WO2017168170A1 - Cellule progénitrice immunomodulatrice (imp) exprimant un ou plusieurs lymphocytes parmi les lymphocytes cd3, cd3e, cd8, cd8b, cd4, cd5, cd6 et cd7 - Google Patents

Cellule progénitrice immunomodulatrice (imp) exprimant un ou plusieurs lymphocytes parmi les lymphocytes cd3, cd3e, cd8, cd8b, cd4, cd5, cd6 et cd7 Download PDF

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WO2017168170A1
WO2017168170A1 PCT/GB2017/050917 GB2017050917W WO2017168170A1 WO 2017168170 A1 WO2017168170 A1 WO 2017168170A1 GB 2017050917 W GB2017050917 W GB 2017050917W WO 2017168170 A1 WO2017168170 A1 WO 2017168170A1
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cells
population
detectable levels
cell
imp
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Ajan Reginald
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Cell Therapy Limited
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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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells

Definitions

  • CD3E CD8. CD8B. CD4. CD5. CD6 AND CD7
  • the invention relates to immuno-modulatory progenitor (IMP) cells expressing one or more of
  • Mesodermal cells are derived from a number of tissues and act as the supportive structure for other cell types. Bone marrow for instance is made of both haematopoietic and mesenchymal derived cells. Two principle mesenchymal cell types have been previously described and characterized, namely (i) mesenchymal stem cells (MSCs) and their precursors and (ii) mesenchymal precursor cells (MPCs) found in the bone marrow.
  • MSCs mesenchymal stem cells
  • MSCs mesenchymal stem cells
  • MSCs are multipotent, adult stem cells. MSCs differentiate to form the different specialised cells found in the skeletal tissues. For example, they can differentiate into cartilage cells (chondrocytes), bone cells (osteoblasts) and fat cells (adipocytes).
  • MSCs are used in a variety of therapies, such as the treatment of Age-related Macular
  • MSCs myocardial infarct
  • ASD myocardial Degeneration
  • myocardial infarct Once administered to the patient, the MSCs typically migrate (or home) to the damaged tissue and exert their therapeutic effects through paracrine signaling and by promoting survival, repair and regeneration of the neighbouring cells in the damaged tissue.
  • This invention relates to a novel cell type that has not been previously identified or isolated, the immuno-modulatory progenitor cell.
  • This IMP cell is quite distinct and different to both MSCs and MPCs in its composition, function and characteristics which impart an enhanced immuno-modulatory capacity.
  • the inventors have surprisingly identified a new immuno-modulatory progenitor (IMP) cell having a specific marker expression pattem.
  • the IMP cell expresses (a) MIC A/B, CD304 (Neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like receptor 9), CD363 (Sphingosine-1 -phosphate receptor 1), CD99, CD181 (C-X-C chemokine receptor type 1; CXCR1), epidermal growth factor receptor (EGF-R), CXCR2 and CD126 and (b) one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • the IMP cell expresses significantly greater amounts of these markers than a mesenchymal stem cell (MSC).
  • the IMP cell preferably expresses CD66e, CD 121b, CD 122, CD 164, CD172a, CD203c, CD264, CD270, CD328, CD358, HLA-ABC, Notch2, T cell receptor (TCR) gamma delta, CD360 and CD1 lb.
  • the IMP cell preferably expresses significantly greater amounts of these markers than a mesenchymal stem cell (MSC).
  • the IMP cell preferably does not express detectable levels of FMC7 and ITGB7.
  • the IMP cells of the invention can be isolated from mononuclear cells (MCs), such as peripheral blood MCs.
  • the IMP cells are capable of efficiently migrating to and repairing damaged tissues. In particular, they are capable of homing, adherence, transmigration, proliferation, angiogenic effects and paracrine signalling.
  • the invention provides an immuno-modulatory progenitor (IMP) cell, wherein the cell expresses detectable levels of (a) MIC A/B, CD304 (Neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like receptor 9), CD363 (Sphingosine-1 -phosphate receptor 1), CD99, CD181 (C-X-C chemokine receptor type 1; CXCRl), epidermal growth factor receptor (EGF-R), CXCR2 and CD 126 and (b) one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • MIC A/B CD304 (Neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like receptor 9), CD363 (Sphingosine-1 -phosphate receptor 1), CD99, CD181 (C-X-C chemokine receptor type 1; CXCRl), epidermal growth factor receptor (EGF-R), CXCR2 and CD
  • the invention further provides an IMP cell, wherein the cell expresses detectable levels of MIC A/B, CD304 (Neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like receptor 9), CD363 (Sphingosine-1 -phosphate receptor 1), CD99, CD181 (C-X-C chemokine receptor type 1; CXCRl), epidermal growth factor receptor (EGF-R), CXCR2, CD126, CD66e, CD121b, CD122, CD164, CD172a, CD203c, CD264, CD270, CD328, CD358, HLA-ABC, Notch2, T cell receptor (TCR) gamma delta, CD360 and CD1 lb and wherein the cell does not express detectable levels of FMC7 and ITGB7.
  • CD304 Neuropilin 1
  • CD178 FAS ligand
  • CD289 Toll-like receptor 9
  • CD363 Sphingosine-1 -phosphate receptor 1
  • CD99 CD181
  • the invention also provides:
  • EGF-R epidermal growth factor receptor
  • composition comprising (a) an IMP cell of the invention or a population of the invention and (b) a pharmaceutically acceptable carrier or diluent, one or more liposomes and/or one or more microbubbles;
  • a pharmaceutical composition comprising (a) an IMP cell of the invention or a population of the invention; (b) an immune cell; (c) an antigen; and (d) a pharmaceutically acceptable carrier or diluent; a method of producing a population of IMP cells of the invention, comprising (a) culturing mononuclear cells (MCs) under conditions which induce the MCs to differentiate into IMP cells and (b) harvesting and culturing those IMP cells which have an expression partem as defined above and thereby producing a population of the invention;
  • a method of repairing a damaged tissue in a patient comprising administering to the patient a population of the invention or a pharmaceutical composition of the invention, wherein the population or composition comprises a therapeutically effective number of cells, and thereby treating the damaged tissue in the patient;
  • a population of the invention or a pharmaceutical composition of the invention for use in a method of treating a cardiac, bone, cartilage, tendon, ligament, liver, kidney or lung injury or disease in a patient;
  • an in vitro method of increasing the activity of cytotoxic, helper or gamma delta T cells in response to an antigen comprising incubating the T cells with the antigen and a population of the invention under conditions which increase the activity of the T cells;
  • an in vitro method of increasing the activity of regulatory T cells in response to an antigen comprising incubating the T cells with the antigen and a population of the invention under conditions which increase the activity of the T cells; primed regulatory T cells produced using the above method;
  • an in vitro method of decreasing the activity of cytotoxic, helper or gamma delta T cells in response to an antigen comprising incubating the T cells with the antigen and a population of the invention under conditions which decrease the activity of the T cells;
  • an in vitro method of decreasing the activity of regulatory T cells in response to an antigen comprising incubating the T cells with the antigen and a population of the invention under conditions which decrease the activity of the T cells;
  • an in vivo method of increasing the activity of cytotoxic, helper or gamma delta T cells in response to an antigen comprising administering a population or pharmaceutical composition of the invention to a subject under conditions which increase the activity of the T cells;
  • an in vivo method of increasing the activity of regulatory T cells in response to an antigen comprising administering a population or pharmaceutical composition of the invention to a subject under conditions which increase the activity of the T cells;
  • an in vivo method of decreasing the activity of cytotoxic, helper or gamma delta T cells in response to an antigen comprising administering a population or pharmaceutical composition of the invention to a subject under conditions which decrease the activity of the T cells;
  • an in vivo method of decreasing the activity of regulatory T cells in response to an antigen comprising administering a population or a pharmaceutical composition of the invention to a subject under conditions which decrease the activity of the T cells;
  • a method of treating a disease by increasing cytotoxic, helper or gamma delta T cell responses to an antigen in a subject comprising administering to the subject:
  • a method of treating a disease by decreasing regulatory T cell responses to an antigen in a subject comprising administering to the subject:
  • a method of treating a disease by decreasing cytotoxic, helper or gamma delta T cell responses to an antigen in a subject comprising administering to the subject:
  • a method of treating a disease by increasing regulatory T cell responses to an antigen in a subject comprising administering to the subject:
  • a method of treating a disease by decreasing cytotoxic, helper or gamma delta T cell responses to an antigen in a subject comprising administering to the subject:
  • a method of treating cancer in a subject comprising administering to the subject the population or pharmaceutical composition of the invention;
  • a method of treating an allergic, autoimmune or immune-mediated disease in a subject comprising administering to the subject the population or pharmaceutical composition of the invention; and a method of improving the potency, viability or stability of CAR T cells, comprising incubating CAR T cells in the presence of a population of the invention;
  • a method of inducing an IMP cell of the invention to express detectable levels of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 comprising (i) editing the genome of the cell and/or (ii) transfecting or transducing the cell with one or more nucleic acid constructs encoding one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 and /or (iii) genotype modulation using magnetic stimulation or electrical stimulation; and
  • a method of producing an IMP cell of the invention comprising providing an IMP cell expressing detectable levels of MIC A/B, CD304 (Neuropilin), CD 178 (FAS ligand), CD289 (Toll -like receptor 9), CD363 (Sphinogine-1 -phosphate receptor 1), CD99, CD181 (C-X-C chemokine receptor type 1; CXCRl), epidermal growth factor receptor (EGF-R), CXCR2 and CD 126 and inducing the cell to express detectable levels of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 by (i) editing the genome of the cell and/or (ii) transfecting or transducing the cell with one or more nucleic acid constructs encoding one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 and /or (iii) genotype modulation using magnetic stimulation or electrical stimulation.
  • the present invention provides an immuno-modulatory progenitor (IMP) cell.
  • the IMP cell expresses detectable levels of MIC A/B, CD304 (Neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like receptor 9), CD363 (Sphingosine-1 -phosphate receptor 1), CD99, CD181 (C-X-C chemokine receptor type 1; CXCRl), epidermal growth factor receptor (EGF-R), CXCR2, and CD126.
  • the IMP cell of the invention also expresses detectable levels of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • the IMP cell may express detectable levels of CD3, CD3e or CD3 and CD3e.
  • the IMP cell may express detectable levels of CD8, CD8b or CD8 and CD8b.
  • the IMP cell may naturally express detectable levels of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • the cell may, when cultured, express one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7without the need to edit the genome or the cell, or transfect or transduce the cell to express one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 may be artificially induced in the cell as discussed below, for instance by editing the genome of the cell, or transfecting or transducing the cell to express one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • the IMP cell expresses detectable levels of CD66e, CD 121b, CD 122, CD 164, CD172a, CD203c, CD264, CD270, CD328, CD358, HLA-ABC, Notch2, T cell receptor (TCR) gamma delta, CD360 and CDl lb and does not express detectable levels of FMC7 and ITGB7.
  • CD66e CD 121b, CD 122, CD 164, CD172a, CD203c, CD264, CD270, CD328, CD358, HLA-ABC, Notch2, T cell receptor (TCR) gamma delta, CD360 and CDl lb and does not express detectable levels of FMC7 and ITGB7.
  • MIC allows adaptation of cells and their immuno-behaviour in an inflammatory context by decreasing their susceptibility to NK killing.
  • CD304 (alternate name Neuropilin 1) is a co-receptor for vascular endothelial growth factor (VEGF) and has roles in angiogenesis, cell survival, migration and invasion.
  • VEGF vascular endothelial growth factor
  • CD 178 (alternate name FAS ligand) maintains cell phenotype and controls differentiation. It is also capable of inducing proliferation of cells.
  • FAS ligand is known primarily in apoptotic signalling, it has been shown that FAS and FAS ligand expressing cells are resistant to FAS -induced apoptosis.
  • CD289 (alternate name Toll-like receptor 9) is involved in the modulation of immune responses and may facilitate cell migration towards a target tissue.
  • CD363 alternative name is Sphingosine-1 -phosphate receptor 1
  • Sphingosine-1 -phosphate receptor 1 Sphingosine-1 -phosphate receptor 1
  • CD363 also promotes angiogenesis, modulates cell homing, modulates trafficking and migration of cells and regulates chemotaxis.
  • CD99 is involved in cell adhesion and transmigration.
  • interleukin-8 receptors There are two classes of interleukin-8 (IL-8) receptors, CXCRl (or CD181) and CXCR2. Both receptors bind IL-8 with high affinity, in contrast to the other CXC chemokines. Functionally, CXCRl and CXCR2 have been shown to play significant roles in proliferation, migration, invasion and angiogenesis. Damaged tissues release a variety of soluble inflammatory factors, such as macrophage migration inhibitory factor (MIF) and interleukin-8, and these factors may attract the IMP cells of the invention (and other inflammatory cells) to the damaged tissue though binding to binding CXCR1 and/or CXCR2.
  • MIF macrophage migration inhibitory factor
  • interleukin-8 interleukin-8
  • EGF-R is involved in cell migration, adhesion and proliferation.
  • CD 126 (alternate name is IL-6R1) increases immune-privilege.
  • CD3 is a T cell co-receptor that helps to activate the T cell. It is a protein complex composed of four distinct chains. In mammals, the complex comprises a CD3y chain, a CD35 chain, and two CD3s chains. These chains associate with the T-cell receptor (TCR) and the ⁇ -chain (zeta-chain) to generate an activation signal in T lymphocytes.
  • TCR T-cell receptor
  • ⁇ -chain zeta-chain
  • CD3 molecules together constitute the TCR complex.
  • the CD3s chains are also known as CD3e, and are encoded by the CD3e gene.
  • CD4 is a glycoprotein found on the surface of immune cells such as T helper cells, monocytes, macrophages and dendritic cells.
  • CD4 is a member of the immunoglobulin superfamily. It has four immunoglobulin domains (Dl to D4) that are exposed on the extracellular surface of the cell. Dl and D3 resemble immunoglobulin variable (IgV) domains. D2 and D4 resemble immunoglobulin constant (IgC) domains.
  • CD4 uses its Dl domain to interact with the ⁇ 2 -domain of MHC class II molecules. T cells expressing CD4 molecules (and not CD8) on their surface, therefore, are specific for antigens presented by MHC II and not by MHC class I (they are MHC class II -restricted). MHC class I contains Beta-2 microglobulin.
  • the short cytoplasmic/intracellular tail (C) of CD4 contains a special sequence of amino acids that allow it to interact with the lck molecule.
  • CD5 is a cluster of differentiation that is found on T cells, and also on a subset of IgM-secreting B cells (B-l cells). CD5 serves to mitigate activating signals from the BCR so that the B-l cells can only be activated by very strong stimuli (such as bacterial proteins) and not by normal tissue proteins. T cells express higher levels of CD5 than B cells. CD5 is upregulated on T cells upon strong activation. CD5 is a good marker for T cells, and about 76% of T cell neoplasm express CD5. CD5 is also found in chronic lymphocytic leukemia and mantle cell lymphoma cells. In the thymus, there is a correlation with CD5 expression and strength of the interaction of the T cell towards self-peptides.
  • CD6 is a protein found on the outer membrane of T cells and some other immune cells.
  • the protein contains three scavenger receptors cysteine-rich (SRCR) domains and a binding site for an activated leukocyte adhesion molecule. CD6 is important for the continuation of T cell activation.
  • SRCR scavenger receptors cysteine-rich
  • CD7 is a transmembrane protein which is a member of the immunoglobulin superfamily. CD7 is found on thymocytes and mature T cells. It plays an essential role in T cell interactions, and also in T-cell/B-cell interaction during early lymphoid development.
  • CD66e (alternative name Carcinoembryonic Antigen-related Cell Adhesion Molecule 5, CEACAM-5) functions as a calcium independent adhesion molecule through homophilic and heterophilic interactions with CEACAM-1. CD66e promotes cell migration, invasion and adhesion, and blocks apoptosis following loss of extra-cellular matrix (ECM) anchorage (anoikis).
  • ECM extra-cellular matrix
  • CD121b (alternative name Interleukin 1 receptor type II, IL1R2) binds interleukin alpha
  • Interleukin 4 is reported to antagonize the activity of interleukin 1 by inducing the expression and release of this cytokine.
  • Interleukin 2 binds to the IL-2 receptor, which has three forms. These three forms are generated by different combinations of three different proteins, often referred to as "chains”: a (alpha) (also called IL-2Ra, CD25, or Tac antigen), ⁇ (beta) (also called IL-2R ⁇ $, or CD 122), and ⁇ (gamma) (also called IL-2Ry, yc, common gamma chain, or CD132).
  • IL-2 and its receptor have important roles in key functions of the immune system, such as tolerance and immunity. The effects of IL-2 and its receptor are primarily mediated via their direct effects on T cells.
  • CD 164 is also known as sialomucin core protein 24, and functions as a cell adhesion molecule.
  • Sialomucins are a heterogeneous group of secreted or membrane-associated mucins that appear to play two key but opposing roles in vivo, firstly as cytoprotective or antiadhesive agents and secondly as adhesion receptors.
  • CD 164 may serve as a signalling receptor that regulates proliferation, adhesion and migration in progenitor cells.
  • CD 164 may also associate with the chemokine receptor CXCR4, possibly as a co-receptor for the CXCR4 ligand SDF-lalpha.
  • CD 172a (alternative name signal regulatory protein a, SIRP a) is regulatory membrane glycoprotein from the SIRP family expressed mainly by myeloid cells and also by stem cells or neurons. SIRP a acts as inhibitory receptor and interacts with the broadly expressed transmembrane protein CD47 (also known as "don't eat me” signal). This interaction negatively controls effector function of innate immune cells such as host cell phagocytosis.
  • SIRP a alternative name signal regulatory protein a, SIRP a
  • CD47 also known as "don't eat me” signal
  • CD264 is a membrane receptor for CD253 (TNF-related apoptosis-inducing ligand, TRAIL) and is thought to act as a decoy receptor by competing for binding with other TRAIL receptors and inhibiting TRAIL-induced apoptosis. CD264 does not induce apoptosis, and has been shown to play an inhibitory role in TRAIL-induced cell apoptosis.
  • TRAIL TNF-related apoptosis-inducing ligand
  • CD270 is a type I transmembrane protein and a member of the TNFR-TNF superfamily.
  • CD270 interaction on T cells provides a costimulatory signal via CD270 signalling.
  • CD270 has been reported to be involved in the induction of cytokines and matrix metalloproteinases.
  • CD328 (alternative name sialic acid-binding Ig-like lectin 7, SIGLEC7) is a putative adhesion molecule that mediates sialic-acid dependent binding to cells. CD328 mediates the inhibition of the cytotoxic function of natural killer (NK) cells. CD328 also inhibits the differentiation of CD34+ cell precursors towards the myelomonocytic cell lineage, and the in vitro proliferation of leukemic myeloid cells in vitro.
  • NK natural killer
  • TCR-gamma delta is a T cell receptor (TCR) comprising gamma and delta TCR chains. TCRs discriminate foreign from self-peptides presented by major histocompatibility complex (MHC) class II molecules and essential for effective adaptive immune responses. T cells expressing TCR-gamma delta are known as gamma-delta T cells. Gamma delta T cells have shown anti -tumour and
  • HLA-ABC is a type of human MHC class I cell surface receptor.
  • Notch 2 regulates the determination of cell fate.
  • CD360 is the IL-21R. It is involved in direct inhibitory effects on the anti-presenting function of dendritic cells.
  • CD1 lb positively regulates TLR4-induced signaling pathways in dendritic cells, but not in macrophages.
  • the IMP cells may express detectable levels of CD3, CD3e, CD8, CD66b and CLIP.
  • CD8 is a T-cell co-receptor.
  • CD66b is a granulocyte activation and adhesion marker.
  • CLIP Class Il-associated invariant chain peptide
  • the IMP cells of the invention may advantageously be used to repair damaged tissues in patients.
  • the IMP cells are capable of efficiently migrating (or homing) to a damaged tissue and exerting anti-inflammatory effects in the tissue. This is discussed in more detail below.
  • One of the most important abilities of the IMP cells is to migrate (or home) to injured sites, which involves chemotaxis. This is based on chemokine-signalling and utilises mechanisms such as rolling, adhesion and transmigration.
  • the anti-inflammatory effects of the IMP cells promote survival, repair and regeneration of the neighbouring cells in the damaged tissue.
  • the cells are also able to exert paracrine effects such as the secretion of angiogenic, chemotactic and anti-apoptotic factors. This is also discussed in more detail below.
  • the IMP cells of the invention may advantageously be used to treat a disease in a subject.
  • the IMP cells may be used to treat cancer in a subject.
  • the IMP cells may also be used to treat an allergic, autoimmune or immune-mediated disease in a subject.
  • the IMP cells of the invention may treat disease via their direct effects.
  • the IMP cells may kill cells via contact-dependent cell lysis.
  • the IMP cells kill tumour cells via contact-dependent cell lysis.
  • the IMP cells may also secrete molecules that act on other cells. Such molecules may affect cell metabolism, proliferation, survival, function or signalling.
  • the IMP cells may secrete pro-inflammatory cytokines and/or anti-inflammatory cytokines.
  • the IMP cells may secrete pro-apoptotic molecules and/or anti-apoptotic molecules.
  • the IMP cells of the invention may modulate immune responses.
  • the IMP cells may have immuno-modulatory effects.
  • the IMPs may increase or decrease the activity of immune cells such as cytotoxic T cells, helper T cells, gamma delta T cells and regulatory T cells.
  • the IMP cells of the invention may therefore be used to treat disease in a subject by increasing or decreasing T cell responses. This is discussed in more detail below.
  • the IMP cells of the invention may be used to modulate T cell activity in response to an antigen in vitro or in vivo. Accordingly, the IMP cells may be used to produce a population of T cells having a modified activity in response to an antigen. For instance, the IMP cells may be used to produce a population of primed or suppressed T cells. The primed or suppressed T cells may be used to treat a disease in a subject. Specifically, the primed or suppressed T cells may be used to treat disease in a subject by increasing or decreasing T cell activity. The primed or suppressed T cells may be administered to the subject alone or in combination with the IMP cells. The IMP cells of the invention may also be used in a method of improving the potency, viability and/or the stability of chimeric antigen receptor (CAR) T cells. This is also discussed in more detail below.
  • CAR chimeric antigen receptor
  • the IMP cells are produced from mononuclear cells (MCs), such as peripheral MCs, taken from an individual, such as a human individual. Since the IMP cells are produced from MCs, they may be produced easily (such as from peripheral blood) and may be autologous for the patient to be treated and thereby avoid the risk of immunological rejection by the patient.
  • MCs mononuclear cells
  • the IMP cells may be produced easily (such as from peripheral blood) and may be autologous for the patient to be treated and thereby avoid the risk of immunological rejection by the patient.
  • IMP cells of the invention can therefore be made in large numbers.
  • the IMP cells of the invention are produced from MCs, they are substantially homologous and may be autologous. They also avoid donor-to-donor variation, which frequently occurs with MSCs. Numerous populations of IMP cells of the invention can be produced from a single sample taken from the patient before any other therapy, such as chemotherapy or radiotherapy, has begun. Therefore, the IMP cells of the invention can avoid any of the detrimental effects of those treatments.
  • IMP cells of the invention can be made quickly. IMP cells can be produced from MCs in less than 30 days, such as in about 22 days.
  • the IMP cells of the invention are typically produced from human MCs.
  • the IMP cells of the invention are therefore typically human.
  • the IMP cells may be produced from MCs from other animals or mammals, for instance from commercially farmed animals, such as horses, cattle, sheep or pigs, from laboratory animals, such as mice or rats, or from pets, such as cats, dogs, rabbits or guinea pigs.
  • the IMP cells of the invention can be identified as immunomodulatory progenitor cells using standard methods known in the art, including expression of lineage restricted markers, structural and functional characteristics.
  • the IMP cells will express detectable levels of cell surface markers known to be characteristic of IMPs. These are discussed below.
  • the IMP cells of the invention are capable of successfully completing differentiation assays in vitro to confirm that they are of mesodermal lineage.
  • Such assays include, but are not limited to, adipogenic differentiation assays, osteogenic differentiation assays and neurogenic differentiation assays (Zaim M et al Ann Hematol. 2012 Aug;91(8): 1175-86).
  • the IMP cells of the invention are not stem cells. In particular, they are not MSCs. They are terminally differentiated. Although they can be forced under the right conditions in vitro to differentiating, for instance into cartilage or bone cells, they typically do not differentiate in vivo.
  • the IMP cells of the invention have their effects by migrating to the damaged tissue and exerting paracrine signalling in the damaged tissue.
  • the IMP cells are preferably capable of inducing antiinflammatory effects in the damaged tissue. This is discussed in more detail below.
  • the IMP cells of the invention are typically characterised by a spindle-shaped morphology.
  • the IMP cells are typically fibroblast-like, i.e. they have a small cell body with a few cell processes that are long and thin.
  • the cells are typically from about 10 to about 20 ⁇ in diameter.
  • the IMP cells of the invention are distinguished from known cells, including MSCs, via their marker expression partem.
  • the IMPs express detectable levels of (a) MIC A/B, CD304 (Neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like receptor 9), CD363 (Sphingosine-1 -phosphate receptor 1), CD99, CD181 (C-X-C chemokine receptor type 1; CXCR1), epidermal growth factor receptor (EGF- R), CXCR2 and CD126, and (b) one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • the IMPs preferably express an increased amount of these markers compared with MSCs.
  • the IMP cells preferably express detectable levels of CD66e, CD121b, CD122, CD164, CD172a, CD203c, CD264, CD270, CD328, CD358, HLA-ABC, Notch2, T cell receptor (TCR) gamma delta, CD360 and CD1 lb.
  • the IMPs preferably express an increased amount of these markers compared with MSCs. This can be determined by comparing the expression level/amount of the markers in an IMP of the invention with the expression level/amount in an MSC using the same technique under the same conditions.
  • Suitable MSCs are commercially available.
  • the MSC used for comparison is preferably a human MSC. Human MSCs are commercially available from Mesoblast® Ltd, Osiris Therapeutics® Inc. or Lonza®. The human MSC is preferably obtained from Lonza®. Such cells were used for the comparison in the Example.
  • the MSC may be derived from any of the animals or mammals discussed above.
  • the IMP cells preferably do not express detectable levels of FMC7 and ITGB7.
  • the IMP cells preferably express an increased amount of one or more of MIC A/B, CD304 (Neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like receptor 9), CD363 (Sphingosine-1 -phosphate receptor 1), CD99, CD181 (C-X-C chemokine receptor type 1; CXCR1), epidermal growth factor receptor (EGF-R), CXCR2 and CD126 compared with a MSC.
  • the IMP cells preferably express an increased amount of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 compared with a MSC.
  • the IMP cells preferably express an increased amount of all of MIC A/B, CD304 (Neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like receptor 9), CD363 (Sphingosine-1 -phosphate receptor 1), CD99, CD181 (C-X-C chemokine receptor type 1; CXCR1), epidermal growth factor receptor (EGF-R), CXCR2, and CD126, and one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 compared with a MSC.
  • the IMP cells preferably express an increased amount of one or more of CD66e, CD121b, CD122, CD164, CD172a, CD264, CD270, CD328, CD358, HLA-ABC, Notch2, T cell receptor (TCR) gamma delta, CD360 and CDl lb compared with a MSC.
  • the IMP cells preferably express an increased amount of all of these markers compared with a MSC.
  • the IMP cells preferably express detectable levels of ⁇ CD3 ⁇ ; ⁇ CD3e ⁇ ; ⁇ CD8 ⁇ ; ⁇ CD8b ⁇ ; ⁇ CD4 ⁇ ; ⁇ CD5 ⁇ ; ⁇ CD6 ⁇ ; ⁇ CD7 ⁇ ; ⁇ CD3,CD3e ⁇ ; ⁇ CD3,CD8 ⁇ ; ⁇ CD3,CD8b ⁇ ; ⁇ CD3,CD4 ⁇ ;
  • the IMP cells express an increased amount of one or more of any of the combinations above compared with a MSC.
  • the IMP cell preferably expresses detectable levels of CD3 and/or CD3e.
  • the IMP cell may express detectable levels of CD3, CD3e or CD3 and CD3e.
  • This cell preferably further expresses detectable levels of any number and combination of CD8, CD8b, CD4, CD5, CD6 and CD7 above (i.e. any combination in ⁇ above which does not include CD3 and/or CD3e).
  • the IMP cell preferably expresses detectable levels of CD8 and/or CD8b.
  • the IMP cell may express detectable levels of CD8, CD8b or CD8 and CD8b.
  • This cell preferably further expresses detectable levels of any number and combination of CD3, CD3e, CD4, CD5, CD6 and CD7 (i.e. any combination in ⁇ above which does not include CD8 and/or CD8b).
  • the IMP cell preferably expresses detectable levels of CD4. This cell preferably further expresses detectable levels of any number and combination of one or more of CD3, CD3e, CD8, CD8b,
  • CD5, CD6 and CD7 i.e. any combination in ⁇ above which does not include CD4.
  • the IMP cell preferably expresses detectable levels of CD5.
  • This cell preferably further expresses detectable levels of any number and combination of one or more of CD3, CD3e, CD8, CD8b, CD4, CD6 and CD7 (i.e. any combination in ⁇ above which does not include CD5).
  • the IMP cell preferably expresses detectable levels of CD6. This cell preferably further expresses detectable levels of any number and combination of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5 and CD7 (i.e. any combination in ⁇ above which does not include CD6).
  • the IMP cell preferably expresses detectable levels of CD7. This cell preferably further expresses detectable levels of any number and combination of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5 and CD6 (i.e. any combination in ⁇ above which does not include CD7).
  • the IMP cell may naturally express detectable levels of one or more of CD3, CD3e, CD8,
  • CD8b, CD4, CD5, CD6 and CD7 the cell may, when cultured, express detectable levels of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 without the need to edit the genome or the cell, or transfect or transduce the cell to express detectable levels one or more of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • the expression of detectable levels of one or more of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 may be artificially induced in the cell as discussed below, for instance by editing the genome of the cell, or transfecting or transducing the cell to express detectable levels of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • the IMP cell preferably expresses detectable levels of CD3 and/or CD3e.
  • the IMP cell may express detectable levels of CD3, CD3e or CD3 and CD3e.
  • This cell preferably does not express detectable levels of any number and combination of CD8, CD8b, CD4, CD5, CD6 and CD7 above (i.e. any combination in ⁇ above which does not include CD3 and/or CD3e).
  • the IMP cell preferably expresses detectable levels of CD4. This cell preferably does not express detectable levels of any number and combination of one or more of CD3, CD3e, CD8, CD8b, CD5, CD6 and CD7 (i.e. any combination in ⁇ above which does not include CD4).
  • the IMP cell preferably expresses detectable levels of CD5. This cell preferably does not express detectable levels of any number and combination of one or more of CD3, CD3e, CD8, CD8b, CD4, CD6 and CD7 (i.e. any combination in ⁇ above which does not include CD5).
  • the IMP cell preferably expresses detectable levels of CD6. This cell preferably does not express detectable levels of any number and combination of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5 and CD7 (i.e. any combination in ⁇ above which does not include CD6).
  • the IMP cell preferably expresses detectable levels of CD7.
  • the cell preferably does not express detectable levels of any number and combination of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5 and CD6 (i.e. any combination in ⁇ above which does not include CD7).
  • Suitable methods include, but are not limited to, immunocytochemistry, immunoassays, flow cytometry, such as fluorescence activated cells sorting (FACS), and polymerase chain reaction (PCR), such as reverse transcription PCR (RT-PCR).
  • Suitable immunoassays include, but are not limited to, Western blotting, enzyme-linked immunoassays (ELISA), enzyme-linked immunosorbent spot assays (ELISPOT assays), enzyme multiplied immunoassay techniques, radioallergosorbent (RAST) tests, radioimmunoassays, radiobinding assays and immunofluorescence.
  • HT-FACS high-throughput FACS
  • Antibodies and fluorescently-labelled antibodies for all of the various markers discussed herein are commercially-available.
  • the IMP cells of the invention preferably demonstrate an antibody mean fluorescence intensity
  • MFI of at least 330, such as at least 350 or at least 400, for MIC A/B, an MFI of at least 210, such as at least 250 or at least 300, for CD304 (Neuropilin 1), an MFI of at least 221, such as at least 250 or at least 300, for CD178 (FAS ligand), an MFI of at least 186, such as at least 200 or at least 250, for CD289 (Toll-like receptor 9), an MFI of at least 181, such as at least 200 or at least 250, for CD363 (Sphingosine-1 -phosphate receptor 1), an MFI of at least 184, such as at least 200 or at least 250, for CD99, an MFI of at least 300, such as at least 350 or at least 400, for CD181 (C-X-C chemokine receptor type 1; CXCR1), an MFI of at least 173, such as at least 200 or at least 250, for epidermal growth factor receptor (EGF-R), an MFI of at least 236, such
  • the IMP cells demonstrate an MFI of at least 200, such as at least 250 or at least 300, for CD66e, an MFI of at least 250, such as at least 300 or at least 350, for CD121b, an MFI of at least 200, such as at least 250 or at least 300, for CD122, an MFI of at least 150, such as at least 200 or at least 250, for CD164, an MFI of at least 200, such as at least 250 or at least 300, for CD172a, an MFI of at least 150, such as at least 200 or at least 250, for CD203c, an MFI of at least 200, such at least 250 or at least 300, for CD264, an MFI of at least 200, such as at least 250 or at least 300, for CD270, an MFI of at least 200, such as at least 250 or at least 300, for CD328, an MFI of at least 250, such as at least 300 or at least 350, for CD358, an MFI of at least 1500, such as at least 1600 or at least 1700, for HLA-
  • the IMP cells When the IMP cells express detectable levels of CD3, the IMP cells preferably demonstrate a MFI of at least 249, such as at least 300 or at least 350, for CD3. When the IMP cells express detectable levels of CD3e, the IMP cells preferably demonstrate a MFI of at least 236, such as at least 250, at least 300 or at least 350 for CD3e. When the IMP cells express detectable levels of CD4, the IMP cells preferably demonstrate an MFI of at least 235, such as at least 250 or at least 300, for CD4. When the IMP cells express detectable levels of CD5, the IMP cells preferably demonstrate an MFI of at least 215, such as at least 250 or at least 300, for CD5.
  • the IMP cells When the IMP cells express detectable levels of CD6, the IMP cells preferably demonstrate an MFI of at least 253, such as at least 300 or at least 350, for CD6.
  • the IMP cells When the IMP cells express detectable levels of CD7, the IMP cells preferably demonstrate an MFI of at least 182, such as at least 200 or at least 250, for CD7.
  • the IMP cells When the IMP cells express detectable levels of CD8 the IMP cells preferably demonstrate an MFI of at least 242, such as at least 250 or at least 300, for CD8.
  • the IMP cells When the IMP cells express detectable levels of CD8b the IMP cells preferably demonstrate an MFI of at least 311, such as at least 350 or at least 400, for CD8b.
  • Mean fluorescent intensity is a measure of intensity, time average energy flux measured in watts per square metre. It is an SI unit.
  • the MFI for each marker is typically measured using HT- FACS.
  • the MFI for each marker is preferably measured using HT-FACS as described in the Example.
  • the IMP cells of the invention typically express detectable levels of one or more of the other markers shown in Table 1 in the Example.
  • the IMP cells may express detectable levels of any number and combination of those markers.
  • the IMP cells of the invention express levels of one or more of the other markers shown in Table 1 in the Example, apart from FMC7 and ITGB8.
  • CD49d, CD146, CD340, Notch 2 CD49b, CD63, CD58, CD44, CD49c, CD105, CD166, HLA-ABC, CD13, CD29, CD49e, CD73, CD81, CD90, CD98, CD147, CD151 and CD276.
  • the IMP cells preferably express detectable levels of one or more of CD156b, CD61, CD202b, CD130, CD148, CD288, CD337, SSEA-4, CD349 and CD140b.
  • the IMP cells more preferably express detectable levels of one or more of CD156b, CD61, CD202b, CD130, CD148, CD288, CD337, SSEA-4, CD349, CD140b, CD10, CD111, CD267, CD47, CD273, CD51/CD61, CD49f, CD49d, CD146, CD55, CD340, CD91, Notch 2, CD175s, CD82, CD49b, CD95, CD63, CD245, CD58, CD108, B2-microglobulin, CD155, CD298, CD44, CD49c, CD105, CD166, CD230, HLA-ABC, CD13, CD29, CD49e, CD59, CD73, CD81, CD90, CD98, CD147, CD151 and CD276.
  • the IMP cells may
  • the IMP cells preferably express detectable levels of one or more of CD72, CD133, CD192,
  • the IMP cells preferably express detectable levels of all of these markers.
  • the IMP cells of the invention are preferably capable of migrating to a specific, damaged tissue in a patient.
  • the cells when the cells are administered to a patient having a damaged tissue, the cells are capable of migrating (or homing) to the damaged tissue.
  • This is advantageous because it means that the cells can be infused via standard routes, for instance intravenously, and will then target the site of damage.
  • the cells do not have to be delivered to the damaged tissue.
  • the damage may be due to injury or disease as discussed in more detail below.
  • the specific tissue is preferably cardiac, bone, cartilage, tendon, ligament, liver, kidney or lung tissue. This applies not only to migration, but also adherence, transmigration, proliferation, antiinflammatory effects and angiogenesis as discussed in more detail below.
  • the ability of the IMP cells of the invention to migrate to damaged tissue may be measured using standard assays known in the art. Suitable methods include, but are not limited to, genomic reverse transcription polymerase chain reaction (RT-PCR with or without reporter genes) and labelling techniques.
  • RT-PCR genomic reverse transcription polymerase chain reaction
  • labelling techniques include, but are not limited to, labelling techniques.
  • RT-PCR is the most straightforward and simple means to trace the IMP cells of the invention within a patient.
  • a transduced transgene or individual donor markers can be used for this purpose and transplanted cell-specific signals have been obtained in several patient studies. The results are generally semi-quantitative.
  • the IMP cells of the invention may be stained with a dye of interest, such as a fluorescent dye, and may be monitored in the patient via the signal from the dye.
  • a dye of interest such as a fluorescent dye
  • Migration is typically determined by measuring the number of cells that arrive at the damaged tissue. It may also be measured indirectly by observing the numbers of cells that have accumulated in the lungs (rather than the damaged tissue).
  • Damaged heart tissue releases inflammatory chemokines and cytokines, such as stromal cell- derived factor-1 (SDF-1), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), granulocyte- colony-stimulating factor (G-CSF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF).
  • chemokines and cytokines such as stromal cell- derived factor-1 (SDF-1), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), granulocyte- colony-stimulating factor (G-CSF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF).
  • myocardial infarct increases the levels of VEGF and erythropoietin (EPO).
  • CXCR4 binds to its ligand SDF-1 and so IMP cells of the invention expressing CXCR4
  • the IMP cells of the invention preferably express detectable levels of TGF-beta 3, bone morphogenetic protein-6 (BMP-6), SOX-9, Collagen-2, CD117 (c-kit), chemokine (C-C motif) ligand 12 (CCL12), CCL7, interleukin-8 (IL-8), platelet-derived growth factor-A (PDGF-A), PDGF-B, PDGF-C, PDGF-D, macrophage migration inhibitory factor (MIF), IGF- 1, hepatocyte growth factor (HGF), PDGF-Ra, PDGF-R , CXCR4, C-C chemokine receptor type 1 (CCR1), IGF-1 receptor (IGF-1R), hepatocyte growth factor receptor (HGFR), CXCL12 and NFkappaB.
  • the bone-homing IMP cells of the invention preferably express an increased amount of one or more of, or even all of, these factors compared with mesenchymal stem cells MSC
  • the IMP cells of the invention are preferably capable of adhering to a specific, damaged tissue in a patient.
  • Adherence and adhesion assay are known in the art (Humphries, Methods Mol Biol. 2009;522:203-10).
  • the IMP cells of the invention are preferably capable of transmigrating through the vascular endothelium to a specific, damaged tissue in a patient.
  • Transmigration assays are known in the art (Muller and Luscinskas, Methods Enzymol. 2008; 443: 155-176).
  • the IMP cells of the invention are preferably capable of proliferating in a specific, damaged tissue in a patient.
  • Cell proliferation assays are well known in the art. Such assays are commercially available, for instance from Life Technologies®.
  • the IMP cells of the invention are preferably capable of promoting angiogenesis in a specific, damaged tissue in a patient.
  • Angiogenesis assays are known in the art (Auerback et al. , Clin Chem. 2003 Jan;49(l):32-40).
  • the IMP cells of the invention are preferably capable of having pro-inflammatory or antiinflammatory effects in a damaged tissue of a patient.
  • the ability of the IMP cells of the invention to have pro-inflammatory or anti-inflammatory effects may also be measured using standard assays known in the art. Suitable methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs) for the secretion of cytokines, enhanced mixed leukocyte reactions and up-regulation of co-stimulatory molecules and maturation markers, measured by flow cytometry. Specific methods that may be used are disclosed in the Example.
  • ELISAs enzyme-linked immunosorbent assays
  • the cytokines measured are typically interleukins, such as interleukin-8 (IL-8), selectins, adhesion molecules, such as Intercellular Adhesion Molecule-1 (ICAM-1), and chemoattractant proteins, such as monocyte chemotactic protein- 1 (MCP-1) and tumour necrosis factor alpha (TNF-alpha).
  • IL-8 interleukin-8
  • selectins selectins
  • adhesion molecules such as Intercellular Adhesion Molecule-1 (ICAM-1)
  • chemoattractant proteins such as monocyte chemotactic protein- 1 (MCP-1) and tumour necrosis factor alpha (TNF-alpha).
  • MCP-1 monocyte chemotactic protein- 1
  • TNF-alpha tumour necrosis factor alpha
  • the IMP cells preferably secrete detectable levels of one or more of interleukin-6 (IL-6), IL-8, C-X-C motif chemokine 10 (CXCL10; interferon gamma-induced protein 10; IP-10), Chemokine (C-C motif) ligand 2 (CCL2; monocyte chemotactic protein-1; MCP-1) and Chemokine (C-C motif) ligand 5 (CCL5; regulated on activation, normal T cell expressed and secreted; RANTES).
  • IL-6 interleukin-6
  • IL-8 C-X-C motif chemokine 10
  • CXCL10 interferon gamma-induced protein 10
  • IP-10 interferon gamma-induced protein 10
  • CCL2 Chemokine (C-C motif) ligand 2
  • CCL5 Chemokine (C-C motif) ligand 5 (CCL5; regulated on activation, normal T cell expressed and secreted; RANTES).
  • the IMP cells preferably secrete an increased amount of one or more of IL-6, IL-8, IP- 10, MCP-1 and RANTES compared with a MSC.
  • the IMP cells may secrete an increased amount of any number and combination of these factors.
  • the IMP cells preferably secrete an increased amount of all of these markers.
  • the IMP cells preferably secrete a decreased amount of interleukin-10 (IL-10) and/or IL-12 compared with a mesenchymal stem cell MSC.
  • IL-10 and IL-12 are pro-inflammatory cytokines.
  • the IMP cells of the invention are more preferably capable of migrating to a damaged tissue in a patient and having anti-inflammatory effects in the damaged tissue. This allows the damage to be repaired effectively and reduces the number of cells that need to be administered.
  • the IMP cells of the invention will express a variety of different other markers over and above those discussed above. Some of these will assist the IMP cells will their ability to migrate to a damaged tissue and have anti-inflammatory effects once there. Any of the IMP cells of the invention may further express detectable levels of one or more of (i) insulin-like growth factor- 1 (IGF-1), (ii) IGF-1 receptor; (iii) C-C chemokine receptor type 1 (CCR1), (iv) stromal cell-derived factor-1 (SDF- 1), (v) hypoxia-inducible factor-1 alpha (HIF-1 alpha), (vi) Aktl and (vii) hepatocyte growth factor (HGF) and/or granulocyte colony-stimulating factor (G-CSF).
  • IGF-1 insulin-like growth factor- 1
  • IGF-1 receptor IGF-1 receptor
  • CCR1 C-C chemokine receptor type 1
  • SDF- 1 stromal cell-derived factor-1
  • HGF hypoxia-inducible
  • IGF-1 receptors promote migration capacity towards an IGF-1 gradient.
  • One of the mechanisms by which IGF-1 increases migration is by up-regulating CXCR4 on the surface of the cells, which makes them more sensitive to SDF-1 signaling. This is discussed above.
  • CCR1 is the receptor for CCL7 (previously known as MCP3) increases homing and engraftment capacity of MSCs (and so would be expected to have the same effect for the IMP cells of the invention) and can increase the capillary density in injured myocardium through paracrine signalling.
  • HIF-1 alpha activates pathways that increase oxygen delivery and promote adaptive pro- survival responses.
  • target genes of HIF-1 alpha are erythropoietin (EPO), endothelin and VEGF (with its receptor Flk-1).
  • EPO erythropoietin
  • VEGF VEGF
  • Flk-1 VEGF
  • IMP cells that express or express an increased amount of HIF- 1 alpha will have upregulated expression of paracrine stimuli of for example several vasculogenic growth factors that may promote a more therapeutic subtype.
  • the IMP cells of the invention can be preconditioned into a more therapeutic subtype by culturing them under hypoxic conditions (less than 20% oxygen), such as for example about 2% or about 0% oxygen.
  • Aktl is an intracellular serine/threonine protein kinase that plays a key role in multiple cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription and cell migration. Overexpression of Aktl has been shown to prevent rat MSCs from undergoing apoptosis and will have the same effect in the IMP cells of the invention. Protection from apoptosis will enhance the therapeutic effect of the IMP cells.
  • HGF and G-CSF exhibit synergistic effects in this regard.
  • MSCs that have a high expression of HGF and its receptor c-met also have an increased migratory capacity into the damaged tissue, achieved through hormonal, paracrine and autocrine signaling. The same will be true for the IMP cells of the invention expressing HGF and/or G-CSF.
  • the IMP cells may express detectable levels off one or more of (i) to (vii) defined above.
  • the IMP cells of the invention preferably express an increased amount of one or more of (i) to (vii) compared with MSCs. Quantitative assays for cell markers are described above. The detectable expression of these markers and their level of expression may be measured as discussed above.
  • any of the IMP cells of the invention may express detectable levels of one or more of (i) vascular endothelial growth factor (VEGF), (ii) transforming growth factor beta (TGF-beta), (iii) insulin-like growth factor- 1 (IGF-1), (iv) fibroblast growth factor (FGF), (v) tumour necrosis factor alpha (TNF-alpha), (vi) interferon gamma (IFN -gamma) and (vii) interleukin-1 alpha (IL-1 alpha).
  • VEGF vascular endothelial growth factor
  • TGF-beta transforming growth factor beta
  • IGF-1 insulin-like growth factor- 1
  • FGF fibroblast growth factor
  • TNF-alpha tumour necrosis factor alpha
  • IFN -gamma interferon gamma
  • IL-1 alpha interleukin-1 alpha
  • the IMP cells may express detectable levels of one or more of (i) to (vii).
  • the IMP cells of the invention may express an increased amount of one or more of (i) to (vii) compared with MSCs.
  • Quantitative assays for cell markers are described above. The detectable expression of these markers and their level of expression may be measured as discussed above.
  • any combination of one or more of (i) to (vii) may be expressed or expressed in an increased amount.
  • the IMP cells may express detectable levels of, or express an increased amount of, (i); (ii); (iii);
  • the IMP cells of the invention preferably express and/or secrete detectable levels of interferon gamma (IFN-gamma).
  • IFN-gamma interferon gamma
  • the IMP cells of the invention preferably express and/or secrete an increased amount of IFN-gamma compared with a MSC. IFN-gamma expression or secretion may be determined using the methods set out above.
  • the IMP cells of the invention preferably also express detectable levels of, LIF and/or platelet-derived growth factor (PDGF) receptors.
  • the IMP cells of the invention preferably express an increased amount of LIF and/or platelet-derived growth factor (PDGF) receptors compared with mesenchymal stem cells.
  • the PDGF receptors are preferably PDGF-A receptors and/or PSDGF-B receptors. MSCs that have high expression of these receptors can migrate effectively into areas in which platelets have been activated, such as wounds and thrombotic vessels. The same will be true of IMP cells expressing or expressing an increased amount of the receptors.
  • the IMP cells of the invention are preferably capable of immuno-modulation.
  • Immuno- modulation is the modulation of an immune response or of the activity of an immune cell. Immuno- modulation may be achieved by a variety of mechanisms, For instance, the IMP cells may secrete cytokines or inflammatory mediators that alter act on immune cells to alter their activity. The IMP cells may also signal to immune cells by other means. For example, ligands on the IMP cells may bind to receptors on target immune cells, triggering a signalling cascade. Methods for measuring cytokine secretion and marker (ligand) expression are discussed above. Methods of measuring immune cells signalling and activity are well known in the art. The IMP cells preferably use the same pathways as T cells to regulate immune responses.
  • the IMP cells of the invention are preferably autologous.
  • the cells are preferably derived from the patient into which the cells will be administered.
  • the IMP cells are preferably allogeneic.
  • the cells are preferably derived from a patient that is immunologically compatible with the patient into which the cells will be administered.
  • An IMP cell of the invention may be isolated, substantially isolated, purified or substantially purified.
  • the IMP cell is isolated or purified if it is completely free of any other components, such as culture medium, other cells of the invention or other cell types.
  • the IMP cell is substantially isolated if it is mixed with carriers or diluents, such as culture medium, which will not interfere with its intended use.
  • the IMP cell of the invention may be present in a growth matrix or immobilized on a surface as discussed below.
  • IMP cells of the invention may be isolated using a variety of techniques including antibody- based techniques.
  • Cells may be isolated using negative and positive selection techniques based on the binding of monoclonal antibodies to those surface markers which are present on the IMP cell (see above).
  • the IMP cells may be separated using any antibody-based technique, including fluorescent activated cell sorting (FACS) and magnetic bead separation.
  • FACS fluorescent activated cell sorting
  • the IMP cells may be treated ex vivo.
  • the cells may be loaded or transfected with a therapeutic or diagnostic agent and then used therapeutically in the methods of the invention.
  • the invention also provides a population of two or more IMP cells of the invention. Any number of cells may be present in the population.
  • the population of the invention preferably comprises at least about 5 x 10 5 IMP cells of the invention.
  • the population more preferably comprises at least about 1 x 10 6 , at least about 2 x 10 6 , at least about 2.5 2 x 10 6 , at least about 5 x 10 6 , at least about 1 x 10 7 , at least about 2 x 10 7 , at least about 5 x 10 7 , at least about 1 x 10 8 or at least about 2 x 10 8 IMP cells of the invention.
  • the population may comprise at least about 1.0 x 10 7 , at least about 1.0 x 10 8 , at least about 1.0 x 10 9 , at least about 1.0 x 10 10 , at least about 1.0 x 10 11 or at about least 1.0 x 10 12 IMP cells of the invention or even more.
  • the population comprising two or more IMP cells of the invention may comprise other cells in addition to the IMP cells of the invention. However, at least 70% of the cells in the population are preferably IMP cells of the invention. More preferably, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 97%, at least about 98% or at least about 99% of the cells in the population are IMP cells of the invention.
  • the invention also provides specific populations of IMP cells.
  • the invention provides a population of immuno-modulatory progenitor (IMP) cells, wherein
  • CD8b CD4, CD5, CD6 and CD7.
  • At least 50%, such as least 50%, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or at least 85% of the cells express one or more of CD3 and/or CD3e, such as CD3, CD3e or CD3 and CD3e.
  • CD3 and/or CD3e such as CD3, CD3e or CD3 and CD3e.
  • at least 90%, at least 94% or at least 94.3% of the cells in the population express detectable levels of CD3.
  • at least 90%, at least 94% or at least 94.6% of the cells in the population express detectable levels of CD3e.
  • At least 50%, such as least 50%, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or at least 85% of the cells express one or more of CD8 and/or CD8b, such as CD8, CD8b or CD8 and CD8b.
  • CD8 and/or CD8b such as CD8, CD8b or CD8 and CD8b.
  • at least 87% or at least 87.3% of the cells in the population express detectable levels of CD8.
  • At least 90%, at least 92% or at least 92.4% of the cells in the population express detectable levels of CD8b.
  • At least 90%, at least 95%, at least 99% or at least 99.1% of the cells in the population express detectable levels of CD4.
  • At least 90%, at least 91% or at least 91.7% of the cells in the population express detectable levels of CD5.
  • At least 90%, at least 93% or at least 93.3% of the cells in the population express detectable levels of CD6.
  • At least 90%, at least 95%, at least 99% or at least 99.6% of the cells in the population express detectable levels of CD7.
  • the cells expressing CD3 and the cells expressing CD3e may be the same cells or different cells. Therefore, some cells in the population may express (a) CD3 but not CD3e, (b) CD3e but not CD3 or (c) both CD3 and CD3e.
  • the population may thus contain (a); (b); (c), (a) and (b); (a) and (c); (b) and (c); or (a), (b) and (c).
  • the cells expressing CD8 and the cells expressing CD8b may be the same cells or different cells. Therefore, some cells in the population may express (a) CD8 but not CD8b, (b) CD8b but not CD8 or (c) both CD8 and CD8b.
  • the population may thus contain (a); (b); (c), (a) and (b); (a) and (c); (b) and (c); or (a), (b) and (c).
  • cells in the population may naturally express detectable levels of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • cells in the population may, when cultured, express detectable levels of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 without the need to edit the genome of the cells, or transfect or transduce the cells to express detectable levels one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • the expression of detectable levels of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 may be artificially induced in cells in the population as discussed below, for instance by editing the genome of the cell, or transfecting or transducing the cell to express detectable levels of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • the invention provides a population of immuno-modulatory progenitor (IMP) cells, wherein
  • the population may have any number and combination of (xi) to (xviii), such as any number and combination of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 shown above.
  • the cells expressing CD3 and the cells expressing CD3e may be the same cells or different cells as set out above. If, in (xiii) and (xiv), at least 50% of the cells in the population express CD8 and at least 50% of the cells in the population express CD8b, the cells expressing CD8 and the cells expressing CD8b may be the same cells or different cells as set out above.
  • the cells in any of these populations may further express detectable levels of any of the markers discussed above with reference to the IMP of the invention.
  • the cells in any of these populations may have any of the advantageous properties of the IMP cells discussed above.
  • At least 90%, such as at least 95%, of the cells in the population preferably express detectable levels of one or more of CDIO, CDl l l, CD267, CD47, CD273, CD51/CD61, CD49f, CD49d, CD146, CD55, CD340, CD91, Notch2, CD175s, CD82, CD49b, CD95, CD63, CD245, CD58, CD108, B2- microglobulin, CD155, CD298, CD44, CD49c, CD105, CD166, CD230, HLA-ABC, CD13, CD29, CD49e, CD59, CD73, CD81, CD90, CD98, CD147, CD151 and CD276. At least 90%, such as at least 95%, of the cells in the population may express detectable levels of any number and combination of these markers. At least 90%, such as at least 95%, of the cells in the population preferably express detectable levels of all of these markers.
  • At least 80%, such as at least 85%, of the cells in the population preferably express detectable levels of one or more of CD156b, CD61, CD202b, CD130, CD148, CD288, CD337, SSEA-4, CD349 and CD 140b. At least 80%, such as at least 85%, of the cells in the population may express detectable levels of any number and combination of these markers. At least 80%, such as at least 85%, of the cells in the population preferably express detectable levels of all of these markers.
  • At least 70%, such as at least 75%, of the cells in the population preferably express detectable levels of one or more of CD318, CD351, CD286, CD46, CD119 and CD 132. At least 70%, such as at least 75%, of the cells in the population may express detectable levels of any number and combination of these markers. At least 70%, such as at least 75%, of the cells in the population preferably express detectable levels of all of these markers.
  • At least 50% such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 94% or at least 94.3% of the cells in the population express detectable levels of CD3.
  • at least 50% such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 94% or at least 94.6% of the cells in the population express detectable levels of CD3e.
  • At least 50%, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% of the cells in the population express CD7.
  • at least 50%, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 87% of the cells in the population express CD8.
  • at least 50%, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 87% of the cells in the population express CD8b.
  • the population may express any combination of one or more of CD3, CD3e, CD4, CD5, CD6, CD7, CD8 and CD8b discussed above.
  • 1% or fewer, such as 0.5% or fewer, of the cells in the population preferably express detectable levels of one or more of CD72, CD133, CD192, CD207, CD144, CD41b, FMC7, CD75, CD37, CD158a, CD172b, CD282, CD100, CD94, CD39, CD158b, CD40, CD35, CD15, PAC-1, CD48, CD278, CD103, CD209, CD197, HLA-DM, CD20, CD74, CD87, CD129, CDw329, CD57, CD163, TPBG, CD206, CD243 (BD), CD19, CD52, CD184, CD107b, CD138, CD50, HLA-DR, CD158e2, CD64, DCIR, CD45, CLA, CD38, CD45RB, CD34, CD101, CD2, CD41a, CD69, CD136, CD62P, TCR alpha beta, CD16b, CDla, ITGB7, CD154, CD70, CDw218a, CD137,
  • 1% or fewer, such as 0.5% or fewer, of the cells in the population may express detectable levels of any number and combination of these markers. 1% or fewer, such as 0.5% or fewer, of the cells in the population preferably express detectable levels of all of these markers.
  • 10% or fewer, such as 8% or fewer, 7% or fewer, 6% or fewer, 5% or fewer, 4% or fewer, 3% or fewer or 2% or fewer of the cells in the population express detectable levels of one or more of CD4, CD5, CD6, CD7, CD8 and CD8b, as set out above.
  • the at least 50% of the cells in the population express detectable levels of CD3 and/or CD3e, 10% or fewer, such as 8% or fewer, 7% or fewer, 6% or fewer, 5% or fewer, 4% or fewer, 3% or fewer or 2% or fewer of the cells in the population preferably express detectable levels of one or more of CD4, CD5, CD6, CD7, CD8 and CD8b, as set out above.
  • 10% or fewer, such as 8% or fewer, 7% or fewer, 6% or fewer, 5% or fewer, 4% or fewer, 3% or fewer or 2% or fewer of the cells in the population preferably express detectable levels of one or more of CD3, CD3e, CD4, CD5, CD6 and CD7, as set out above.
  • the at least 50% of the cells in the population express detectable levels of CD4, 10% or fewer, such as 8% or fewer, 7% or fewer, 6% or fewer, 5% or fewer, 4% or fewer, 3% or fewer or 2% or fewer of the cells in the population preferably express detectable levels of one or more of CD3, CD3e, CD5, CD6, CD7, CD8 and CD8b, as set out above.
  • the at least 50% of the cells in the population express detectable levels of CD5, 10% or fewer, such as 8% or fewer, 7% or fewer, 6% or fewer, 5% or fewer, 4% or fewer, 3% or fewer or 2% or fewer of the cells in the population preferably express detectable levels of one or more of CD3, CD3e, CD4, CD6, CD7, CD8 and CD8b, as set out above.
  • the at least 50% of the cells in the population express detectable levels of CD6, 10% or fewer, such as 8% or fewer, 7% or fewer, 6% or fewer, 5% or fewer, 4% or fewer, 3% or fewer or 2% or fewer of the cells in the population preferably express detectable levels of one or more of CD3, CD3e, CD4, CD5, CD7, CD8 and CD8b, as set out above.
  • the at least 50% of the cells in the population express detectable levels of CD7, 10% or fewer, such as 8% or fewer, 7% or fewer, 6% or fewer, 5% or fewer, 4% or fewer, 3% or fewer or 2% or fewer of the cells in the population preferably express detectable levels of one or more of CD3, CD3e, CD4, CD5, CD6, CD8 and CD8b, as set out above.
  • the populations preferably comprise at least 5,000 cells, such as at least 6,000, at least 7,000, at least 8,000, at least 9,000, at least 10,000, at least 20,000, at least 30,000 or at least 40,000 cells.
  • populations may comprise any of the number of cells discussed above. Any of the populations of cells disclosed herein may be diluted with other cells before use. For instance, the population may be combined with patient blood, mononuclear cells (MCs), MSCs, progenitor cells of the mesodermal lineage (PMLs) or a combination thereof. PMLs are disclosed in PCT/GB2012/051600 (published as WO 2013/005053).
  • the populations of the invention are advantageous for therapy as discussed below. This ability to produce populations comprising large numbers of IMP cells of the invention is one of the key advantages of the invention.
  • the invention allows the treatment of patients with a population of cells of which most, if not all, migrate efficiently to the tissue of interest and have anti -inflammatory effects once there. This allows the use of a low cell-dose and avoids off-target side effects and volume-related side effects.
  • the population of the invention is preferably homologous.
  • all of the IMP cells in the population are preferably genotypically and phenotypically identical.
  • the population is preferably autologous or allogeneic as defined above.
  • the population can also be semi-allogeneic.
  • Semi-allogeneic populations are typically produced from mononuclear cells from two or more patients that are immunologically compatible with the patient into which the population will be administered.
  • all of the cells in the population are preferably genetically identical or sufficiently genetically identical that the population is immunologically compatible with the patient into which the population will be administered.
  • the IMP cells of the invention may be derived from a patient, they may be autologous with the patient to be treated (i.e. genetically identical with the patient or sufficiently genetically identical that they are compatible for administration to the patient).
  • the population is typically cultured in vitro. Techniques for culturing cells are well known to a person skilled in the art.
  • the cells are may be cultured under standard conditions of 37°C, 5% CO2 in medium without serum.
  • the cells are preferably cultured under low oxygen conditions as discussed in more detail below.
  • the cells may be cultured in any suitable flask or vessel, including wells of a flat plate such as a standard 6 well plate. Such plates are commercially available from Fisher scientific, VWR suppliers, Nunc, Starstedt or Falcon.
  • the wells typically have a capacity of from about lml to about 4ml.
  • the flask, vessel or wells within which the population is contained or cultured may be modified to facilitate handling of the IMP cells.
  • the flask, vessel or wells may be modified to facilitate culture of the cells, for instance by including a growth matrix.
  • the flask, vessel or wells may be modified to allow attachment of the IMP cells or to allow immobilization of the IMP cells onto a surface.
  • One or more surfaces may be coated with extracellular matrix proteins such as laminin or collagen or any other capture molecules that bind to the cells and immobilize or capture them on the surface(s).
  • the population may be modified ex vivo using any of the techniques described herein. For instance, the population may be transfected or loaded with therapeutic or diagnostics agents. The population may then be used in the methods of treatment discussed in more detail below.
  • the invention also provides a method for producing a population of the invention.
  • the method involves culturing mononuclear cells (MCs) under conditions which induce the MCs to differentiate into IMP cells.
  • the method then involves harvesting and culturing the IMP cells which express detectable levels of (a) MIC A/B, CD304 (Neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like receptor 9), CD363 (Sphingosine-1 -phosphate receptor 1), CD99, CD181 (C-X-C chemokine receptor type 1; CXCRl), epidermal growth factor receptor (EGF-R), CXCR2 and CD 126, and (b) one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • the method involves harvesting and culturing the IMP cells which express detectable levels of MIC A/B, CD304 (Neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like receptor 9), CD363 (Sphingosine-1 -phosphate receptor 1), CD99, CD 181 (C-X-C chemokine receptor type 1 ; CXCRl ), epidermal growth factor receptor (EGF- R), CXCR2, CD126, CD3, CD3e, CD66e, CD121b, CD122, CD164, CD172a, CD203c, CD264, CD270, CD328, CD358, HLA-ABC, Notch2, T cell receptor (TCR) gamma delta, CD360 and CD1 lb and which do not express detectable levels of FMC7 and ITGB7.
  • the harvested cells may express detectable levels of or increased amounts of any of the markers and factors described above with reference to the cells of the invention.
  • the MCs may be primary MCs isolated from bone marrow.
  • the MCs are preferably peripheral blood MCs (PBMCs), such as lymphocytes, monocytes and/or macrophages.
  • PBMCs can be isolated from blood using a hydrophilic polysaccharide, such as Ficoll®.
  • Ficoll-Paque® a commercially-available density medium
  • the MCs Before they are cultured, the MCs may be exposed to a mesenchymal stem cell enrichment cocktail.
  • the cocktail preferably comprises antibodies that recognise CD14, CD19, CD38, (which are present on unwanted cells) and a component of red blood cells. Such a cocktail cross links unwanted cells with red blood cells forming immunorosettes which may be removed from the wanted MCs.
  • a preferred cocktail is RosetteSep®.
  • Conditions suitable for inducing MCs to differentiate into mesenchymal cells are known in the art. For instance, suitable conditions are disclosed in Capelli, C, et al. (Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts. Bone Marrow Transplantation, 2007. 40: p. 785-791). These conditions may also be used to induce MCs to differentiate into IMP cells in accordance with the invention.
  • the MCs are may be cultured under standard conditions of 37°C, 5% CO2 in medium without serum. MCs are typically seeded at a density of lxl 0 5 cells cm 2 .
  • the method preferably comprises culturing MCs with plasma lysate to induce the MCs to differentiate into IMP cells.
  • Platelet lysate refers to the combination of natural growth factors contained in platelets that has been released through lysing those platelets. Lysis can be accomplished through chemical means (i.e. CaCh ), osmotic means (use of distilled H2O) or through freezing/thawing procedures. Platelet lysate can be derived from whole blood as described in U.S. Pat. No. 5,198,357. Platelet lysate is preferably prepared as described in PCT/GB12/052911 (published as WO
  • the plasma lysate is preferably human plasma lysate.
  • step (a) of the method of the invention comprises culturing MCs in a medium comprising platelet lysate for sufficient time to induce the MCs to differentiate into IMP cells.
  • the sufficient time is typically from about 15 to about 25 days, preferably about 22, 23, 24 or 25 days.
  • the medium preferably comprises about 20% or less platelet lysate by volume, such as about 15% or less by volume or about 10% or less by volume.
  • the medium preferably comprises from about 5% to about 20% of platelet lysate by volume, such as from about 10% to about 15% by volume.
  • the medium preferably comprises about 10% of platelet lysate by volume.
  • step (a) of the method of the invention comprises exposing
  • the MCs to a mesenchymal enrichment cocktail and then culturing the MCs in a medium comprising platelet lysate for sufficient time to induce the MCs to differentiate into IMP cells.
  • the sufficient time is typically from about 15 to about 25 days, preferably about 22 days.
  • the medium is preferably Minimum Essential Medium (MEM).
  • MEM is commercially available from various sources including Sigma- Aldrich.
  • the medium preferably further comprises one or more of heparin, L-glutamine and penicillin/streptavidin (P/S).
  • P/S penicillin/streptavidin
  • the L-glutamine may be replaced with GlutaMAX® (which is commercially-available from Life Technologies).
  • CXCR4 cytokine-dependent and is increased when cells are exposed to stem cell factor (SCF), interleukin-6 (IL-6), Flt-3 ligand, hepatocyte growth factor (HGF) and IL-3.
  • SCF stem cell factor
  • IL-6 interleukin-6
  • Flt-3 Flt-3 ligand
  • HGF hepatocyte growth factor
  • the medium may comprise one or more of (i) SCF, (ii) IL-6, (iii) Flt-3 ligand, (iv) hepatocyte growth factor and (v) IL-3, such as (i); (ii); (iii); (iv); (v); (i) and (ii); (i) and (iii); (i) and (iv); (i) and (v); (ii) and (iv); (iii) and (iv); (iii) and (iv); (iii) and (v); (iv) and (v); (i), (ii) and (ii); (i), (ii) and (v); (iv) and (v); (i), (ii) and (ii); (i), (ii) and (iv); (iv); (i), (ii) and (iv); (i), (iii); (i), (iv);
  • any of (i) to (v) may be present at from about from about 10 to about about 150 ng/ml.
  • Step (a) preferably comprises culturing the MCs under conditions which allow the IMP cells to adhere. Suitable conditions are discussed in more detail above.
  • the MCs are preferably cultured under low oxygen conditions.
  • the MCs are preferably cultured at less than about 20% oxygen (O2), such as less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2% or less than about 1% oxygen (O2).
  • O2 oxygen
  • the MCs are most preferably cultured in the presence of platelet lysate and under low oxygen conditions. This combination mimics the natural conditions in the damaged tissue and so result in healthier and more therapeutically potent cells.
  • Conventional cell culture is performed in 20% or 21% oxygen (approximately the atmospheric content) but there is no place in the human body that has this oxygen level.
  • the epithelial cells in the lungs would "see” this oxygen level, but once the oxygen is dissolved and leaves the lungs, it decreases to around 17%. From there, it decreases even further to about 1-2% in the majority of the tissues, but being as low as 0.1% in avascular tissues such as the cartilage in the joints.
  • step (a) the method preferably comprises culturing the MCs under conditions which induce the MCs to differentiate into immuno-modulatory progenitor (iMP) cells. This is described in
  • the iMP cells express detectable levels of MIC A/B, CD304 (Neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like receptor 9), CD363, (Sphingosine-1 -phosphate receptor 1), CD99, CD181 (C-X-C chemokine receptor type 1; CXCRl), epidermal growth factor receptor (EGF-R), CXCR2 and CD 126.
  • the iMP cells also typically express detectable levels of CD29, CD44, CD73, CD90, CD 105 and CD271 and do not express detectable levels of CD14, CD34 and CD45.
  • Platelet lysate is preferably prepared as described in International Patent Application No. PCT/GB12/052911 (published as WO 2013/076507). For instance, it may be prepared by subjecting a population of platelets to at least one freeze-thaw cycle, wherein the freeze portion of each cycle is carried out at a temperature lower than or equal to -78 °C.
  • the platelet lysate is preferably prepared by subjecting a population of platelets to four freeze-thaw cycles, wherein the freeze portion of each cycle is carried out at a temperature lower than or equal to - 78 °C, for instance using liquid nitrogen.
  • the method preferably further comprises culturing the MCs cells under conditions which epigenetically modify the MCs to form the IMP cells of the invention, namely IMP cells which express detectable levels of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • the method preferably comprises culturing the MCs under conditions which induce the MCs to differentiate into immuno-modulatory progenitor (iMP) cells of the invention.
  • the conditions may comprise one or more of (a) to (f) in the Table below.
  • CD4 Culture medium comprises an anti-CD4 antibody
  • CD5 Co-culture with B cells and culture medium comprises less than about 20 pg/ml interleukin-10 (IL-10)
  • CD6 Co-culture with T cells and culture medium comprises greater than about 200 pg/ml IL-10
  • CD7 Culture medium comprises greater than about 300 pg/ml interleukin-15 (IL-15)
  • CD8 and/or CD8b Culture medium comprises greater than about 200 pg/ml IL-10
  • T cells and B cells in any of (a), (c) and (d) may be any of those discussed below.
  • Anti-CD4 antibodies for (b) are commercially available, such as from BioLegend, Abeam and R&D Systems.
  • the culture medium preferably comprises less than about 15 pg/ml IL-10 or less than about 10 pg/ml IL-10.
  • the culture medium in (c) preferably comprises from about 1 to about 20 pg/ml IL-10, from about 2 to about 15 pg/ml IL-10 or from about 5 to about 10 pg/ml IL-10.
  • the culture medium preferably comprises greater than about 300 pg/ml IL-10, greater than about 400 pg/ml IL-10 or greater than about 500 pg/ml IL-10.
  • the culture medium in (d) and/or (f) preferably comprises from about 200 to about 900 pg/ml IL-10, from about 250 to about 750 pg/ml IL-10 or from about 300 to about 500 pg/ml IL-10.
  • the culture medium preferably comprises greater than about 400 pg/ml IL-15, greater than about 500 pg/ml IL-15 or greater than about 600 pg/ml IL-15.
  • the culture medium in (d) and/or (f) preferably comprises from about 300 to about 900 pg/ml IL-15, from about 350 to about 750 pg/ml IL- 15 or from about 400 to about 600 pg/ml IL- 15.
  • the conditions may comprise any number and combination of (a) to (f), such as ⁇ a ⁇ ; ⁇ f ⁇ ; ⁇ b ⁇ ; ⁇ c ⁇ ; ⁇ d ⁇ ; ⁇ e ⁇ ; ⁇ a,f ⁇ ; ⁇ a,b ⁇ ; ⁇ a,c ⁇ ; ⁇ a,d ⁇ ; ⁇ a,e ⁇ ; ⁇ f,b ⁇ ; ⁇ f,c ⁇ ; ⁇ f,d ⁇ ; ⁇ f,e ⁇ ; ⁇ b,c ⁇ ; ⁇ b,d ⁇ ; ⁇ b,e ⁇ ; ⁇ c,d ⁇ ;
  • step (b) the method further comprises harvesting and culturing IMP cells which have the necessary marker expression pattern as discussed above.
  • the IMP cells having the necessary marker expression partem may be harvested using any antibody -based technique, including fluorescent activated cell sorting (FACS) and magnetic bead separation. FACS is preferred. HT-FACS is more preferred. Any of the methods for culturing IMP cells disclosed in relation to step (a) equally apply to step (b). In particular, the cells are cultured in step (b) in the presence of platelet lysate and under low oxygen conditions as discussed above in relation to step (a).
  • FACS fluorescent activated cell sorting
  • HT-FACS is more preferred.
  • IMP cells of the invention particularly suitable for administration to patients.
  • the MCs are preferably obtained from a patient or an allogeneic donor.
  • the invention also provides a method for producing a population of the invention that is suitable for administration to a patient, wherein the method comprises culturing MCs obtained from the patient under conditions which induce the MCs to differentiate into IMP cells and (b) harvesting and culturing those progenitor cells which have an expression pattern as defined above and thereby producing a population of the invention that is suitable for administration to the patient.
  • the population will be autologous with the patient and therefore will not be rejected upon implantation.
  • the invention also provides a population of the invention that is suitable for administration to a patient and is produced in this manner.
  • the invention provides a method for producing a population of the invention that is suitable for administration to a patient, wherein the method comprises culturing MCs obtained from a different patient that is immunologically compatible with the patient into which the cells will be administered under conditions which induce the MCs to differentiate into IMP cells and (b) harvesting and culturing those IMP cells which have an expression pattern as defined above and thereby producing a population of the invention that is suitable for administration to the patient.
  • the population will be allogeneic with the patient and therefore will reduce the chance of rejection upon implantation.
  • the invention also provides a population of the invention that is suitable for administration to a patient and is produced in this manner.
  • the IMP cells or population of the invention may be used in an in vitro method of regulating the activity of immune cells.
  • the IMP cells may regulate the activity of any immune cells, such as T cells, B cells, dendritic cells, neutrophils, basophils, mast cells, eosinophils, innate lymphoid cells (ILCs), natural killer (NK) cells, monocytes, macrophages, megakaryocytes, thymocytes or platelets.
  • the IMP cells are used to regulate the activity of T cells. More preferably, the IMP cells are used to regulate the activity of helper T (Th) cells, cytotoxic T cells, regulatory T cells (Treg), gamma delta T cells or natural killer T (NKT) cells.
  • cytotoxic, helper or gamma delta T cells are preferred. Any reference to cytotoxic, helper or gamma delta T cells herein may refer to (i) cytotoxic T cells, (ii) helper T cells, (iii) gamma delta T cells, (iv) cytotoxic T cells and helper T cells, (v) helper T cells and gamma delta T cells, (vi) cytotoxic T cells and gamma delta T cells or (vii) cytotoxic T cells, helper T cells and gamma delta T cells.
  • the IMP cells of the invention express one or more of CD3, CD3e, CD8, CD8b, CD4, CD5,
  • CD3 is a T cell co-receptor that helps to activate the T cell.
  • CD3e is a particular change in the four-chain CD3 complex.
  • CD3 and CD3e each have an important function in T cell activation.
  • Expression of CD3 and/or CD3e by the IMP cells of the invention facilitates the regulation of T cells. Such expression may improve the interaction of the IMPs of the invention with the T cells that they target. Such expression may also improve intracellular and/or paracrine signalling between the IMP cells of the invention and T cells. Therefore, the IMP cells of the invention have an improved ability to regulate T cell activity, function and/or proliferation. This improved ability to regulate T cells may enhance the T cells' and/or the IMP cells' anti-leukaemic and/or anti-metastatic effect. It may also help to maintain T cell specificity, reducing off-target or bystander damage.
  • CD8 and CD8b are T cell co-receptors that helps to activate the T cell.
  • CD8 and/or CD8b by the IMP cells of the invention facilitates the regulation of T cells.
  • Such expression may improve the interaction of the IMPs of the invention with the T cells that they target.
  • Such expression may also improve intracellular and/or paracrine signalling between the IMP cells of the invention and T cells. Therefore, the IMP cells of the invention have an improved ability to regulate T cell activity, function and/or proliferation. This improved ability to regulate T cells may enhance the T cells' and/or the IMP cells' anti-leukaemic and/or anti-metastatic effect. It may also help to maintain T cell specificity, reducing off-target or bystander damage.
  • CD4 is a glycoprotein found on the surface of immune cells such as T cells, monocytes, macrophages and dendritic cells.
  • CD4 is, in particular, found on the surface of T helper cells.
  • Expression of CD4 by the IMP cells of the invention thus facilitates the regulation of T cells. Such expression may improve the interaction of the IMPs of the invention with the T cells that they target. Such expression may also improve intracellular and/or paracrine signalling between the IMP cells of the invention and T cells. Therefore, the IMP cells of the invention have an improved ability to regulate T cell activity, function and/or proliferation. This improved ability to regulate T cells may enhance the T cells' and/or the IMP cells' anti-leukaemic and/or anti-metastatic effect.
  • IMP cells expressing CD4 may also have a similarly improved ability to regulate other CD4-expressing immune cells.
  • CD5 is found on T cells, B-l cells, neoplastic T cells, chronic lymphocytic leukemia cells and mantle cell lymphoma cells. In the thymus, there is a correlation with CD5 expression and strength of the interaction of the T cell towards self-peptides. Expression of CD5 by the IMP cells of the invention facilitates the regulation of T cells and B cells such as B-l cells. Such expression may improve the interaction of the IMPs of the invention with the T cells and B cells that they target.
  • Such expression may also improve intracellular and/or paracrine signalling between the IMP cells of the invention and T cells and/or B cells. Therefore, the IMP cells of the invention have an improved ability to regulate T cell and B cell activity, function and/or proliferation. This improved ability to regulate T cells may enhance the T cells' and/or the IMP cells' anti-leukaemic and/or anti- metastatic effect. It may also help to maintain T cell specificity, reducing off-target or bystander damage. Furthermore, expression of CD5 by IMP cells may facilitate IMP cell interaction and/or signalling with neoplastic T cells, chronic lymphocytic leukemia cells and mantle cell lymphoma cells. CD5-expressing IMPs may therefore have an improved ability to directly regulate such cells, and thus improved anti-leukemic and/or anti-metastatic effect.
  • CD6 is important for the continuation of T cell activation.
  • Expression of CD6 by the IMP cells of the invention facilitates the regulation of T cells. Such expression may improve the interaction of the IMPs of the invention with the T cells that they target. Such expression may also improve intracellular and/or paracrine signalling between the IMP cells of the invention and T cells. Therefore, the IMP cells of the invention have an improved ability to regulate T cell activity, function and/or proliferation. This improved ability to regulate T cells may enhance the T cells' and/or the IMP cells' anti-leukaemic and/or anti-metastatic effect. It may also help to maintain T cell specificity, reducing off-target or bystander damage.
  • CD7 is a transmembrane protein that plays an essential role in T cell interactions.
  • Expression of CD7 by the IMP cells of the invention thus facilitates the regulation of T cells.
  • Such expression may improve the interaction of the IMPs of the invention with the T cells that they target.
  • Such expression may also improve intracellular and/or paracrine signalling between the IMP cells of the invention and T cells. Therefore, the IMP cells of the invention have an improved ability to regulate T cell activity, function and/or proliferation. This improved ability to regulate T cells may enhance the T cells' and/or the IMP cells' anti-leukaemic and/or anti-metastatic effect. It may also help to maintain T cell specificity, reducing off-target or bystander damage.
  • the method may comprise incubating the immune cells with a population of the invention under conditions which regulate the activity of the immune cells.
  • the conditions may increase the activity of the immune cells.
  • the incubation may take place in the presence of lipopolysaccharide.
  • the entire period that the immune cells are incubated with a population of the invention may take place in the present of lipopolysaccharide.
  • the immune cells are incubated with a population of the invention and lipopolysaccharide for a period of one hour.
  • the conditions may decrease the activity of the immune cells.
  • the incubation may take place in the presence of poly I:C.
  • the entire period that the immune cells are incubated with a population of the invention may take place in the present of poly I:C.
  • the immune cells are incubated with a population of the invention and poly I:C for a period of 24 hours.
  • the activity of the immune cells may be evaluated during or after incubation.
  • the presence or secretion of pro-inflammatory cytokines or other mediators, or a reduction in the presence or secretion of anti-inflammatory cytokines may indicate that the activity of the immune cells has increased.
  • the presence or secretion of anti-inflammatory cytokines or other mediators, or a reduction in the presence or secretion of pro-inflammatory cytokines may indicate that the activity of the immune cells has decreased.
  • the phenotype of the population of the invention may be evaluated before, during or after incubation.
  • the presence or secretion of pro-inflammatory cytokines or other mediators, or a reduction in the presence or secretion of anti-inflammatory cytokines may indicate that the IMP cells have a pro-inflammatory phenotype and are primed to increase the activity of the immune cells.
  • the presence or secretion of anti-inflammatory cytokines or other mediators, or a reduction in the presence or secretion of pro-inflammatory cytokines may indicate that the IMP cells have an antiinflammatory phenotype and are primed to decrease the activity of the immune cells
  • the method may further comprise incubating the immune cells with an antigen.
  • the response to be modulated may be a response to the antigen.
  • the response may be antigen-specific.
  • the invention provides an in vitro method of increasing the activity of cytotoxic, helper or gamma delta T cells in response to an antigen.
  • Gamma delta T cells are preferred.
  • T cell proliferation and/or cytokine secretion may be measured in response to stimulation (e.g. with the antigen, or with antibodies that bind to the TCR and/or co-stimulatory receptors).
  • stimulation e.g. with the antigen, or with antibodies that bind to the TCR and/or co-stimulatory receptors.
  • activation e.g.
  • the method may comprise the step of incubating the T cells with the antigen and a population of the invention.
  • the incubation may be carried out under conditions which increase the activity of the T cells. Such conditions are discussed above and below.
  • the invention also provides primed cytotoxic, helper or gamma delta T cells produced according to this in vitro method. Gamma delta T cells are preferred.
  • Primed T cells are T cells that will robustly respond to an antigen following further contact with the antigen.
  • the invention also provides an in vitro method of increasing the activity of regulatory T cells in response to an antigen. Methods of measuring T cell activity are discussed above.
  • the method may comprise incubating the T cells with the antigen and a population of the invention. The incubation may be carried out under conditions which increase the activity of the T cells. Such conditions are discussed above and below.
  • the invention further provides primed regulatory T cells produced according to this in vitro method.
  • the invention provides an in vitro method of decreasing the activity of cytotoxic, helper or gamma delta T cells in response to an antigen.
  • Gamma delta T cells are preferred.
  • Methods of measuring T cell activity are discussed above.
  • the method may comprise incubating the T cells with the antigen and a population of the invention. The incubation may be carried out under conditions which decrease the activity of the T cells. Such conditions are discussed above and below.
  • the invention further provides suppressed cytotoxic, helper or gamma delta T cells produced according to this in vitro method.
  • Gamma delta T cells are preferred.
  • Suppressed T cells are T cells that sub- normally respond to an antigen following further contact with the antigen.
  • the invention also provides an in vitro method of decreasing the activity of regulatory T cells in response to an antigen. Methods of measuring T cell activity are discussed above.
  • the method may comprise incubating the T cells with the antigen and a population of the invention. The incubation may be carried out under conditions which decrease the activity of the T cells. Such conditions are discussed above and below.
  • the invention further provides suppressed regulatory T cells produced according to this method.
  • the T cells may be concurrently incubated with the antigen and a population of the invention.
  • the T cells may be incubated with the antigen and the population of the invention separately.
  • the T cells may be incubated with the antigen and then incubated with the population of the invention.
  • the T cells may be incubated with the population of the invention and then incubated with the antigen.
  • the T cells may be incubated with the antigen to form a T cell/antigen culture.
  • the population of the invention may then be added to the T cell/antigen culture after a period of time has elapsed.
  • the T cells may be incubated with the population of the invention to form a T cell/population culture.
  • the antigen may then be added to the T cell/population culture after a period of time has elapsed.
  • the period of time may be anything from 30 seconds to 3 days.
  • the period of time may be 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 1 day, 2 days or 3 days.
  • the antigen provided to the T cells may be any antigen to which the T cells respond.
  • the antigen may be an antigen that is found on tumour cells.
  • the antigen may also be an antigen found on cells that are present within a healthy or diseased individual.
  • the antigen may be one that is associated with autoimmune disease, such as autoimmune encephalomyelitis.
  • the antigen may alternatively be an antigen that is found on a pathogenic agent, such a bacteria, a virus or a protozoa.
  • the antigen may be an environmental antigen, such as an allergen.
  • the antigen is an antigen that is associated with atopic dermatitis, allergic airway inflammation or perennial allergic rhinitis.
  • any of the above in vitro methods may further comprise incubating the T cells with antigen presenting cells.
  • Antigen presenting cells suitable for use in the in vitro methods of the invention include professional antigen presenting cells such as dendritic cells, B cells, macrophages, monocytes, activated epithelial cells, as well as non-professional antigen presenting cells.
  • the T cells are preferably incubated with dendritic cells.
  • cytokines may be added to the cell culture.
  • IL-10 may be added to the cell culture. IL-10 can efficiently enhance immune responses and can skew the outcome towards a stronger Thl response.
  • Thl cytokines/mediators such as IL-2, IL-12, IFN-gamma or IgA may be added to skew the immune response towards a Thl response.
  • Th2 cytokines such as IL-4, IL-5, IL-5, IL-10 or alpha interferon may be added to skew the immune response towards a Th2 response.
  • the oxygen saturation of the culture may be varied.
  • the culture temperature may be varied.
  • the composition of the culture medium may be varied. The culture may be carried out in different vessels.
  • the IMPs may influence T cell activity in a variety of ways. For example, there may be interplay or cross-talk between IMP cell function and T cell function For instance, there may interplay or cross-talk between IMP cell-mediated inhibition of T-cell function, and T cell cytotoxic attack of IMP cells. Alternatively, there may be interplay or cross-talk between IMP cell cytotoxic attach of T cells, and T cell-mediated inhibition of IMP function. The balance of the interactions may determine whether there is a net increase or net decrease in T cell activation following incubation with IMP cells. The IMP cell-T-cell interaction may involve a positive feedback mechanism.
  • This mechanism may be mediated by interactions between ligands expressed on the IMP cells and receptors expressed on the T cells, or vice versa.
  • the positive feedback mechanism involves the activation of natural killer group 2d (NKG2D) on the T cells.
  • NKG2S is an activating receptor that is found on NK cells and T cells. Its ligands are stress-induced proteins such as MIC- A and MIC-B, both of which are expressed in low amounts on IMP cells.
  • the IMP cells may also be able to alter the T-cell phenotype, and suppress T cell cytokine secretion and cytotoxicity.
  • Indoleamine-pyrrole 2,3-dioxygenase and prostaglandin E2 are thought to be key mediators of IMP -induced inhibition of T cells.
  • micro-environment is of importance for IMP cell and T cell function, and for the interaction between these cell types.
  • a microenvironment rich in IFN-gamma may protect IMP cells from being attacked and destroyed by T cells.
  • the IMP cells may therefore secrete IFN-gamma to promote their own longevity and assist their immuno-modulatory function.
  • the IMP cells or population of the invention may be used in an in vivo method of regulating the activity of immune cells.
  • the IMP cells may regulate the activity of any immune cells, such as T cells, B cells, dendritic cells, neutrophils, basophils, mast cells, eosinophils, innate lymphoid cells (ILCs), natural killer (NK) cells, monocytes, macrophages, megakaryocytes, thymocytes or platelets.
  • the IMP cells are used to regulate the activity of T cells. More preferably, the IMP cells are used to regulate the activity of helper T (Th) cells, cytotoxic T cells, regulatory T cells (Treg), gamma delta T cells or natural killer T (NKT) cells. Gamma delta T cells are preferred.
  • the method may comprise administering a population or pharmaceutical composition of the invention to a subject under conditions which regulate the activity of the immune cells.
  • the conditions may increase the activity of the immune cells.
  • the conditions may decrease the activity of the immune cells.
  • the invention provides an in vivo method of increasing the activity of cytotoxic, helper or gamma delta T cells in response to an antigen.
  • Gamma delta T cells are preferred.
  • Methods of measuring T cell activity are discussed above.
  • the method may comprise administering a population or pharmaceutical composition of the invention to a subject. The administration may take place under conditions which increase the activity of the T cells. Such conditions are discussed in more detail below.
  • the invention further provides primed cytotoxic, helper or gamma delta T cells produced according to this method. Gamma delta T cells are preferred. Primed T cells are as defined above.
  • the invention also provides an in vivo method of increasing the activity of regulatory T cells in response to an antigen. Methods of measuring T cell activity are discussed above.
  • the method may comprise administering a population or pharmaceutical composition of the invention to a subject. The administration may take place under conditions which increase the activity of the T cells. Such conditions are discussed in more detail below.
  • the invention further provides primed regulatory T cells produced according to this method.
  • the invention provides an in vivo method of decreasing the activity of cytotoxic, helper or gamma delta T cells in response to an antigen.
  • Gamma delta T cells are preferred.
  • Methods of measuring T cell activity are discussed above.
  • the method may comprise administering a population or pharmaceutical composition of the invention to a subject. The administration may take place under conditions which decrease the activity of the T cells. Such conditions are discussed in more detail below.
  • the invention further provides suppressed cytotoxic, helper or gamma delta T cells produced according to this method.
  • Gamma delta T cells are preferred. Suppressed T cells are as defined above.
  • any of the above in vivo methods may further comprise administering the antigen to the subject.
  • the antigen may be administered before, at the same time as, or after the population or pharmaceutical composition is administered to the subject.
  • the antigen may be administered the subject from 1 to 28 days, such as 3 to 25 days, 6 to 22 days, 9 to 18 days or 12 to 15 days, before or after the population or pharmaceutical composition is administered.
  • the antigen may be administered the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days before or after the population or pharmaceutical composition is administered.
  • the antigen may be administered to the subject on one occasion.
  • the antigen may be administered to the subject on at least two occasions, such as at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 occasions.
  • the interval between the occasions may be from 1 to 28 days, such as 3 to 25 days, 6 to 22 days, 9 to 18 days or 12 to 15 days.
  • the interval between occasions is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days.
  • the IMP cells may be administered to the subject on one occasion.
  • the IMP cells may be administered to the subject on at least two occasions, such as at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 occasions.
  • the interval between the occasions may be from 1 to 28 days, such as 3 to 25 days, 6 to 22 days, 9 to 18 days or 12 to 15 days.
  • the interval between occasions is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days.
  • An adjuvant may be administered to the individual before, at the same time as, or after the antigen.
  • Suitable adjuvants are known in the art. These include but are not limited to alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, paraffin oil, killed Bordetella pertussis, Mycobacterium bovis, bacterial toxoids, squalene, thimerosal, detergents, plant saponins such as those from Quillaja, Soybean and Polygala senega, cytokines such as IL-1, IL-2 and IL-12, Freund's complete adjuvant and Freund's incomplete adjuvant.
  • the antigen may be any antigen to which the T cells respond.
  • the antigen may be an antigen that is found on tumour cells.
  • the antigen may be an one that is associated with autoimmune disease, such as autoimmune encephalomyelitis.
  • the antigen may also be an antigen that is found on the subject's own cells.
  • the antigen may be an antigen that is found on the cells of another healthy or diseased individual.
  • the antigen is one that is found on the cells of another individual but that is not found on the subject's own cells.
  • the antigen may alternatively be an antigen that is found on a pathogenic agent, such a bacteria, a virus or a protozoa.
  • the antigen may be an environmental antigen, such as an allergen.
  • the antigen is an antigen that is associated with atopic dermatitis, allergic airway inflammation or perennial allergic rhinitis.
  • the outcome of administration of the population of the invention is dependent on the conditions under which the population of the invention is administered.
  • Such conditions may pre-exist in the subject.
  • the conditions may naturally exist in the healthy state.
  • the conditions may be associated with disease in the subject.
  • the conditions may be induced in the subject prior to, concurrently with, or after administration of the population.
  • the conditions may be induced by administering one or more substances to the subject.
  • substances may include drugs, vaccines, antibodies, antigens, adjuvants, cytokines, nucleic acids, peptides, proteins and cells.
  • a Thl and/or Th2 immune response may pre-exist in the subject or be induced in the subject.
  • Thl responses may be enhanced by cytokines/mediators such as IL-2, IL-12, IFN- gamma, and IgA (an immunoglobulin that supports mucosal immunity).
  • Th2 immune responses may be enhanced by IL4, IL-5, IL-6 and IL-10. Accordingly, one or more of these cytokines/mediators such as IL-2, IL-12, IFN- gamma, and IgA (an immunoglobulin that supports mucosal immunity).
  • Th2 immune responses may be enhanced by IL4, IL-5, IL-6 and IL-10. Accordingly, one or more of these cytokines/mediators such as IL-2, IL-12, IFN- gamma, and IgA (an immunoglobulin that supports mucosal immunity).
  • Th2 immune responses may be enhanced by IL4, IL-5, IL-6 and IL-10. Accordingly, one or more of these cytokines/mediators such as IL-2, IL-12, IFN
  • cytokines/mediators may be present in the subject prior to administration of the population. One or more of these cytokines/mediators may be administered to the subject prior to, concurrently with, or after administration of the population.
  • administration of the population of the invention affects the Thl/Th2 balance in the subject.
  • a failure of the Thl arm of the immune system and an overactive Th2 arm is implicated in a wide variety of chronic illnesses. These include acquired immune deficiency syndrome (AIDS), chronic fatigue immune dysfunction (CFIDS), Candidiasis, allergies, Multiple Chemical Sensitivities (MCS), viral hepatitis, Gulf War Syndrome (GWS), cancer, etc.
  • AIDS acquired immune deficiency syndrome
  • CFDSS chronic fatigue immune dysfunction
  • MCS Multiple Chemical Sensitivities
  • GWS Gulf War Syndrome
  • cancer etc.
  • AIDS it has been reported that as HIV infection progresses from the asymptomatic stage to advanced disease, the immune response shifts from a more effective Thl response to an ineffective Th2 response.
  • Thl and Th2 arms of the immune system may diminish or ablate many of the symptoms associated with the chronic illnesses set out above.
  • the IMP cells of the invention may be used in a method of therapy of the human or animal body.
  • the invention provides an IMP cell of the invention or a population of the invention for use in a method of treatment of the human or animal body by therapy.
  • the invention concerns using the IMP cells of the invention or a population of the invention to repair a damaged tissue in a patient.
  • the invention also concerns using the IMP cells of the invention or a population of the invention to treat a cardiac, bone, cartilage, tendon, ligament, liver, kidney or lung injury or disease in the patient.
  • the invention provides a method of repairing a damaged tissue in a patient, comprising administering to the patient a population of the invention, wherein the population comprises a therapeutically effective number of cells, and thereby treating the damaged tissue in the patient.
  • the invention also provides a population of the invention for use in repairing a damaged tissue in the patient.
  • the invention also provides use of a population of the invention in the manufacture of a medicament for repairing a damaged tissue in a patient.
  • the tissue is preferably derived from the mesoderm.
  • the tissue is more preferably cardiac, bone, cartilage, tendon, ligament, liver, kidney or lung tissue.
  • the damage to the tissue may be caused by injury or disease.
  • the injury or disease is preferably a cardiac, bone, cartilage, tendon, ligament, liver, kidney or lung injury or disease in a patient.
  • the invention therefore provides a method of treating a cardiac, bone, cartilage, tendon, ligament, liver, kidney or lung injury or disease in a patient, comprising administering to the patient a population of the invention, wherein the population comprises a therapeutically effective number of cells, and thereby treating the injury or disease in the patient.
  • the invention also provides a population of the invention for use in treating a cardiac, bone, cartilage, tendon, ligament, liver, kidney or lung injury or disease in a patient.
  • the invention also provides use of a population of the invention in the manufacture of a medicament for treating a cardiac, bone, cartilage, tendon, ligament, liver, kidney or lung injury or disease in a patient.
  • the cardiac injury or disease is preferably selected from myocardial infarct (MI), left ventricular hypertrophy, right ventricular hypertrophy, emboli, heart failure, congenital heart deficit, heart valve disease, arrhythmia and myocarditis.
  • MI myocardial infarct
  • left ventricular hypertrophy left ventricular hypertrophy
  • right ventricular hypertrophy emboli
  • heart failure congenital heart deficit
  • heart valve disease arrhythmia
  • arrhythmia myocarditis
  • MI increases the levels of VEGF and EPO released by the myocardium. Furthermore, MI is associated with an inflammatory reaction and infarcted tissue also releases macrophage migration inhibitory factor (MIF), interleukin (IL-6) and KC/Gro-alpha. CCL7 (previously known as MCP3),
  • CXCLl, CXCL2 are significantly upregulated in the heart following myocardial infarct (MI) and might be implicated in regulating engraftment and homing of MSCs to infarcted myocardium.
  • MI myocardial infarct
  • IL-8 was shown to highly up-regulate gene expression primarily in the first 2 days post-MI. Remarkably, the increased IL-8 expression was located predominantly in the infarcted area and the border zone, and only to a far lesser degree in the spared myocardium.
  • CXCR2 By activating CXCR2, MIF displays chemokine-like functions and acts as a major regulator of inflammatory cell recruitment and atherogenesis.
  • the bone disease or injury is preferably selected from fracture, Salter-Harris fracture, greenstick fracture, bone spur, craniosynostosis, Coffin-Lowry syndrome, fibrodysplasia ossificans progressive, fibrous dysplasia, Fong Disease (or Nail-patella syndrome), hypophosphatasia, Klippel- Feil syndrome, Metabolic Bone Disease, Nail-patella syndrome, osteoarthritis, osteitis deformans (or Paget's disease of bone), osteitis fibrosa cystica (or Osteitis fibrosa or Von Recklinghausen's disease of bone), osteitis pubis, condensing osteitis (or osteitis condensans), osteitis condensans ilii, osteochondritis dissecans, osteogenesis imperfecta, osteomalacia, osteomyelitis, osteopenia, osteopetrosis, osteoporosis, osteonecrosis, porotic hyperosto
  • the bone cancer can be Ewing sarcoma, multiple myeloma, osteosarcoma (giant tumour of the bone), osteochondroma or osteoclastoma.
  • the metastatic cancer that results in a bone lesion can be breast cancer, prostate cancer, kidney cancer, lung cancer and/or adult T-cell leukemia.
  • the IMP cells in the population preferably express detectable levels of CD29, CD44, CD73, CD90, CD105, CD271, CXCR1, CXCR2 and CXCR4 and do not express detectable levels of CD14, CD34 and CD45.
  • the IMP cells in the population more preferably express detectable levels of CD29, CD44, CD73, CD90, CD105, CD271, TGF-beta 3, bone morphogenetic protein-6 (BMP-6), SOX-9, Collagen- 2, CD117 (c-kit), chemokine (C-C motif) ligand 12 (CCL12), CCL7, interleukin-8 (IL-8), platelet- derived growth factor-A (PDGF-A), PDGF-B, PDGF-C, PDGF-D, macrophage migration inhibitory factor (MIF), IGF-1, hepatocyte growth factor (HGF), PDGF-Ra, PDGF ⁇ , CXCR4, C-C chemokine receptor type 1 (CCR1), IGF-1 receptor (IGF-1R), hepatocyte growth factor receptor (HGFR), CXCL12 and NFkappaB and do not express detectable levels of CD14, CD34 and CD45.
  • BMP-6 bone morphogenetic protein-6
  • SOX-9
  • the disease or disorder may be periodontal disease, endometriosis or meniscal tears.
  • the invention concerns using the IMP cells of the invention, a population of the invention, or the pharmaceutical composition of the invention to treat disease by modulating immune cell responses.
  • the immune cells are preferably T cells.
  • the invention also concerns using the IMP cells of the invention, a population of the invention or the pharmaceutical composition of the invention to treat cancer in a subject.
  • the invention further concerns using the IMP cells of the invention, a population of the invention or the pharmaceutical composition of the invention to treat an allergic or autoimmune disease in a subject.
  • the invention provides a method of treating a disease by increasing cytotoxic, helper or gamma delta T cell responses to an antigen in a subject, the method comprising administering to the subject: (a) the population of the invention or the pharmaceutical composition of the invention; (b) the population of the invention or the pharmaceutical composition of the invention, and the primed cytotoxic, helper or gamma delta T cells of the invention; or (c) the primed cytotoxic, helper or gamma delta T cells of the invention.
  • the invention also provides a method of treating a disease by decreasing regulatory T cell responses to an antigen in a subject, the method comprising administering to the subject: (a) the population of the invention or the pharmaceutical composition of the invention; (b) the population of the invention or the pharmaceutical composition of the invention and the suppressed regulatory T cells of the invention; or (c) the suppressed regulatory T cells of the invention.
  • the invention further provides a method of treating a disease by decreasing regulatory T cell responses to an antigen in a subject, the method comprising administering to the subject: (a) the population of the invention or the pharmaceutical composition of the invention and the primed cytotoxic, helper or gamma delta T cells of the invention; or (b) the primed cytotoxic, helper or gamma delta T cells of the invention.
  • the disease may be any disease in which the subject may benefit from increased cytotoxic, helper or gamma delta T cell responses or decreased regulatory T cell response to an antigen.
  • the disease is preferably cancer.
  • the cancer is anal cancer, bile duct cancer
  • cholangiocarcinoma bladder cancer, blood cancer, bone cancer, bowel cancer, brain tumours, breast cancer, colorectal cancer, cervical cancer, endocrine tumours, eye cancer (such as ocular melanoma), fallopian tube cancer, gall bladder cancer, head and/or neck cancer, Kaposi's sarcoma, kidney cancer, larynx cancer, leukaemia, liver cancer, lung cancer, lymph node cancer, lymphoma, melanoma, mesothelioma, myeloma, neuroendocrine tumours, ovarian cancer, oesophageal cancer, pancreatic cancer, penis cancer, primary peritoneal cancer, prostate cancer, Pseudomyxoma peritonei, skin cancer, small bowel cancer, soft tissue sarcoma, spinal cord tumours, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, trachea cancer, unknown primary cancer, vagina cancer, vulva cancer or endometrial cancer
  • the leukaemia is preferably acute lymphoblastic leukaemia, acute myeloid leukaemia, chronic lymphocytic leukaemia or chronic myeloid leukaemia.
  • the lymphoma is preferably Hodgkin lymphoma or non-Hodgkin lymphoma.
  • the cancer is preferably primary cancer or secondary cancer.
  • the invention also provides a method of treating cancer in a subject, the method comprising administering to the subject a population of the invention or the pharmaceutical composition of the invention.
  • the cancer is preferably a cancer that is mentioned above with reference to treating a disease by regulating T cell responses.
  • the invention provides a method of treating a disease by decreasing cytotoxic, helper or gamma delta T cell responses to an antigen in a subject, the method comprising administering to the subject: (a) the population of the invention or the pharmaceutical composition of the invention;
  • the invention also provides a method of treating a disease by increasing regulatory T cell responses to an antigen in a subject, the method comprising administering to the subject: (a) the population of the invention or the pharmaceutical composition of the invention; (b) the population of the invention or the pharmaceutical composition of the invention, and the primed regulatory T cells of the invention; or (c) the primed regulatory T cells of the invention.
  • the invention further provides a method of treating a disease by decreasing cytotoxic, helper or gamma delta T cell responses to an antigen in a subject, the method comprising administering to the subject: (a) the population of the invention or the pharmaceutical composition of the invention and the primed regulatory T cells of the invention; or (b) the primed regulatory T cells according to of the invention.
  • the disease may be any disease in which the subject may benefit from decreased cytotoxic, helper or gamma delta T cell responses or increased regulatory T cell responses to an antigen.
  • the disease is preferably an allergic disease. More preferably, the disease is atopic dermatitis, allergic airway inflammation or perennial allergic rhinitis.
  • the disease is preferably an autoimmune disease.
  • the disease may be alopecia areata, autoimmune
  • encephalomyelitis autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), autoimmune juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune myocarditis, multiple sclerosis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma/systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, autoimmune thyroiditis, uveitis or vitiligo.
  • the disease is preferably autoimmune encephalomyelitis. In other cases, the disease is preferably an immune-mediated
  • the invention provides a method of treating an allergic, autoimmune or immune-mediated disease in a subject, the method comprising administering to the subject the population of the invention or the pharmaceutical composition of the invention.
  • the allergic, autoimmune or immune-mediated disease is preferably a disease that is mentioned above with reference to treating a disease by regulating T cell responses.
  • the antigen may be any antigen to which the T cells respond.
  • the antigen may be an antigen that is found on tumour cells.
  • the antigen may be an one that is associated with autoimmune disease, such as autoimmune encephalomyelitis.
  • the antigen may also be an antigen that is found on the subject's own cells.
  • the antigen may be an antigen that is found on the cells of another healthy or diseased individual.
  • the antigen is one that is found on the cells of another individual but that is not found on the subject's own cells.
  • the antigen may alternatively be an antigen that is found on a pathogenic agent, such a bacteria, a virus or a protozoa.
  • the antigen may be an environmental antigen, such as an allergen.
  • the antigen is an antigen that is associated with atopic dermatitis, allergic airway inflammation or perennial allergic rhinitis.
  • the method may involve administering the T cells of the invention to the subject.
  • the T cells are preferably autologous or allogeneic.
  • the T cells are preferably chimeric antigen receptor (CAR) T cells.
  • CAR T cells are described in more detail below.
  • the number of T cells administered to the subject is preferably a therapeutically effective number. For example, 0.2 x 10 6 , 0.25 x 10 6 , 0.5 x 10 6 , 1.5 x 10 6 , 4.0 x 10 6 or 5.0 x 10 6 T cells per kg of subject may be
  • T cells may be administered.
  • the number of T cells to be administered may be from 10 5 to 10 9 , preferably from 10 6 to 10 8 .
  • up to 2 x 10 8 T cells are administered to each subject.
  • the method may also involve administering both (i) the population of the invention or the pharmaceutical composition of the invention, and (ii) the T cells of the invention to the subject.
  • the population or pharmaceutical composition of the invention may be administered
  • the population or pharmaceutical composition of the invention may be administered before or after the T cells of the invention.
  • the population or pharmaceutical composition of the invention may be administered the subject from 1 to 28 days, such as 3 to 25 days, 6 to 22 days, 9 to 18 days or 12 to 15 days, before or after the T cells of the invention are administered.
  • the population or pharmaceutical composition of the invention may be administered the subject up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 21, up to 22, up to 23, up to 24, up to 25, up to 26, up to 27 or up to 28 days before or after the T cells of the invention are administered.
  • the population of the invention and/or the pharmaceutical composition of the invention and/or the T cells of the invention may be administered to the subject on one occasion.
  • the population of the invention and/or the pharmaceutical composition of the invention and/or the T cells of the invention n may be administered to the subject on at least two occasions, such as at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 occasions.
  • the interval between the occasions may be from 1 to 28 days, such as 3 to 25 days, 6 to 22 days, 9 to 18 days or 12 to 15 days.
  • the interval between occasions is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days.
  • the IMP cells may secrete cytokines.
  • the IMP cells preferably secrete pro-inflammatory cytokines or anti-inflammatory cytokines. Secretion of cytokines by IMP cells is discussed in more detail above.
  • the IMP cells may also secrete molecules that regulate apoptosis. Preferably, the IMP cells secrete pro-apoptotic or anti-apoptotic molecules.
  • the IMP cells may secrete or express pro-apoptotic molecules such as Notch2, cadherin 11 (CDH11), CD81, CD95, CD230, CD295, CD55, CD82, LBTR, beta 2-microglubulin and/or DR6.
  • Notch2 signalling is known to induce apoptosis.
  • CD82, CD95, CD230, CD81 and beta 2- microglobulin are also known to induce apoptosis.
  • Enhanced CD295 expression marks apoptotic cells.
  • LTBR activates multiple signalling pathways leading to the expression of adhesion molecules and chemokines, and cell death.
  • DR6 is also known as CD358 or TNFRSF21, and is a member of the tumour necrosis factor receptor superfamily. DR6 activates nuclear factor kappa-B and mitogen- activated protein kinase 8 and induces cell apoptosis.
  • the IMP cells may secrete anti-apoptotic molecules such as CD66e (CEACAM-5), CD264, CD63, CD 120a and/or CD 105.
  • CD66e promotes tumour cell migration, invasion, adhesion, and metastasis, and contributes to tumour formation by maintaining cellular proliferation in the presence of differentiation stimuli and by blocking apoptosis following loss of ECM anchorage.
  • CD264 has been shown to play an inhibitory role in TNF-related apoptosis-inducing ligand (TRAIL)-induced cell apoptosis.
  • CD63 is bound by TIMP-1, leading to activation of intracellular signal transduction pathways and inhibition of apoptosis.
  • CD120a is phosphorylated to recruit Bcl-2 and protect against apoptosis.
  • the IMP cells may secrete or express other molecules that regulate apoptosis, such as CD44 and/or CD59.
  • CD59 has been shown to regulate apoptosis of human lung cancer cells.
  • the IMP cells preferably target cells by contact-dependent cell lysis.
  • the IMP cells may attack tumour cells by contact-dependent cell lysis. Mechanisms of IMP action are discussed in more detail above.
  • the IMP cells of the invention are preferably derived from the patient or an allogeneic donor. Deriving the IMP cells of the invention from the patient should ensure that the IMP cells are themselves not rejected by the patient's immune system. Any difference between the donor and recipient will ultimately cause clearance of the IMP cells, but not before they have repaired at least a part of the damaged tissue.
  • the invention concerns administering to the patient a therapeutically effective number of IMP cells of the invention to the patient.
  • a therapeutically effective number is a number which ameliorates one or more symptoms of the damage, disease or injury.
  • a therapeutically effective number is preferably a number which repairs the damaged tissue or treats the disease or injury. Suitable numbers are discussed in more detail below.
  • the IMP cells of the invention may be administered to any suitable patient.
  • the patient is generally a human patient.
  • the patient may be any of the animals or mammals mentioned above with reference to the source of the IMP cells.
  • the patient may be an infant, a juvenile or an adult.
  • the patient may be known to have a damaged tissue or is suspected of having a damaged tissue.
  • the patient may be susceptible to, or at risk from, the relevant disease or injury. For instance, the patient may be genetically predisposed to heart failure.
  • the invention may be used in combination with other means of, and substances for, repairing damaged tissue , providing pain relief or treating disease.
  • the IMP cells of the invention may be administered simultaneously, sequentially or separately with other substances which are intended for repairing the damaged tissue or for providing pain relief.
  • the IMP cells may be used in combination with existing treatments for damaged tissue and may, for example, be simply mixed with such treatments.
  • the invention may be used to increase the efficacy of existing treatments of damaged tissue.
  • the invention preferably concerns the use of IMP cells loaded or transfected with a therapeutic and/or diagnostic agent.
  • a therapeutic agent may help to repair the damaged tissue.
  • a diagnostic agent such as a fluorescent molecule, may help to identify the location of the IMP cells in the patient.
  • the IMP cells may be loaded or transfected using any method known in the art. The loading of IMP cells may be performed in vitro or ex vivo. In each case, the IMP cells may simply be in contact with the agent in culture. Alternatively, the IMP cells may be loaded with an agent using delivery vehicle, such as liposomes. Such vehicles are known in the art.
  • the transfection of IMP cells may be performed in vitro or ex vivo. Alternatively, stable transfection may be perfomed at the MC stage allowing IMP cells expressing the transgene to be differentiated from them.
  • the IMP cells are transfected with a nucleic acid encoding the agent. For instance, viral particles or other vectors encoding the agent may be employed. Methods for doing this are known in the art.
  • the nucleic acid gives rise to expression of the agent in the IMP cells.
  • the nucleic acid molecule will preferably comprise a promoter which is operably linked to the sequences encoding the agent and which is active in the IMP cells or which can be induced in the IMP cells.
  • the nucleic acid encoding the agent may be delivered via a viral particle.
  • the viral particle may comprise a targeting molecule to ensure efficient transfection.
  • the targeting molecule will typically be provided wholly or partly on the surface of the virus in order for the molecule to be able to target the virus to the IMP cells.
  • the virus may, for example, be a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a vaccinia virus or a herpes simplex virus.
  • the virus may be a lentivirus.
  • the lentivirus may be a modified HIV virus suitable for use in delivering genes.
  • the lentivirus may be a SIV, FIV, or equine infectious anemia virus (EQIA) based vector.
  • the virus may be a moloney murine leukaemia virus (MMLV).
  • the viruses used in the invention are preferably replication deficient.
  • Viral particles do not have to be used. Any vector capable of transfecting the IMP cells of the invention may be used, such as conventional plasmid DNA or RNA transfection.
  • Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents.
  • transfection agents include cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example,
  • the cell may be loaded or tranfected under suitable conditions.
  • the cell and agent or vector may, for example, be contacted for between five minutes and ten days, preferably from an hour to five days, more preferably from five hours to two days and even more preferably from twelve hours to one day.
  • the invention also provides IMP cells which have been loaded or transfected with an agent as discussed above. Such IMP cells may be used in the therapeutic embodiments of the invention.
  • MCs may be recovered from a patient, converted into IMP cells using the invention, loaded or transfected in vitro and then returned to the same patient.
  • the IMP cells employed in the invention will be autologous cells and fully matched with the patient.
  • the cells employed in the invention are recovered from a patient and utilised ex vivo and subsequently returned to the same patient.
  • the invention additionally provides a pharmaceutical composition comprising an IMP cell of the invention or a population of the invention in combination with a pharmaceutically acceptable carrier or diluent, (ii) one or more lipsomes and/or (iii) one or more microbubbles.
  • the composition may comprise (i); (ii); (iii); (i) and (ii); (i) and (iii); (ii) and (iii); or (i), (ii) and (iii).
  • the IMP cell or population are preferably contained with the one or more liposomes and/or one or more microbubbles. Any number of liposomes and/or microbubbles may be present. Any of the numbers discussed above with reference to the population of the invention are equally application to the lipsomes and/or microbubbles.
  • a lipsome or microbubble may contain one IMP cell or more than one IMP cell.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising (i) an IMP cell of the invention or a population of the invention in combination with a pharmaceutically acceptable carrier or diluent, (ii) one or more immune cells and/or (iii) one or more antigens.
  • the composition may comprise (i); (ii); (iii); (i) and (ii); (i) and (iii); (ii) and (iii); or (i), (ii) and (iii).
  • the immune cell may be any immune cell, such as those discussed above. In some aspects, the immune cell may be a T-cell, a gamma delta T-cell or an NK cell.
  • the antigen may be any antigen, such as any of the antigens discussed above.
  • the composition may comprise any of the IMP cells or populations mentioned herein and, in some embodiments, the nucleic acid molecules, vectors, or viruses described herein.
  • the invention provides a method of repairing a damaged tissue in a patient comprising administering to the patient an effective amount of a pharmaceutical composition of the invention. Any of the therapeutic embodiments discussed above equally apply to this embodiment.
  • compositions of the invention may be formulated using any suitable method.
  • Formulation of cells with standard pharmaceutically acceptable carriers and/or excipients may be carried out using routine methods in the pharmaceutical art. The exact nature of a formulation will depend upon several factors including the cells to be administered and the desired route of
  • the cells may be administered by any route. Suitable routes include, but are not limited to, intravenous, intramuscular, intraperitoneal or other appropriate administration routes.
  • the damaged tissue is cardiac tissue
  • the cells may be administered via an endomyocardial, epimyocardial, intraventicular, intracoronary, retrograde coronary sinus, intra-arterial, intra-pericardial or intravenous route.
  • the damaged tissue is bone
  • the cells may be administered via an intraosseous route or to the site of the injury, such as a fracture, or disease.
  • the damaged tissue is cartilage, tendon, ligament, liver, kidney or lung tissue
  • the cells may be administered directly into the tissue.
  • the damaged tissue is lung tissue
  • the cells may be introduced via an intra-pulmonary route.
  • the damaged tissue is liver or kidney
  • the cells may be introduced via an intra-peritoneal route.
  • the cells are preferably administered intravenously.
  • compositions may be prepared together with a physiologically acceptable carrier or diluent.
  • a physiologically acceptable carrier or diluent typically, such compositions are prepared as liquid suspensions of cells.
  • the cells may be mixed with an excipient which is pharmaceutically acceptable and compatible with the active ingredient.
  • excipients are, for example, water, saline, dextrose, glycerol, of the like and combinations thereof.
  • compositions of the invention may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance effectiveness.
  • the composition preferably comprises human serum albumin.
  • Plasma-Lyte A® is a sterile, nonpyrogenic isotonic solution for intravenous administration.
  • Each 100 mL contains 526 mg of Sodium Chloride, USP (NaCl); 502 mg of Sodium Gluconate (C6H1 lNa07); 368 mg of Sodium Acetate Trihydrate, USP (C2H3Na02 » 3H20); 37 mg of Potassium Chloride, USP (KC1); and 30 mg of Magnesium Chloride, USP (MgC12 » 6H20). It contains no antimicrobial agents.
  • the pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).
  • the IMP cells may be contained within one or more liposomes and/or one or more
  • Suitable liposomes are known in the art. Suitable liposomes are disclosed in, for example, Akbarzadeh et al. Nanoscale Research Letters 2013, 8: 102 and Crystala et al. International Journal Of Pharmaceutical And Chemical Sciences, 2012, 1(1): 1-10. Suitable lipids for use in forming liposomes are discussed below with reference to microbubbles.
  • Microbubbles are bubbles smaller than one millimetre in diameter and larger than one micrometre in diameter.
  • the microbubble used in the present invention is preferably 8 ⁇ or less in diameter, such as 7 ⁇ or less in diameter, 6 ⁇ or less in diameter, 5 ⁇ or less in diameter, 4 ⁇ or less in diameter, 3 ⁇ or less in diameter or 2 ⁇ or less in diameter.
  • the microbubble may be formed from any substance.
  • the general composition of a microbubble is a gas core stabilised by a shell.
  • the gas core may comprise air or a heavy gas, such as perfiuorocarbon, nitrogen or perflouropropane. Heavy gases are less water soluble and so are less likely to leak out from the microbubble leading to microbubble dissolution. Microbubbles with heavy gas cores typically last longer in circulation.
  • the shell may be formed from any material.
  • the shell material preferably comprises a protein, a surfactant, a lipid, a polymer or a mixture thereof.
  • Suitable proteins include but are not limited to, albumin, lysozyme and avidin. Proteins within the shell may be chemically-crosslinked, for instance by cysteine-cysteine linkage. Other crosslinkages are known in the art.
  • Suitable surfactants include, but are not limited to, sorbitan monopalmitate (such as SPAN-40), polysorbate detergents (such as TWEEN-40), mixtures of SPAN-40 and TWEEN-40 and sucrose stearate (mono- and di-ester).
  • Suitable polymers include, but are not limited to, alginate polymers, double ester polymers of ethylidene, the copolymer poly(D,L-lactide-co-glycolide) (PLGA), polyvinyl alcohol) (PVA), the copolymer polyperfiuorooctyloxycaronyl-poly(lactic acid) (PLA-PFO) and other block copolymers.
  • Block copolymers are polymeric materials in which two or more monomer sub-units that are polymerized together to create a single polymer chain. Block copolymers typically have properties that are contributed by each monomer sub-unit.
  • a block copolymer may have unique properties that polymers formed from the individual sub-units do not possess.
  • Block copolymers can be engineered such that one of the monomer sub-units is hydrophobic (i.e. lipophilic), whilst the other sub- unit(s) are hydrophilic whilst in aqueous media.
  • the block copolymer may possess amphiphilic properties and may form a structure that mimics a biological membrane.
  • the block copolymer may be a diblock (consisting of two monomer sub-units), but may also be constructed from more than two monomer sub-units to form more complex arrangements that behave as amphipiles.
  • the copolymer may be a triblock, tetrablock or pentablock copolymer.
  • Block copolymers may also be constructed from sub-units that are not classed as lipid sub-materials; for example a hydrophobic polymer may be made from siloxane or other non-hydrocarbon based monomers.
  • the hydrophilic subsection of block copolymer can also possess low protein binding properties, which allows the creation of a membrane that is highly resistant when exposed to raw biological samples.
  • This head group unit may also be derived from non-classical lipid head-groups.
  • the lipid composition is chosen such that the microbubble has the required properties, such surface charge, packing density or mechanical properties.
  • the lipid composition can comprise one or more different lipids.
  • the lipid composition can contain up to 100 lipids.
  • the lipid composition preferably contains 1 to 10 lipids.
  • the lipid composition may comprise naturally-occurring lipids and/or artificial lipids.
  • Suitable interfacial moieties include, but are not limited to, naturally-occurring interfacial moieties, such as glycerol-based or ceramide-based moieties.
  • Suitable hydrophobic tail groups include, but are not limited to, saturated hydrocarbon chains, such as lauric acid (ft-Dodecanolic acid), myristic acid (w-Tetradecononic acid), palmitic acid (w-Hexadecanoic acid), stearic acid (w-Octadecanoic) and arachidic (w-Eicosanoic); unsaturated hydrocarbon chains, such as oleic acid (cw-9-Octadecanoic); and branched hydrocarbon chains, such as phytanoyl.
  • the length of the chain and the position and number of the double bonds in the unsaturated hydrocarbon chains can vary.
  • the length of the chains and the position and number of the branches, such as methyl groups, in the branched hydrocarbon chains can vary.
  • the hydrophobic tail groups can be linked to the interfacial moiety as an ether or an ester.
  • the lipids can also be chemically -modified.
  • the head group or the tail group of the lipids may be chemically-modified.
  • Suitable lipids whose head groups have been chemically -modified include, but are not limited to, PEG-modified lipids, such as l,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N - [Methoxy (Poly ethylene glycol)-2000] ; functionalised PEG Lipids, such as l,2-Distearoyl-sn-Glycero-3 Phosphoethanolamine-N-[Biotinyl(Polyethylene Glycol)2000]; and lipids modified for conjugation, such as l,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(succinyl) and 1,2-Dipalmitoyl-sn- Glycero-3-Phosphoethanolamine-N-(Biotinyl).
  • Suitable lipids whose tail groups have been chemically - modified include, but are not limited to, polymerisable lipids, such as l,2-bis(10,12-tricosadiynoyl)-sn- Glycero-3-Phosphocholine; fluorinated lipids, such as l-Palmitoyl-2-(16-Fluoropalmitoyl)-sn-Glycero- 3-Phosphocholine; deuterated lipids, such as l,2-Dipalmitoyl-D62-sn-Glycero-3-Phosphocholine; and ether linked lipids, such as l,2-Di-0-phytanyl-sn-Glycero-3-Phosphocholine.
  • the lipids may be chemically-modified or functionalised to facilitate coupling of the ligands, receptors ro antibodies as discussed above.
  • the microbubble shell is preferably formed from a phospholipid. Suitable phospholipids are known in the art.
  • lipid shell microbubble formulations such as Definity (Lantheus Medical Imaging) and Sonovue® (Bracco Diagnostics).
  • the microbubble may also be formed from a polymer-surfactant hybrid that involves forming polyelectrolyte multilayer (PEM) shells on a preformed microbubble.
  • PEM polyelectrolyte multilayer
  • the preformed microbubble is coated with a charged surfactant or protein layer, which serves as a substrate for PEM deposition.
  • the layer-by-layer assembly technique is used to sequentially adsorb oppositely charged polyions to the microbubble shell.
  • PEM can be deposited onto microbubbles using poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS) for the polyion pair.
  • PEM microbubbles with phospholipid containing the cationic headgroup trimethylammonium propane (TAP) as the underlying shell and DNA and poly(L-lysine) (PLL) as the polyion pair have also been developed.
  • the microbubble is typically formed by providing an interface between a gas and a
  • the microbubble may be loaded with the IMP cells after formation of the microbubble or during formation of the microbubble.
  • Any suitable number of cells may be administered to a subject. For example, at least, or about,
  • 0.2 x 10 6 , 0.25 x 10 6 , 0.5 x 10 6 , 1.5 x 10 6 , 4.0 x 10 6 or 5.0 x 10 6 cells per kg of patient may
  • the number of cells of the invention to be administered may be from 10 5 to 10 9 , preferably from 10 6 to 10 8 .
  • the number of cells of the invention to be administered may be from 10 5 to 10 9 , preferably from 10 6 to 10 8 .
  • up to 2 x 10 8 IMP cells are administered to each patient.
  • culture medium may be present to facilitate the survival of the cells.
  • the cells of the invention may be provided in frozen aliquots and substances such as DMSO may be present to facilitate survival during freezing. Such frozen cells will typically be thawed and then placed in a buffer or medium either for maintenance or for administration.
  • T cells play a key role in many immune responses.
  • T cells are important for cell- mediated immunity to cancer cells.
  • Cancer cells use many strategies to evade the host immune response. For example, cancer cells may downregulate antigens that are targeted by T cells, or may express antigens that are only weakly immunogenic. In addition, many tumours create a
  • T cells can be genetically modified in order to increase their anti-tumour responses, thereby enhancing tumour immunity.
  • a T cell can be induced to express a chimeric antigen receptor (CAR) specific for an antigen present on cancer cells.
  • CAR chimeric antigen receptor
  • a CAR typically comprises an antigen-binding region, a transmembrane domain, and at least one intracellular domain.
  • the antigen-binding region confers the specificity of the CAR and is often derived from an antibody.
  • CARs specific for almost any antigen can be engineered.
  • the transmembrane domain anchors the CAR to the T cell.
  • the intracellular domain induces T cell signalling, leading to activation, persistence and effector function.
  • T cell activation requires the T cell to interact with an antigen presenting cell.
  • T cell receptor recognises a peptide antigen associated with MHC molecule present on the antigen presenting cell. This means that traditional T cell activation relies on antigen uptake, processing and presentation by antigen presenting cells.
  • CAR-expressing T cells can be activated in the absence of an interaction with an MHC molecule.
  • the antigen-binding region binds to the target antigen
  • signalling events are triggered via the CAR intracellular domain(s) and the CAR T cell becomes activated.
  • CAR T cells may recognise antigens other than proteins or peptides.
  • CARs may recognise carbohydrate and glycolipid structures that are typically expressed on the surface of cancer cells. CARs can therefore redirect the effector functions of a T cell towards any protein or non-protein target expressed on the cell surface as long as an antibody or similar targeting domain is available.
  • CAR T cells are produced by inducing CAR expression in T cells isolated from a subject.
  • T cells can be isolated from blood or other tissues and modified to express CARs.
  • CAR T cells produced in this way are generally administered autologously.
  • the resultant CAR T cells are administered to the same subject as that from which they were derived.
  • allogeneic CAR T cells i.e. CAR T cells that are derived from a subject that is immunologically compatible with the subject into which the cells are administered
  • a bank of CAR T cells directed to particular antigens could be generated and maintained for use in the treatment of an array of MHC-mismatched subjects.
  • allogeneic CAR T cells are less stable and therefore less viable than autologous CAR T cells.
  • the present invention provides a method of improving the potency, viability or stability of CAR T cells, comprising incubating CAR T cells in the presence of a population of the invention.
  • the method of the invention gives rise to CAR T cells with improved persistence and function, which may be therefore be administered autologously or allogeneically.
  • the more stable phenotype increases the efficacy of the CAR T cells and reduces off-target effects.
  • the CAR T cells produced according to the method of the invention remain more specifically targeted to the relevant antigen and are thus safer for use in vivo.
  • the method of the invention may improve the in vitro and/or in vivo potency, viability or stability of the CAR T cells.
  • Methods for evaluating T cell potency, viability and stability are well known in the art.
  • Incubation of the CAR T cells with the population of the invention may also comprise co-culture of the CAR T cells with the population.
  • the CAR T cells and the population may be co- cultured for at least 12 hours, at least 24 hours , at least 48 hours, at least 72 hours or at least 96 hours.
  • Techniques for culturing cells are well known in the art.
  • the cells are may be cultured under standard conditions of 37°C, 5% CO2 in medium without serum.
  • the cells may be cultured in any suitable flask or vessel, including wells of a flat plate such as a standard 6 well plate. Such plates are commercially available from Fisher scientific, VWR suppliers, Nunc, Starstedt or Falcon.
  • the wells typically have a capacity of from about 1ml to about 4ml.
  • the flask, vessel or wells within which the population is contained or cultured may be modified to facilitate handling of the cells.
  • the flask, vessel or wells may be modified to facilitate culture of the cells, for instance by including a growth matrix.
  • the flask, vessel or wells may be modified to allow attachment of the cells or to allow immobilization of the cells onto a surface.
  • One or more surfaces may be coated with extracellular matrix proteins such as laminin or collagen or any other capture molecules that bind to the cells and immobilize or capture them on the surface(s).
  • the incubation may take place in the presence of antigen presenting cells, T cell activator beads, or one or more antibodies.
  • the antigen presenting cell are preferably dendritic cells.
  • the antibodies are preferably anti-CD3 and/or anti-CD28.
  • the incubation may also take place in the presence of an antigen.
  • the antigen is preferably the antigen for which the CAR T cells are specific.
  • Other substances that may be provided during the incubation step are cytokines, nucleic acids, peptides, proteins and other types of cells.
  • One or more IMP cells of the invention may form part of a hybrid composition as disclosed in PCT/GB2015/051672 and are preferably administered to a patient as part of such a composition.
  • the invention provides a hybrid composition, which comprises:
  • the hybrid composition of the invention comprises one or more biocompatible fibres.
  • a fibre is biocompatible if it does not cause any adverse reactions or side effects when contacted with a damaged tissue.
  • the composition may comprise only one fibre.
  • the composition typically comprises more than one fibre, such at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 500 fibres, at least 1000 fibres or even more fibres.
  • biocompatible fibres are known in the art.
  • the one or more biocompatible fibres may be natural or synthetic.
  • Preferred biocompatible fibres include, but are not limited to, cellulose fibres, collagen fibres, collagen-glycosaminoglycan fibres, gelatin fibres, silk fibroin fibres, one or more fibrin fibres, chitosan fibres, starch fibres, alginate fibres, hyaluronan fibres, poloaxmer fibres or a combination thereof.
  • the glycosaminoglycan is preferably chondroitin.
  • the cellulose is preferably carboxymethylcellulose, hydroxypropylmethylcellulose or methylcellulose.
  • the poloaxmer is preferably pluronic acid, optionally Pluronic F-127.
  • the population of fibres may be homogenous. In other words, all of the fibres in the population may be the same type of fibre, e.g. cellulose fibres. Alternatively, the population of fibres may be heterogeneous. In other words, the population of fibres may contain different types of fibre, such cellulose fibres and collagen fibres.
  • the one or more fibres may be any length.
  • the one or more fibres are preferably
  • the length of one or more fibres is preferably designed such that the composition can penetrate a damaged tissue to a prescribed depth.
  • the one or more fibres may be any length.
  • the lower limit of the length of the one or more fibres is typically determined by the diameter of the one or more therapeutic cells. Suitable lengths include, but are not limited to, at least 1 ⁇ in length, at least ⁇ in length, at least ⁇ in length, at least 500 ⁇ in length, at least 1mm in length, at least 10mm (1cm) in length, at least 100mm (10cm) in length, at least 500mm (50cm) in length or at least 1000mm (100cm or lm) in length.
  • the one or more fibres may be even longer.
  • the one or more fibres may be up to 5m or 10m in length, for instance if being used to repair damage along the human intestinal tract, or even longer if being used in larger animals, such as horses.
  • the length of the one or more fibres is typically determined by their intended use and/or their ability to be manipulated, for instance by a surgeon, by a robot or via some other means, such as magnetically.
  • the one or more fibres may be charged.
  • the one or more fibres are preferably positively- charged.
  • the one or more fibres are preferably negatively-charged.
  • the one or more fibres may be magnetic.
  • the one or more fibres may be modified to include one or more magnetic atoms or groups. This allows magnetic targeting of the composition.
  • the magnetic atoms or groups may be paramagnetic or superparamagnetic. Suitable atoms or groups include, but are not limited to, gold atoms, iron atoms, cobalt atoms, nickel atoms and a metal chelating groups, such as nitrilotriacetic acid, containing any of these atoms.
  • the composition also comprises one or more biocompatible components.
  • the one or more biocompatible components (i) attach the one or more therapeutic cells to the one or more fibres and/or embed the one or more therapeutic cells and the one or more fibres and/or (ii) are capable of attaching the composition to a tissue.
  • the one or more biocompatible components may (a) attach the one or more therapeutic cells to the one or more fibres, (b) embed the one or more therapeutic cells and the one or more fibres, (c) be capable of attaching the composition to a tissue, (d) attach the one or more therapeutic cells to the one or more fibres and embed the one or more therapeutic cells and the one or more fibres, (e) attach the one or more therapeutic cells to the one or more fibres and be capable of attaching the composition to a tissue, (f) embed the one or more therapeutic cells and the one or more fibres and be capable of attaching the composition to a tissue or (g) attach the one or more therapeutic cells to the one or more fibres, embed the one or more therapeutic cells and the one or more fibres and be capable of attaching the composition to a tissue.
  • a component is biocompatible if it does not cause any adverse reactions or side effects when contacted with a damaged tissue.
  • the composition typically comprises only one component or two components.
  • the composition may comprise more than two components, such as at least 3, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50 components or even more components.
  • the one or more biocompatible components preferably comprise a biocompatible adhesive which attaches the one or more therapeutic cells to the one or more fibres.
  • the biocompatible adhesive may attach the one or more therapeutic cells (a) on the surface of the one or more fibres, (b) within the one or more fibres or (c) both on the surface of and within the one or more fibres.
  • the biocompatible adhesive may be natural or synthetic. Suitable biocompatible adhesives are known in the art. Suitable adhesives include, but are not limited to, fibrin, fibrin gel, integrin, integrin gel, cadherin and cadherin gel.
  • the one or more biocompatible components preferably comprise a biocompatible gel which embeds the one or more therapeutic cells and the one or more fibres.
  • Suitable biocompatible gels are known in the art.
  • the biocompatible gel may be natural or synthetic.
  • Preferred biocompatible gels include, but are not limited to, a cellulose gel, a collagen gel, a gelatin gel, a fibrin gel, a chitosan gel, a starch gel, an alginate gel, a hyaluronan gel, an agarose gel, a poloaxmer gel or a combination thereof.
  • the cellulose gel may be formed from any of the celluloses discussed above.
  • the cellulose polymer concentration is preferably from about 1.5% (w/w) to about 4.0% (w/w), such as from about 2.0% (w/w) to about 3.0% (w/w).
  • the cellulose polymer preferably has a molecular weight of from about 450,000 to about 4,000,000, such as from about 500,000 to about 3,500,000, from about 500,000 to about 3,000,000 or from about 750,000 to about 2,500,000 or from about 1000,000 to about 2,000,000.
  • the poloaxmer gel is preferably a pluronic acid gel, optionally a Pluronic F-127 gel.
  • the adhesive and/or gel preferably has a viscosity in the range of 1000 to 500,000 mPa » s (cps) at room temperature, such as from about 1500 to about 450,000 mPa » s at room temperature, from about 2000 to about 400,000 mPa » s at room temperature, from about 2500 to about 350,000 mPa » s at room temperature, from about 5000 to about 300,000 mPa » s at room temperature, from about 10,000 to about 250,000 mPa » s at room temperature, from about 50,000 to about 200,000 mPa » s at room temperature or from about 50,000 to about 150,000 mPa » s at room temperature.
  • cps viscosity at room temperature
  • Viscosity is a measure of the resistance of the adhesive and/or gel to being deformed by either shear stress or tensile stress. Viscosity can be measured using any method known in the art. Suitable methods include, but are not limited to, using a viscometer or a rheometer.
  • Room temperature is typically from about 18 °C to about 25 °C, such as from about 19 °C to about 24 °C or from about 20 °C to about 23 °C or from about 21 °C to about 22 °C.
  • temperature is preferably any of 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C and 25 °C.
  • Viscosity is most preferably measured at 25 °C.
  • the one or more biocompatible components preferably comprises a biocompatible adhesive which attaches the one or more therapeutic cells to the one or more fibres and a biocompatible gel which embeds the one or more therapeutic cells and the one or more fibres.
  • the composition may comprise a fibrin gel which attaches the one or more therapeutic cells to the one or more fibres and a cellulose gel which embeds the one or more therapeutic cells and the one or more fibres.
  • biocompatible gel preferably comprises platelet lysate.
  • the adhesive and/or the gel may be a platelet lystae gel.
  • Platelet lysate refers to the combination of natural growth factors contained in platelets that has been released through lysing those platelets. Lysis can be accomplished through chemical means (i.e. CaCk ), osmotic means (use of distilled H2O) or through freezing/thawing procedures. Platelet lysate can be derived from whole blood as described in U.S. Pat. No. 5,198,357. Platelet lysate is preferably prepared as described in PCT/GB 12/052911 (published as WO
  • 2013/076507 it may be prepared by subjecting a population of platelets to at least one freeze-thaw cycle, wherein the freeze portion of each cycle is carried out at a temperature lower than or equal to - 78 °C.
  • the platelet lysate is preferably prepared by subjecting a population of platelets to four freeze-thaw cycles, wherein the freeze portion of each cycle is carried out at a temperature lower than or equal to - 78 °C, for instance using liquid nitrogen.
  • the adhesive and/or gel preferably comprises (a) platelet lysate, (b) at least one
  • composition is an aqueous gel having a viscosity in the range of 1000 to 500,000 mPa » s (cps) at room temperature.
  • pharmaceutically acceptable polymer is preferably cellulose or a poloaxmer. It may be any of the celluloses and poloaxmers discussed above.
  • the platelet lysate is preferably human platelet lysate. Platelet lysate is discussed in more detail above.
  • the hybrid composition may be contained within one or more liposomes or one or more microbubbles. Such structures are known in the art.
  • CD3e Induction of Expression of one or more of CD3.
  • CD3e CD8.
  • CD8b CD4.
  • CD5. CD6 and CD7
  • CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 may be artificially induced in a cell expressing the IMP markers MIC A/B, CD304
  • CD178 FAS ligand
  • CD289 Toll-like receptor 9
  • CD363 Sphingosine-1 -phosphate receptor 1
  • CD99 CD181
  • CD181 C-X-C chemokine receptor type 1; CXCR1
  • EGF-R epidermal growth factor receptor
  • CXCR2 CXCR2 and CD 126.
  • These cells are the IMP cells described in International Patent Application No. PCT/GB2015/051673 (WO 2015/189587). Expression of any combination of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 as discussed above may be induced. Artificial induction may be achieved by various means.
  • the expression of one or more of CD8, CD8b, CD4, CD5, CD6 and CD7 may be artificially induced in an IMP cell of the invention expressing CD3 and/or CD3e.
  • the expression of one or more of CD3, CD3e, CD4, CD5, CD6 and CD7 may be artificially induced in an IMP cell of the invention expressing CD8 and/or CD8b.
  • the expression of one or more of CD3, CD3e, CD8, CD8b, CD5, CD6 and CD7 may be artificially induced in an IMP cell of the invention expressing CD4.
  • the expression of one or more of CD3, CD3e, CD8, CD8b, CD4, CD6 and CD7 may be artificially induced in an IMP cell of the invention expressing CD5.
  • CD3, CD3e, CD8, CD8b, CD4, CD5 and CD7 may be artificially induced in an IMP cell of the invention expressing CD6.
  • the expression of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5 and CD6 may be artificially induced in an IMP cell of the invention expressing CD7.
  • the invention therefore provides a method of producing an IMP cell of the invention, comprising providing an IMP cell expressing detectable levels of MIC A/B, CD304 (Neuropilin), CD178 (FAS ligand), CD289 (Toll-like receptor 9), CD363 (Sphinogine-1 -phosphate receptor 1), CD99, CD181 (C-X-C chemokine receptor type 1; CXCR1), epidermal growth factor receptor (EGF- R), CXCR2 and CD126 and inducing the cell to express detectable levels of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 by (i) editing the genome of the cell and/or (ii) transfecting or transducing the cell with one or more nucleic acid constructs encoding one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 and /or (iii) genotype modulation using magnetic stimulation or electrical stimulation.
  • CD304 Neuropilin
  • the invention also provides a method of inducing an IMP cell which expresses detectable levels of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 to express detectable levels of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 of which it does not already express detectable levels, comprising (i) editing the genome of the cell and/or (ii) transfecting or transducing the cell with one or more nucleic acid constructs encoding one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 and /or (iii) genotype modulation using magnetic stimulation or electrical stimulation.
  • the expression of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 may be induced by editing the genome of the cell.
  • Gene editing may be used alone or in combination with transfection and/or transduction as described below to induce expression of these markers.
  • Gene editing is a type of genetic engineering in which one or more nucleic acid (such as two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 15 or more, 20 or more, 25 or more, or 50 or more nucleic acids) is inserted, deleted or replaced in the genome of an organism using engineered nucleases. These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome.
  • the induced DSBs may be repaired through nonhomologous end joining (NHEJ) or homology directed repair (HDR), resulting in targeted mutations or edits, or the targeted insertion of a desired sequence.
  • any gene editing technique or combination or gene editing techniques may be used to induce expression of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • the genome of the cell is edited using one or more gene editing techniques selected from (a) zinc finger nuclease (ZFN) technology, (b) transcription activator-like effector nuclease (TALEN) technology, (c) clustered regularly-interspace short palindromic repeats (CPJSPR)/ CRISPR-associated gene (Cas) technology, (d) homology directed repair (HDR) and (e) non-homologous end joining (NHEJ).
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • CPJSPR clustered regularly-interspace short palindromic repeats
  • Cas CRISPR-associated gene
  • HDR homology directed repair
  • NHEJ non-homologous end joining
  • the genome of the IMP cell may be edited using two or more, such as three or more, or four or more gene editing techniques selected from (a) ZFN technology, (b) TALEN, (c) CRISPR/ Cas technology, (d) HDR and (e) NHEJ. Any combination of gene editing techniques may be used.
  • the genome of the cell may be edited using (a); (b); (c); (d); (e); (a) and, (b); (a) and , (c); (a) and, (d); (a) and , (e;) (b) and, (c); (b) and, (d); (b) and, (e); (c) and, (d); (c) and, (e;) (d) and , (e); (a), (b) and, (c); (a), (b), and (d); (a), (b) and, (e); (a), (c) and, (d); (a), (c) and, (e); (a), (d) and, (e); (b), (c) and, (d); (b), (c) and, (d); (b), (c) and, (e); (b), (d) and, (e); (c), (d) and, (e); (a), (b), (c) and, (e); (b), (d) and, (e);
  • the first gene editing technique may be used to introduce one or more site-specific DSBs in the genome.
  • the second gene editing technique may be used to repair the DSB in such as way as to modify the sequence and/or expression of the gene in which the DSB occurs.
  • the second gene editing technique may be used to repair the DSB by inserting a desired sequence or construct at the site specific DSB. Any combination of first and second gene editing techniques may be used.
  • the genome of the IMP cell may be edited using (i) and (iv); (i) and (v); (ii) and (iv); (iii) and (iv); (iii) and (iv); or (iii) and (v).
  • the targeted mutations or edits may themselves lead to the expression of one or more of CD3,
  • the mutation or edit may disrupt a part of the genome that suppresses or inhibits the expression of one or more of these markers. Removal of the negative regulation of marker expression may lead to expression of the one or more marker.
  • the mutation or edit may occur in or positively influence a part of the genome that promotes the expression of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 . Enhancing the positive regulation of marker expression in this way may lead to expression of the one or more markers.
  • NHEJ may be particularly useful for inducing marker expression in this manner. NHEJ is error-prone, and it is very likely that mutations will be generated at the site of the DSB when NHEJ is used to repair the DSB.
  • Gene editing may also be used to insert desired genetic material (e.g. a construct or sequence) in a site-specific manner at the point of the DSB.
  • desired genetic material e.g. a construct or sequence
  • gene editing may be used to insert all of or part of a nucleic acid construct encoding one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 at a particular point in the genome.
  • nucleic acid constructs are described in detail below.
  • gene editing may be used to insert all of or part of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 15 or more, 20 or more 25 or more or 50 or more constructs.
  • All or part of any particular type of construct may be inserted at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 15 or more, 20 or more 25 or more or 50 or more DSB points in the genome.
  • the nucleic acid construct may be supplied to the cell by transfection and/or transformation, as set out above.
  • HDR may be particularly useful for inserting desired genetic material at the site of the DSB.
  • HDR is dependent on a homologous sequence to repair a DSB. Therefore, desired genetic material may be inserted within a sequence that is homologous to the flanking sequence of the DSB. When this material is used as a template by the HDR system, the desired change is incorporated within the genomic region of interest.
  • the construct or part thereof may be inserted such that the expression of the marker(s) it encodes is controlled by a regulatory element that pre-exists in the genome.
  • the construct may be inserted downstream of the promoter for a gene that is constitutively expressed in the IMP cells, such that the markers encoded by the construct are also constitutively expressed.
  • the expression of the marker(s) encoded by the construct may be controlled by one or more regulatory elements present in the construct.
  • the regulatory elements may be inducible regulatory elements.
  • the cell may be induced to express one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7 by transfecting and/or transducing the cell with one or more nucleic acid constructs encoding one or more of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • the cell may be transfected or transduced with two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 15 or more, 20 or more 25 or more or 50 or more constructs.
  • the constructs may be the same or different.
  • Each construct may encode two or more, three or more or four or more of one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7
  • the construct may comprise two or more, such as five or more, ten or more or twenty or more, sequences encoding a particular marker.
  • each construct may comprise two or more sequences encoding one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7.
  • the construct may comprise DNA and/or RNA.
  • the DNA may be double-stranded DNA (dsDNA) or single stranded DNA (ssDNA).
  • the RNA may be double-stranded RNA (dsRNA) or single stranded RNA (ssRNA).
  • transfection may be used to describe non- virus-mediated nucleic acid transfer.
  • a human artificial chromosome and/or naked RNA and/or siRNA may be used to transfect the cell with the one or more constructs.
  • Human artificial chromosomes are described in e.g. Kazuki et al., Mol. Ther. 19(9): 1591-1601 (2011), and Kouprina et al., Expert Opinion on Drug Delivery 11(4): 517-535 (2014).
  • Alternative non-viral delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome.
  • Methods of non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, poly cation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024.
  • the preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al, Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al, Bioconjugate Chem.
  • a viral vector may be used to transduce the cell with the one or more constructs.
  • Conventional viral based expression systems could include retroviral, lentivirus, adenoviral, adeno-associated (AAV) and herpes simplex virus (HSV) vectors for gene transfer. Methods for producing and purifying such vectors are know in the art. Since AAV is a DNA virus, the construct is preferably DNA if AAV or rAAV is used.
  • Nanoparticle delivery systems may be used to transfect the cell with the one or more constructs.
  • Such delivery systems include, but are not limited to, lipid-based systems, liposomes, micelles, microvesicles, exosomes, and gene gun.
  • nanoparticles that can deliver RNA see, e.g., Alabi et al, Proc Natl Acad Sci U S A. 2013 Aug 6;110(32): 12881-6; Zhang et al., Adv Mater. 2013 Sep 6;25(33):4641-5; Jiang et al, Nano Lett. 2013 Mar 13;13(3): 1059-64; Karagiannis et al, ACS Nano.
  • Genotype modulation using magnetic stimulation is described in Sutton and Schuman (2006), Repeated magnetic stimulation induces Long Term Potentiation that is associated with altered gene expressions and protein synthesis ; and Mulleret al, (2000), Long-term repetitive transcranial magnetic stimulation increases the expression of brain-derived neurotrophic factor and cholecystokinin mRNA, but not neuropeptide tyrosine mRNA in specific areas of rat brain, Neuropsychopharmacology, 23: 205-215, doi:10.1016/S0893-133X(00)00099-3.
  • Genotype modulation using electrical stimulation is described in Simis et al. (2013), Motor cortex-induced plasticity by noninvasive brain stimulation: a comparison between transcranial direct current stimulation and transcranial magnetic stimulation, Neuroreport, 24: 973-975,
  • CD4, CD5, CD6 and CD7 may be used alone or in any combination, such as (i) editing the genome of the cell; (ii) transfecting or transducing the cell with one or more nucleic acid constructs encoding one or more of CD3, CD3e, CD8, CD8b, CD4, CD5, CD6 and CD7; (iii) genotype modulation using magnetic stimulation or electrical stimulation; (i) and (ii); (i) and (iii); (ii) and (iii); or (i), (ii) and (iii).
  • the methods may be performed concurrently. Alternatively, the methods may be performed sequentially.
  • a bone marrow sample was diluted with Hank Buffered Saline Solution and layered over Ficoll-Paque for the isolation of mononuclear cells (MCs) by centrifugation. The MCs were then re- suspended in Hank Buffered Saline Solution and counted using 0.4% trypan blue exclusion assay to assess cellular viability. Cells were seeded in T25 flasks (in 5 ml of cell culture media, MEM,
  • IMP cells were harvested using cell dissociating solution according to manufacturer's instructions. Cells were cryopreserved in passage 2 in culture media supplemented with 10% dimethyl sulfoxide to -80°C and stored in liquid nitrogen for later use.
  • High-throughput fluorescence activated cell sorting (HT-FACS) analysis is a high-throughput screening platform which can rapidly characterize the cell surface phenotype of cells in suspension, with over 370 cell surface markers currently in the panel. This platform has undergone extensive validation and has been performed on many types of human tissues and cells.
  • the panel consists of 370 human cell surface-specific antibodies arrayed in 96-well plates.
  • ioMP cells are the subject of the International Patent Application No. PCT/GB2016/052447.
  • IMP cells are the subject of International Patent Application No. PCT/GB2015/051673 (WO 2015/189587).
  • the high-throughput-FACS (HT-FACS) platform allows the screening of up to 370 surface antigens.
  • PB-MSCs PB-MSCs
  • CTL media 37°C, 5% C02
  • Cells were grown until confluence of 80-90% changing the media every 2-3 days.
  • the media was removed and cells were washed twice with PBS.
  • Cells were treated with 3 ml of Trypsin 0.25% until detached. Eight ml of media were added to inactivate the trypsin and cells were collected by centrifugation at 400g for 5 min.
  • Cells were re- suspended in 5 ml of media and seeded in a T175 cm2 flask containing 30 mL of CTL media (37°C, 5% C02). Between 8 to 10 T175 cm2 flasks at 80-90% confluence were required to harvest 20-30 million cells (at passage 4) for the HT-FACS screening. In order to obtain a sufficient number of flow cytometry "events" per antibody, approximately 20 million viable cells is optimal. To collect the cells, the media was removed and cells were washed twice with PBS. Cells were treated with 5 ml of Trypsin 0.25% until detached. Media was added (8 ml) to inactivate the trypsin and collect the cells.
  • HBSS Hank's Balanced Salt Solution minus calcium/magnesium, supplemented with 2mM EDTA and 1% BSA).
  • exclusion dye 0.2% trypan blue
  • CDH3 1.93 326 2.93 198 0.475 257
  • CDw218a 2.97 361 0.384 192 0 ⁇
  • CD80 5.98 432 2.94 208 4.57 536
  • CD81 100 5254 100 3950 99.9 5920
  • CD135 2.42 309 5.18 206 0.575 686
  • CD 140b 100 4987 89.1 695 97.8 1922
  • CD 147 100 5563 100 4780 100 3704
  • CD150 3.18 338 0.467 217 0.364 204 CD151 100 14835 100 10207 99.9 9421
  • CD243 0.0112 141 0.208 203 0 ⁇ CD244 0.336 363 0.548 195 0 ⁇
  • CD276 100 11060 100 4110 97.8 1749
  • CD292 2.83 281 2.39 223 0.522 244
  • CD300a 5.45 321 1.82 188 0.222 184
  • CD324 7.15 327 17.2 268 0.387 1206
  • CD363 1.28 350 18.7 242 0.757 337
  • a luminex assay was used to quantitate different cytokines in the conditioned media from Lonza cells and IMP cell cultures. Data is shown in pg ⁇ g of RNA, this is to standardise the data relevant to the number of cells in culture.
  • Example 1 was repeated using MCs in the same culture conditions, except for the modifications (a) to (f) shown in the Table below. On day 8 the media was changed. Cells were monitored daily for observation of IMP-like cells, which took about 22 to 25 days from seeding of MCs to appear. Once present, IMP cells were harvested using cell dissociating solution according to manufacturer's instructions.

Abstract

L'invention concerne des cellules progénitrices immunomodulatrices (IMP) exprimant un ou plusieurs lymphocytes parmi les lymphocytes CD3, CD3E, CD8, CD8B, CD4, CD5, CD6 et CD7, et leur utilisation à des fins thérapeutiques.
PCT/GB2017/050917 2016-03-31 2017-03-31 Cellule progénitrice immunomodulatrice (imp) exprimant un ou plusieurs lymphocytes parmi les lymphocytes cd3, cd3e, cd8, cd8b, cd4, cd5, cd6 et cd7 WO2017168170A1 (fr)

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GB201605504 2016-03-31
GB201605502 2016-03-31
GB201605507 2016-03-31
GB1605499.1 2016-03-31
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Citations (2)

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Publication number Priority date Publication date Assignee Title
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WO1998015616A1 (fr) * 1996-10-07 1998-04-16 Fordham University Procedes de generation in vitro de lymphocytes t cytotoxiques
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