPKKASVEQLQKIRDLIAIERSSRLDGSN KSHKVKSSQQPAASTQLPTTPSSNPSGLNQHTRNRQGQSSDPPSASTVAADSAILE VLQSNIQHVLVYENPALQEKALACIPVQELKRKSQEKLSRARKLDKGINISDEDFLLL ELLHWFKEEFFHWVNNVLCSKCGGQTRSRDRSLLPSDDELKWGAKEVEDHYCDA CQFSNRFPRYNNPEKLLETRCGRCGEWANCFTLCCRAVGFEARYVWDYTDHVWT EVYSPSQQRWLHCDACEDVCDKPLLYEIGWGKKLSYVIAFSKDEVVDVTW
  • PNGase hydrolyses the b-aspartylglucosaminyl bond between the core-chitobiose region of an N-linked glycan and an asparagine (Asn) residue, converting Asn to aspartate (Asp), which results in the release of glycan moieties from glycoproteins or glycopeptides.
  • Asn asparagine
  • Asp aspartate
  • the peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase) is functional at neutral pH.
  • the glycosidase hydrolyses a b-aspartylglucosaminyl bond between the core-chitobiose region of an N-linked glycan and an asparagine (Asn) residue.
  • the glycosidase is peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), or a variant thereof.
  • the glycosidase is human peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), or a variant thereof.
  • the PNGase variant is a truncated PNGase. In one embodiment, the PNGase lacks a PUB domain. In another embodiment the PNGase lacks a PAW domain. In a further embodiment, the PNGase lacks or both a PUB and a PAW domain.
  • Exemplary PUB truncated human PNGase (SEQ ID NO: 2): KASVEQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQPAASTQLPTTPSSNPSGLNQH TRNRQGQSSDPPSASTVAADSAILEVLQSNIQHVLVYENPALQEKALACIPVQELKR KSQEKLSRARKLDKGINISDEDFLLLELLHWFKEEFFHWVNNVLCSKCGGQTRSRD RSLLPSDDELKWGAKEVEDHYCDACQFSNRFPRYNNPEKLLETRCGRCGEWANC FTLCCRAVGFEARYVWDYTDHVWTEVYSPSQQRWLHCDACEDVCDKPLLYEIGW GKKLSYVIAFSKDEVVDVTWRYSCKHEEVIARRTKVKEALLRDTINGLNKQRQLFLS ENRRKELLQRIIVELVEFISPKTPKPGELGGRISGSVAWRVARGEMGLQRKETLFI
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, or a fragment thereof.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, or a fragment thereof.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3, or a fragment thereof.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34, or a fragment thereof.
  • the polypeptides of the foregoing may be combined with other sequence features.
  • the glycosidases of the invention may comprise a signal peptide sequence.
  • the glycosidases of the invention may comprise a signal peptide and a tag.
  • Further exemplary glycosidase sequences are set out below: Exemplary human PNGase with CD8 signal peptide (SEQ ID NO: 4): MALPVTALLLPLALLLHAARPAAAALGSSSGSASPAVAELCQNTPETFLEASKLLLT YADNILRNPNDEKYRSIRIGNTAFSTRLLPVRGAVECLFEMGFEEGETHLIFPKKASV EQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQPAASTQLPTTPSSNPSGLNQHTRNR QGQSSDPPSASTVAADSAILEVLQSNIQHVLVYENPALQEKALACIPVQELKRKSQE KLSRARKLDKGINISDEDFLLLELLHWFKEEFFHWVNNVLCSKCGGQTRSRDRSLLP SDDELKWGAKE
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4, 6 or 9, or a fragment thereof.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7 or 10, or a fragment thereof.
  • the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8 or 11, or a fragment thereof.
  • the glycosidase is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 5, 12, 13, and/or 14, or a fragment thereof.
  • the glycosidase is encoded by a polynucleotide sequence comprising or consisting of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one or more of SEQ ID NO: 5, 12, 13, and/or 14, or a fragment thereof.
  • Chimeric antigen receptors (CAR) “Chimeric antigen receptor” or "CAR” or “CARs” as used herein refers to engineered receptors which can confer an antigen specificity onto cells (for example T cells such as naive T cells, central memory T cells, effector memory T cells or combinations thereof).
  • CARs are also known as artificial T-cell receptors, chimeric T-cell receptors or chimeric immunoreceptors.
  • the CARs of the invention comprise an antigen-specific targeting region, an extracellular domain, a transmembrane domain, optionally one or more co-stimulatory domains, and an intracellular signaling domain.
  • Antigen-specific targeting domain The antigen-specific targeting domain provides the CAR with the ability to bind to the target antigen of interest.
  • the antigen-specific targeting domain preferably targets an antigen of clinical interest against which it would be desirable to trigger an effector immune response that results in cell killing.
  • the antigen-specific targeting domain may be any protein or peptide that possesses the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or tumor protein, or a component thereof).
  • the antigen-specific targeting domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest.
  • Illustrative antigen-specific targeting domains include antibodies or antibody fragments or derivatives, extracellular domains of receptors, ligands for cell surface molecules/receptors, or receptor binding domains thereof, and tumor binding proteins.
  • the antigen-specific targeting domain is, or is derived from, an antibody.
  • An antibody-derived targeting domain can be a fragment of an antibody or a genetically engineered product of one or more fragments of the antibody, which fragment is involved in binding with the antigen.
  • examples include a variable region (Fv), a complementarity determining region (CDR), a Fab, a single chain antibody (scFv), a heavy chain variable region (VH), a light chain variable region (VL) and a camelid antibody (VHH).
  • the binding domain is a single chain antibody (scFv).
  • the scFv may be murine, human or humanized scFv.
  • CDR complementarity determining region
  • the heavy chain variable region and the light chain variable region each contain 3 CDRs.
  • Heavy chain variable region or “VH” refers to the fragment of the heavy chain of an antibody that contains three CDRs interposed between flanking stretches known as framework regions, which are more highly conserved than the CDRs and form a scaffold to support the CDRs.
  • Light chain variable region or “VL” refers to the fragment of the light chain of an antibody that contains three CDRs interposed between framework regions.
  • Fv refers to the smallest fragment of an antibody to bear the complete antigen binding site.
  • An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.
  • Single-chain Fv antibody or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence.
  • Antibodies that specifically bind a tumor cell surface molecule can be prepared using methods well known in the art.
  • Such methods include phage display, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce human antibodies.
  • Phage display libraries of partially or fully synthetic antibodies are available and can be screened for an antibody or fragment thereof that can bind to the target molecule.
  • Phage display libraries of human antibodies are also available. Once identified, the amino acid sequence or polynucleotide sequence coding for the antibody can be isolated and/or determined.
  • antigens which may be targeted by the CAR of the invention include but are not limited to antigens expressed on cancer cells and antigens expressed on cells associated with various hematologic diseases, autoimmune diseases, inflammatory diseases and infectious diseases.
  • the selection of the targeting domain will depend on the type of cancer to be treated, and may target tumor antigens.
  • a tumor sample from a subject may be characterized for the presence of certain biomarkers or cell surface markers.
  • breast cancer cells from a subject may be positive or negative for each of Her2Neu, Estrogen receptor, and/or the Progesterone receptor.
  • a tumor antigen or cell surface molecule is selected that is found on the individual subject's tumor cells.
  • the antigen-specific targeting domain targets a cell surface molecule that is found on tumor cells and is not substantially found on normal tissues, or restricted in its expression to non-vital normal tissues.
  • antigens specific for cancer which may be targeted by the CAR of the invention include but are not limited to any one or more of carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, ROR1, mesothelin, c-Met, GD-2, and MAGE A3 TCR, 4-1BB, 5T4, adenocarcinoma antigen, alpha- fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), CCR4, CD152, CD200, CD22, CD19, CD22, CD123, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44, CD44 v6, CD51, CD52, CD56, CD74, CD80, CS-1, CEA, CNT0888, CTLA-4, DR5, EGFR
  • antigens specific for cancer which may be targeted by a CAR include but are not limited to any one or more of mesothelin, EGFRvIII, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-l, CD33, GD2, GD3, BCMA, Tn Ag, prostate specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-l3Ra2, interleukin-11 receptor a (IL-l lRa), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor- beta (PDGFR-beta), SSEA-4, CD20, Folate receptor alpha (FRa), ERBB2 (Her2/neu), MUC1, epidermal growth factor receptor (EGFR), NCAM, Prostase, PAP, EFF2M, Ephrin B2, IGF-I receptor, CAIX
  • Antigens specific for inflammatory diseases which may be targeted by the CAR of the invention include but are not limited to any one or more of AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin- 1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (a chain of IL-2 receptor), CD3, CD4, CD5, IFN- ⁇ , IFN- ⁇ , IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin ⁇ 4, integrin ⁇ 4 ⁇ 7, Lama glama, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAb ⁇ 7, scleroscin, SOST, TGF ⁇ 1, TNF-a or VEGF-A.
  • Antigens specific for neuronal disorders which may be targeted by the CAR of the invention include but are not limited to any one or more of beta amyloid or MABT5102A. Further antigens which may be targeted by the CAR of the invention include but are not limited to any one or more of IL-1 ⁇ or CD3. Antigens specific for cardiovascular diseases which may be targeted by the CARs of the invention include but are not limited to any one or more of C5, cardiac myosin, CD41 (integrin alpha-IIb), fibrin II, beta chain, ITGB2 (CD18) and sphingosine-1-phosphate.
  • antigens which may be targeted by the CARs of the invention include but are not limited to Claudin18.2, GRP78, AFP peptide/A2, CD70, CD133, CD147, cMet, DLL3, EGFR806, FBP, ICAM1, MG7, p32, CS1 (SLAMF7 or CD319), CXCR5, CD318, TSPAN8, CD66c, CD229, LMP1, CD276, CD138, AXL, CD147, CLDN6, DLL3, DR5, gp100, LeY, MMP2, MUC16, MUC16ecto, NECTIN4, NKG2D, NKG2DL, ROR2, TM4SF1, TnMUC1, CD7, CD99, TRBC1, CCR9, Siglec-6, CD229, APRIL.
  • the antigen-specific binding domain specifically binds to a tumor antigen.
  • the polynucleotide codes for a single chain Fv that specifically binds CD44v6.
  • the polynucleotide codes for a single chain Fv that specifically binds CEA.
  • Co-stimulatory domain The CAR of the invention may also comprise one or more co-stimulatory domains. This domain may enhance cell proliferation, cell survival and development of memory cells.
  • Each co-stimulatory domain comprises the co-stimulatory domain of any one or more of, for example, members of the TNFR super family, CD28, CD137 (4-1BB), CD134 (OX40), DaplO, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-1, TNFR-II, Fas, CD30, CD40 or combinations thereof.
  • Co-stimulatory domains from other proteins may also be used with the CAR of the invention. Additional co-stimulatory domains will be apparent to those of skill in the art.
  • the co-stimulatory domain is a CD28 co-stimulatory domain.
  • Intracellular signaling domain The CAR of the invention may also comprise an intracellular signaling domain.
  • This domain may be cytoplasmic and may transduce the effector function signal and direct the cell to perform its specialized function.
  • intracellular signaling domains include, but are not limited to, ⁇ chain of the T-cell receptor or any of its homologs (e.g., ⁇ chain, Fc ⁇ R1 ⁇ and ⁇ chains, MB1 (Ig ⁇ ) chain, B29 (Ig ⁇ ) chain, etc.), CD3 polypeptides ( ⁇ , ⁇ and ⁇ ), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28.
  • ⁇ chain of the T-cell receptor or any of its homologs e.g., ⁇ chain, Fc ⁇ R1 ⁇ and ⁇ chains, MB1 (Ig ⁇ ) chain, B29 (Ig ⁇ ) chain, etc.
  • the intracellular signaling domain may be human CD3 zeta chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, immunoreceptor tyrosine- based activation motif (ITAM) bearing cytoplasmic receptors or combinations thereof. Additional intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.
  • Transmembrane domain The CAR of the invention may also comprise a transmembrane domain.
  • the transmembrane domain may comprise the transmembrane sequence from any protein which has a transmembrane domain, including any of the type I, type II or type III transmembrane proteins.
  • the transmembrane domain of the CAR of the invention may also comprise an artificial hydrophobic sequence.
  • the transmembrane domains of the CARs of the invention may be selected so as not to dimerize. Additional transmembrane domains will be apparent to those of skill in the art.
  • transmembrane (TM) regions used in CAR constructs are: 1) The CD28 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41; Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35; Casucci et al, Blood, 2013, Nov 14;122(20):3461-72.); 2) The OX40 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41); 3) The 41BB TM region (Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35); 4) The CD3 zeta TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41; Savoldo B, Blood, 2009, Jun 18;113(25):6392-402.); 5) The CD8a TM region (Maher et al, Nat
  • the transmembrane domain is a CD28 transmembrane domain.
  • Spacer domain The CAR of the invention may comprise an extracellular spacer domain.
  • the extracellular spacer domain may be attached to the antigen-specific targeting region and the transmembrane domain.
  • the spacer is an IgG1-derived hinge spacer.
  • the CAR of the present invention may comprise an extracellular spacer which comprises at least part of the extracellular domain of human low affinity nerve growth factor (LNGFR) or a derivative thereof. LNGFR is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of transduced gene expression by immunofluorescence, with single cell resolution.
  • LNGFR human low affinity nerve growth factor
  • the CAR of the invention comprises a truncated LNGFR (also known as ⁇ LNGFR).
  • LNGFR also known as ⁇ LNGFR
  • the LNGFR used in the present invention is truncated in its intracytoplasmic domain.
  • the LNGFR spacer of the present invention comprises at least part of the extracellular domain or a derivative thereof but lacks the intracellular domain of LNGFR.
  • the extracellular domain may comprise amino acids 29 – 250 of LNGFR or a derivative thereof.
  • Exemplary human LNGFR [UNIPROT accession P08138, TNR16_HUMAN] (SEQ ID NO: 15): MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQ PCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCA YGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPC LPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPE QDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKRWNS CKQNKQGANSRPVNQTPPPEGEKLHSDSGISVDSQSLHDQQPHTQTASGQALKG DGGLYSSLPPAKREEVEKLLNGSAGDTWRHLAGELGYQPEHIDSFTHEACPVRALL A
  • the spacer comprises at least part of a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the extracellular domain of LNGFR (e.g., SEQ ID NO: 16). In one embodiment, the spacer comprises at least part of a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to amino acids 29-250 of the LNGFR protein (e.g., SEQ ID NO: 15). In one embodiment, the spacer comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 16.
  • the spacer comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to amino acids 29-250 of SEQ ID NO: 15.
  • Exemplary LNGFR spacer (SEQ ID NO: 31) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPC KPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFS CQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEE
  • Exemplary LNGFR spacer (SEQ ID NO: 32) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPC KPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFS CQDKQNTVCEECPDGTYSDEAARAADAECEEIPGR
  • LNGFR comprises 4 TNFR-Cys domains (TNFR-Cys 1, TNFR-Cys 2, TNFR-Cys 3 and TNFR-Cys 4). Sequences of the domains are exemplified below: TNFR-Cys 1 (SEQ ID NO: 17) ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC TNFR-Cys 2 (SEQ ID NO: 18) PCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVC TNFR-Cys 3 (SEQ ID NO: 19) RCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVC TNFR-Cys 4 (SEQ ID NO: 20) ECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAEC
  • the spacer comprises TNFR-Cys 1, 2 and 3 domains or fragments or derivatives thereof.
  • the spacer comprises the TNFR-Cys 1, 2, 3 and 4 domains or fragments or derivatives thereof.
  • the spacer comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or 100% identity to TNFR-Cys 1 (SEQ ID NO: 17), a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or 100% identity to TNFR- Cys 2 (SEQ ID NO: 18), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or 100% identity to TNFR-Cys 3 (SEQ ID NO: 19).
  • the spacer may further comprise a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or 100% identity to TNFR-Cys 4 (SEQ ID NO: 20). Rather than comprise the full TNFR-Cys 4 domain, the spacer may comprise a TNFR-Cys 4 domain with the following amino acids deleted from said domain: NHVDPCLPCTVCEDTERQLRECTRW (SEQ ID NO: 21). In one embodiment, the NHVDPCLPCTVCEDTERQLRECTRW amino acids are replaced with the following amino acids: ARA. In one embodiment the spacer lacks the LNGFR serine/threonine-rich stalk.
  • the spacer comprises the LNGFR serine/threonine-rich stalk.
  • the spacer may comprise or consist of a sequence of SEQ ID NO: 17 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 17.
  • the spacer may comprise or consist of a sequence of SEQ ID NO: 18 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 18.
  • the spacer may comprise or consist of a sequence of SEQ ID NO: 19 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 19.
  • the spacer may comprise or consist of a sequence of SEQ ID NO: 20 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 20.
  • the spacer may comprise or consist of a sequence of SEQ ID NO: 31 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 31.
  • the spacer may comprise or consist of a sequence of SEQ ID NO: 32 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 32.
  • the spacer may comprise or consist of a sequence of SEQ ID NO: 33 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 33.
  • the spacer may confer properties to the CAR such that it allows for immunoselection of cells, preferably T-cells, expressing said CAR.
  • the CAR of the present invention e.g. comprising the spacer referred to herein
  • the CAR of the present invention e.g.
  • the spacer referred to herein preferably enables T-cells expressing the CAR to mediate therapeutically significant anti-cancer effects against a cancer that the CAR is designed to target.
  • the CAR of the present invention e.g. comprising the spacer referred to herein
  • An exemplary CAR of the present invention comprising the LNGFR-based spacer may avoid activation of unwanted and potentially toxic off-target immune responses and may allow CAR-expressing T cells to persist in vivo without being prematurely cleared by the host immune system.
  • the present invention also encompasses the use of variants, derivatives, homologues and fragments of the spacer elements described herein.
  • the CAR is an anti-CD44v6 CAR (44v6.28z).
  • the CAR comprises an IgG1-derived hinge spacer, a CD28 transmembrane and costimulatory domain and a CD3 ⁇ endodomain Exemplary 44v6.28z CAR protein sequence (SEQ ID NO: 22): MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCSASSSINYIYWLQQK PGQAPRILIYLTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLT FGGGTKVEIKRGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYD MSWVRQAPGKGLEWVSTISSGGSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAE DTAVYYCARQGLDYWGRGTLVTVSSGPVEPKSCDKTHTCPPCPPLIKFWVLVVVG GV
  • the CAR is encoded by a polynucleotide sequence comprising or consisting of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23, or a fragment thereof.
  • Signal peptides A signal peptide is a short peptide that functions in protein targeting and translocation. Signal peptides are co-translated as part of a longer polypeptide, which they direct the targeting of. Signal peptides may direct a polypeptide towards a secretory pathway, i.e., for secretion, or via another intracellular pathway for additional processing.
  • Signal peptides may also be referred to as a signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide.
  • Signal peptides may be grafted onto polypeptides with which they are not naturally associated, in order to direct the new modified peptide to a specific cellular compartment, or along a desired pathway, e.g., a secretory pathway.
  • grafted it is meant that a signal peptide may form a fusion protein with any one or more polypeptide.
  • signal peptides may be combined with glycosidases according to the invention.
  • Glycosidases of the invention may comprise a signal peptide, such as an heterologous signal peptide.
  • the signal peptide directs the secretion of the glycosidase.
  • the signal peptide and glycosidase comprise a fusion protein.
  • a signal peptide may be cleaved, e.g., by a signal peptidase, from the polypeptide of which it is a component.
  • the signal peptide and glycosidase may transiently comprise a fusion protein.
  • the signal peptide is cleaved from the glycosidase in a cell.
  • the glycosidase is secreted from a cell following cleavage of the signal peptide.
  • the signal peptide may be selected from the group consisting of: a CD8a signal peptide, an IgG variable region heavy chain signal peptide, a GM- CSF/CSF signal peptide, an Ig kappa chain V-III region VG signal peptide and a CSFR2A signal peptide.
  • the signal peptide is a CD8 signal peptide.
  • the CD8 signal peptide may also be referred to as CD8a.
  • the signal peptide comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24, or a fragment thereof.
  • the signal peptide comprises SEQ ID NO: 24.
  • the signal peptide consists of SEQ ID NO: 24.
  • the signal peptide is a IgG variable heavy signal peptide.
  • the signal peptide comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 25, or a fragment thereof.
  • the signal peptide comprises SEQ ID NO: 25.
  • the signal peptide consists of SEQ ID NO: 25.
  • the signal peptide is a GM-CFS/CSF signal peptide.
  • Exemplary GM-CFS/CSF signal peptide [residues 1 – 17; Uniprot accession P04141] (SEQ ID NO: 26): MWLQSLLLLGTVACSIS
  • the signal peptide comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 26, or a fragment thereof.
  • the signal peptide comprises SEQ ID NO: 26.
  • the signal peptide consists of SEQ ID NO: 26.
  • the signal peptide is an Ig kappa chain V-III region VG signal peptide.
  • Exemplary Ig kappa chain V-III region VG signal peptide [residues 1 – 20; Uniprot accession P04433] (SEQ ID NO: 27): MEAPAQLLFLLLLWLPDTTG
  • the signal peptide comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 27, or a fragment thereof.
  • the signal peptide comprises SEQ ID NO: 27. In one embodiment, the signal peptide consists of SEQ ID NO: 27. In one embodiment, the signal peptide is a CSFR2A signal peptide. Exemplary CSFR2A signal peptide [residues 1 – 22; NCBI ref.
  • the signal peptide comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 28, or a fragment thereof.
  • the signal peptide comprises SEQ ID NO: 28.
  • the signal peptide consists of SEQ ID NO: 28.
  • Tags and linkers The polynucleotide and polypeptide sequences of the invention may further comprise tag and/or linker sequences.
  • Linkers At both the polynucleotide and polypeptide levels, certain functional sequence elements may be separated by linkers.
  • Linkers typically comprise a short polynucleotide or polypeptide sequence. Linkers may be used to physically separate functional sequences in order, e.g., to improve the functionality of said sequences.
  • the polynucleotide of the invention comprises one or more linker sequences.
  • the sequence encoding the glycosidase is separated from another functional sequence by a linker.
  • the glycosidase of the invention comprises one or more linker sequences.
  • the linker is a GS linker, or a GGS linker.
  • Tags Both the polynucleotides and polypeptides of the invention may comprise tags. Tags are typically sequences that facilitate the detection or isolation of the molecule to which they are attached. Tags may be particularly useful in experimental studies utilising the polynucleotides or polypeptides of the invention.
  • the polynucleotide of the invention comprises one or more tag sequences.
  • the glycosidase of the invention comprises one or more tag sequences.
  • the tag is a MYC tag.
  • the tag comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 29 or 30, or a fragment thereof.
  • the tag comprises SEQ ID NO: 29 or 30.
  • the tag consists of SEQ ID NO: 29 or 30.
  • Expression control sequences The polynucleotide of the invention may comprise one or more expression control sequence.
  • the nucleic acid sequence encoding the glycosidase or CAR is operably linked to one or more expression control sequence.
  • an “expression control sequence” may refer to a nucleotide sequence which controls expression of a transgene, e.g. to facilitate and/or increase expression.
  • the expression control sequence and the transgene may be in any suitable arrangement in the polynucleotide, providing that the expression control sequence is operably linked to the transgene (e.g. nucleic acid sequence encoding the glycosidase or CAR).
  • Promoters In some embodiments, the expression control sequence is a promoter. Any suitable promoter may be used, the selection of which may be readily made by the skilled person.
  • the promoter sequence may be constitutively active (i.e. operational in any host cell background), or alternatively may be active only in a specific host cell environment, thus allowing for targeted expression of the nucleotide of interest (e.g. glycosidase and/or CAR) in a particular cell type (e.g. a tissue-specific promoter).
  • the promoter may show inducible expression in response to presence of another factor, for example a factor present in a host cell.
  • the promoter should be functional in the target cell background.
  • the polynucleotide further comprises a promoter operably linked to the nucleic acid sequence encoding the glycosidase.
  • the polynucleotide further comprises a promoter operably linked to the nucleic acid sequence encoding the CAR. In some embodiments, the polynucleotide further comprises a promoter operably linked to the nucleic acid sequence encoding the glycosidase and the nucleic acid sequence encoding the CAR. In some embodiments, the promoter is a constitutive promoter. In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR are operably linked to one or more promoter(s). In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR are operably linked to the same promoter(s).
  • the nucleotide sequences encoding the glycosidase and the CAR may share a promoter such that their expression may be regulated by a single regulatory sequence.
  • the nucleotide sequences encoding the glycosidase and the CAR are independently operably linked to one or more promoter(s).
  • the nucleotide sequences encoding the glycosidase and the CAR may each be operably linked to separate promoter such that their expression may be independently regulated by independent regulatory sequences.
  • the nucleotide sequences encoding the glycosidase and the CAR are operably linked to separate promoter(s).
  • the glycosidase and the CAR are encoded in opposing directions.
  • the glycosidase and the CAR are encoded in opposing directions and are independently operably linked to separate promoters. In one embodiment, the glycosidase and the CAR are encoded in the same direction.
  • the promoter is selected from the group consisting of: a cytomegalovirus promoter (CMV), a human phosphoglycerate kinase promoter (PGK), an EF-1 ⁇ promoter and an inducible NFAT promoter. In one embodiment, the promoter is a cytomegalovirus (CMV) promoter.
  • the promoter is a minimal cytomegalovirus (mCMV) promoter (mCMV, see, for example, Amendola (2005) Nat Biotech 23: 108-116).
  • mCMV minimal cytomegalovirus
  • the promoter is human phosphoglycerate kinase (PGK) promoter.
  • PGK human phosphoglycerate kinase
  • the promoter is an EF-1 ⁇ promoter.
  • the promoter is an inducible NFAT promoter.
  • the inducible module may be composed of a synthetic NFAT response element usually comprising repetitions of the consensus NFAT binding site placed upstream of a minimal promoter.
  • Proteins as used herein includes single-chain polypeptide molecules as well as multiple-polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means.
  • polypeptide and peptide as used herein refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds.
  • protein is also intended to encompass glycoproteins or glycopeptides, for example, glycoproteins/peptides, that are the target of the glycosidases herein.
  • the proteins of the invention include any of the proteins disclosed herein with a methionine at the N-terminus.
  • Polynucleotides of the invention may, for example, comprise DNA or RNA. They may be single-stranded or double-stranded. It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that the skilled person may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed. The polynucleotides may be modified by any method available in the art.
  • Polynucleotides such as DNA polynucleotides may be produced recombinantly, synthetically or by any means available to the skilled person. They may also be cloned by standard techniques. Longer polynucleotides will generally be produced using recombinant means, for example using polymerase chain reaction (PCR) cloning techniques. This may involve making a pair of primers (e.g.
  • a vector is a tool that allows or facilitates the transfer of an entity from one environment to another.
  • some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g. a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell.
  • the vector may serve the purpose of maintaining the heterologous nucleic acid (DNA or RNA) within the cell, facilitating the replication of the vector comprising a segment of nucleic acid or facilitating the expression of the protein encoded by a segment of nucleic acid.
  • Vectors may be non-viral or viral. Examples of vectors used in recombinant nucleic acid techniques include, but are not limited to, plasmids, mRNA molecules (e.g.
  • the vector may also be, for example, a naked nucleic acid (e.g. DNA). In its simplest form, the vector may itself be a nucleotide of interest.
  • the vectors used in the invention may be, for example, plasmid, mRNA or virus vectors and may include a promoter for the expression of a polynucleotide and optionally a regulator of the promoter. Vectors comprising polynucleotides used in the invention may be introduced into cells using a variety of techniques known in the art, such as transfection, transformation and transduction.
  • Non-viral delivery systems include but are not limited to DNA transfection methods.
  • transfection includes a process using a non-viral vector to deliver a gene to a target cell.
  • Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14: 556) and combinations thereof.
  • Transfection of cells with mRNA vectors can be achieved, for example, using nanoparticles, such as liposomes.
  • the vector is comprised in a nanoparticle.
  • the nanoparticle is a polymeric nanoparticle, inorganic nanoparticle or lipid nanoparticle.
  • the nanoparticle is a liposome.
  • the nanoparticle may be targeted to a specific cell type(s) using one or more ligand displayed on its surface.
  • polynucleotide delivery is transposon mediated.
  • the polynucleotide is an mRNA.
  • the mRNA may be comprised in a nanoparticle.
  • Viral vectors In preferred embodiments, the vector is a viral vector.
  • the viral vector may be in the form of a viral vector particle.
  • the viral vector may be, for example, a retroviral, lentiviral, adeno-associated viral (AAV) or adenoviral vector.
  • the vector is a lentiviral vector.
  • the vector is an AAV vector.
  • Retroviral and lentiviral vectors A retroviral vector may be derived from or may be derivable from any suitable retrovirus.
  • retroviruses include murine leukaemia virus (MLV), human T-cell leukaemia virus (HTLV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukaemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29) and avian erythroblastosis virus (AEV).
  • MMV murine leukaemia virus
  • HTLV human T-cell leukaemia virus
  • MMTV mouse mammary tumour virus
  • RSV Rous sarcoma virus
  • Retroviruses may be broadly divided into two categories, “simple” and “complex”. Retroviruses may be even further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses. A review of these retroviruses is presented in Coffin et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63. The basic structure of retrovirus and lentivirus genomes share many common features such as a 5’ LTR and a 3’ LTR.
  • a packaging signal to enable the genome to be packaged
  • a primer binding site to enable integration into a host cell genome
  • gag, pol and env genes encoding the packaging components – these are polypeptides required for the assembly of viral particles.
  • Lentiviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell.
  • these genes are flanked at both ends by regions called long terminal repeats (LTRs).
  • LTRs are responsible for proviral integration and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes.
  • the LTRs themselves are identical sequences that can be divided into three elements: U3, R and U5.
  • U3 is derived from the sequence unique to the 3’ end of the RNA.
  • R is derived from a sequence repeated at both ends of the RNA.
  • U5 is derived from the sequence unique to the 5’ end of the RNA.
  • the sizes of the three elements can vary considerably among different retroviruses. In a defective retroviral vector genome gag, pol and env may be absent or not functional. In a typical retroviral vector, at least part of one or more protein coding regions essential for replication may be removed from the virus. This makes the viral vector replication-defective. Lentivirus vectors are part of the larger group of retroviral vectors.
  • lentiviruses can be divided into primate and non-primate groups.
  • primate lentiviruses include but are not limited to human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS); and simian immunodeficiency virus (SIV).
  • non-primate lentiviruses examples include the prototype “slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
  • VMV visna/maedi virus
  • CAEV caprine arthritis-encephalitis virus
  • EIAV equine infectious anaemia virus
  • FIV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • the lentivirus family differs from retroviruses in that lentiviruses have the capability to infect both dividing and non-dividing cells (Lewis et al. (1992) EMBO J. 11: 3053-8; Lewis et al. (1994) J. Virol.68: 510-6).
  • a lentiviral vector is a vector which comprises at least one component part derivable from a lentivirus. Preferably, that component part is involved in the biological mechanisms by which the vector infects cells, expresses genes or is replicated.
  • the lentiviral vector may be a “primate” vector.
  • the lentiviral vector may be a “non-primate” vector (i.e. derived from a virus which does not primarily infect primates, especially humans).
  • non-primate lentiviruses may be any member of the family of lentiviridae which does not naturally infect a primate.
  • the viral vector used in the present invention has a minimal viral genome.
  • minimal viral genome it is to be understood that the viral vector has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in WO 1998/017815.
  • the plasmid vector used to produce the viral genome within a host cell/packaging cell will have sufficient lentiviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle which is capable of infecting a target cell, but is incapable of independent replication to produce infectious viral particles within the final target cell.
  • the vector lacks a functional gag-pol and/or env gene and/or other genes essential for replication.
  • the plasmid vector used to produce the viral genome within a host cell/packaging cell will also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a host cell/packaging cell.
  • These regulatory sequences may be the natural sequences associated with the transcribed viral sequence (i.e. the 5’ U3 region), or they may be a heterologous promoter, such as another viral promoter (e.g. the CMV promoter).
  • the vectors may be self-inactivating (SIN) vectors in which the viral enhancer and promoter sequences have been deleted. SIN vectors can be generated and transduce non-dividing cells in vivo with an efficacy similar to that of wild-type vectors.
  • the transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus should prevent mobilisation by replication- competent virus. This should also enable the regulated expression of genes from internal promoters by eliminating any cis-acting effects of the LTR.
  • LTR long terminal repeat
  • the vectors may be integration-defective.
  • Integration defective lentiviral vectors can be produced, for example, either by packaging the vector with catalytically inactive integrase (such as an HIV integrase bearing the D64V mutation in the catalytic site) or by modifying or deleting essential att sequences from the vector LTR, or by a combination of the above.
  • Cells The invention provides a cell comprising the polynucleotide or product, the vector, or the glycosidase of the invention.
  • the cell of the invention may comprise any suitable cell type from any suitable organism or subject. In one embodiment the cell is a mammalian cell. In another embodiment, the cell is a human cell. In one embodiment, the cell is a cell from a subject.
  • the subject is a human subject.
  • the cell is a T cell.
  • the cell is a natural killer (NK) cell.
  • the cell is a hematopoietic stem cell (HSC).
  • the cell is a hematopoietic stem and/or progenitor cell (HSPC).
  • the cell is a tumor-infiltrating lymphocyte (TIL).
  • the cell is an invariant-NK T cell, a cytokine-induced killer cell (CIK) or a macrophage.
  • TILs are T cells that can be isolated from a tumor. TILs are enriched in natural T cells that recognize the tumor antigens.
  • Isolated TILs can be expanded and modified, such as transduced with a polynucleotide or vector according to the invention, ex vivo and re- introduced to a tumor or subject. Since TILs may naturally have specificity for tumor cells, they may not require a CAR for targeting. Thus, in one embodiment, the TIL does not comprise the CAR. In one embodiment, the TIL comprises the glycosidase. Variants, derivatives, analogues, homologues, and fragments In addition to the specific proteins and polynucleotides mentioned herein, the invention also encompasses the use of variants, derivatives, analogues, homologues and fragments thereof.
  • a variant of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions.
  • a variant sequence can be obtained by addition, deletion, substitution, modification, replacement and/or variation of at least one residue present in the naturally-occurring protein.
  • derivative as used herein in relation to proteins or polypeptides of the invention includes any substitution of, variation of, modification of, replacement of, deletion of and/or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide substantially retains at least one of its endogenous functions.
  • analogue as used herein in relation to polypeptides or polynucleotides includes any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics.
  • amino acid substitutions may be made, for example from 1, 2 or 3 to 10 or 20 substitutions provided that the modified sequence substantially retains the required activity or ability.
  • Amino acid substitutions may include the use of non-naturally occurring analogues. Proteins used in the invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues as long as the endogenous function is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.
  • Conservative substitutions may be made, for example according to the table below.
  • a homologous sequence may include an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95% or 97% or 99% identical to the subject sequence.
  • the homologues will comprise the same active sites etc. as the subject amino acid sequence.
  • a homologous sequence may include a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95% or 97% or 99% identical to the subject sequence.
  • homology can also be considered in terms of similarity, in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • reference to a sequence which has a percent identity to any one of the SEQ ID NOs disclosed herein refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to.
  • Homology comparisons can be conducted by eye or, more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percentage homology or identity between two or more sequences. Percentage homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum percentage homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties.
  • a suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al. (1984) Nucleic Acids Res.12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid – Ch.18), FASTA (Atschul et al. (1990) J. Mol. Biol.
  • BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program.
  • Another tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187-8). Although the final percentage homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix – the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • “Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full- length polypeptide or polynucleotide. Such variants may be prepared using standard recombinant DNA techniques such as site- directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5’ and 3’ flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made.
  • flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut.
  • the DNA is then expressed in accordance with the invention to make the encoded protein.
  • Codon optimisation The polynucleotides used in the invention may be codon-optimised. Codon optimisation has previously been described in WO 1999/41397 and WO 2001/79518. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type.
  • compositions The polynucleotides, proteins, vectors, and cells of the invention may be formulated for administration to subjects with a pharmaceutically-acceptable carrier, diluent or excipient. Suitable carriers and diluents include isotonic saline solutions, for example phosphate- buffered saline, and potentially contain human serum albumin.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, a surfactant, such as pluronic acid (PF68) 0.001% may be used. In some cases, serum albumin may be used in the composition.
  • PF68 pluronic acid
  • the active ingredient may be in the form of an aqueous solution, which is pyrogen-free, and has suitable pH, isotonicity and stability.
  • aqueous solution which is pyrogen-free, and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection or Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included as required.
  • the medicament may be included in a pharmaceutical composition which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art. Handling of the cell therapy products is preferably performed in compliance with FACT-JACIE International Standards for cellular therapy.
  • the invention provides the polynucleotide, product, vector, glycosidase, cell, composition or pharmaceutical composition of the invention for use in therapy.
  • the invention provides the polynucleotide, product, vector, glycosidase, cell, composition or pharmaceutical composition of the invention for use in the treatment of cancer.
  • All references herein to treatment include curative, palliative and prophylactic treatment.
  • the treatment of mammals, particularly humans, is preferred. Both human and veterinary treatments are within the scope of the invention.
  • the method of treatment provides the polynucleotide, product, vector, glycosidase, cell, composition or pharmaceutical composition of the invention to a tumor.
  • the polynucleotide or product, the vector, the glycosidase, or the cell according to the invention for use in therapy.
  • the polynucleotide or product, the vector, the glycosidase, or the cell according to the invention for use in the treatment of cancer.
  • the cancer is a solid tumor.
  • the cancer is a solid or haematopoietic or lymphoid tumor.
  • the cancer is a haematopoietic or lymphoid tumor.
  • the solid tumor is selected from the group consisting of: colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the group consisting
  • the haematopoietic or lymphoid tumor is selected from the group consisting of: chronic lymphocytic leukemia (CLL), acute leukemias, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitfs lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin’s lymphoma, Ho
  • the cancer is a neuroendocrine tumor.
  • a method of producing a CAR cell comprising introducing the polynucleotide or product, the vector, or the glycosidase into a cell.
  • the cell is a CAR-T cell.
  • the method or use wherein the therapeutic activity of the CAR cell or CAR-T cell is improved.
  • the therapeutic activity is target cell killing.
  • a method of treatment comprising producing a CAR cell according to the method of the invention, and administering the CAR cell to a subject in need thereof.
  • Administration the polynucleotide, product, vector, glycosidase, cell, composition or pharmaceutical composition is administered to a subject locally. Local administration may include administration to the tumor of interest. When a glycosidase is locally administered to a tumor, the glycosidase may lack a signal peptide.
  • the polynucleotide, product, vector, glycosidase, cell, composition or pharmaceutical composition is administered to a tumor.
  • the polynucleotide, product, vector, glycosidase, cell, composition or pharmaceutical composition is administered to a subject systemically.
  • systemic delivery or “systemic administration” as used herein means that the agent of the invention is administered into the circulatory system, for example to achieve broad distribution of the agent.
  • topical or local administration restricts the delivery of the agent to a localised area, e.g. a tumor.
  • the polynucleotide, product, vector, glycosidase, cell, composition or pharmaceutical composition is administered in a nanoparticle that targets T cells in vivo. Dosage The skilled person can readily determine an appropriate dose of an agent of the invention to administer to a subject.
  • a physician will determine the actual dosage that will be most suitable for an individual patient, which will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of the invention.
  • Subject refers to either a human or non-human animal. Examples of non-human animals include vertebrates, for example mammals, such as non- human primates (particularly higher primates), dogs, rodents (e.g.
  • mice, rats or guinea pigs), pigs and cats The non-human animal may be a companion animal. Preferably, the subject is a human.
  • the skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed. Preferred features and embodiments of the invention will now be described by way of non- limiting examples.
  • the practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T.
  • T cells were derived from peripheral blood of healthy donors after gradient centrifugation. All procedures were approved by the Institutional Review Board of IRCCS San Raffaele Scientific Institute and were compliant with all relevant ethical regulations. T cells were activated with CD3/CD28 beads (Gibco, 40203D) at a 3:1 ratio, transduced at day 2 and cultured in RPMI- 1640 with interleukin (IL)-7 and IL-15 (5 ng/ml, Peprotech, 200-07, 200-15). At day 6, beads were removed and CAR T cells were expanded in complete medium until Day 21.
  • CD3/CD28 beads Gibco, 40203D
  • IL-7 and IL-15 5 ng/ml, Peprotech, 200-07, 200-15
  • Tumor pancreatic cell line (T3M-4) and pulmonary adenocarcinoma cell line (PC9) were cultured in RPMI 1640 (Euroclone, ECB90062L) and detached with TrypLE Express enzyme (Gibco).
  • 293T cells were employed for virus production and kept in Iscove’s Modified Dulbecco’s Medium (IMDM, Euroclone, ECB2072L). 293T cells were detached using trypsin-EDTA (Euroclone).
  • PNGase constructs The construct “wtPNGase” was generated by cloning the wild-type PNGase sequence (UniProt Q96IV0-1) into a bidirectional lentiviral vector.
  • wtPNGase was produced by GeneArt (Thermo Fisher) and cloned under the direct control of the human phosphoglycerate kinase promoter (PGK), whereas the GFP marker gene was placed under the control of a minimal core promoter derived from the cytomegalovirus (minCMV).
  • the construct “sPNGase” was generated by adding the CD8 (UniProt P01732) leader sequence (signal peptide) at the N-terminus of the wtPNGase and cloned into the bidirectional lentiviral vector as previously described for wtPNGase.
  • CAR constructs 44v6.28 ⁇ was generated by cloning the antigen-specific single chain fragment variable (scFv, BIWA-8 mAb) in frame into an original CAR incorporating an IgG1-derived hinge spacer, a CD28 transmembrane and costimulatory domain and a CD3 ⁇ endodomain (Savoldo (2011) J Clin Invest 121: 1822-1826).
  • CAR cDNA was produced by GeneArt (Thermo Fisher) and cloned into a bidirectional lentiviral vector.
  • CAR constructs were placed under the direct control of the human phosphoglycerate kinase promoter (PGK) in place of the ⁇ NGFR marker gene, whereas ⁇ NGFR was substituted to GFP under the control of a minimal core promoter derived from the cytomegalovirus (minCMV) (Amendola (2005) Nat Biotech 23: 108- 116).
  • Viral supernatants were produced in 293T packaging cells. 44v6.28 ⁇ _sPNGase was generated by cloning the secreted PNGase sequence (sPNGase) into the bidirectional lentiviral vector platform under the control of minCMV promoter.
  • CAR T cells were co-cultured with target cells at different effector:target (E:T) ratios in RPMI- 1640 fully supplemented in the absence of cytokines. After 24 hours, supernatants were collected and analyzed with the LEGENDplex bead-based cytokine immunoassay (BioLegend, 740724). After 4 days (tumors) or 3 days (primary keratinocytes), surviving cells were counted using Flow-Count Fluorospheres (Beckman Coulter, 7547053) and analyzed by flow cytometry. T cells that were untransduced or transduced with an irrelevant CAR (19.28 ⁇ ) were used as a control.
  • E:T effector:target
  • Elimination index was calculated as follows: 1 – (number of residual target cells with experimental CAR T cells / number of residual target cells with control T cells). Flow cytometry Samples were washed with phosphate-buffered saline (PBS) containing 1% fetal bovine serum (FBS) and stained at 4°C for 20min. Prior to use, all antibodies were validated and titrated for the optimal on target/off target activity on human peripheral blood cells or tumor cell lines.
  • PBS phosphate-buffered saline
  • FBS 1% fetal bovine serum
  • CD3 allophycocyanin (APC)-Cy7 clone UCHT1, BioLegend, 344818
  • CD4 phycoerythrin Pe, clone RPA- T4, BioLegend, 300508)
  • CD8 Peridinin-Chlorophyll-Protein PerCP, clone SK1, BioLegend, 344708)
  • CD45 APC-Cy7 clone HI30, BioLegend, 304014
  • CD45 PE-Cy7 clone HI30, BioLegend, 304016
  • CD45 BV510 clone 30-F11, BioLegend, 103137
  • CD45RA fluorescein isothiocyanate FITC, clone HI100, BioLegend, 98300
  • branched N-glycan surface expression analysis cells were incubated with 50mg/mL biotinylated PHA-L for 1 hour at room temperature, washed and incubated with streptavidin (PE- or APC-conjugated, BioLegend, 405203, 405207).
  • streptavidin PE- or APC-conjugated, BioLegend, 405203, 405207.
  • Relative Fluorescent Intensity was calculated as the ratio of the mean fluorescence intensities (MFI) of a specific fluorophore-conjugated antibody over a fluorophore-conjugated control. Either secondary antibodies or control isotypes were used as control. Data were collected using FACS Canto (BD Biosciences) and analyzed with FlowJo Software.
  • the inventors designed a modified form of the enzyme comprising a CD8 signal peptide to direct the newly synthesized PNGase protein to the cell membrane.
  • sPNGase secreted form of PNGase
  • the first construct carried the wild-type PNGase (wtPNGase; Uniprot Q96IV0-1) under the control of the PGK promoter, while the expression of eGFP (i.e., marker gene) was controlled by the minimal CMV promoter (mCMV) ( Figure 1a).
  • the second construct comprised the same components, with the exception that the wtPNGase was replaced with the sPNGase ( Figure 1b).
  • T3M-4 tumor cells were independently transduced with the two lentiviral vectors and Phytohemagglutinin-L lectin (PHA-L) was utilized to assay the tumor’s glyco-phenotype.
  • the inventors sought to optimize the design of the human PNGase in order to reduce the transgene size in an effort to improve virus production, and improve the de- glycosylation activity toward native proteins.
  • the wild- type enzyme includes domains responsible interaction with proteins involved in ERAD, but are dispensable for the catalytic activity.
  • N-terminal PUB domain which associates with the cytosolic p97, favoring the extraction of ubiquitinated-misfolded proteins from the ER to the cytosol, and with the UBL domain of cytosolic HR23 that interacts with the 26S proteasome, delivering proteins for degradation.
  • the PAW domain is located at the C- terminus and binds high mannose N-glycans.
  • the catalytic core is located between the two domains, which is responsible for the release of N-glycan moieties from glycoproteins and comprises a transglutaminase-like (TG) core defined by a catalytic triad of cysteine, histidine, and aspartic acid, and a pair of CXXC motifs involved in Zn-binding.
  • TG transglutaminase-like
  • PNGase mutants lacking either domain were designed for their capacity to de-glycosylate native (or folded), rather than unfolded, glycoproteins as compared to the wild-type PNGase enzyme.
  • An IgG variable heavy signal peptide and a MYC tag were included to improve secretion and detection.
  • EXAMPLE 2 44v6.28z_sPNGase CAR-T cells de-glycosylate T3M-4 tumor cells. Having assessed the functionality of sPNGase in de-glycosylating surface proteins, the inventors next designed an all-in-one construct in which both an anti-CD44v6 CAR (44v6.28z) and the secreted PNGase (sPNGase) were co-expressed (44v6.28z_sPNGase). A bidirectional lentiviral platform was utilised, in which sPNGase was expressed under the control of the minimal CMV promoter (mCMV) while the expression of 44v6.28z was driven by the PGK promoter ( Figure 2a).
  • mCMV minimal CMV promoter
  • Either 44v6.28z_sPNGase or control CD19 cells (19.28z) were co-cultured with 44v6 ko T3M- 4 cells at 1:5 effector-to-target ratio for 48 hours and binding to PHA-L lectin was used as read-out to assess the glyco-phenotype of tumor cells. Strikingly, tumor cells displayed a marked drop in PHA-L binding upon co-culture with 44v6.28z_sPNGase as compared to control 19.28z cells, suggestive of effective deglycosylation mediated by sPNGase, in the context of a CAR T-cell. EXAMPLE 3: PNGase expression does not alter CAR-T cells phenotype.
  • 44v6.28z_sPNGase expanded similarly to 44v6.28z cells ( Figure 3a, b) and displayed an equivalent CD4/CD8 ratio ( Figure 3c), together with a marked enrichment of stem cell memory cells (TSCM, CD45RA + CD62L + , Figure 3d).
  • EXAMPLE 4 44v6.28z_sPNGase CAR-T cells display superior killing of pancreatic adenocarcinoma cells. After assessing the de-glycosylating capacity of 44v6.28z_sPNGase cells, their killing ability, as compared to standard 44v6.28z cells, against the pancreatic adenocarcinoma cell line T3M-4 was assessed.
  • EXAMPLE 5 44v6.28z_sPNGase CAR-T cells display superior killing of lung adenocarcinoma cell lines.
  • co-culture assays were performed in which 44v6.28z_sPNGase cells were challenged against the CD44v6 + lung adenocarcinoma PC9 cells.
  • 44v6.28z_sPNGase cells sensitized tumor cells to recognition and displayed a significant increase in tumor targeting, marked as improved killing ( Figure 4a, b), higher expansion ( Figure 4c) and activation ( Figure 4d, e) compared to 44v6.28z control cells.
  • a polynucleotide comprising (a) a nucleotide sequence encoding a glycosidase; and (b) a nucleotide sequence encoding a chimeric antigen receptor (CAR).
  • a product comprising (a) a first polynucleotide comprising a nucleotide sequence encoding a glycosidase; and (b) a second polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR).
  • PNGase paragine amidase
  • the glycosidase comprises or consists of: (a) a sequence that has at least 70% sequence identity to SEQ ID NO: 1, or a fragment thereof; (b) a sequence that has at least 70% sequence identity to SEQ ID NO: 2, or a fragment thereof; (c) a sequence that has at least 70% sequence identity to SEQ ID NO: 3, or a fragment thereof; or (d) a sequence that has at least 70% sequence identity to SEQ ID NO: 34, or
  • CMV cytomegalovirus promoter
  • PGK human phosphoglycerate kinase promoter
  • EF-1 ⁇ promoter an EF-1 ⁇ promoter
  • a vector comprising the polynucleotide or product of any one of paras 1 to 11, optionally wherein the vector is a viral vector, optionally wherein the vector is a lentiviral vector or adeno-associated viral (AAV) vector.
  • glycosidase wherein the glycosidase is peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), and wherein the PNGase: (a) comprises a signal peptide; and/or (b) lacks a PUB domain, a PAW domain, or both. 14.
  • a polynucleotide comprising a nucleotide sequence encoding the glycosidase of para 13 or para 14. 16.
  • a vector comprising the polynucleotide of para 15 or para 16 optionally wherein the vector is a viral vector, optionally wherein the vector is a lentiviral vector or adeno- associated viral (AAV) vector. 18.
  • a cell comprising the polynucleotide or product according to any one of paras 1 to 11,15 and 16, the vector of para 12 or 17, or the glycosidase of para 13 or 14, wherein the cell is optionally a T cell.
  • a method of treatment comprising producing a CAR cell according to the method of para 23 or para 24 and administering the CAR cell to a subject in need thereof.

Abstract

The present invention relates to deglycosylating enzymes and modified versions thereof. The invention also provides deglycosylating enzymes alongside cell-binding molecules, such as CARs, for improving the therapeutic activity of CAR-containing cells. Methods and uses involving the deglycosylating enzymes of the invention are also provided.

Description

ENZYMES FIELD OF THE INVENTION The present invention relates to deglycosylating enzymes and modified versions thereof. The invention also provides combinations of deglycosylating enzymes and binding molecules, such as CARs, for example for improving the therapeutic activity of CAR-containing cells. Methods and uses involving the deglycosylating enzymes of the invention are also provided. BACKGROUND TO THE INVENTION Chimeric antigen receptors (CARs) are synthetic biology molecules commonly constructed by fusing an antigen-binding moiety, often derived from a tumor-reactive monoclonal antibody, with intracellular signaling domains derived from T lymphocytes. Clinical testing of CAR-T cell therapies has increased rapidly in recent years, leading to marketing authorizations for different CAR-T cell products targeting either CD19 or BCMA in relapsed/refractory B-cell lymphomas, B-cell acute lymphoblastic leukemia of children and young adults and multiple myeloma. Despite the successes of CAR-T therapies, objective response rates in patients with solid tumors are far less frequent and improving therapeutic efficacy against these malignancies represents one of the biggest challenges in the field. The relative resistance of solid tumors to CAR-T cells highlights the need for improved understanding of different determinants of CAR-T cell activity and therapeutic efficacy. It has previously been demonstrated that N-glycans may protect tumors from CAR-T cells by interfering with immunological synapse formation and by promoting the interaction between co-inhibitory molecules, such as PD-1 and PD-L1. Besides fostering mechanisms of resistance by tumor cells, N-glycans might also support the inhibitory function exerted by the tumor microenvironment (TME). Moreover, it has been observed that blocking N-glycan synthesis in tumor cells with glycosylation inhibitors, such as the glucose/mannose analogue 2-deoxi-2-glucose (2DG), resulted in higher CAR-T cell activation, improved tumor cell lysis, and superior antitumor activity in different xenograft mouse models (Greco et al. (2022) Sci Trans Med 14: eabg3072). Despite their successes, CAR-T therapies may still have limited efficacy in certain therapeutic scenarios, for example, when treating solid tumors. Thus, there is a significant need for improved CAR-T therapies that overcome limitations associated with conventional therapeutic approaches. SUMMARY OF THE INVENTION The present inventors have developed deglycosylating enzymes (glycosidases) and that may be used in combination with tumor specific CAR-T cells to create single CAR T cell agents that are endowed de-glycosylating activity. Despite most glycanases being unsuitable lysosomal enzymes that function at an acidic pH, the inventors have surprisingly developed and validated a modified glycosidase (a peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase; PNGase) that is functional in the extracellular space and may deglycosylate proteins exposed on the surface of tumor cells. The inventors have demonstrated in model studies that CAR T cells engineered to express the glycosidase display improved killing of tumor cells, such as pancreatic adenocarcinoma cells and lung adenocarcinoma cells. In one aspect, there is provided a polynucleotide comprising (a) a nucleotide sequence encoding a glycosidase; and (b) a nucleotide sequence encoding a chimeric antigen receptor (CAR). In one aspect, there is provided a product comprising (a) a first polynucleotide comprising a nucleotide sequence encoding a glycosidase; and (b) a second polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR). The product may be, for example, a kit or a composition. In one aspect, there is provided a kit comprising (a) a first polynucleotide comprising a nucleotide sequence encoding a glycosidase; and (b) a second polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR). In another aspect, there is provided a composition comprising (a) a first polynucleotide comprising a nucleotide sequence encoding a glycosidase; and (b) a second polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR). In one embodiment, the first polynucleotide is comprised in a first vector and the second polynucleotide is comprised in a second vector. In one embodiment, the glycosidase is a secreted glycosidase. In one embodiment, the glycosidase is secreted from a cell. In another embodiment, the cell is a CAR cell, i.e., it displays a chimeric antigen receptor. Thus, in one embodiment, the glycosidase is secreted by a CAR cell. Glycosidases may comprise a signal peptide, to facilitate their secretion. In one embodiment, the glycosidase comprises a signal peptide. The signal peptide may be cleaved from the glycosidase during its export from a cell. In a preferred embodiment, the nucleotide sequence encoding a glycosidase further encodes a signal peptide operably linked to the glycosidase. In one embodiment, the signal peptide is selected from the group consisting of: a CD8 signal peptide, an IgG variable region heavy chain signal peptide, an Ig kappa chain V-III region VG signal peptide, a GM-CFS/CSF signal peptide, and a CSFR2A signal peptide. In one embodiment, the signal peptide is a CD8 signal peptide. In one embodiment, the signal peptide is an IgG variable region heavy chain signal peptide. In one embodiment, the signal peptide comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence as set forth in the group consisting of SEQ ID NOs: 24 – 28 or a fragment thereof. In one embodiment, the glycosidase is functional in the extracellular environment. In one embodiment, the glycosidase hydrolyses cell-surface displayed glycoproteins or glycopeptides. In one embodiment, the glycosidase has a substrate that is an N-linked glycan. In one embodiment, the glycosidase is an N-glycanase. In one embodiment, the glycosidase hydrolyses a bond (e.g. a b-aspartylglucosaminyl bond) between an N-linked glycan and an asparagine (Asn) residue. In one embodiment, the glycosidase hydrolyses a bond (e.g. a b-aspartylglucosaminyl bond) between a core- chitobiose region of an N-linked glycan and an asparagine (Asn) residue. In one embodiment, the glycosidase is peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), or a variant thereof. In another embodiment, the glycosidase is human peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), or a variant thereof. In one embodiment, the PNGase variant is a truncated PNGase. In one embodiment, the PNGase lacks a PUB domain. In another embodiment the PNGase lacks a PAW domain. In a further embodiment, the PNGase lacks both a PUB and a PAW domain. In one embodiment, the glycosidase comprises or consists of: (a) a sequence that has at least 70% sequence identity to SEQ ID NO: 1, or a fragment thereof; (b) a sequence that has at least 70% sequence identity to SEQ ID NO: 2, or a fragment thereof; (c) a sequence that has at least 70% sequence identity to SEQ ID NO: 3, or a fragment thereof; or (d) a sequence that has at least 70% sequence identity to SEQ ID NO: 34, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34, or a fragment thereof. In one embodiment, the CAR is a 44v6.28z CAR. In another embodiment, the CAR comprises or consists of a sequence with at least 70% sequence identity to SEQ ID NO: 22. In another embodiment, the CAR is encoded by a polynucleotide sequence that comprises or consists of a sequence with at least 70% sequence identity to SEQ ID NO: 23. In one embodiment, the CAR comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22, or a fragment thereof. In one embodiment, the CAR is encoded by a polynucleotide sequence that comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23, or a fragment thereof. In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR are operably linked to one or more promoter(s). In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR are operably linked to the same promoter(s). In another embodiment, the nucleotide sequences encoding the glycosidase and the CAR are independently operably linked to one or more promoter(s). In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR are operably linked to separate promoter(s). In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR are in opposing directions. In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR are in opposing directions and are independently operably linked to separate promoters. In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR in the same direction. In one embodiment, the promoter is selected from the group consisting of: a cytomegalovirus promoter (CMV), a human phosphoglycerate kinase promoter (PGK), an EF-1α promoter and an inducible NFAT promoter. In one embodiment, the promoter is a cytomegalovirus (CMV) promoter. In another embodiment, the promoter is a minimal cytomegalovirus (mCMV) promoter. In one aspect, there is provided a vector comprising the polynucleotide or product according to the invention. In one embodiment, the vector is a viral vector. In one embodiment, the vector is a lentiviral vector. In one embodiment, the lentiviral vector is a bidirectional lentiviral vector. In one embodiment, the vector is an adeno-associated viral (AAV) vector. In a further aspect, there is provided a glycosidase, wherein the glycosidase is a peptide-N(4)- (N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), and wherein the PNGase: (a) comprises a signal peptide; and/or (b) lacks a PUB domain, a PAW domain, or both. In a further aspect, there is provided a glycosidase, wherein the glycosidase is a peptide-N(4)- (N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), and wherein the PNGase: (a) lacks a signal peptide; and/or (b) lacks a PUB domain, a PAW domain, or both. In a preferred embodiment, the glycosidase comprises a signal peptide. In one embodiment, the glycosidase lacks a signal peptide. In one embodiment, the glycosidase is secreted from a cell. In one embodiment, the glycosidase lacks a PUB domain. In one embodiment, the glycosidase lacks a PAW domain. In one embodiment, the glycosidase lacks both a PUB domain and a PAW domain. In one embodiment, the glycosidase comprises a signal peptide and lacks a PUB domain. In one embodiment, the glycosidase comprises a signal peptide and lacks a PAW domain. In a preferred embodiment, the glycosidase comprises a signal peptide and lacks a PUB domain and a PAW domain. In one embodiment, the glycosidase lacks a signal peptide and lacks a PUB domain. In one embodiment, the glycosidase lacks a signal peptide and lacks a PAW domain. In one embodiment, the glycosidase lacks a signal peptide and lacks a PUB domain and a PAW domain. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity to SEQ ID NO: 6, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6, or a fragment thereof. In one embodiment, the glycosidase comprises a signal peptide and lacks a PUB domain. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity to SEQ ID NO: 7, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7, or a fragment thereof. In one embodiment, the glycosidase comprises a signal peptide and lacks a PAW domain. In one embodiment, the glycosidase comprises a signal peptide and lacks a PUB domain and a PAW domain. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity to SEQ ID NO: 8, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of: (a) a sequence that has at least 70% sequence identity to SEQ ID NO: 1, 4, 6, or 9, or a fragment thereof; (b) a sequence that has at least 70% sequence identity to SEQ ID NO: 2, 7, or 10, or a fragment thereof; (c) a sequence that has at least 70% sequence identity to SEQ ID NO: 3, 8, or 11 or a fragment thereof; or (d) a sequence that has at least 70% sequence identity to SEQ ID NO: 34 or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, 4, 6, or 9, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, 7, or 10, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3, 8, or 11, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34, or a fragment thereof. In one aspect, there is provided a polynucleotide comprising a nucleotide sequence encoding the glycosidase of the invention. In one embodiment, the glycosidase is encoded by a polynucleotide that comprises or consists of: (a) a sequence that has at least 70% sequence identity to SEQ ID NO: 5 or 12, or a fragment thereof; (b) a sequence that has at least 70% sequence identity to SEQ ID NO: 13, or a fragment thereof; or (c) a sequence that has at least 70% sequence identity to SEQ ID NO: 14 or a fragment thereof. In one embodiment, the glycosidase is encoded by a polynucleotide that comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5 or 12, or a fragment thereof. In one embodiment, the glycosidase is encoded by a polynucleotide that comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13, or a fragment thereof. In one embodiment, the glycosidase is encoded by a polynucleotide that comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14 or a fragment thereof. In one aspect, there is provided a vector comprising the polynucleotide according to the invention. In one embodiment, the vector is a non-viral vector. In one embodiment, the vector is a nanoparticle. In one embodiment, the vector is a viral vector. In one embodiment, the vector is a lentiviral vector. In one embodiment, the lentiviral vector is a bidirectional lentiviral vector. In one embodiment, the vector is an adeno-associated viral (AAV) vector. In one embodiment, the polynucleotide or vector comprises a promoter that is operably linked to the nucleotide sequence encoding the glycosidase. In one embodiment, the promoter is a PGK promoter. In one aspect, there is provided a cell comprising the polynucleotide or product, the vector, or the glycosidase according to the invention. In one embodiment, the cell is a eukaryotic cell, such as a mammalian cell. In one embodiment, the cell is selected from the group consisting of a rodent cell, such as a mouse or rat cell, a feline cell, a canine cell, and a human cell. In a preferred embodiment, the cell is a human cell. In one embodiment the cell is an immune cell. In one embodiment the cell is a T cell. In one aspect, there is provided the polynucleotide or product, the vector, the glycosidase, or the cell according to the invention, for use in therapy. In one aspect, there is provided the polynucleotide or product, the vector, the glycosidase, or the cell according to the invention, for use in the treatment of cancer. In one embodiment, the cancer is a solid tumor. In one aspect, there is provided use of the polynucleotide or product, the vector, the glycosidase, or the cell according to the invention, for improving CAR cell activity. In one aspect, there is provided a method of producing a CAR cell, comprising introducing the polynucleotide or product, the vector, or the glycosidase according to the invention into a cell. In a further embodiment, the therapeutic activity of the CAR cell or CAR-T cell is improved. In one embodiment, the therapeutic activity is target cell killing. In one aspect, there is provided a method of treatment comprising producing a CAR cell according to the method of the invention, and administering the CAR cell to a subject in need thereof. In one embodiment, the subject is a human subject. In one embodiment, the subject has cancer. It is also contemplated that the glycosidase of the invention may be used in combination with T cell receptors (TCRs), thus additional aspects of the invention relate to the use of the glycosidase with a TCR instead of the CAR as disclosed herein. In one aspect, there is provided a polynucleotide comprising (a) a nucleotide sequence encoding a glycosidase; and (b) a nucleotide sequence encoding a chimeric antigen receptor (CAR). In one aspect, there is provided a product comprising (a) a first polynucleotide comprising a nucleotide sequence encoding a glycosidase; and (b) a second polynucleotide comprising a nucleotide sequence encoding a T cell receptor (TCR). The product may be, for example, a kit or a composition. In one aspect, there is provided a kit comprising (a) a first polynucleotide comprising a nucleotide sequence encoding a glycosidase; and (b) a second polynucleotide comprising a nucleotide sequence encoding a T cell receptor (TCR). In another aspect, there is provided a composition comprising (a) a first polynucleotide comprising a nucleotide sequence encoding a glycosidase; and (b) a second polynucleotide comprising a nucleotide sequence encoding a T cell receptor (TCR). In some embodiments, the cell of the invention may be a tumour-infiltrating lymphocyte (TIL). DESCRIPTION OF THE DRAWINGS FIGURE 1. Generation and evaluation of engineered PNGase. Schematic representation of lentiviral vectors cloned to generate T3M-4 model cell lines expressing (a) wild-type PNGase (wtPNGase) or (b) secreted PNGase (sPNGase). (c) Flow- cytometry profile of PHA-L binding to T3M-4 transduced to express wtPNGase compared to control untransduced cells. (d) Flow-cytometry profile of PHA-L binding to T3M-4 transduced to express sPNGase compared to control untransduced cells. FIGURE 2. T cells co-expressing 44v6.28z CAR and secreted PNGase reduce binding of PHA-L lectin to tumor cells. (a) Schematics of the lentiviral bidirectional vector expressing the anti-CD44v6 CAR (44v6.28z) and the secreted PNGase enzyme (sPNGase; 44v6.28z_sPNGase) and further schematic of PNGase deglycosylation. The Phytohemagglutinin-L (PHA-L) target motif is shown (dark box). (b) Frequency of PHA-L binding to CD44v6 knocked-out T3M-4 cells after 48hours co-culture with either 44v6.28z_sPNGase or control CD19 CAR-T cells (19.28z) at 1:5 effector-to-target ratio (n=3 technical replicates). P values (*P < 0.05) were determined by t-test. Data are mean ± s.e.m. FIGURE 3. Expansion and phenotype of 44v6.28z_sPNGase CAR-T cells. (a, b) CD3 counts and fold increase during CAR-T cell manufacture. Fold increase was calculated over day 2 from T cell activation with αCD3/CD28 beads (n=2 donors). Frequency of CD4 and CD8 cells (c) and of memory subsets (d) at the end of manufacturing. TSCM, stem cell memory (CD62L+CD45RA+); TCM, central memory (CD62L+CD45RA-); Tem, effector memory (CD62L-CD45RA-); Temra, terminal effector memory (CD62L-CD45RA+). FIGURE 4.44v6.28z_sPNGase CAR-T cells improve killing of T3M-4 pancreatic adenocarcinoma cells in co-culture. (a) Representative flow-cytometry plot showing killing of T3M-4 cells after co-culture with 44v6.28z, 44v6.28z_sPNGase or untransduced (UT) cells as a control, at 1:5 effector-to-target (E:T) ratio. (b) Killing of T3M-4 cells at different E:T ratios (n=2 donors and 3 technical replicates). Killing is expressed as Elimination Index with respect to control UT. IFN-γ production (c) and CAR-T cell counts (d) at 1:10 E:T ratio. (d) Frequency of HLA-DR expression at 1:5 E:T ratio. (e) Relative fluorescence intensity (RFI) of CD69 expression at 1:5 E:T ratio. P values (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) were determined by two-way ANOVA (b) and t-test (c-f). Data are mean ± s.e.m. FIGURE 5.44v6.28z_sPNGase CAR-T cells improve killing of PC9 lung adenocarcinoma cells in co-culture. (a) Representative flow-cytometry plot showing killing of PC9 cells after co-culture with 44v6.28z, 44v6.28z_sPNGase or untransduced (UT) cells as control at 1:5 effector-to-target (E:T) ratio. (b) Killing of PC9 cells at different E:T ratios (n=1 donor and 3 technical replicates). Killing is expressed as Elimination Index with respect to control UT. (c) CAR-T cell counts at 1:10 E:T ratio. (d) Frequency of HLA-DR expression at 1:5 E:T ratio. (e) Relative fluorescence intensity (RFI) of CD69 expression at 1:5 E:T ratio. P values (*P < 0.05) were determined by two-way ANOVA (b) and t-test (c – e). Data are mean ± s.e.m. DETAILED DESCRIPTION OF THE INVENTION The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including” or “includes”; or “containing” or “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or steps. The terms “comprising”, “comprises” and “comprised of” also include the term “consisting of”. It will be understood that when referring to a protein or polypeptide herein, the same may equally be applied to a polynucleotide encoding the same, and, where relevant (i.e., when referring to a coding sequence within a polynucleotide) vice versa. Glycosidases Glycosidases (or glycoside hydrolases/glycosyl hydrolases) are enzymes that catalyse the hydrolysis of glycosidic bonds in sugars. Glycosidases that act upon carbohydrates that are bound to proteins, e.g., as N- or O-linked glycans, may also be referred to herein as deglycosylating enzymes as they catalyse removal of glycans or sugar moieties. Glycosidases are abundant and diverse and similarly have diverse functionality. For example, glycosidases may vary in size, structure, target specificity, catalytic activity, and the conditions under which they are catalytically active. For example, while some glycosidases may catalyse the removal of individual sugars at the termini of glycan structures, others catalyse the removal of entire glycans by the targeted hydrolysis of the inner-most sugar-protein bond. In either scenario, the enzymatic removal of sugars may be considered to be deglycosylation. Glycosidases function endogenously in diverse biological processes, and in different cellular compartments, under different conditions. Preferably, the glycosidase of the invention is functional, i.e., enzymatically active, in the extracellular space. Preferably, such an enzyme will be able to deglycosylate proteins in the extracellular environment, such as those exposed on the surface of cells, e.g., tumor cells. It will be understood that enzymes may function over a range of conditions, e.g., pH, with varying activity. An enzyme according to the invention may be used under non-optimal conditions and yet still retain functionality, i.e., an enzyme does not need to be optimally functioning to still be functional and retain the activity of deglycosylation. In one embodiment, the glycosidase is functional in the extracellular environment. Functionality in the extracellular environment may be a natural property of the enzyme, or it may be introduced e.g., by mutation, to an enzyme, for example constituting a variant. Glycosidases that are functional in the extracellular environment may catalyze the removal of glycans (or “act upon” glycans) from diverse substrates. In one embodiment, the glycosidase deglycosylates glycoproteins or glycopeptides. In one embodiment, the glycosidase deglycosylates cell-surface displayed glycoproteins or glycopeptides. In one embodiment, the glycosidase has a substrate that is an N-linked glycan. In one embodiment, the glycosidase deglycosylates N-linked glycans. In one embodiment, the glycosidase is an N-glycanase. In one embodiment, the glycosidase has a substrate that is an O-linked glycan. In one embodiment, the glycosidase deglycosylates O-linked glycans. In one embodiment, the glycosidase is an O-glycanase. In one embodiment, the glycosidase catalyzes the removal of one or more sugar moieties. In one embodiment, the glycosidase catalyzes the removal of an entire glycan chain. As used herein, “a glycan” may refer to an entire glycan structure, or fragments of said structure, such as individual sugars (mono-, di-, poly-saccharides). Exemplary glycosidases An exemplary glycosidase is peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), such as human PNGase. Further, modified versions of human PNGase described herein may also be considered exemplary and may be used in the present invention. In one embodiment, the glycosidase is PNGase. In one embodiment, the glycosidase is human PNGase. Exemplary human PNGase [Uniprot (Q96IV0-1)] (SEQ ID NO: 1): AAAALGSSSGSASPAVAELCQNTPETFLEASKLLLTYADNILRNPNDEKYRSIRIGNT AFSTRLLPVRGAVECLFEMGFEEGETHLIFPKKASVEQLQKIRDLIAIERSSRLDGSN KSHKVKSSQQPAASTQLPTTPSSNPSGLNQHTRNRQGQSSDPPSASTVAADSAILE VLQSNIQHVLVYENPALQEKALACIPVQELKRKSQEKLSRARKLDKGINISDEDFLLL ELLHWFKEEFFHWVNNVLCSKCGGQTRSRDRSLLPSDDELKWGAKEVEDHYCDA CQFSNRFPRYNNPEKLLETRCGRCGEWANCFTLCCRAVGFEARYVWDYTDHVWT EVYSPSQQRWLHCDACEDVCDKPLLYEIGWGKKLSYVIAFSKDEVVDVTWRYSCK HEEVIARRTKVKEALLRDTINGLNKQRQLFLSENRRKELLQRIIVELVEFISPKTPKPG ELGGRISGSVAWRVARGEMGLQRKETLFIPCENEKISKQLHLCYNIVKDRYVRVSN NNQTISGWENGVWKMESIFRKVETDWHMVYLARKEGSSFAYISWKFECGSVGLKV DSISIRTSSQTFQTGTVEWKLRSDTAQVELTGDNSLHSYADFSGATEVILEAELSRG DGDVAWQHTQLFRQSLNDHEENCLEIIIKFSDL In one embodiment, the glycosidase is a variant of human PNGase. PNGase hydrolyses the b-aspartylglucosaminyl bond between the core-chitobiose region of an N-linked glycan and an asparagine (Asn) residue, converting Asn to aspartate (Asp), which results in the release of glycan moieties from glycoproteins or glycopeptides. Unlike many deglycosylating enzymes, such as those that are lysosomally resident and optimally functional at acidic pH, the peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase) is functional at neutral pH. In one embodiment, the glycosidase hydrolyses a b-aspartylglucosaminyl bond between the core-chitobiose region of an N-linked glycan and an asparagine (Asn) residue. In one embodiment, the glycosidase is peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), or a variant thereof. In another embodiment, the glycosidase is human peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), or a variant thereof. In one embodiment, the PNGase variant is a truncated PNGase. In one embodiment, the PNGase lacks a PUB domain. In another embodiment the PNGase lacks a PAW domain. In a further embodiment, the PNGase lacks or both a PUB and a PAW domain. Exemplary PUB truncated human PNGase (SEQ ID NO: 2): KASVEQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQPAASTQLPTTPSSNPSGLNQH TRNRQGQSSDPPSASTVAADSAILEVLQSNIQHVLVYENPALQEKALACIPVQELKR KSQEKLSRARKLDKGINISDEDFLLLELLHWFKEEFFHWVNNVLCSKCGGQTRSRD RSLLPSDDELKWGAKEVEDHYCDACQFSNRFPRYNNPEKLLETRCGRCGEWANC FTLCCRAVGFEARYVWDYTDHVWTEVYSPSQQRWLHCDACEDVCDKPLLYEIGW GKKLSYVIAFSKDEVVDVTWRYSCKHEEVIARRTKVKEALLRDTINGLNKQRQLFLS ENRRKELLQRIIVELVEFISPKTPKPGELGGRISGSVAWRVARGEMGLQRKETLFIPC ENEKISKQLHLCYNIVKDRYVRVSNNNQTISGWENGVWKMESIFRKVETDWHMVYL ARKEGSSFAYISWKFECGSVGLKVDSISIRTSSQTFQTGTVEWKLRSDTAQVELTG DNSLHSYADFSGATEVILEAELSRGDGDVAWQHTQLFRQSLNDHEENCLEIIIKFSD L Exemplary PUB and PAW truncated human PNGase (SEQ ID NO: 3): KASVEQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQPAASTQLPTTPSSNPSGLNQH TRNRQGQSSDPPSASTVAADSAILEVLQSNIQHVLVYENPALQEKALACIPVQELKR KSQEKLSRARKLDKGINISDEDFLLLELLHWFKEEFFHWVNNVLCSKCGGQTRSRD RSLLPSDDELKWGAKEVEDHYCDACQFSNRFPRYNNPEKLLETRCGRCGEWANC FTLCCRAVGFEARYVWDYTDHVWTEVYSPSQQRWLHCDACEDVCDKPLLYEIGW GKKLSYVIAFSKDEVVDVTWRYSCKHEEVIARRTKVKEALLRDTINGLNKQRQLFLS ENRRKELLQRIIVELVEFISPKTPKPG Exemplary PAW truncated human PNGase (SEQ ID NO: 34): AAAALGSSSGSASPAVAELCQNTPETFLEASKLLLTYADNILRNPNDEKYRSIRIGNT AFSTRLLPVRGAVECLFEMGFEEGETHLIFPKKASVEQLQKIRDLIAIERSSRLDGSN KSHKVKSSQQPAASTQLPTTPSSNPSGLNQHTRNRQGQSSDPPSASTVAADSAILE VLQSNIQHVLVYENPALQEKALACIPVQELKRKSQEKLSRARKLDKGINISDEDFLLL ELLHWFKEEFFHWVNNVLCSKCGGQTRSRDRSLLPSDDELKWGAKEVEDHYCDA CQFSNRFPRYNNPEKLLETRCGRCGEWANCFTLCCRAVGFEARYVWDYTDHVWT EVYSPSQQRWLHCDACEDVCDKPLLYEIGWGKKLSYVIAFSKDEVVDVTWRYSCK HEEVIARRTKVKEALLRDTINGLNKQRQLFLSENRRKELLQRIIVELVEFISPKTPKPG In one embodiment, the glycosidase comprises or consists of: (a) a sequence that has at least 70% sequence identity to SEQ ID NO: 1, or a fragment thereof; (b) a sequence that has at least 70% sequence identity to SEQ ID NO: 2, or a fragment thereof; (c) a sequence that has at least 70% sequence identity to SEQ ID NO: 3, or a fragment thereof; or (d) a sequence that has at least 70% sequence identity to SEQ ID NO: 34, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34, or a fragment thereof. The polypeptides of the foregoing may be combined with other sequence features. For example, the glycosidases of the invention may comprise a signal peptide sequence. Further, the glycosidases of the invention may comprise a signal peptide and a tag. Further exemplary glycosidase sequences are set out below: Exemplary human PNGase with CD8 signal peptide (SEQ ID NO: 4): MALPVTALLLPLALLLHAARPAAAALGSSSGSASPAVAELCQNTPETFLEASKLLLT YADNILRNPNDEKYRSIRIGNTAFSTRLLPVRGAVECLFEMGFEEGETHLIFPKKASV EQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQPAASTQLPTTPSSNPSGLNQHTRNR QGQSSDPPSASTVAADSAILEVLQSNIQHVLVYENPALQEKALACIPVQELKRKSQE KLSRARKLDKGINISDEDFLLLELLHWFKEEFFHWVNNVLCSKCGGQTRSRDRSLLP SDDELKWGAKEVEDHYCDACQFSNRFPRYNNPEKLLETRCGRCGEWANCFTLCC RAVGFEARYVWDYTDHVWTEVYSPSQQRWLHCDACEDVCDKPLLYEIGWGKKLS YVIAFSKDEVVDVTWRYSCKHEEVIARRTKVKEALLRDTINGLNKQRQLFLSENRRK ELLQRIIVELVEFISPKTPKPGELGGRISGSVAWRVARGEMGLQRKETLFIPCENEKI SKQLHLCYNIVKDRYVRVSNNNQTISGWENGVWKMESIFRKVETDWHMVYLARKE GSSFAYISWKFECGSVGLKVDSISIRTSSQTFQTGTVEWKLRSDTAQVELTGDNSL HSYADFSGATEVILEAELSRGDGDVAWQHTQLFRQSLNDHEENCLEIIIKFSDL CD8 Signal peptide Exemplary nucleotide sequence encoding human PNGase with CD8 signal peptide (SEQ ID NO: 5): ATGGCCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACG CCGCCAGACCCGCCGCCGCCGCCCTGGGCAGCAGCAGCGGCAGCGCCAGCC CCGCCGTGGCCGAGCTGTGCCAGAACACCCCCGAGACCTTCCTGGAGGCCAG CAAGCTGCTGCTGACCTACGCCGACAACATCCTGAGAAACCCCAACGACGAGA AGTACAGAAGCATCAGAATCGGCAACACCGCCTTCAGCACCAGACTGCTGCCC GTGAGAGGCGCCGTGGAGTGCCTGTTCGAGATGGGCTTCGAGGAGGGCGAGA CCCACCTGATCTTCCCCAAGAAGGCCAGCGTGGAGCAGCTGCAGAAGATCAGA GACCTGATCGCCATCGAGAGAAGCAGCAGACTGGACGGCAGCAACAAGAGCCA CAAGGTGAAGAGCAGCCAGCAGCCCGCCGCCAGCACCCAGCTGCCCACCACC CCCAGCAGCAACCCCAGCGGCCTGAACCAGCACACCAGAAACAGACAGGGCC AGAGCAGCGACCCCCCCAGCGCCAGCACCGTGGCCGCCGACAGCGCCATCCT GGAGGTGCTGCAGAGCAACATCCAGCACGTGCTGGTGTACGAGAACCCCGCC CTGCAGGAGAAGGCCCTGGCCTGCATCCCCGTGCAGGAGCTGAAGAGAAAGA GCCAGGAGAAGCTGAGCAGAGCCAGAAAGCTGGACAAGGGCATCAACATCAGC GACGAGGACTTCCTGCTGCTGGAGCTGCTGCACTGGTTCAAGGAGGAGTTCTT CCACTGGGTGAACAACGTGCTGTGCAGCAAGTGCGGCGGCCAGACCAGAAGC AGAGACAGAAGCCTGCTGCCCAGCGACGACGAGCTGAAGTGGGGCGCCAAGG AGGTGGAGGACCACTACTGCGACGCCTGCCAGTTCAGCAACAGATTCCCCAGA TACAACAACCCCGAGAAGCTGCTGGAGACCAGATGCGGCAGATGCGGCGAGT GGGCCAACTGCTTCACCCTGTGCTGCAGAGCCGTGGGCTTCGAGGCCAGATAC GTGTGGGACTACACCGACCACGTGTGGACCGAGGTGTACAGCCCCAGCCAGC AGAGATGGCTGCACTGCGACGCCTGCGAGGACGTGTGCGACAAGCCCCTGCT GTACGAGATCGGCTGGGGCAAGAAGCTGAGCTACGTGATCGCCTTCAGCAAGG ACGAGGTGGTGGACGTGACCTGGAGATACAGCTGCAAGCACGAGGAGGTGAT CGCCAGAAGAACCAAGGTGAAGGAGGCCCTGCTGAGAGACACCATCAACGGC CTGAACAAGCAGAGACAGCTGTTCCTGAGCGAGAACAGAAGAAAGGAGCTGCT GCAGAGAATCATCGTGGAGCTGGTGGAGTTCATCAGCCCCAAGACCCCCAAGC CCGGCGAGCTGGGCGGCAGAATCAGCGGCAGCGTGGCCTGGAGAGTGGCCA GAGGCGAGATGGGCCTGCAGAGAAAGGAGACCCTGTTCATCCCCTGCGAGAAC GAGAAGATCAGCAAGCAGCTGCACCTGTGCTACAACATCGTGAAGGACAGATA CGTGAGAGTGAGCAACAACAACCAGACCATCAGCGGCTGGGAGAACGGCGTGT GGAAGATGGAGAGCATCTTCAGAAAGGTGGAGACCGACTGGCACATGGTGTAC CTGGCCAGAAAGGAGGGCAGCAGCTTCGCCTACATCAGCTGGAAGTTCGAGTG CGGCAGCGTGGGCCTGAAGGTGGACAGCATCAGCATCAGAACCAGCAGCCAG ACCTTCCAGACCGGCACCGTGGAGTGGAAGCTGAGAAGCGACACCGCCCAGG TGGAGCTGACCGGCGACAACAGCCTGCACAGCTACGCCGACTTCAGCGGCGC CACCGAGGTGATCCTGGAGGCCGAGCTGAGCAGAGGCGACGGCGACGTGGCC TGGCAGCACACCCAGCTGTTCAGACAGAGCCTGAACGACCACGAGGAGAACTG CCTGGAGATCATCATCAAGTTCAGCGACCTGTGA Exemplary human PNGase with IgG variable heavy signal peptide (SEQ ID NO: 6): MEFGLSWVFLVALLRGVQCAAAALGSSSGSASPAVAELCQNTPETFLEASKLLLTY ADNILRNPNDEKYRSIRIGNTAFSTRLLPVRGAVECLFEMGFEEGETHLIFPKKASVE QLQKIRDLIAIERSSRLDGSNKSHKVKSSQQPAASTQLPTTPSSNPSGLNQHTRNR QGQSSDPPSASTVAADSAILEVLQSNIQHVLVYENPALQEKALACIPVQELKRKSQE KLSRARKLDKGINISDEDFLLLELLHWFKEEFFHWVNNVLCSKCGGQTRSRDRSLLP SDDELKWGAKEVEDHYCDACQFSNRFPRYNNPEKLLETRCGRCGEWANCFTLCC RAVGFEARYVWDYTDHVWTEVYSPSQQRWLHCDACEDVCDKPLLYEIGWGKKLS YVIAFSKDEVVDVTWRYSCKHEEVIARRTKVKEALLRDTINGLNKQRQLFLSENRRK ELLQRIIVELVEFISPKTPKPGELGGRISGSVAWRVARGEMGLQRKETLFIPCENEKI SKQLHLCYNIVKDRYVRVSNNNQTISGWENGVWKMESIFRKVETDWHMVYLARKE GSSFAYISWKFECGSVGLKVDSISIRTSSQTFQTGTVEWKLRSDTAQVELTGDNSL HSYADFSGATEVILEAELSRGDGDVAWQHTQLFRQSLNDHEENCLEIIIKFSDL IgG heavy signal peptide Exemplary human PNGase with IgG variable heavy signal peptide and MYC tag with N- terminal “GS” linker (SEQ ID NO: 9): MEFGLSWVFLVALLRGVQCAAAALGSSSGSASPAVAELCQNTPETFLEASKLLLTY ADNILRNPNDEKYRSIRIGNTAFSTRLLPVRGAVECLFEMGFEEGETHLIFPKKASVE QLQKIRDLIAIERSSRLDGSNKSHKVKSSQQPAASTQLPTTPSSNPSGLNQHTRNR QGQSSDPPSASTVAADSAILEVLQSNIQHVLVYENPALQEKALACIPVQELKRKSQE KLSRARKLDKGINISDEDFLLLELLHWFKEEFFHWVNNVLCSKCGGQTRSRDRSLLP SDDELKWGAKEVEDHYCDACQFSNRFPRYNNPEKLLETRCGRCGEWANCFTLCC RAVGFEARYVWDYTDHVWTEVYSPSQQRWLHCDACEDVCDKPLLYEIGWGKKLS YVIAFSKDEVVDVTWRYSCKHEEVIARRTKVKEALLRDTINGLNKQRQLFLSENRRK ELLQRIIVELVEFISPKTPKPGELGGRISGSVAWRVARGEMGLQRKETLFIPCENEKI SKQLHLCYNIVKDRYVRVSNNNQTISGWENGVWKMESIFRKVETDWHMVYLARKE GSSFAYISWKFECGSVGLKVDSISIRTSSQTFQTGTVEWKLRSDTAQVELTGDNSL HSYADFSGATEVILEAELSRGDGDVAWQHTQLFRQSLNDHEENCLEIIIKFSDLGSE QKLISEEDL IgG heavy signal peptide MYC tag with N-terminal “GS” linker Exemplary nucleotide sequence encoding human PNGase with IgG variable heavy signal peptide and MYC tag with N-terminal “GS” linker (SEQ ID NO: 12): ATGGAATTTGGATTGTCATGGGTGTTTCTTGTCGCATTGCTTAGGGGAGTCCAA TGTGCTGCTGCAGCGTTGGGGTCTTCCTCTGGATCTGCAAGTCCTGCGGTCGC TGAATTGTGTCAAAATACACCAGAGACGTTCTTGGAAGCAAGTAAACTCCTTCTT ACGTATGCCGATAATATATTGCGAAATCCAAATGATGAAAAGTATAGGTCTATTC GCATAGGTAATACTGCTTTCTCCACTCGCTTGCTCCCAGTTCGCGGTGCAGTAG AATGTTTGTTTGAAATGGGATTTGAAGAAGGAGAAACTCATTTGATATTTCCTAA GAAAGCGTCCGTCGAACAATTGCAGAAAATTAGGGATTTGATAGCTATTGAAAG GTCTTCACGCCTCGATGGATCTAATAAAAGTCATAAAGTAAAATCTTCCCAACAA CCGGCTGCTAGTACACAATTGCCTACGACACCATCCAGTAATCCTTCAGGGCTT AATCAACATACAAGGAATCGGCAAGGTCAAAGTTCTGATCCGCCTAGTGCATCT ACGGTTGCTGCAGATAGTGCTATTCTCGAAGTTCTTCAATCTAATATTCAACATG TCCTTGTTTATGAAAATCCAGCTCTCCAAGAGAAAGCTCTCGCATGTATACCAGT CCAAGAATTGAAACGTAAATCCCAAGAGAAATTATCACGCGCACGGAAACTCGA TAAAGGGATTAATATATCCGACGAAGATTTTCTTCTCCTCGAACTTTTACACTGG TTTAAAGAAGAATTCTTTCATTGGGTAAATAATGTCTTATGTTCTAAATGTGGCGG ACAAACTCGCAGTCGCGATCGATCACTCCTGCCTAGTGATGATGAATTGAAATG GGGTGCTAAAGAAGTCGAAGATCATTATTGTGATGCTTGTCAATTTTCAAATCGC TTTCCGCGGTATAATAATCCGGAGAAACTCTTGGAAACACGCTGTGGACGCTGT GGGGAATGGGCGAATTGTTTTACATTATGTTGTCGGGCTGTTGGATTTGAAGCG AGGTATGTCTGGGATTATACAGATCATGTCTGGACAGAAGTTTATTCCCCATCCC AACAACGTTGGCTCCATTGTGATGCTTGTGAAGATGTTTGTGATAAACCGTTGCT TTATGAAATTGGATGGGGTAAGAAATTGTCATATGTCATTGCATTTTCCAAAGAT GAAGTCGTCGATGTTACTTGGCGTTATTCTTGTAAACATGAAGAAGTTATTGCGC GAAGGACAAAAGTTAAAGAAGCGCTTCTCCGCGATACAATTAATGGTCTCAATA AACAACGGCAACTCTTTCTCAGTGAAAATAGGCGCAAAGAACTCTTACAAAGGA TTATTGTCGAACTTGTCGAATTTATTTCTCCTAAAACACCAAAACCTGGAGAATT GGGAGGGAGGATTTCAGGATCAGTTGCTTGGCGAGTTGCGCGTGGAGAAATGG GGTTACAACGCAAAGAAACTTTGTTTATACCATGCGAAAATGAAAAGATTTCCAA ACAACTCCACTTGTGTTATAATATTGTAAAAGATCGGTATGTCCGGGTTTCAAAC AATAATCAAACTATATCAGGGTGGGAAAATGGGGTCTGGAAAATGGAATCTATAT TTCGAAAAGTAGAAACAGATTGGCATATGGTTTATCTTGCGCGCAAAGAAGGAA GCTCATTTGCTTATATATCATGGAAATTTGAATGTGGGTCCGTTGGATTGAAAGT TGATAGTATAAGTATTCGCACTAGTTCTCAAACATTTCAAACTGGAACTGTCGAA TGGAAACTCCGAAGTGATACAGCACAAGTCGAATTGACTGGAGATAATTCTCTT CATTCCTATGCTGATTTTAGTGGAGCGACTGAAGTAATTCTCGAAGCAGAATTGA GTCGGGGAGATGGTGATGTCGCTTGGCAACACACTCAACTGTTTAGGCAATCTC TTAATGATCATGAAGAAAATTGTCTTGAAATAATTATTAAATTTTCAGATCTCGGG TCCGAACAGAAATTAATTTCTGAAGAGGATTTGTAA Exemplary PUB truncated human PNGase with IgG variable heavy signal peptide (SEQ ID NO: 7): MEFGLSWVFLVALLRGVQCKASVEQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQP AASTQLPTTPSSNPSGLNQHTRNRQGQSSDPPSASTVAADSAILEVLQSNIQHVLV YENPALQEKALACIPVQELKRKSQEKLSRARKLDKGINISDEDFLLLELLHWFKEEFF HWVNNVLCSKCGGQTRSRDRSLLPSDDELKWGAKEVEDHYCDACQFSNRFPRYN NPEKLLETRCGRCGEWANCFTLCCRAVGFEARYVWDYTDHVWTEVYSPSQQRWL HCDACEDVCDKPLLYEIGWGKKLSYVIAFSKDEVVDVTWRYSCKHEEVIARRTKVK EALLRDTINGLNKQRQLFLSENRRKELLQRIIVELVEFISPKTPKPGELGGRISGSVA WRVARGEMGLQRKETLFIPCENEKISKQLHLCYNIVKDRYVRVSNNNQTISGWENG VWKMESIFRKVETDWHMVYLARKEGSSFAYISWKFECGSVGLKVDSISIRTSSQTF QTGTVEWKLRSDTAQVELTGDNSLHSYADFSGATEVILEAELSRGDGDVAWQHTQ LFRQSLNDHEENCLEIIIKFSDL IgG heavy signal peptide Exemplary PUB truncated human PNGase and MYC tag with N-terminal “GS” linker (SEQ ID NO: 10): MEFGLSWVFLVALLRGVQCKASVEQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQP AASTQLPTTPSSNPSGLNQHTRNRQGQSSDPPSASTVAADSAILEVLQSNIQHVLV YENPALQEKALACIPVQELKRKSQEKLSRARKLDKGINISDEDFLLLELLHWFKEEFF HWVNNVLCSKCGGQTRSRDRSLLPSDDELKWGAKEVEDHYCDACQFSNRFPRYN NPEKLLETRCGRCGEWANCFTLCCRAVGFEARYVWDYTDHVWTEVYSPSQQRWL HCDACEDVCDKPLLYEIGWGKKLSYVIAFSKDEVVDVTWRYSCKHEEVIARRTKVK EALLRDTINGLNKQRQLFLSENRRKELLQRIIVELVEFISPKTPKPGELGGRISGSVA WRVARGEMGLQRKETLFIPCENEKISKQLHLCYNIVKDRYVRVSNNNQTISGWENG VWKMESIFRKVETDWHMVYLARKEGSSFAYISWKFECGSVGLKVDSISIRTSSQTF QTGTVEWKLRSDTAQVELTGDNSLHSYADFSGATEVILEAELSRGDGDVAWQHTQ LFRQSLNDHEENCLEIIIKFSDLGSEQKLISEEDL IgG heavy signal peptide MYC tag with N-terminal “GS” linker Exemplary nucleotide sequence encoding PUB truncated human PNGase and MYC tag with N-terminal “GS” linker (SEQ ID NO: 13): TCACAGGTCCTCCTCGCTGATCAGCTTCTGCTCGCTGCCCAGGTCGCTGAACTT GATGATGATCTCCAGGCAGTTCTCCTCGTGGTCGTTCAGGCTCTGTCTGAACAG CTGGGTGTGCTGCCAGGCCACGTCGCCGTCGCCTCTGCTCAGCTCGGCCTCCA GGATCACCTCGGTGGCGCCGCTGAAGTCGGCGTAGCTGTGCAGGCTGTTGTC GCCGGTCAGCTCCACCTGGGCGGTGTCGCTTCTCAGCTTCCACTCCACGGTGC CGGTCTGGAAGGTCTGGCTGCTGGTTCTGATGCTGATGCTGTCCACCTTCAGG CCCACGCTGCCGCACTCGAACTTCCAGCTGATGTAGGCGAAGCTGCTGCCCTC CTTTCTGGCCAGGTACACCATGTGCCAGTCGGTCTCCACCTTTCTGAAGATGCT CTCCATCTTCCACACGCCGTTCTCCCAGCCGCTGATGGTCTGGTTGTTGTTGCT CACTCTCACGTATCTGTCCTTCACGATGTTGTAGCACAGGTGCAGCTGCTTGCT GATCTTCTCGTTCTCGCAGGGGATGAACAGGGTCTCCTTTCTCTGCAGGCCCAT CTCGCCTCTGGCCACTCTCCAGGCCACGCTGCCGCTGATTCTGCCGCCCAGCT CGCCGGGCTTGGGGGTCTTGGGGCTGATGAACTCCACCAGCTCCACGATGATT CTCTGCAGCAGCTCCTTTCTTCTGTTCTCGCTCAGGAACAGCTGTCTCTGCTTG TTCAGGCCGTTGATGGTGTCTCTCAGCAGGGCCTCCTTCACCTTGGTTCTTCTG GCGATCACCTCCTCGTGCTTGCAGCTGTATCTCCAGGTCACGTCCACCACCTCG TCCTTGCTGAAGGCGATCACGTAGCTCAGCTTCTTGCCCCAGCCGATCTCGTAC AGCAGGGGCTTGTCGCACACGTCCTCGCAGGCGTCGCAGTGCAGCCATCTCTG CTGGCTGGGGCTGTACACCTCGGTCCACACGTGGTCGGTGTAGTCCCACACGT ATCTGGCCTCGAAGCCCACGGCTCTGCAGCACAGGGTGAAGCAGTTGGCCCAC TCGCCGCATCTGCCGCATCTGGTCTCCAGCAGCTTCTCGGGGTTGTTGTATCTG GGGAATCTGTTGCTGAACTGGCAGGCGTCGCAGTAGTGGTCCTCCACCTCCTT GGCGCCCCACTTCAGCTCGTCGTCGCTGGGCAGCAGGCTTCTGTCTCTGCTTC TGGTCTGGCCGCCGCACTTGCTGCACAGCACGTTGTTCACCCAGTGGAAGAAC TCCTCCTTGAACCAGTGCAGCAGCTCCAGCAGCAGGAAGTCCTCGTCGCTGAT GTTGATGCCCTTGTCCAGCTTTCTGGCTCTGCTCAGCTTCTCCTGGCTCTTTCTC TTCAGCTCCTGCACGGGGATGCAGGCCAGGGCCTTCTCCTGCAGGGCGGGGT TCTCGTACACCAGCACGTGCTGGATGTTGCTCTGCAGCACCTCCAGGATGGCG CTGTCGGCGGCCACGGTGCTGGCGCTGGGGGGGTCGCTGCTCTGGCCCTGTC TGTTTCTGGTGTGCTGGTTCAGGCCGCTGGGGTTGCTGCTGGGGGTGGTGGG CAGCTGGGTGCTGGCGGCGGGCTGCTGGCTGCTCTTCACCTTGTGGCTCTTGT TGCTGCCGTCCAGTCTGCTGCTTCTCTCGATGGCGATCAGGTCTCTGATCTTCT GCAGCTGCTCCACGCTGGCCTTCAGGAAGGTCTCGGGGGTGTTCTGGCACAGC TCGGCCACGGCGGGGCTGGCGCTGCCGCTGCTGCTGCCCAGGGCGGCGGCG GCGCACTGCACGCCTCTCAGCAGGGCCACCAGGAACACCCAGCTCAGGCCGA ACTCCAT Exemplary PUB and PAW truncated human PNGase with IgG variable heavy signal peptide (SEQ ID NO: 8): MEFGLSWVFLVALLRGVQCKASVEQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQP AASTQLPTTPSSNPSGLNQHTRNRQGQSSDPPSASTVAADSAILEVLQSNIQHVLV YENPALQEKALACIPVQELKRKSQEKLSRARKLDKGINISDEDFLLLELLHWFKEEFF HWVNNVLCSKCGGQTRSRDRSLLPSDDELKWGAKEVEDHYCDACQFSNRFPRYN NPEKLLETRCGRCGEWANCFTLCCRAVGFEARYVWDYTDHVWTEVYSPSQQRWL HCDACEDVCDKPLLYEIGWGKKLSYVIAFSKDEVVDVTWRYSCKHEEVIARRTKVK EALLRDTINGLNKQRQLFLSENRRKELLQRIIVELVEFISPKTPKPG IgG heavy signal peptide Exemplary PUB and PAW truncated human PNGase and MYC tag with N-terminal “GS” linker (SEQ ID NO: 11): MEFGLSWVFLVALLRGVQCKASVEQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQP AASTQLPTTPSSNPSGLNQHTRNRQGQSSDPPSASTVAADSAILEVLQSNIQHVLV YENPALQEKALACIPVQELKRKSQEKLSRARKLDKGINISDEDFLLLELLHWFKEEFF HWVNNVLCSKCGGQTRSRDRSLLPSDDELKWGAKEVEDHYCDACQFSNRFPRYN NPEKLLETRCGRCGEWANCFTLCCRAVGFEARYVWDYTDHVWTEVYSPSQQRWL HCDACEDVCDKPLLYEIGWGKKLSYVIAFSKDEVVDVTWRYSCKHEEVIARRTKVK EALLRDTINGLNKQRQLFLSENRRKELLQRIIVELVEFISPKTPKPGGSEQKLISEEDL IgG heavy signal peptide MYC tag with N-terminal “GS” linker Exemplary nucleotide sequence encoding PUB and PAW truncated human PNGase and MYC tag with N-terminal “GS” linker (SEQ ID NO: 14): ATGGAGTTCGGCCTGAGCTGGGTGTTCCTGGTGGCCCTGCTGAGAGGCGTGCA GTGCAAGGCCAGCGTGGAGCAGCTGCAGAAGATCAGAGACCTGATCGCCATCG AGAGAAGCAGCAGACTGGACGGCAGCAACAAGAGCCACAAGGTGAAGAGCAG CCAGCAGCCCGCCGCCAGCACCCAGCTGCCCACCACCCCCAGCAGCAACCCC AGCGGCCTGAACCAGCACACCAGAAACAGACAGGGCCAGAGCAGCGACCCCC CCAGCGCCAGCACCGTGGCCGCCGACAGCGCCATCCTGGAGGTGCTGCAGAG CAACATCCAGCACGTGCTGGTGTACGAGAACCCCGCCCTGCAGGAGAAGGCCC TGGCCTGCATCCCCGTGCAGGAGCTGAAGAGAAAGAGCCAGGAGAAGCTGAG CAGAGCCAGAAAGCTGGACAAGGGCATCAACATCAGCGACGAGGACTTCCTGC TGCTGGAGCTGCTGCACTGGTTCAAGGAGGAGTTCTTCCACTGGGTGAACAAC GTGCTGTGCAGCAAGTGCGGCGGCCAGACCAGAAGCAGAGACAGAAGCCTGC TGCCCAGCGACGACGAGCTGAAGTGGGGCGCCAAGGAGGTGGAGGACCACTA CTGCGACGCCTGCCAGTTCAGCAACAGATTCCCCAGATACAACAACCCCGAGA AGCTGCTGGAGACCAGATGCGGCAGATGCGGCGAGTGGGCCAACTGCTTCAC CCTGTGCTGCAGAGCCGTGGGCTTCGAGGCCAGATACGTGTGGGACTACACCG ACCACGTGTGGACCGAGGTGTACAGCCCCAGCCAGCAGAGATGGCTGCACTG CGACGCCTGCGAGGACGTGTGCGACAAGCCCCTGCTGTACGAGATCGGCTGG GGCAAGAAGCTGAGCTACGTGATCGCCTTCAGCAAGGACGAGGTGGTGGACGT GACCTGGAGATACAGCTGCAAGCACGAGGAGGTGATCGCCAGAAGAACCAAGG TGAAGGAGGCCCTGCTGAGAGACACCATCAACGGCCTGAACAAGCAGAGACAG CTGTTCCTGAGCGAGAACAGAAGAAAGGAGCTGCTGCAGAGAATCATCGTGGA GCTGGTGGAGTTCATCAGCCCCAAGACCCCCAAGCCCGGCGGCAGCGAGCAG AAGCTGATCAGCGAGGAGGACCTGTGA In one embodiment, the glycosidase comprises or consists of: (a) a sequence that has at least 70% sequence identity to SEQ ID NO: 4, 6 or 9, or a fragment thereof; (b) a sequence that has at least 70% sequence identity to SEQ ID NO: 7 or 10, or a fragment thereof; or (c) a sequence that has at least 70% sequence identity to SEQ ID NO: 8 or 11, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4, 6 or 9, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7 or 10, or a fragment thereof. In one embodiment, the glycosidase comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8 or 11, or a fragment thereof. In one embodiment, the glycosidase is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 5, 12, 13, and/or 14, or a fragment thereof. In one embodiment, the glycosidase is encoded by a polynucleotide sequence comprising or consisting of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one or more of SEQ ID NO: 5, 12, 13, and/or 14, or a fragment thereof. Chimeric antigen receptors (CAR) “Chimeric antigen receptor" or "CAR" or "CARs" as used herein refers to engineered receptors which can confer an antigen specificity onto cells (for example T cells such as naive T cells, central memory T cells, effector memory T cells or combinations thereof). CARs are also known as artificial T-cell receptors, chimeric T-cell receptors or chimeric immunoreceptors. Preferably the CARs of the invention comprise an antigen-specific targeting region, an extracellular domain, a transmembrane domain, optionally one or more co-stimulatory domains, and an intracellular signaling domain. Antigen-specific targeting domain The antigen-specific targeting domain provides the CAR with the ability to bind to the target antigen of interest. The antigen-specific targeting domain preferably targets an antigen of clinical interest against which it would be desirable to trigger an effector immune response that results in cell killing. The antigen-specific targeting domain may be any protein or peptide that possesses the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or tumor protein, or a component thereof). The antigen-specific targeting domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest. Illustrative antigen-specific targeting domains include antibodies or antibody fragments or derivatives, extracellular domains of receptors, ligands for cell surface molecules/receptors, or receptor binding domains thereof, and tumor binding proteins. In a preferred embodiment, the antigen-specific targeting domain is, or is derived from, an antibody. An antibody-derived targeting domain can be a fragment of an antibody or a genetically engineered product of one or more fragments of the antibody, which fragment is involved in binding with the antigen. Examples include a variable region (Fv), a complementarity determining region (CDR), a Fab, a single chain antibody (scFv), a heavy chain variable region (VH), a light chain variable region (VL) and a camelid antibody (VHH). In a preferred embodiment, the binding domain is a single chain antibody (scFv). The scFv may be murine, human or humanized scFv. "Complementarity determining region" or "CDR" with regard to an antibody or antigen- binding fragment thereof refers to a highly variable loop in the variable region of the heavy chain or the light chain of an antibody. CDRs can interact with the antigen conformation and largely determine binding to the antigen (although some framework regions are known to be involved in binding). The heavy chain variable region and the light chain variable region each contain 3 CDRs. "Heavy chain variable region" or "VH" refers to the fragment of the heavy chain of an antibody that contains three CDRs interposed between flanking stretches known as framework regions, which are more highly conserved than the CDRs and form a scaffold to support the CDRs. "Light chain variable region" or "VL" refers to the fragment of the light chain of an antibody that contains three CDRs interposed between framework regions. "Fv" refers to the smallest fragment of an antibody to bear the complete antigen binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain. "Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence. Antibodies that specifically bind a tumor cell surface molecule can be prepared using methods well known in the art. Such methods include phage display, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce human antibodies. Phage display libraries of partially or fully synthetic antibodies are available and can be screened for an antibody or fragment thereof that can bind to the target molecule. Phage display libraries of human antibodies are also available. Once identified, the amino acid sequence or polynucleotide sequence coding for the antibody can be isolated and/or determined. Examples of antigens which may be targeted by the CAR of the invention include but are not limited to antigens expressed on cancer cells and antigens expressed on cells associated with various hematologic diseases, autoimmune diseases, inflammatory diseases and infectious diseases. With respect to targeting domains that target cancer antigens, the selection of the targeting domain will depend on the type of cancer to be treated, and may target tumor antigens. A tumor sample from a subject may be characterized for the presence of certain biomarkers or cell surface markers. For example, breast cancer cells from a subject may be positive or negative for each of Her2Neu, Estrogen receptor, and/or the Progesterone receptor. A tumor antigen or cell surface molecule is selected that is found on the individual subject's tumor cells. Preferably the antigen-specific targeting domain targets a cell surface molecule that is found on tumor cells and is not substantially found on normal tissues, or restricted in its expression to non-vital normal tissues. Further antigens specific for cancer which may be targeted by the CAR of the invention include but are not limited to any one or more of carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, ROR1, mesothelin, c-Met, GD-2, and MAGE A3 TCR, 4-1BB, 5T4, adenocarcinoma antigen, alpha- fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), CCR4, CD152, CD200, CD22, CD19, CD22, CD123, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44, CD44 v6, CD51, CD52, CD56, CD74, CD80, CS-1, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N- glycolylneuraminic acid, NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, cadherin CDH-17 or vimentin. Further antigens specific for cancer which may be targeted by a CAR include but are not limited to any one or more of mesothelin, EGFRvIII, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-l, CD33, GD2, GD3, BCMA, Tn Ag, prostate specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-l3Ra2, interleukin-11 receptor a (IL-l lRa), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor- beta (PDGFR-beta), SSEA-4, CD20, Folate receptor alpha (FRa), ERBB2 (Her2/neu), MUC1, epidermal growth factor receptor (EGFR), NCAM, Prostase, PAP, EFF2M, Ephrin B2, IGF-I receptor, CAIX, FMP2, gplOO, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sFe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CFDN6, GPRC5D, CXORF61, CD97, CD l79a, AFK, Polysialic acid, PFAC1, GloboH, NY-BR-l, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO- l, LAGE-la, MAGE-A1, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-l, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B 1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY- TES 1, LCK, AKAP-4, SSX2, RAGE-l, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1. Antigens specific for inflammatory diseases which may be targeted by the CAR of the invention include but are not limited to any one or more of AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin- 1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (a chain of IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, Lama glama, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAb β7, scleroscin, SOST, TGF β1, TNF-a or VEGF-A. Antigens specific for neuronal disorders which may be targeted by the CAR of the invention include but are not limited to any one or more of beta amyloid or MABT5102A. Further antigens which may be targeted by the CAR of the invention include but are not limited to any one or more of IL-1β or CD3. Antigens specific for cardiovascular diseases which may be targeted by the CARs of the invention include but are not limited to any one or more of C5, cardiac myosin, CD41 (integrin alpha-IIb), fibrin II, beta chain, ITGB2 (CD18) and sphingosine-1-phosphate. Further antigens which may be targeted by the CARs of the invention include but are not limited to Claudin18.2, GRP78, AFP peptide/A2, CD70, CD133, CD147, cMet, DLL3, EGFR806, FBP, ICAM1, MG7, p32, CS1 (SLAMF7 or CD319), CXCR5, CD318, TSPAN8, CD66c, CD229, LMP1, CD276, CD138, AXL, CD147, CLDN6, DLL3, DR5, gp100, LeY, MMP2, MUC16, MUC16ecto, NECTIN4, NKG2D, NKG2DL, ROR2, TM4SF1, TnMUC1, CD7, CD99, TRBC1, CCR9, Siglec-6, CD229, APRIL. Preferably, the antigen-specific binding domain specifically binds to a tumor antigen. In a specific embodiment, the polynucleotide codes for a single chain Fv that specifically binds CD44v6. In a specific embodiment, the polynucleotide codes for a single chain Fv that specifically binds CEA. Co-stimulatory domain The CAR of the invention may also comprise one or more co-stimulatory domains. This domain may enhance cell proliferation, cell survival and development of memory cells. Each co-stimulatory domain comprises the co-stimulatory domain of any one or more of, for example, members of the TNFR super family, CD28, CD137 (4-1BB), CD134 (OX40), DaplO, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-1, TNFR-II, Fas, CD30, CD40 or combinations thereof. Co-stimulatory domains from other proteins may also be used with the CAR of the invention. Additional co-stimulatory domains will be apparent to those of skill in the art. In some embodiments, the co-stimulatory domain is a CD28 co-stimulatory domain. Intracellular signaling domain The CAR of the invention may also comprise an intracellular signaling domain. This domain may be cytoplasmic and may transduce the effector function signal and direct the cell to perform its specialized function. Examples of intracellular signaling domains include, but are not limited to, ζ chain of the T-cell receptor or any of its homologs (e.g., η chain, FcεR1γ and β chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), CD3 polypeptides (∆, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, 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, immunoreceptor tyrosine- based activation motif (ITAM) bearing cytoplasmic receptors or combinations thereof. Additional intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. Transmembrane domain The CAR of the invention may also comprise a transmembrane domain. The transmembrane domain may comprise the transmembrane sequence from any protein which has a transmembrane domain, including any of the type I, type II or type III transmembrane proteins. The transmembrane domain of the CAR of the invention may also comprise an artificial hydrophobic sequence. The transmembrane domains of the CARs of the invention may be selected so as not to dimerize. Additional transmembrane domains will be apparent to those of skill in the art. Examples of transmembrane (TM) regions used in CAR constructs are: 1) The CD28 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41; Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35; Casucci et al, Blood, 2013, Nov 14;122(20):3461-72.); 2) The OX40 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41); 3) The 41BB TM region (Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35); 4) The CD3 zeta TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41; Savoldo B, Blood, 2009, Jun 18;113(25):6392-402.); 5) The CD8a TM region (Maher et al, Nat Biotechnol, 2002, Jan;20(1):70-5.; Imai C, Leukemia, 2004, Apr;18(4):676-84; Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35; Milone et al, Mol Ther, 2009, Aug;17(8):1453-64.). In some embodiments, the transmembrane domain is a CD28 transmembrane domain. Spacer domain The CAR of the invention may comprise an extracellular spacer domain. The extracellular spacer domain may be attached to the antigen-specific targeting region and the transmembrane domain. In some embodiments, the spacer is an IgG1-derived hinge spacer. The CAR of the present invention may comprise an extracellular spacer which comprises at least part of the extracellular domain of human low affinity nerve growth factor (LNGFR) or a derivative thereof. LNGFR is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of transduced gene expression by immunofluorescence, with single cell resolution. Thus, fluorescence activated cell sorter analysis of expression of LNGFR may be performed in transduced cells to study gene expression. Further details on analysis using LNGFR may be found in Mavilio (1994) Blood 83, 1988-1997. In one embodiment, the CAR of the invention comprises a truncated LNGFR (also known as ∆LNGFR). Preferably the LNGFR used in the present invention is truncated in its intracytoplasmic domain. Such a truncation is described in Mavilio (1994). Thus, preferably the LNGFR spacer of the present invention comprises at least part of the extracellular domain or a derivative thereof but lacks the intracellular domain of LNGFR. The extracellular domain may comprise amino acids 29 – 250 of LNGFR or a derivative thereof. Exemplary human LNGFR [UNIPROT accession P08138, TNR16_HUMAN] (SEQ ID NO: 15): MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQ PCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCA YGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPC LPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPE QDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKRWNS CKQNKQGANSRPVNQTPPPEGEKLHSDSGISVDSQSLHDQQPHTQTASGQALKG DGGLYSSLPPAKREEVEKLLNGSAGDTWRHLAGELGYQPEHIDSFTHEACPVRALL ASWATQDSATLDALLAALRRIQRADLVESLCSESTATSPV Exemplary extracellular domain of the human LNGFR [UNIPROT accession P08138, TNR16_HUMAN, position 29 – 250] (SEQ ID NO: 16) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPC KPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFS CQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIP GRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTT DN Preferably the LNGFR lacks the signal peptide. In one embodiment, the spacer comprises at least part of a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the extracellular domain of LNGFR (e.g., SEQ ID NO: 16). In one embodiment, the spacer comprises at least part of a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to amino acids 29-250 of the LNGFR protein (e.g., SEQ ID NO: 15). In one embodiment, the spacer comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 16. In one embodiment, the spacer comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to amino acids 29-250 of SEQ ID NO: 15. Exemplary LNGFR spacer (SEQ ID NO: 31) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPC KPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFS CQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEE Exemplary LNGFR spacer (SEQ ID NO: 32) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPC KPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFS CQDKQNTVCEECPDGTYSDEAARAADAECEEIPGRWITRSTPPEGSDSTAPSTQE PEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDN Exemplary LNGFR spacer (SEQ ID NO: 33) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPC KPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFS CQDKQNTVCEECPDGTYSDEAARAADAECEE In one embodiment the spacer comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 31 – 33. LNGFR comprises 4 TNFR-Cys domains (TNFR-Cys 1, TNFR-Cys 2, TNFR-Cys 3 and TNFR-Cys 4). Sequences of the domains are exemplified below: TNFR-Cys 1 (SEQ ID NO: 17) ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC TNFR-Cys 2 (SEQ ID NO: 18) PCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVC TNFR-Cys 3 (SEQ ID NO: 19) RCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVC TNFR-Cys 4 (SEQ ID NO: 20) ECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAEC In one embodiment, the spacer comprises TNFR-Cys 1, 2 and 3 domains or fragments or derivatives thereof. In another embodiment, the spacer comprises the TNFR-Cys 1, 2, 3 and 4 domains or fragments or derivatives thereof. In one embodiment the spacer comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or 100% identity to TNFR-Cys 1 (SEQ ID NO: 17), a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or 100% identity to TNFR- Cys 2 (SEQ ID NO: 18), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or 100% identity to TNFR-Cys 3 (SEQ ID NO: 19). The spacer may further comprise a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or 100% identity to TNFR-Cys 4 (SEQ ID NO: 20). Rather than comprise the full TNFR-Cys 4 domain, the spacer may comprise a TNFR-Cys 4 domain with the following amino acids deleted from said domain: NHVDPCLPCTVCEDTERQLRECTRW (SEQ ID NO: 21). In one embodiment, the NHVDPCLPCTVCEDTERQLRECTRW amino acids are replaced with the following amino acids: ARA. In one embodiment the spacer lacks the LNGFR serine/threonine-rich stalk. In another embodiment the spacer comprises the LNGFR serine/threonine-rich stalk. The spacer may comprise or consist of a sequence of SEQ ID NO: 17 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 17. The spacer may comprise or consist of a sequence of SEQ ID NO: 18 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 18. The spacer may comprise or consist of a sequence of SEQ ID NO: 19 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 19. The spacer may comprise or consist of a sequence of SEQ ID NO: 20 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 20. The spacer may comprise or consist of a sequence of SEQ ID NO: 31 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 31. The spacer may comprise or consist of a sequence of SEQ ID NO: 32 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 32. The spacer may comprise or consist of a sequence of SEQ ID NO: 33 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 33. The spacer may confer properties to the CAR such that it allows for immunoselection of cells, preferably T-cells, expressing said CAR. The CAR of the present invention (e.g. comprising the spacer referred to herein) preferably enables T-cells expressing the CAR to proliferate in the presence of cells expressing the antigen for which the CAR is designed. The CAR of the present invention (e.g. comprising the spacer referred to herein) preferably enables T-cells expressing the CAR to mediate therapeutically significant anti-cancer effects against a cancer that the CAR is designed to target. The CAR of the present invention (e.g. comprising the spacer referred to herein) is preferably suitable for facilitating immunoselection of cells transduced with said CAR. An exemplary CAR of the present invention comprising the LNGFR-based spacer may avoid activation of unwanted and potentially toxic off-target immune responses and may allow CAR-expressing T cells to persist in vivo without being prematurely cleared by the host immune system. As described herein, the present invention also encompasses the use of variants, derivatives, homologues and fragments of the spacer elements described herein. Exemplary CAR In one embodiment, the CAR is an anti-CD44v6 CAR (44v6.28z). In some embodiments, the CAR comprises an IgG1-derived hinge spacer, a CD28 transmembrane and costimulatory domain and a CD3ζ endodomain Exemplary 44v6.28z CAR protein sequence (SEQ ID NO: 22): MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCSASSSINYIYWLQQK PGQAPRILIYLTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLT FGGGTKVEIKRGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYD MSWVRQAPGKGLEWVSTISSGGSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAE DTAVYYCARQGLDYWGRGTLVTVSSGPVEPKSCDKTHTCPPCPPLIKFWVLVVVG GVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA AYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR Exemplary nucleotide sequence encoding 44v6.28z CAR (SEQ ID NO: 23): ATGGAAGCCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCCGACAC CACCGGCGAGATCGTGCTGACACAGAGCCCCGCCACCCTGTCTCTGAGCCCTG GCGAGAGAGCCACCCTGAGCTGTAGCGCCAGCAGCAGCATCAACTACATCTAC TGGCTGCAGCAGAAGCCCGGCCAGGCCCCCAGAATCCTGATCTACCTGACCAG CAACCTGGCCAGCGGCGTGCCCGCCAGATTTTCTGGCAGCGGCAGCGGCACC GACTTCACCCTGACCATCAGCAGCCTGGAACCCGAGGACTTCGCCGTGTACTA CTGCCTGCAGTGGTCCAGCAACCCCCTGACCTTCGGCGGAGGCACCAAGGTG GAAATCAAGCGGGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGAGGTGCAGCT GGTGGAAAGCGGCGGAGGCCTGGTCAAGCCTGGCGGCAGCCTGAGACTGAGC TGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTCCGACA GGCTCCAGGCAAGGGACTGGAATGGGTGTCCACCATCAGCAGCGGCGGCAGC TACACCTACTACCTGGACAGCATCAAGGGCCGGTTCACCATCAGCCGGGACAA CGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACC GCCGTCTACTACTGTGCCCGGCAGGGCCTCGACTACTGGGGCAGAGGCACCC TGGTCACCGTGTCCAGTGGACCGGTCGAGCCCAAGAGCTGCGACAAGACCCAC ACCTGTCCCCCCTGCCCCCCCTTAatTAAAttTTGGGTGCTGGTGGTGGTTGGTG GAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGT GAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCC GCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGA CTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCG CGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGA GAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGG GAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAA GATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGA GGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGA CACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCTAA In one embodiment, the CAR comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22, or a fragment thereof. In one embodiment, the CAR is encoded by a polynucleotide sequence comprising or consisting of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23, or a fragment thereof. Signal peptides A signal peptide is a short peptide that functions in protein targeting and translocation. Signal peptides are co-translated as part of a longer polypeptide, which they direct the targeting of. Signal peptides may direct a polypeptide towards a secretory pathway, i.e., for secretion, or via another intracellular pathway for additional processing. Signal peptides may also be referred to as a signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide. Signal peptides may be grafted onto polypeptides with which they are not naturally associated, in order to direct the new modified peptide to a specific cellular compartment, or along a desired pathway, e.g., a secretory pathway. Here, by “grafted” it is meant that a signal peptide may form a fusion protein with any one or more polypeptide. Preferably, signal peptides may be combined with glycosidases according to the invention. Glycosidases of the invention may comprise a signal peptide, such as an heterologous signal peptide. In one embodiment, the signal peptide directs the secretion of the glycosidase. In one embodiment, the signal peptide and glycosidase comprise a fusion protein. A signal peptide may be cleaved, e.g., by a signal peptidase, from the polypeptide of which it is a component. In one embodiment, the signal peptide and glycosidase may transiently comprise a fusion protein. In one embodiment, the signal peptide is cleaved from the glycosidase in a cell. In one embodiment, the glycosidase is secreted from a cell following cleavage of the signal peptide. Any signal peptide capable of directing the secretion of a glycosidase according to the invention may be used. Online databases exist that provide signal peptide sequences which can be readily accessed and searched, for example http://www.signalpeptide.de/index.php?m=listspdb_mammalia. Exemplary signal peptides Glycosidases may be modified by the addition of a signal peptide to facilitate their secretion. Any signal peptide that may drive the secretion of a glycosidase according to the invention may be suitable for use. Without limitation, the signal peptide may be selected from the group consisting of: a CD8a signal peptide, an IgG variable region heavy chain signal peptide, a GM- CSF/CSF signal peptide, an Ig kappa chain V-III region VG signal peptide and a CSFR2A signal peptide. In one embodiment, the signal peptide is a CD8 signal peptide. The CD8 signal peptide may also be referred to as CD8a. Exemplary CD8 signal peptide [residues 1 – 12; Uniprot accession P01732] (SEQ ID NO: 24): MALPVTALLLPLALLLHAARP In one embodiment, the signal peptide comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24, or a fragment thereof. In one embodiment, the signal peptide comprises SEQ ID NO: 24. In one embodiment, the signal peptide consists of SEQ ID NO: 24. In one embodiment, the signal peptide is a IgG variable heavy signal peptide. Exemplary IgG variable heavy signal peptide [residues 1 – 19; Uniprot accession P01768] (SEQ ID NO: 25): MEFGLSWVFLVALLRGVQC In one embodiment, the signal peptide comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 25, or a fragment thereof. In one embodiment, the signal peptide comprises SEQ ID NO: 25. In one embodiment, the signal peptide consists of SEQ ID NO: 25. In one embodiment, the signal peptide is a GM-CFS/CSF signal peptide. Exemplary GM-CFS/CSF signal peptide [residues 1 – 17; Uniprot accession P04141] (SEQ ID NO: 26): MWLQSLLLLGTVACSIS In one embodiment, the signal peptide comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 26, or a fragment thereof. In one embodiment, the signal peptide comprises SEQ ID NO: 26. In one embodiment, the signal peptide consists of SEQ ID NO: 26. In one embodiment, the signal peptide is an Ig kappa chain V-III region VG signal peptide. Exemplary Ig kappa chain V-III region VG signal peptide [residues 1 – 20; Uniprot accession P04433] (SEQ ID NO: 27): MEAPAQLLFLLLLWLPDTTG In one embodiment, the signal peptide comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 27, or a fragment thereof. In one embodiment, the signal peptide comprises SEQ ID NO: 27. In one embodiment, the signal peptide consists of SEQ ID NO: 27. In one embodiment, the signal peptide is a CSFR2A signal peptide. Exemplary CSFR2A signal peptide [residues 1 – 22; NCBI ref. sequence NP_758452.1] (SEQ ID NO: 28): MLLLVTSLLLCELPHPAFLLIP In one embodiment, the signal peptide comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 28, or a fragment thereof. In one embodiment, the signal peptide comprises SEQ ID NO: 28. In one embodiment, the signal peptide consists of SEQ ID NO: 28. Tags and linkers The polynucleotide and polypeptide sequences of the invention may further comprise tag and/or linker sequences. Linkers At both the polynucleotide and polypeptide levels, certain functional sequence elements may be separated by linkers. Linkers typically comprise a short polynucleotide or polypeptide sequence. Linkers may be used to physically separate functional sequences in order, e.g., to improve the functionality of said sequences. In one embodiment, the polynucleotide of the invention comprises one or more linker sequences. In one embodiment, the sequence encoding the glycosidase is separated from another functional sequence by a linker. In one embodiment, the glycosidase of the invention comprises one or more linker sequences. In one embodiment, the linker is a GS linker, or a GGS linker. Tags Both the polynucleotides and polypeptides of the invention may comprise tags. Tags are typically sequences that facilitate the detection or isolation of the molecule to which they are attached. Tags may be particularly useful in experimental studies utilising the polynucleotides or polypeptides of the invention. In one embodiment, the polynucleotide of the invention comprises one or more tag sequences. In one embodiment, the glycosidase of the invention comprises one or more tag sequences. In one embodiment, the tag is a MYC tag. Exemplary MYC tag (SEQ ID NO: 29): EQKLISEEDL Exemplary MYC tag with “GS” linker (SEQ ID NO: 30): GSEQKLISEEDL In one embodiment, the tag comprises or consists of a sequence that has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 29 or 30, or a fragment thereof. In one embodiment, the tag comprises SEQ ID NO: 29 or 30. In one embodiment, the tag consists of SEQ ID NO: 29 or 30. Expression control sequences The polynucleotide of the invention may comprise one or more expression control sequence. Suitably, the nucleic acid sequence encoding the glycosidase or CAR is operably linked to one or more expression control sequence. As used herein an “expression control sequence” may refer to a nucleotide sequence which controls expression of a transgene, e.g. to facilitate and/or increase expression. The expression control sequence and the transgene may be in any suitable arrangement in the polynucleotide, providing that the expression control sequence is operably linked to the transgene (e.g. nucleic acid sequence encoding the glycosidase or CAR). Promoters In some embodiments, the expression control sequence is a promoter. Any suitable promoter may be used, the selection of which may be readily made by the skilled person. The promoter sequence may be constitutively active (i.e. operational in any host cell background), or alternatively may be active only in a specific host cell environment, thus allowing for targeted expression of the nucleotide of interest (e.g. glycosidase and/or CAR) in a particular cell type (e.g. a tissue-specific promoter). The promoter may show inducible expression in response to presence of another factor, for example a factor present in a host cell. In any event, where the vector is administered for therapy, it is preferred that the promoter should be functional in the target cell background. In some embodiments, the polynucleotide further comprises a promoter operably linked to the nucleic acid sequence encoding the glycosidase. In some embodiments, the polynucleotide further comprises a promoter operably linked to the nucleic acid sequence encoding the CAR. In some embodiments, the polynucleotide further comprises a promoter operably linked to the nucleic acid sequence encoding the glycosidase and the nucleic acid sequence encoding the CAR. In some embodiments, the promoter is a constitutive promoter. In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR are operably linked to one or more promoter(s). In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR are operably linked to the same promoter(s). The nucleotide sequences encoding the glycosidase and the CAR may share a promoter such that their expression may be regulated by a single regulatory sequence. In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR are independently operably linked to one or more promoter(s). The nucleotide sequences encoding the glycosidase and the CAR may each be operably linked to separate promoter such that their expression may be independently regulated by independent regulatory sequences. In one embodiment, the nucleotide sequences encoding the glycosidase and the CAR are operably linked to separate promoter(s). In one embodiment, the glycosidase and the CAR are encoded in opposing directions. In one embodiment, the glycosidase and the CAR are encoded in opposing directions and are independently operably linked to separate promoters. In one embodiment, the glycosidase and the CAR are encoded in the same direction. In one embodiment, the promoter is selected from the group consisting of: a cytomegalovirus promoter (CMV), a human phosphoglycerate kinase promoter (PGK), an EF-1α promoter and an inducible NFAT promoter. In one embodiment, the promoter is a cytomegalovirus (CMV) promoter. In another embodiment, the promoter is a minimal cytomegalovirus (mCMV) promoter (mCMV, see, for example, Amendola (2005) Nat Biotech 23: 108-116). In one embodiment, the promoter is human phosphoglycerate kinase (PGK) promoter. In one embodiment, the promoter is an EF-1α promoter. In one embodiment, the promoter is an an inducible NFAT promoter. The inducible module may be composed of a synthetic NFAT response element usually comprising repetitions of the consensus NFAT binding site placed upstream of a minimal promoter. Proteins The term “protein” as used herein includes single-chain polypeptide molecules as well as multiple-polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means. The terms “polypeptide” and “peptide” as used herein refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds. The term protein is also intended to encompass glycoproteins or glycopeptides, for example, glycoproteins/peptides, that are the target of the glycosidases herein. The proteins of the invention include any of the proteins disclosed herein with a methionine at the N-terminus. Polynucleotides Polynucleotides of the invention may, for example, comprise DNA or RNA. They may be single-stranded or double-stranded. It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that the skilled person may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed. 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 lifespan of the polynucleotides of the invention. Polynucleotides such as DNA polynucleotides may be produced recombinantly, synthetically or by any means available to the skilled person. They may also be cloned by standard techniques. Longer polynucleotides will generally be produced using recombinant means, for example using polymerase chain reaction (PCR) cloning techniques. This may involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking the target sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture with an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector. Vectors A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the invention, and by way of example, some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g. a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. The vector may serve the purpose of maintaining the heterologous nucleic acid (DNA or RNA) within the cell, facilitating the replication of the vector comprising a segment of nucleic acid or facilitating the expression of the protein encoded by a segment of nucleic acid. Vectors may be non-viral or viral. Examples of vectors used in recombinant nucleic acid techniques include, but are not limited to, plasmids, mRNA molecules (e.g. in vitro transcribed mRNAs), chromosomes, artificial chromosomes and viruses. The vector may also be, for example, a naked nucleic acid (e.g. DNA). In its simplest form, the vector may itself be a nucleotide of interest. The vectors used in the invention may be, for example, plasmid, mRNA or virus vectors and may include a promoter for the expression of a polynucleotide and optionally a regulator of the promoter. Vectors comprising polynucleotides used in the invention may be introduced into cells using a variety of techniques known in the art, such as transfection, transformation and transduction. Several such techniques are known in the art, for example infection with recombinant viral vectors, such as retroviral, lentiviral (e.g. integration-defective lentiviral), adenoviral, adeno- associated viral, baculoviral and herpes simplex viral vectors; direct injection of nucleic acids and biolistic transformation. Non-viral delivery systems include but are not limited to DNA transfection methods. Here, transfection includes a process using a non-viral vector to deliver a gene to a target cell. Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14: 556) and combinations thereof. Transfection of cells with mRNA vectors can be achieved, for example, using nanoparticles, such as liposomes. In some embodiments, the vector is comprised in a nanoparticle. In some embodiments, the nanoparticle is a polymeric nanoparticle, inorganic nanoparticle or lipid nanoparticle. In some embodiments, the nanoparticle is a liposome. The nanoparticle may be targeted to a specific cell type(s) using one or more ligand displayed on its surface. In one embodiment, polynucleotide delivery is transposon mediated. In one embodiment, the polynucleotide is an mRNA. The mRNA may be comprised in a nanoparticle. Viral vectors In preferred embodiments, the vector is a viral vector. The viral vector may be in the form of a viral vector particle. The viral vector may be, for example, a retroviral, lentiviral, adeno-associated viral (AAV) or adenoviral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an AAV vector. Retroviral and lentiviral vectors A retroviral vector may be derived from or may be derivable from any suitable retrovirus. A large number of different retroviruses have been identified. Examples include murine leukaemia virus (MLV), human T-cell leukaemia virus (HTLV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukaemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29) and avian erythroblastosis virus (AEV). A detailed list of retroviruses may be found in Coffin et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63. Retroviruses may be broadly divided into two categories, “simple” and “complex”. Retroviruses may be even further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses. A review of these retroviruses is presented in Coffin et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63. The basic structure of retrovirus and lentivirus genomes share many common features such as a 5’ LTR and a 3’ LTR. Between or within these are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome, and gag, pol and env genes encoding the packaging components – these are polypeptides required for the assembly of viral particles. Lentiviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell. In the provirus, these genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for proviral integration and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes. The LTRs themselves are identical sequences that can be divided into three elements: U3, R and U5. U3 is derived from the sequence unique to the 3’ end of the RNA. R is derived from a sequence repeated at both ends of the RNA. U5 is derived from the sequence unique to the 5’ end of the RNA. The sizes of the three elements can vary considerably among different retroviruses. In a defective retroviral vector genome gag, pol and env may be absent or not functional. In a typical retroviral vector, at least part of one or more protein coding regions essential for replication may be removed from the virus. This makes the viral vector replication-defective. Lentivirus vectors are part of the larger group of retroviral vectors. A detailed list of lentiviruses may be found in Coffin et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758- 63. Lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include but are not limited to human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS); and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototype “slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV). The lentivirus family differs from retroviruses in that lentiviruses have the capability to infect both dividing and non-dividing cells (Lewis et al. (1992) EMBO J. 11: 3053-8; Lewis et al. (1994) J. Virol.68: 510-6). In contrast, other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue. A lentiviral vector, as used herein, is a vector which comprises at least one component part derivable from a lentivirus. Preferably, that component part is involved in the biological mechanisms by which the vector infects cells, expresses genes or is replicated. The lentiviral vector may be a “primate” vector. The lentiviral vector may be a “non-primate” vector (i.e. derived from a virus which does not primarily infect primates, especially humans). Examples of non-primate lentiviruses may be any member of the family of lentiviridae which does not naturally infect a primate. Preferably, the viral vector used in the present invention has a minimal viral genome. By “minimal viral genome” it is to be understood that the viral vector has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in WO 1998/017815. Preferably, the plasmid vector used to produce the viral genome within a host cell/packaging cell will have sufficient lentiviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle which is capable of infecting a target cell, but is incapable of independent replication to produce infectious viral particles within the final target cell. Preferably, the vector lacks a functional gag-pol and/or env gene and/or other genes essential for replication. However, the plasmid vector used to produce the viral genome within a host cell/packaging cell will also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a host cell/packaging cell. These regulatory sequences may be the natural sequences associated with the transcribed viral sequence (i.e. the 5’ U3 region), or they may be a heterologous promoter, such as another viral promoter (e.g. the CMV promoter). The vectors may be self-inactivating (SIN) vectors in which the viral enhancer and promoter sequences have been deleted. SIN vectors can be generated and transduce non-dividing cells in vivo with an efficacy similar to that of wild-type vectors. The transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus should prevent mobilisation by replication- competent virus. This should also enable the regulated expression of genes from internal promoters by eliminating any cis-acting effects of the LTR. The vectors may be integration-defective. Integration defective lentiviral vectors (IDLVs) can be produced, for example, either by packaging the vector with catalytically inactive integrase (such as an HIV integrase bearing the D64V mutation in the catalytic site) or by modifying or deleting essential att sequences from the vector LTR, or by a combination of the above. Cells The invention provides a cell comprising the polynucleotide or product, the vector, or the glycosidase of the invention. The cell of the invention may comprise any suitable cell type from any suitable organism or subject. In one embodiment the cell is a mammalian cell. In another embodiment, the cell is a human cell. In one embodiment, the cell is a cell from a subject. In one embodiment, the subject is a human subject. In one embodiment, the cell is a T cell. In one embodiment, the cell is a natural killer (NK) cell. In one embodiment, the cell is a hematopoietic stem cell (HSC). In one embodiment, the cell is a hematopoietic stem and/or progenitor cell (HSPC). In one embodiment, the cell is a tumor-infiltrating lymphocyte (TIL). In one embodiment, the cell is an invariant-NK T cell, a cytokine-induced killer cell (CIK) or a macrophage. TILs are T cells that can be isolated from a tumor. TILs are enriched in natural T cells that recognize the tumor antigens. Isolated TILs can be expanded and modified, such as transduced with a polynucleotide or vector according to the invention, ex vivo and re- introduced to a tumor or subject. Since TILs may naturally have specificity for tumor cells, they may not require a CAR for targeting. Thus, in one embodiment, the TIL does not comprise the CAR. In one embodiment, the TIL comprises the glycosidase. Variants, derivatives, analogues, homologues, and fragments In addition to the specific proteins and polynucleotides mentioned herein, the invention also encompasses the use of variants, derivatives, analogues, homologues and fragments thereof. In the context of the invention, a variant of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and/or variation of at least one residue present in the naturally-occurring protein. The term “derivative” as used herein in relation to proteins or polypeptides of the invention includes any substitution of, variation of, modification of, replacement of, deletion of and/or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide substantially retains at least one of its endogenous functions. The term “analogue” as used herein in relation to polypeptides or polynucleotides includes any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics. Typically, amino acid substitutions may be made, for example from 1, 2 or 3 to 10 or 20 substitutions provided that the modified sequence substantially retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues. Proteins used in the invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine. Conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
Figure imgf000048_0001
The term “homologue” as used herein means an entity having a certain homology with the wild type amino acid sequence or the wild type nucleotide sequence. The term “homology” can be equated with “identity”. A homologous sequence may include an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95% or 97% or 99% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity. A homologous sequence may include a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95% or 97% or 99% identical to the subject sequence. Although homology can also be considered in terms of similarity, in the context of the present invention it is preferred to express homology in terms of sequence identity. Preferably, reference to a sequence which has a percent identity to any one of the SEQ ID NOs disclosed herein refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to. Homology comparisons can be conducted by eye or, more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percentage homology or identity between two or more sequences. Percentage homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues. Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the nucleotide sequence may cause the following codons to be put out of alignment, thus potentially resulting in a large reduction in percent homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local homology. However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum percentage homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al. (1984) Nucleic Acids Res.12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid – Ch.18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187-8). Although the final percentage homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix – the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62. Once the software has produced an optimal alignment, it is possible to calculate percentage homology, preferably percentage sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. “Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full- length polypeptide or polynucleotide. Such variants may be prepared using standard recombinant DNA techniques such as site- directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5’ and 3’ flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used. Codon optimisation The polynucleotides used in the invention may be codon-optimised. Codon optimisation has previously been described in WO 1999/41397 and WO 2001/79518. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. It is also possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available. Compositions The polynucleotides, proteins, vectors, and cells of the invention may be formulated for administration to subjects with a pharmaceutically-acceptable carrier, diluent or excipient. Suitable carriers and diluents include isotonic saline solutions, for example phosphate- buffered saline, and potentially contain human serum albumin. Materials used to formulate a pharmaceutical composition should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may be determined by the skilled person according to the route of administration. The pharmaceutical composition is typically in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, a surfactant, such as pluronic acid (PF68) 0.001% may be used. In some cases, serum albumin may be used in the composition. For injection, the active ingredient may be in the form of an aqueous solution, which is pyrogen-free, and has suitable pH, isotonicity and stability. The skilled person is well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection or Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included as required. For delayed release, the medicament may be included in a pharmaceutical composition which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art. Handling of the cell therapy products is preferably performed in compliance with FACT-JACIE International Standards for cellular therapy. Methods of treatment In one aspect, the invention provides the polynucleotide, product, vector, glycosidase, cell, composition or pharmaceutical composition of the invention for use in therapy. In one aspect, the invention provides the polynucleotide, product, vector, glycosidase, cell, composition or pharmaceutical composition of the invention for use in the treatment of cancer. All references herein to treatment include curative, palliative and prophylactic treatment. The treatment of mammals, particularly humans, is preferred. Both human and veterinary treatments are within the scope of the invention. In some embodiments, the method of treatment provides the polynucleotide, product, vector, glycosidase, cell, composition or pharmaceutical composition of the invention to a tumor. In one aspect, there is provided the polynucleotide or product, the vector, the glycosidase, or the cell according to the invention, for use in therapy. In one aspect, there is provided the polynucleotide or product, the vector, the glycosidase, or the cell according to the invention, for use in the treatment of cancer. In one embodiment, the cancer is a solid tumor. In one embodiment, the cancer is a solid or haematopoietic or lymphoid tumor. In one embodiment, the cancer is a haematopoietic or lymphoid tumor. In one embodiment, the solid tumor is selected from the group consisting of: colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angio genesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers. In one embodiment, the haematopoietic or lymphoid tumor is selected from the group consisting of: chronic lymphocytic leukemia (CLL), acute leukemias, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitfs lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin’s lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or preleukemia, combinations of said cancers, and metastatic lesions of said cancers. In one embodiment, the cancer is a neuroendocrine tumor. In one aspect, there is provided use of the polynucleotide or product, the vector, the glycosidase, or the cell according to the invention, for improving CAR cell activity. In one aspect, there is provided a method of producing a CAR cell, comprising introducing the polynucleotide or product, the vector, or the glycosidase into a cell. In one embodiment, the cell is a CAR-T cell. In a further embodiment, there is provided the method or use, wherein the therapeutic activity of the CAR cell or CAR-T cell is improved. In one embodiment, the therapeutic activity is target cell killing. In one aspect, there is provided a method of treatment comprising producing a CAR cell according to the method of the invention, and administering the CAR cell to a subject in need thereof. Administration In some embodiments, the polynucleotide, product, vector, glycosidase, cell, composition or pharmaceutical composition is administered to a subject locally. Local administration may include administration to the tumor of interest. When a glycosidase is locally administered to a tumor, the glycosidase may lack a signal peptide. In preferred embodiments, the polynucleotide, product, vector, glycosidase, cell, composition or pharmaceutical composition is administered to a tumor. In some embodiments, the polynucleotide, product, vector, glycosidase, cell, composition or pharmaceutical composition is administered to a subject systemically. The term “systemic delivery” or “systemic administration” as used herein means that the agent of the invention is administered into the circulatory system, for example to achieve broad distribution of the agent. In contrast, topical or local administration restricts the delivery of the agent to a localised area, e.g. a tumor. In some embodiments, the polynucleotide, product, vector, glycosidase, cell, composition or pharmaceutical composition is administered in a nanoparticle that targets T cells in vivo. Dosage The skilled person can readily determine an appropriate dose of an agent of the invention to administer to a subject. Typically, a physician will determine the actual dosage that will be most suitable for an individual patient, which will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of the invention. Subject The term “subject” as used herein refers to either a human or non-human animal. Examples of non-human animals include vertebrates, for example mammals, such as non- human primates (particularly higher primates), dogs, rodents (e.g. mice, rats or guinea pigs), pigs and cats. The non-human animal may be a companion animal. Preferably, the subject is a human. The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed. Preferred features and embodiments of the invention will now be described by way of non- limiting examples. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch.9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O’D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the disclosed polypeptides, polynucleotides, vectors, cells, compositions, uses and methods of the invention will be apparent to the skilled person without departing from the scope and spirit of the invention. Although the invention has been disclosed in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the disclosed modes for carrying out the invention, which are obvious to the skilled person are intended to be within the scope of the following claims. EXAMPLES MATERIAL AND METHODS Cells and cell culture T cells were derived from peripheral blood of healthy donors after gradient centrifugation. All procedures were approved by the Institutional Review Board of IRCCS San Raffaele Scientific Institute and were compliant with all relevant ethical regulations. T cells were activated with CD3/CD28 beads (Gibco, 40203D) at a 3:1 ratio, transduced at day 2 and cultured in RPMI- 1640 with interleukin (IL)-7 and IL-15 (5 ng/ml, Peprotech, 200-07, 200-15). At day 6, beads were removed and CAR T cells were expanded in complete medium until Day 21. Tumor pancreatic cell line (T3M-4) and pulmonary adenocarcinoma cell line (PC9) were cultured in RPMI 1640 (Euroclone, ECB90062L) and detached with TrypLE Express enzyme (Gibco). 293T cells were employed for virus production and kept in Iscove’s Modified Dulbecco’s Medium (IMDM, Euroclone, ECB2072L). 293T cells were detached using trypsin-EDTA (Euroclone). All media were supplemented with penicillin/streptomycin (100UI/ml; Lonza, DE17-602E), glutamine (2mM; Lonza, LOBE17-605E) and 10% FBS (fetal bovine serum, Carlo Erba, FA30WS1810500). All cells were routinely tested for mycoplasma contamination by PCR (Mycoplasmacheck, Eurofins Genomics), and were negative. Generation of PNGase constructs The construct “wtPNGase” was generated by cloning the wild-type PNGase sequence (UniProt Q96IV0-1) into a bidirectional lentiviral vector. Briefly, wtPNGase was produced by GeneArt (Thermo Fisher) and cloned under the direct control of the human phosphoglycerate kinase promoter (PGK), whereas the GFP marker gene was placed under the control of a minimal core promoter derived from the cytomegalovirus (minCMV). The construct “sPNGase” was generated by adding the CD8 (UniProt P01732) leader sequence (signal peptide) at the N-terminus of the wtPNGase and cloned into the bidirectional lentiviral vector as previously described for wtPNGase. Generation of CAR constructs 44v6.28 ^ was generated by cloning the antigen-specific single chain fragment variable (scFv, BIWA-8 mAb) in frame into an original CAR incorporating an IgG1-derived hinge spacer, a CD28 transmembrane and costimulatory domain and a CD3 ^ endodomain (Savoldo (2011) J Clin Invest 121: 1822-1826). CAR cDNA was produced by GeneArt (Thermo Fisher) and cloned into a bidirectional lentiviral vector. Briefly, CAR constructs were placed under the direct control of the human phosphoglycerate kinase promoter (PGK) in place of the ΔNGFR marker gene, whereas ΔNGFR was substituted to GFP under the control of a minimal core promoter derived from the cytomegalovirus (minCMV) (Amendola (2005) Nat Biotech 23: 108- 116). Viral supernatants were produced in 293T packaging cells. 44v6.28 ^_sPNGase was generated by cloning the secreted PNGase sequence (sPNGase) into the bidirectional lentiviral vector platform under the control of minCMV promoter. In vitro functional assays CAR T cells were co-cultured with target cells at different effector:target (E:T) ratios in RPMI- 1640 fully supplemented in the absence of cytokines. After 24 hours, supernatants were collected and analyzed with the LEGENDplex bead-based cytokine immunoassay (BioLegend, 740724). After 4 days (tumors) or 3 days (primary keratinocytes), surviving cells were counted using Flow-Count Fluorospheres (Beckman Coulter, 7547053) and analyzed by flow cytometry. T cells that were untransduced or transduced with an irrelevant CAR (19.28 ^) were used as a control. Elimination index was calculated as follows: 1 – (number of residual target cells with experimental CAR T cells / number of residual target cells with control T cells). Flow cytometry Samples were washed with phosphate-buffered saline (PBS) containing 1% fetal bovine serum (FBS) and stained at 4°C for 20min. Prior to use, all antibodies were validated and titrated for the optimal on target/off target activity on human peripheral blood cells or tumor cell lines. Mouse anti-human fluorophore-conjugated antibodies specific for: CD3 allophycocyanin (APC)-Cy7 (clone UCHT1, BioLegend, 344818), CD3 fluorescein isothiocyanate (FITC, clone HI100, BioLegend, 300406), CD4 phycoerythrin (Pe, clone RPA- T4, BioLegend, 300508), CD8 Peridinin-Chlorophyll-Protein (PerCP, clone SK1, BioLegend, 344708), CD45 APC-Cy7 (clone HI30, BioLegend, 304014), CD45 PE-Cy7 (clone HI30, BioLegend, 304016), CD45 BV510 (clone 30-F11, BioLegend, 103137), CD45RA fluorescein isothiocyanate (FITC, clone HI100, BioLegend, 983002), CD62L APC (clone DREG-56, BioLegend, 304810), major histocompatibility complex II receptor (HLA-DR) APC-Cy7 (clone L243, BioLegend, 307618), and CD69 APC (clone FN50, BioLegend, 310910) were used.7- AAD (BD, 559925) or 4’,6-diamidino-2-phenylindole (DAPI), were used to determine cell vitality. For branched N-glycan surface expression analysis, cells were incubated with 50mg/mL biotinylated PHA-L for 1 hour at room temperature, washed and incubated with streptavidin (PE- or APC-conjugated, BioLegend, 405203, 405207). Relative Fluorescent Intensity (RFI) was calculated as the ratio of the mean fluorescence intensities (MFI) of a specific fluorophore-conjugated antibody over a fluorophore-conjugated control. Either secondary antibodies or control isotypes were used as control. Data were collected using FACS Canto (BD Biosciences) and analyzed with FlowJo Software. Statistical Analysis All data are presented as mean ± s.e.m. Statistical analysis was performed on GraphPad Prism 8 software. Appropriate statistical tests were used as described in the figure legends. Biological and technical replicates are indicated in figure legends. Differences with a P value <0.05 were considered statistically significant. EXAMPLE 1: PNGase de-glycosylates native glycoproteins expressed on tumor cell surface. PNGase is naturally resident in the cytosol. Thus, in order to generate a glycosidase that could be secreted, such as from an “armoured” CAR-T cell, the inventors designed a modified form of the enzyme comprising a CD8 signal peptide to direct the newly synthesized PNGase protein to the cell membrane. To assess whether the secreted form of PNGase (sPNGase) would de-glycosylate tumor glycoproteins, the inventors designed two different bidirectional lentiviral constructs. The first construct carried the wild-type PNGase (wtPNGase; Uniprot Q96IV0-1) under the control of the PGK promoter, while the expression of eGFP (i.e., marker gene) was controlled by the minimal CMV promoter (mCMV) (Figure 1a). The second construct comprised the same components, with the exception that the wtPNGase was replaced with the sPNGase (Figure 1b). To test the de-glycosylating activity of the constructs, T3M-4 tumor cells were independently transduced with the two lentiviral vectors and Phytohemagglutinin-L lectin (PHA-L) was utilized to assay the tumor’s glyco-phenotype. Importantly, while wtPNGase was expected to faithfully recapitulate endogenous enzymatic activity, and therefore to induce the expression of de-glycosylated surface protein as a result of cytosolic localization and activity of wtPNGase, T3M-4 cells transduced with wtPNGase showed a significant decrease in PHA-L binding, suggesting de-glycosylation (Figure 1c). Surprisingly, this trend was also observed with T3M-4 cells transduced with sPNGase, suggestive that sPNGase retained its enzymatic functionality and was able to de-glycosylate surface glycoproteins upon secretion by tumor cells in an autocrine/paracrine manner (Figure 1d). Following the surprising and successful extracellular deglycosylation mediated by the sPNGase, the inventors sought to optimize the design of the human PNGase in order to reduce the transgene size in an effort to improve virus production, and improve the de- glycosylation activity toward native proteins. As PNGase is physiologically active on unfolded substrates during ER-associated degradation of misfolded glycoproteins reactions, the wild- type enzyme includes domains responsible interaction with proteins involved in ERAD, but are dispensable for the catalytic activity. This is the case of the N-terminal PUB domain which associates with the cytosolic p97, favoring the extraction of ubiquitinated-misfolded proteins from the ER to the cytosol, and with the UBL domain of cytosolic HR23 that interacts with the 26S proteasome, delivering proteins for degradation. The PAW domain is located at the C- terminus and binds high mannose N-glycans. The catalytic core is located between the two domains, which is responsible for the release of N-glycan moieties from glycoproteins and comprises a transglutaminase-like (TG) core defined by a catalytic triad of cysteine, histidine, and aspartic acid, and a pair of CXXC motifs involved in Zn-binding. PNGase mutants lacking either domain were designed for their capacity to de-glycosylate native (or folded), rather than unfolded, glycoproteins as compared to the wild-type PNGase enzyme. An IgG variable heavy signal peptide and a MYC tag were included to improve secretion and detection. EXAMPLE 2: 44v6.28z_sPNGase CAR-T cells de-glycosylate T3M-4 tumor cells. Having assessed the functionality of sPNGase in de-glycosylating surface proteins, the inventors next designed an all-in-one construct in which both an anti-CD44v6 CAR (44v6.28z) and the secreted PNGase (sPNGase) were co-expressed (44v6.28z_sPNGase). A bidirectional lentiviral platform was utilised, in which sPNGase was expressed under the control of the minimal CMV promoter (mCMV) while the expression of 44v6.28z was driven by the PGK promoter (Figure 2a). In this system, similarly to the previous lentiviral (LV) constructs employed (example 1), both genes are expressed constitutively. To generate 44v6.28z_sPNGase T cells, human T lymphocytes were stimulated with αCD3/αCD28 beads, transduced with LV vectors with and without the sPNGase gene, and expanded with IL-7/IL- 15. To test the ability of 44v6.28z_sPNGase cells to de-glycosylate tumor cells, T3M-4 cells knocked-out for the expression of the CAR’s antigen CD44v6 (44v6 ko) were used, in order to avoid any possible confounding effect derived from the killing of tumor cells by CAR-T cells. Either 44v6.28z_sPNGase or control CD19 cells (19.28z) were co-cultured with 44v6 ko T3M- 4 cells at 1:5 effector-to-target ratio for 48 hours and binding to PHA-L lectin was used as read-out to assess the glyco-phenotype of tumor cells. Strikingly, tumor cells displayed a marked drop in PHA-L binding upon co-culture with 44v6.28z_sPNGase as compared to control 19.28z cells, suggestive of effective deglycosylation mediated by sPNGase, in the context of a CAR T-cell. EXAMPLE 3: PNGase expression does not alter CAR-T cells phenotype. At present, the impact of glycosylation on the functionality of CAR-T cells is unknown. In order to assess whether the constitutive expression of sPNGase by 44v6.28z_cells could affect their fitness during the manufacturing procedure, factors associated with successful CAR-T cell therapy were analysed. These factors are: the expansion kinetic upon stimulation via αCD3/αCD28 beads; the preservation of both CD4 and CD8 T cell subsets; and the preferential enrichment of early memory T cell compartments in the final cellular product. Importantly, 44v6.28z_sPNGase expanded similarly to 44v6.28z cells (Figure 3a, b) and displayed an equivalent CD4/CD8 ratio (Figure 3c), together with a marked enrichment of stem cell memory cells (TSCM, CD45RA+CD62L+, Figure 3d). EXAMPLE 4: 44v6.28z_sPNGase CAR-T cells display superior killing of pancreatic adenocarcinoma cells. After assessing the de-glycosylating capacity of 44v6.28z_sPNGase cells, their killing ability, as compared to standard 44v6.28z cells, against the pancreatic adenocarcinoma cell line T3M-4 was assessed. Firstly, co-culture experiments were performed with T3M-4 cells at a 1:5 effector-to-target (E:T) ratio. Importantly, while untransduced control T cells (UT) expectedly had no impact on tumor cells vitality, 44v6.28z_sPNGase cells displayed marked and superior killing compared to standard 44v6.28z cells (Figure 4a). To test the potency of this effect, co-culture assays were performed at multiple E:T ratios, which confirmed the improved functionality (Figure 4b). To further investigate the effects from sPNGase-mediated tumor de-glycosylation on global effector functions by CAR-T, several parameters of CAR-T cell functionality were analyzed. These include: the release of inflammatory cytokines; proliferation; and upregulation of surface activation markers. Interestingly, all parameters showed a significant increase as compared to standard 44v6.28z cells (Figure 4 c-f). EXAMPLE 5: 44v6.28z_sPNGase CAR-T cells display superior killing of lung adenocarcinoma cell lines. To test the breadth of the applicability of this strategy, e.g., toward different carcinoma settings, co-culture assays were performed in which 44v6.28z_sPNGase cells were challenged against the CD44v6+ lung adenocarcinoma PC9 cells. Also in this setting, 44v6.28z_sPNGase cells sensitized tumor cells to recognition and displayed a significant increase in tumor targeting, marked as improved killing (Figure 4a, b), higher expansion (Figure 4c) and activation (Figure 4d, e) compared to 44v6.28z control cells.
EMBODIMENTS Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (paras). 1. A polynucleotide comprising (a) a nucleotide sequence encoding a glycosidase; and (b) a nucleotide sequence encoding a chimeric antigen receptor (CAR). 2. A product comprising (a) a first polynucleotide comprising a nucleotide sequence encoding a glycosidase; and (b) a second polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR). 3. The polynucleotide or product of para 1 or para 2, wherein the glycosidase is a secreted glycosidase. 4. The polynucleotide or product of any one of paras 1 to 3, wherein the glycosidase is peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), or a variant thereof. 5. The polynucleotide or product of any one of paras 1 to 4, wherein the glycosidase comprises a signal peptide. 6. The polynucleotide or product of para 5, wherein the signal peptide is selected from the group consisting of: a CD8 signal peptide, a IgG variable region heavy chain signal peptide, an Ig kappa chain V-III region VG signal peptide, a GM-CFS/CSF signal peptide, and a CSFR2A signal peptide. 7. The polynucleotide or product of any one of paras 4 to 6, wherein the variant is a truncated PNGase, optionally wherein the variant lacks a PUB domain, a PAW domain, or both. 8. The polynucleotide or product of any one of paras 1 to 7, wherein the glycosidase comprises or consists of: (a) a sequence that has at least 70% sequence identity to SEQ ID NO: 1, or a fragment thereof; (b) a sequence that has at least 70% sequence identity to SEQ ID NO: 2, or a fragment thereof; (c) a sequence that has at least 70% sequence identity to SEQ ID NO: 3, or a fragment thereof; or (d) a sequence that has at least 70% sequence identity to SEQ ID NO: 34, or a fragment thereof. 9. The polynucleotide or product of any one of paras 1 to 8, wherein the CAR is a 44v6.28z CAR, or comprises or consists of a sequence with at least 70% sequence identity to SEQ ID NO: 22. 10. The polynucleotide or product of any one of paras 1 to 9, wherein the nucleotide sequences encoding the glycosidase and the CAR are operably linked to one or more promoter(s). 11. The polynucleotide or product of any one of paras 1 to 10, wherein the promoter is selected from the group consisting of: a cytomegalovirus promoter (CMV), a human phosphoglycerate kinase promoter (PGK), an EF-1α promoter, and an inducible NFAT promoter. 12. A vector comprising the polynucleotide or product of any one of paras 1 to 11, optionally wherein the vector is a viral vector, optionally wherein the vector is a lentiviral vector or adeno-associated viral (AAV) vector. 13. A glycosidase, wherein the glycosidase is peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), and wherein the PNGase: (a) comprises a signal peptide; and/or (b) lacks a PUB domain, a PAW domain, or both. 14. The glycosidase of para 13, wherein the glycosidase comprises or consists of: (a) a sequence that has at least 70% sequence identity to SEQ ID NO: 1, 4, 6 or 9, or a fragment thereof; (b) a sequence that has at least 70% sequence identity to SEQ ID NO: 2, 7 or 10, or a fragment thereof; (c) a sequence that has at least 70% sequence identity to SEQ ID NO: 3, 8 or 11, or a fragment thereof; or (d) a sequence that has at least 70% sequence identity to SEQ ID NO: 34, or a fragment thereof. 15. A polynucleotide comprising a nucleotide sequence encoding the glycosidase of para 13 or para 14. 16. The polynucleotide of para 15, wherein the polynucleotide comprises or consists of: (a) a sequence that has at least 70% sequence identity to SEQ ID NO: 5 or 12, or a fragment thereof; (b) a sequence that has at least 70% sequence identity to SEQ ID NO: 13, or a fragment thereof; or (c) a sequence that has at least 70% sequence identity to SEQ ID NO: 14, or a fragment thereof. 17. A vector comprising the polynucleotide of para 15 or para 16, optionally wherein the vector is a viral vector, optionally wherein the vector is a lentiviral vector or adeno- associated viral (AAV) vector. 18. The polynucleotide or vector of para 15, 16, or 17, wherein nucleotide sequence encoding the glycosidase is operably linked to a promoter, optionally wherein the promoter is a PGK promoter. 19. A cell comprising the polynucleotide or product according to any one of paras 1 to 11,15 and 16, the vector of para 12 or 17, or the glycosidase of para 13 or 14, wherein the cell is optionally a T cell. 20. The polynucleotide or product according to any one of paras 1 to 11, 15 and 16, the vector of para 12 or 17, the glycosidase of para 13 or 14, or the cell of para 19, for use in therapy. 21. The polynucleotide or product according to any one of paras 1 to 11,15 and 16, the vector of para 12 or 17, the glycosidase of para 13 or 14, or the cell of para 19, for use in the treatment of cancer. 22. Use of the polynucleotide or product according to any one of paras 1 to 11, 15 and 16, the vector of para 12 or 17, the glycosidase of para 13 or 14, or the cell of para 19, for improving CAR cell activity. 23. A method of producing a CAR cell, comprising introducing the polynucleotide or product according to any one of paras 1 to 11, 15 and 16, the vector of para 12 or 17, or the glycosidase of para 13 or 14 into a cell. 24. The use or method of para 22 or 23, wherein the CAR cell is a CAR T cell and wherein the therapeutic activity of the CAR T cell is improved, optionally wherein the therapeutic activity is target cell killing. 25. A method of treatment comprising producing a CAR cell according to the method of para 23 or para 24 and administering the CAR cell to a subject in need thereof.

Claims

CLAIMS 1. A polynucleotide comprising (a) a nucleotide sequence encoding a glycosidase; and (b) a nucleotide sequence encoding a chimeric antigen receptor (CAR). 2. A product comprising (a) a first polynucleotide comprising a nucleotide sequence encoding a glycosidase; and (b) a second polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR). 3. The polynucleotide or product of claim 1 or claim 2, wherein the glycosidase is peptide- N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), or a variant thereof. 4. The polynucleotide or product of any one of claims 1 to 3, wherein the glycosidase comprises a signal peptide. 5. The polynucleotide or product of any one of claims 1 to 4, wherein the glycosidase comprises or consists of: (a) a sequence that has at least 70% sequence identity to SEQ ID NO: 1, or a fragment thereof; (b) a sequence that has at least 70% sequence identity to SEQ ID NO: 2, or a fragment thereof; (c) a sequence that has at least 70% sequence identity to SEQ ID NO: 3, or a fragment thereof; or (d) a sequence that has at least 70% sequence identity to SEQ ID NO: 34, or a fragment thereof. 6. A glycosidase, wherein the glycosidase is peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), and wherein the PNGase: (a) comprises a signal peptide; and/or (b) lacks a PUB domain, a PAW domain, or both. 7. The glycosidase of claim 6, wherein the glycosidase comprises or consists of: (a) a sequence that has at least 70% sequence identity to SEQ ID NO: 1, 4, 6 or 9, or a fragment thereof; (b) a sequence that has at least 70% sequence identity to SEQ ID NO: 2, 7 or 10, or a fragment thereof; (c) a sequence that has at least 70% sequence identity to SEQ ID NO: 3, 8 or 11, or a fragment thereof; or (d) a sequence that has at least 70% sequence identity to SEQ ID NO: 34, or a fragment thereof. 8. A polynucleotide comprising a nucleotide sequence encoding the glycosidase of claim 6 or claim 7. 9. A cell comprising the polynucleotide or product according to any one of claims 1 to 5 or 8, or the glycosidase of claim 6 or 7, wherein the cell is optionally a T cell. 10. The polynucleotide or product according to any one of claims 1 to 5 or 8, the glycosidase of claim 6 or 7, or the cell of claim 9, for use in therapy.
PCT/EP2023/072345 2022-08-12 2023-08-11 Deglycosylation of native glycoproteins expressed on a tumor cell surface WO2024033544A1 (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998017815A1 (en) 1996-10-17 1998-04-30 Oxford Biomedica (Uk) Limited Retroviral vectors
WO1999041397A1 (en) 1998-02-17 1999-08-19 Oxford Biomedica (Uk) Limited Anti-viral vectors
WO2001079518A2 (en) 2000-04-19 2001-10-25 Oxford Biomedica (Uk) Limited Codon optimisation for expression in retrovirus packaging cells
WO2001098332A2 (en) * 2000-06-22 2001-12-27 Incyte Genomics, Inc. Secreted redox proteins
WO2022256620A1 (en) * 2021-06-03 2022-12-08 The Broad Institute, Inc. Novel targets for enhancing anti-tumor immunity

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998017815A1 (en) 1996-10-17 1998-04-30 Oxford Biomedica (Uk) Limited Retroviral vectors
WO1999041397A1 (en) 1998-02-17 1999-08-19 Oxford Biomedica (Uk) Limited Anti-viral vectors
WO2001079518A2 (en) 2000-04-19 2001-10-25 Oxford Biomedica (Uk) Limited Codon optimisation for expression in retrovirus packaging cells
WO2001098332A2 (en) * 2000-06-22 2001-12-27 Incyte Genomics, Inc. Secreted redox proteins
WO2022256620A1 (en) * 2021-06-03 2022-12-08 The Broad Institute, Inc. Novel targets for enhancing anti-tumor immunity

Non-Patent Citations (25)

* Cited by examiner, † Cited by third party
Title
AMENDOLA, NAT BIOTECH, vol. 23, 2005, pages 108 - 116
ATSCHUL ET AL., J. MOL. BIOL., 1990, pages 403 - 410
AUSUBEL, F.M ET AL.: "Current Protocols in Molecular Biology", 1995, JOHN WILEY & SONS
BRENTJENS ET AL., CCR, vol. 13, no. 18 Pt 1, 15 September 2007 (2007-09-15), pages 5426 - 35
CASUCCI ET AL., BLOOD, vol. 122, no. 20, 14 November 2013 (2013-11-14), pages 3461 - 72
COFFIN ET AL.: "Retroviruses", 1997, COLD SPRING HARBOUR LABORATORY PRESS, pages: 758 - 63
DEVEREUX ET AL., NUCLEIC ACIDS RES., vol. 12, 1984, pages 387
FEMS MICROBIOL. LETT, vol. 177, 1999, pages 187 - 50
GAIT, M.J.: "Oligonucleotide Synthesis: A Practical Approach", 1984, IRL PRESS
GRECO ET AL., SCI TRANS MED, vol. 14, 2022, pages 3072
HEARD AMANDA ET AL: "Antigen glycosylation regulates efficacy of CAR T cells targeting CD19", NATURE COMMUNICATIONS, vol. 13, no. 1, 11 June 2022 (2022-06-11), XP093101524, DOI: 10.1038/s41467-022-31035-7 *
IMAI C, LEUKEMIA, vol. 18, no. 4, April 2004 (2004-04-01), pages 676 - 84
LEWIS ET AL., EMBO J., vol. 11, 1992, pages 3053 - 8
LEWIS, J. VIROL, vol. 68, 1994, pages 510 - 6
LILLEY, D.MDAHLBERG, J.E: "Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA", 1992, ACADEMIC PRESS
MAHER ET AL., NAT BIOTECHNOL, vol. 20, no. 1, January 2002 (2002-01-01), pages 70 - 5
MAVILIO, BLOOD, vol. 83, 1994, pages 1988 - 1997
MILONE ET AL., MOL THER, vol. 17, no. 8, August 2009 (2009-08-01), pages 1453 - 64
NAT. BIOTECHNOL., vol. 14, 1996, pages 556
POLAK, J.MMCGEE, J.O'D: "In Situ Hybridization: Principles and Practice", 1990, OXFORD UNIVERSITY PRESS
PULE ET AL., MOL THER, vol. 12, no. 5, November 2005 (2005-11-01), pages 933 - 41
ROE, B.CRABTREE, JKAHN, A: "DNA Isolation and Sequencing: Essential Techniques", 1996, JOHN WILEY & SONS
SAMBROOK, J., FRITSCH, E.F. AND MANIATIS, T: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAVOLDO B, BLOOD, vol. 113, no. 25, 18 June 2009 (2009-06-18), pages 6392 - 402
SAVOLDO, J CLIN INVEST, vol. 121, 2011, pages 1822 - 1826

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