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WO2018127585A1 - Population de lymphocytes t régulateurs monospécifiques avec cytotoxicité pour les lymphocytes b - Google Patents

Population de lymphocytes t régulateurs monospécifiques avec cytotoxicité pour les lymphocytes b Download PDF

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Publication number
WO2018127585A1
WO2018127585A1 PCT/EP2018/050347 EP2018050347W WO2018127585A1 WO 2018127585 A1 WO2018127585 A1 WO 2018127585A1 EP 2018050347 W EP2018050347 W EP 2018050347W WO 2018127585 A1 WO2018127585 A1 WO 2018127585A1
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cells
monospecific
domain
cell population
chimeric receptor
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PCT/EP2018/050347
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English (en)
Inventor
Li Zhou
François Meyer
Tobias Abel
Julie GERTNER-DARDENNE
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Txcell
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Publication of WO2018127585A1 publication Critical patent/WO2018127585A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2501/505—CD4; CD8
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to the field of immunotherapy.
  • the present invention relates to the use of a monospecific regulatory T cell population (monospecific Treg cell population) for treating autoantibody mediated autoimmune diseases, wherein said monospecific Treg cell population is cytotoxic for B cells.
  • the Treg cells of the monospecific Treg cell population of the invention express a chimeric receptor at its surface, wherein said chimeric receptor recognizes an autoantibody on B cells.
  • Autoimmune diseases are caused by an abnormal immune response to autoantigens resulting in damages or dysfunctions in tissues or organs.
  • Two different categories of autoimmune diseases may be distinguished (although not exclusive): those caused by T cells, and those caused by B cells producing antibodies directed to autoantigens (autoantibodies).
  • Autoantibody mediated autoimmune diseases are currently treated by general immune suppression methods.
  • a first example is apheresis, wherein total antibodies are removed from blood of a patient before re-administration of the remaining blood components.
  • a second example is treatment with rituximab, an anti-CD20 monoclonal antibody.
  • CD20 is primarily found on the surface of B cells.
  • Administration of rituximab to a patient thus results in the elimination of B cells.
  • these general immune suppression methods present drawbacks associated with the complete elimination of antibodies or B cells, thus exposing for example the treated patient to increased risks of infection.
  • WO2015/168613 disclosed effector T cells expressing a chimeric autoantibody receptor (CAAR), wherein the expression of said CAAR redirects the cytotoxicity of effector T cells to B cells producing specific autoantibodies.
  • CAAR chimeric autoantibody receptor
  • effector T cells in autoimmune disease patients, i.e., in patients with an over-reactive immune system, may present severe drawbacks, in particular due to the secretion by said cells of specific cytokines, including IFNy or TNFa.
  • the Applicant herein provides evidences supporting the therapeutic use of at least one monospecific regulatory T cell population for treating autoantibody mediated autoimmune diseases. Indeed, the Applicant demonstrated that a monospecific regulatory T cell population wherein the cells are genetically modified for expressing a chimeric receptor directed to an autoantibody on B cells does present cytotoxicity for B cells, whereas they do not before genetic modification.
  • the present invention thus relates to the use of at least one monospecific regulatory T cell population expressing a chimeric receptor recognizing an autoantibody on B cells and presenting cytotoxicity for B cells.
  • the cells of the present invention thus present the advantage of both killing autoreactive B cells and providing a regulatory effect on T cells, in particular in autoimmune diseases with both B and T cells counterpart.
  • the present invention relates to a monospecific Treg cell population, wherein the Treg cells of the population comprise a chimeric receptor, wherein said chimeric receptor recognizes at least one autoantibody on B cells and wherein said Treg cells are cytotoxic for B cells, wherein the autoantibody recognizes at least one autoantigen.
  • the chimeric receptor comprises (i) an extracellular autoantigen, or a variant or fragment of an autoantigen, (ii) optionally an extracellular hinge domain, (iii) optionally a transmembrane domain, and (iv) an intracellular signaling domain, and optionally the chimeric receptor further comprises a tag and/or a leader sequence.
  • the monospecific Treg cell population is cytotoxic for B cells in the conditions of Test A.
  • the cytotoxicity for B cells is chimeric receptor dependent and TCR independent.
  • the B cell is an activated mature B cell.
  • the at least one autoantigen is selected from aquaporin-4 water channel (AQP4), Hu, Ma2, collapsin response-mediator protein 5 (CRMP5), and amphiphysin, voltage-gated potassium channel (VGKC), N-methyl-d-aspartate receptor (NMDAR), a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPAR), Thyroid peroxidase, thyroglobulin, Anti-N-methyl-D-aspartate receptor (NR1 subunit), Rh blood group antigens, I antigen, Dsgl/3, BP180, BP230, Acetylcholine nicotinic postsynaptic receptors, thyrotropin receptors, platelet integrin, GpIIbTIIa, Collagen alpha-3(IV) chain, rheumatoid factor, calpastatin, citrullinated proteins, Myelin basic protein (MBP), Myel
  • the present invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one monospecific Treg cell population of the invention and at least one pharmaceutically acceptable excipient or carrier.
  • the present invention further relates to a medicament comprising at least one monospecific Treg cell population as defined herein.
  • the present invention further relates to a monospecific Treg cell population, a pharmaceutical composition or a medicament of the invention for treating or for use in treating an autoimmune disease in a subject in need thereof.
  • said autoimmune disease is an autoantibody-mediated autoimmune disease.
  • the autoantibody recognizes the autoantigen or variant or fragment thereof comprised in the chimeric receptor.
  • said autoimmune disease is selected from the group comprising uveitis, bullous pemphigoid, lupus (including systemic lupus erythematosus (SLE), lupus nephritis (LN), discoid lupus, lupus erythematosus profundus, Chilbrain lupus erythematosus, tumidus lupus erythematosus, severe systemic lupus erythematosus, acute cutaneous lupus, chronic cutaneous lupus), multiple sclerosis, neuromyelitis optica (NMO), autoimmune limbic encephalitis (LE), Hashimoto's disease, N-methyl-D- aspartate receptor (NMDAR) encephalitis, autoimmune hemolytic anemia, pemphigus vulgaris, myasthenia gravis, Graves' disease, idiopathic thrombocytopenic purpura, Goodpasture
  • said autoimmune disease is selected from the group comprising bullous pemphigoid, lupus (including systemic lupus erythematosus (SLE), lupus nephritis (LN), discoid lupus, lupus erythematosus profundus, Chilbrain lupus erythematosus, tumidus lupus erythematosus, severe systemic lupus erythematosus, acute cutaneous lupus, chronic cutaneous lupus) and multiple sclerosis.
  • SLE systemic lupus erythematosus
  • LN lupus nephritis
  • discoid lupus lupus erythematosus profundus
  • Chilbrain lupus erythematosus Chilbrain lupus erythematosus
  • the autoantigen is desmoglein and the autoimmune disease is Pemphigus Vulgaris.
  • a and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • activation refers to the state of a T cell (e.g., a regulatory T cell) that has been sufficiently stimulated to induce a detectable cellular response. Activation can also be associated with detectable effector function(s) such as cytokine production or suppressive activity.
  • activated regulatory T cells refers to, among other things, regulatory T cells that are capable of suppressing an immune response.
  • the monospecific population is monoclonal, and consequently all the cells of the population recognize the same epitope on the antigen.
  • the monospecific population is polyclonal, and consequently each cell of the population may recognize a different epitope on the antigen.
  • the monospecific Treg cell population of the invention suppresses, inhibits or prevents excessive or unwanted inflammatory responses, such as, for example, autoimmunity or allergic reactions.
  • the monospecific Treg cell population of the invention is a monospecific peripherally derived Treg cell population.
  • peripherally- derived Treg cells include, but are not limited to, Trl cells, TGF- ⁇ secreting Th3 cells, regulatory NKT cells, regulatory ⁇ T cells, regulatory CD8+ T cells, and double negative regulatory T cells.
  • the monospecific Treg cell population of the invention is capable of suppressive activity. In one embodiment, said suppressive activity is contact independent. In one embodiment, the monospecific Treg cell population of the invention presents a suppressive action on effector T cells, preferably said suppressive action is dependent on TCR expression and/or activation. In one embodiment, the monospecific Treg cell population of the invention presents a suppressive action on B cells, preferably said suppressive action is dependent on chimeric receptor expression and/or activation.
  • the monospecific Treg cell population of the invention secretes IL-10 after activation. In one embodiment, the monospecific Treg cell population of the invention secretes an amount of IL-10 of at least about 50 pg/ml, 100 pg/ml, 200 pg/ml, 300 pg/ml, 400 pg/ml, 500 pg/mL or more, or at least about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 thousand pg IL-10/ml or more.
  • the cytokine production of the monospecific Treg cell population is typically evaluated in cultures of cells after activation with polyclonal activators of T lymphocytes such as anti-CD3 + anti-CD28 antibodies or Interleukin-2, PMA + ionomycin.
  • the cytokine production is evaluated in cultures of cells after activation with the antigen to which the monospecific Treg cell population is directed, wherein said antigen is presented by antigen presenting cells.
  • the monospecific Treg cell population is activated for about 48 hours and the cytokine production is evaluated 24 hours after activation.
  • the suppressive activity of the monospecific Treg cell population is IL-10 dependent.
  • the cells of the monospecific Treg cell population are CD4 CD25 + .
  • the cells of the monospecific Treg cell population are CD127 10 . In another embodiment, the cells of the monospecific Treg cell population are
  • the cells of the monospecific Treg cell population are FoxP3 " . In another embodiment, the cells of the monospecific Treg cell population are FoxP3 l0 . In another embodiment, the cells of the monospecific Treg cell population are FoxP3 " /l0 .
  • the cells of the monospecific Treg cell population are CTLA-4 +/" . In another embodiment, the cells of the monospecific Treg cell population are CTLA-4 int .
  • the cells of the monospecific Treg cell population are CD39 + . In one embodiment, the cells of the monospecific Treg cell population are Helios +/" .
  • the cells of the monospecific Treg cell population are Helios lo/" . In another embodiment, the cells of the monospecific Treg cell population are Helios int . In one embodiment, the cells of the monospecific Treg cell population are CD62L 10 . In another embodiment, the cells of the monospecific Treg cell population are CD62L lo/ ⁇ . In another embodiment, the cells of the monospecific Treg cell population are CD62L int . In one embodiment, the cells of the monospecific Treg cell population are VLA4 + . In another embodiment, the cells of the monospecific Treg cell population are VLA4 int . In one another embodiment, the cells of the monospecific Treg cell population are
  • the cells of the monospecific Treg cell population are LFA1 + . In one embodiment, the cells of the monospecific Treg cell population are CD49b +/" .
  • the cells of the monospecific Treg cell population are CD49b int .
  • the cells of the monospecific Treg cell population are ITGb7 l0 . In another embodiment, the cells of the monospecific Treg cell population are ITGb7 int .
  • the cells of the monospecific Treg cell population are PSGL-1 + .
  • the cells of the monospecific Treg cell population are CLA + .
  • the cells of the monospecific Treg cell population are ICOS +/" . In another embodiment, the cells of the monospecific Treg cell population are ICOS + . In one embodiment, the cells of the monospecific Treg cell population are GITR +/ ⁇ . In another embodiment, the cells of the monospecific Treg cell population are GITR lo/int . In another embodiment, the cells of the monospecific Treg cell population are GITR + .
  • the cells of the monospecific Treg cell population are PD-l lo/" . In another embodiment, the cells of the monospecific Treg cell population are PD-l int/+ . In one embodiment, the cells of the monospecific Treg cell population are Perf. In one embodiment, the cells of the monospecific Treg cell population are Perf 10 ' " .
  • the cells of the monospecific Treg cell population are CCR1 +/" .
  • the cells of the monospecific Treg cell population are CCR4 +/" . In one embodiment, the cells of the monospecific Treg cell population are CCR7 . In another embodiment, the cells of the monospecific Treg cell population are
  • the cells of the monospecific Treg cell population are CXCR3 +/ .
  • the cells of the monospecific Treg cell population are CXCR4 +/ -.
  • the cells of the monospecific Treg cell population express Granzyme A and/or Granzyme B.
  • the determination of the expression level of molecules is conducted by flow cytometry, immunofluorescence or image analysis, for example high content analysis.
  • the determination of the expression level of molecules is conducted by flow cytometry.
  • cells are fixed and permeabilized, thereby allowing detecting intracellular proteins .
  • the determination of the expression level of a molecule in a cell population comprises determining the percentage of cells of the cell population expressing the molecule (i.e., cells "+” for the molecule). Preferably, said percentage of cells expressing the molecule is measured by FACS.
  • the terms "expressing (or +)” and “not expressing (or -)” are well known in the art and refer to the expression level of the cell marker of interest, in that the expression level of the cell marker corresponding to "+” is high or intermediate, also referred as "+/-", and the expression level of the cell marker corresponding to "-” is null. In one embodiment, an expression level is "+” if it is superior to 40%.
  • low or “lo” or “lo/-” is well known in the art and refers to the expression level of the cell marker of interest, in that the expression level of the cell marker is low by comparison with the expression level of that cell marker in the population of cells being analyzed as a whole. More particularly, the term “lo” refers to a distinct population of cells that express the cell marker at a lower level than one or more other distinct population of cells. In one embodiment, an expression level is "lo/-" if it ranges from 0 to 20%.
  • high or “hi” or “bright” is well known in the art and refers to the expression level of the cell marker of interest, in that the expression level of the cell marker is high by comparison with the expression level of that cell marker in the population of cells being analyzed as a whole.
  • an expression level is well known in the art and refers to the expression level of the cell marker of interest, in that the expression level of the cell marker is intermediate by comparison with the expression level of that cell marker in the population of cells being analyzed as a whole. In one embodiment, an expression level is "int” if it ranges from 20 to 40%.
  • cells in the top 2, 3, 4, or 5% of staining intensity are designated "hi", with those falling in the top half of the population categorized as being “+”.
  • Those cells falling below 50%>, of fluorescence intensity are designated as “lo” cells and below 5% as “-" cells.
  • the expression level of the cell marker of interest is determined by comparing the Median Fluorescence Intensity (MFI) of the cells from the cell population stained with fluorescently labeled antibody specific for this marker to the fluorescence intensity (FI) of the cells from the same cell population stained with fluorescently labeled antibody with an irrelevant specificity but with the same isotype, the same fluorescent probe and originated from the same specie (referred as Isotype control).
  • MFI Median Fluorescence Intensity
  • FI fluorescence intensity
  • the monospecific Treg cell population is obtained by in vitro differentiation of naive T cells.
  • the monospecific Treg cell population comprises or consists of engineered Treg cells.
  • said engineered monospecific Treg cells population expresses a chimeric receptor (CR) as defined herein, wherein said chimeric receptor recognizes a ligand.
  • the monospecific Treg cell population is TCR-monospecific (i.e. , all the Treg cells recognize the same antigen with their TCR) and CR-monospecific (i.e., all the Treg cells recognize the same antigen with the CR they express).
  • cytotoxic molecule refers to a protein involved in the cytotoxic pathway, preferably to a protein of the serine protease family or to the granzyme family or to granulysin or to perforin.
  • the granzyme family is a family of serine proteases that are released by cytoplasmic vesicles, which can be found for example within cytotoxic T cells and natural killer cells.
  • the protease from the granzyme family is selected from the group comprising granzyme A, granzyme B, granzyme M, granzyme K and granzyme H.
  • Granulysin is a substance first described as released by cytotoxic T cells (CD8) when they are attached to infected body cells.
  • Granulysin may create holes in a target cell membrane, but also induce apoptosis in target cells.
  • Perforin is a cytolytic protein found in the secretory vesicles of Cytotoxic T lymphocytes (CTLs) and NK cells. Upon degranulation, perforin inserts itself into the target cell's plasma membrane, forming a pore.
  • CTLs Cytotoxic T lymphocytes
  • NK cells Upon degranulation, perforin inserts itself into the target cell's plasma membrane, forming a pore.
  • chimeric receptor or CR or CAR refers to one polypeptide or to a set of polypeptides, typically two in the simplest embodiments, which when in an immune cell, provides the cell with specificity for a target ligand and with intracellular signal generation.
  • the set of polypeptides are contiguous with each other.
  • the chimeric receptor is a chimeric fusion protein comprising the set of polypeptides.
  • the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple a ligand binding domain to an intracellular signaling domain.
  • the chimeric receptor comprises an optional leader sequence at the amino -terminus (N-ter) of the chimeric receptor fusion protein. In one embodiment, the chimeric receptor further comprises a leader sequence at the N-terminus of the extracellular ligand binding domain, wherein the leader sequence is optionally cleaved from the ligand binding domain during cellular processing and localization of the chimeric receptor to the cellular membrane.
  • ligand refers to a member of a pair ligand/receptor, and binds to the other member of the pair.
  • signaling domain refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.
  • antibody refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen.
  • Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources.
  • the term “antibody” also includes multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.
  • Antibodies can be multimers of immunoglobulin molecules, such as tetramers of immunoglobulin molecules.
  • the basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ([kappa]) and lambda ([lambda]), based on the amino acid sequences of their constant domains (CL).
  • immunoglobulins can be assigned to different classes or isotypes.
  • immunoglobulins There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha ([alpha]), delta ([delta]), epsilon ([epsilon]), gamma ([gamma]) and mu ([mu]), respectively.
  • the [gamma] and [alpha] classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgGl , IgG2, IgG3, IgG4, IgAl , and IgA2.
  • Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the [alpha] and [gamma] chains and four CH domains for [mu] and [epsilon] isotypes.
  • Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end.
  • the VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • CHI heavy chain
  • the pairing of a VH and VL together forms a single antigen- binding site.
  • An IgM antibody consists of five of the basic heterotetramer units along with an additional polypeptide called a J chain, and therefore, contains ten antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 Daltons.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprised in the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.
  • a monoclonal antibody may be prepared by the hybridoma methodology first described by Kohler et al, Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).
  • a "monoclonal antibody” may also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:581-597 (1991), for example.
  • the monoclonal antibodies herein include "chimeric" antibodies.
  • antibody fragment refers to at least one portion of an intact antibody, preferably the antigen binding region or variable region of the intact antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody.
  • An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23 : 1 126-1 136, 2005).
  • Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies). Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily.
  • the Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CHI).
  • VH variable region domain
  • CHI first constant domain of one heavy chain
  • Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.
  • Pepsin treatment of an antibody yields a single large F(ab') 2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of crosslinking antigen.
  • Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • the "Fc” fragment of an antibody comprises the carboxy-terminal portions of both H chains held together by disulfides.
  • the effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.
  • Fv is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker e.g., a short flexible polypeptide linker
  • an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.
  • antibody heavy chain refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.
  • antibody light chain refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations.
  • Kappa (K) and lambda ( ⁇ ) light chains refer to the two major antibody light chain isotypes.
  • recombinant protein or peptide refers to a protein or peptide (e.g., an antibody) which is generated using recombinant DNA technology, such as, for example, a protein or peptide (e.g. , an antibody) expressed by a bacteriophage or yeast expression system.
  • the term should also be construed to mean a protein or peptide (e.g., an antibody) which has been generated by the synthesis of a DNA molecule encoding the protein or peptide (e.g., the antibody) and which DNA molecule expresses a protein or peptide (e.g. , an antibody), or an amino acid sequence specifying the protein or peptide (e.g. , the antibody), wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.
  • antigen refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immuno logically-competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein.
  • an antigen does not necessarily need to be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen does not necessarily need to be encoded by a "gene” at all. It is readily apparent that an antigen can be synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a cell or a fluid with other biological components. The term "autologous" refers to any material derived from the same individual to whom it is later to be re-introduced.
  • allogeneic refers to any material derived from a different individual of the same specie as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.
  • “Derived from” indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not connote or include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of an intracellular signaling domain that is derived from a CD3zeta molecule, the intracellular signaling domain retains sufficient CD3zeta structure such that is has the required function, namely, the ability to generate a signal under the appropriate conditions.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the biologic function of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into a protein by standard techniques known in the art, such as site-directed mutagenesis and PCR- mediated mutagenesis.
  • amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • Amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
  • amino acids with basic side chains e.g. , lysine, arginine, histidine
  • acidic side chains e.g.
  • aspartic acid, glutamic acid uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • stimulation refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex or chimeric receptor) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex or signal transduction via signaling domains of the chimeric receptor. Stimulation can mediate altered expression of certain molecules.
  • stimulatory molecule refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides the cytoplasmic signaling sequence(s) that regulate activation of the immune cell in a stimulatory way for at least some aspect of the immune cell signaling pathway.
  • the signal is a primary signal that is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • a primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine -based activation motif or IT AM.
  • an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC's) on its surface.
  • MHC's major histocompatibility complexes
  • T-cells may recognize these complexes using their T-cell receptors (TCRs).
  • APCs process antigens and present them to T-cells.
  • intracellular signaling domain refers to an intracellular portion of a molecule.
  • the intracellular signaling domain generates a signal that promotes an immune effector function of the chimeric receptor containing cell.
  • immune effector function in a chimeric receptor-T cell may include cytolytic activity and helper activity, including the secretion of cytokines.
  • zeta or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta” is defined as the protein provided as GenBank Acc. No. BAG36664.1 , or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, and a "zeta stimulatory domain” or alternatively a "CD3-zeta stimulatory domain” or a “TCR- zeta stimulatory domain” is defined as the amino acid residues from the cytoplasmic domain of the zeta chain, or functional derivatives thereof, that are sufficient to functionally transmit an initial signal necessary for T cell activation.
  • the cytoplasmic domain of zeta comprises residues 52 through 164 of GenBank Acc. No. BAG36664.1 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, that are functional orthologs thereof.
  • costimulatory molecule refers to a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response.
  • a costimulatory signaling domain can be the intracellular portion of a costimulatory molecule.
  • a costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors.
  • encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mR A, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase "nucleotide sequence that encodes a protein or a RNA” may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • terapéuticaally effective amount refer to an amount of cells of the monospecific Treg cell population or composition, as described herein effective to achieve a particular biological result.
  • therapeutically effective amount mean a level or amount of agent that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of the targeted disease or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the targeted disease or condition; (3) bringing about ameliorations of the symptoms of the targeted disease or condition; (4) reducing the severity or incidence of the targeted disease or condition; or (5) curing the targeted disease or condition.
  • a therapeutically effective amount may be administered prior to the onset of the targeted disease or condition, for a prophylactic or preventive action. Alternatively, or additionally, the therapeutically effective amount may be administered after initiation of the targeted disease or condition, for a therapeutic action.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • expression refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
  • transfer vector refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term "transfer vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a poly lysine compound, liposome, and the like.
  • Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • lentivirus refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al, Mol. Ther. 17(8): 1453-1464 (2009).
  • Other examples of lentivirus vectors that may be used in the clinic include but are not limited to, the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAXTM vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
  • homologous or identical refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules.
  • two nucleic acid molecules such as, two DNA molecules or two RNA molecules
  • two polypeptide molecules e.g., two amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids,
  • the homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90%> homologous.
  • homologous or “identical” when used in a relationship between the sequences of two or more polypeptides or of two or more nucleic acid molecules, refers to the degree of sequence relatedness between polypeptides or nucleic acid molecules, as determined by the number of matches between strings of two or more amino acid or nucleotide residues. "Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A.
  • Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al, Nucl. Acid. Res. ⁇ 2, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al, J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al, supra). The well-known Smith Waterman algorithm may also be used to determine identity.
  • NCBI National Center for Biotechnology Information
  • Humanized forms of non-human (e.g. , murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary- determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementary- determining region
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • a humanized antibody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance.
  • the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fully human refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or peptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • a preparation of isolated nucleic acid or peptide contains the nucleic acid or peptide at least about 80% pure, at least about 85% pure, at least about 90%> pure, at least about 95% pure, greater than 95% pure, greater than 96%o pure, greater than 97%> pure, greater than 98%> pure, or greater than 99%> pure.
  • An "isolated nucleic acid” is a nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence.
  • the term embraces a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • An "isolated polypeptide” is one that has been identified and separated and/or recovered from a component of its natural environment.
  • nucleic acid bases In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A” refers to adenine, “C” refers to cytosine, “G” refers to guanine, “T” refers to thymine, and “U” refers to uracil.
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.
  • nucleic acid or “polynucleotide” refers to a polymer of nucleotides covalently linked by phosphodiester bonds, such as deoxyribonucleic acids (DNA) or ribonucleic acids (RNA), in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605- 2608 (1985); and Rossolini et al, Mol. Cell. Probes 8:91-98 (1994)).
  • peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • promoter refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
  • promoter/regulatory sequence refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • constitutive promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • inducible promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • a “5' cap” (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the "front" or 5' end of a eukaryotic messenger RNA shortly after the start of transcription.
  • the 5' cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other.
  • RNA polymerase Shortly after the start of transcription, the 5' end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction.
  • the capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.
  • in vitro transcribed RNA refers to RNA, preferably mRNA, that has been synthesized in vitro.
  • the in vitro transcribed RNA is generated from an in vitro transcription vector.
  • the in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.
  • a "poly(A)" is a series of adenosines monophosphate attached to the mRNA.
  • the polyA is between 50 and 5000 adenosines monophosphate, preferably greater than or equal to 64, more preferably greater than or equal to 100, most preferably greater than or equal to 300 or 400 adenosines monophosphate.
  • Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.
  • polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
  • mRNA messenger RNA
  • the 3' poly (A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre -mRNA through the action of an enzyme, polyadenylate polymerase.
  • the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal.
  • Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm.
  • the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase.
  • the cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site.
  • adenosine residues are added to the free 3' end at the cleavage site.
  • transient refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.
  • the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the progression, severity and/or duration of a targeted disease or condition, e.g., an autoimmune condition, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of a targeted disease or condition, e.g. , an autoimmune condition, wherein said amelioration results from the administration of one or more therapies (e.g., one or more therapeutic agents such as a Treg cell of the invention).
  • the terms “treat”, “treatment” and “treating” refer to the amelioration of at least one measurable physical parameter of a targeted disease or condition, e.g., an autoimmune condition.
  • the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a targeted disease or condition, e.g. , an autoimmune condition, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g. , stabilization of a physical parameter, or both.
  • the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the progression, severity and/or duration of a targeted disease or condition, e.g. , an autoimmune disease, or the amelioration of one or more symptoms of a targeted disease or condition, e.g., an autoimmune disease.
  • Treating refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted disease or condition.
  • Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented.
  • a subject is successfully "treated” for a disease or condition if, after receiving a therapeutic amount of a population of cells comprising a chimeric receptor according to the present invention, the subject shows observable and/or measurable improvement in one or more of the following: reduction in the number of pathogenic cells; reduction in the percent of total cells that are pathogenic; and/or relief to some extent, of one or more of the symptoms associated with the specific condition; reduced morbidity and mortality, and/or improvement in quality of life issues.
  • the above parameters for assessing successful treatment and improvement in the condition are readily measurable by routine procedures familiar to a physician.
  • signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
  • a subject is intended to include living organisms in which an immune response can be elicited (e.g. , mammals, human).
  • a subject may be a "patient", i.e., a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of the targeted disease or condition, such as, for example, an inflammatory or autoimmune condition.
  • the subject is an adult (for example a subject above the age of 18).
  • the subject is a child (for example a subject below the age of 18).
  • the subject is a male.
  • the subject is a female.
  • the subject is affected, preferably is diagnosed, with an autoimmune disease, preferably an autoantibody-mediated autoimmune disease.
  • the subject is at risk of developing an autoimmune disease, preferably an autoantibody-mediated autoimmune disease. Examples of risks factor include, but are not limited to, genetic predisposition, or familial history of autoantibody-mediated autoimmune disease.
  • substantially purified cell refers to a cell that is essentially free of other cell types.
  • a substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state.
  • a substantially purified cell refers to a cell which is at least about 75% free, 80% free, or 85% free, and preferably about 90%, 95%, 96%, 97%, 98%, or 99% free, from other cell types with which it is normally associated in its naturally occurring state.
  • a population of substantially purified cells refers to a homogenous population of cells.
  • a population of substantially purified cells refers to a population of cells at least about 75% homogenous, 80%> homogenous, or 85% homogenous, and preferably about 90%, 95%), 96%o, 97%), 98%), or 99% homogenous. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.
  • transfected or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • engineered or “modified” refers to a cell that has been transfected, transformed or transduced.
  • the term “specifically binds,” refers to an antibody, or a ligand, which recognizes and binds with a binding partner present in a sample, but which antibody or ligand does not substantially recognize or bind other molecules in the sample.
  • an "instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention.
  • the instructional material of the kit of the invention may, for example, be affixed to a container which contains the cell of the invention or be shipped together with a container which contains the cell of the invention. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material cell be used cooperatively by the recipient.
  • pharmaceutically acceptable excipient or “pharmaceutically acceptable carrier” refers to an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, for example, FDA Office or EMA.
  • a polynucleotide "variant,” as the term is used herein, is a polynucleotide that typically differs from a polynucleotide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the polynucleotide sequences of the invention and evaluating one or more biological activities of the encoded polypeptide as described herein and/or using any of a number of techniques well known in the art.
  • a polypeptide "variant,” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of techniques well known in the art. Modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics.
  • amino acid sequence of a polypeptide When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant or portion of a polypeptide of the invention, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence.
  • certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of its ability to bind other polypeptides (e.g., antigens) or cells. Since it is the binding capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with similar properties.
  • variant polypeptides of the present invention may contain one or more conservative substitutions.
  • a variant may also, or alternatively, contain nonconservative changes.
  • variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer.
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.
  • the present invention relates to a monospecific Treg cell population, wherein the cells comprise chimeric receptor, wherein said chimeric receptor recognizes an autoantibody on a B cell, and wherein said Treg cells are cytotoxic for B cells, preferably are cytotoxic for B cells expressing at their surface the autoantibody recognized by the chimeric receptor.
  • the Treg cells of Treg cell population of the invention express the chimeric receptor on the cell surface.
  • the Treg cells of Treg cell population of the invention are genetically modified Treg cells that have been genetically modified to express a chimeric receptor recognizing an autoantibody on a B cell.
  • the monospecific Treg cell population of the invention may thus be defined as a redirected monospecific Treg cell population.
  • the term "redirected" refers to a Treg cell carrying a chimeric receptor as described herein, which confers to the Treg cell the ability to bind to and be activated by a ligand that is different from the one the Treg cell is or would have been specific or be activated by.
  • cytotoxicity for B cells results from the expression and/or activation of the chimeric receptor by the cells of the monospecific Treg cell population of the invention.
  • the cells of the monospecific Treg cell population of the invention are preferably non cytotoxic for B cells when not expressing the chimeric receptor recognizing an autoantibody on a B cell.
  • the Applicant demonstrated that the cells of the monospecific Treg cell population of the invention are not cytotoxic for B cell (or present only a low cytotoxic activity for B cells) when not expressing a chimeric receptor as described herein, but become cytotoxic (or more cytotoxic) for B cells when genetically modified for expressing said chimeric receptor recognizing a B cell surface marker (see Examples).
  • the Applicant suggests that redirection of the cells of the monospecific Treg cell population of the invention in order to recognize and be activated by an autoantibody on B cells, results in the activation of a cytotoxicity pathway, thereby providing to the redirected Treg cell of the invention cytotoxic properties against B cells expressing the recognized autoantibody.
  • the cytotoxicity for B cells of the cells of the monospecific Treg cell population of the invention is dependent on the expression and/or activation of the chimeric receptor by the Treg cell.
  • the cytotoxicity for B cells of the Treg cells of the monospecific Treg cell population of the invention is chimeric receptor dependent, i.e., results from the binding (and optionally activation) of the chimeric receptor to an autoantibody presented at the surface of a B cell.
  • the cytotoxicity for B cells of the Treg cells of the monospecific Treg cell population of the invention is TCR independent, i.e., does not result from the binding (and optionally activation) of the TCR expressed by the Treg cell of the invention to a ligand presented at the surface of a B cell.
  • cytotoxicity of the Treg cells of the monospecific Treg cell population of the invention for B cells may be determined by determining the survival of B cells in co-cultures of B cells with Treg cells of the invention.
  • the B cells and Treg cells of the invention are co-cultured in a ratio Treg cells:B cells of 1 :1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10.
  • the B cells express an autoantibody recognized by the autoantigen or variant or fragment thereof comprised in the chimeric receptor expressed by the Treg cells of the monospecific Treg cell population of the invention.
  • Methods for assessing the survival of B cells may rely on determining the percentage of live cells (or alternatively dead cells) and include, for example, flow cytometry analysis to detect live/dead cells, or measure of a compound released in the co-culture medium upon lysis of the B cells by the Treg cells of the monospecific Treg cell population, said B cells having been previously labeled with the compound to be measured.
  • Such "release" methods include, without being limited to, the detection of radioactive compounds, such as for example chromium ( 51 Cr), or the detection of fluorescent dyes, such as for example, calcein-acetoxymethyl (calcein-AM).
  • the dying and dead B cells are specifically labeled with propidium iodide (PI) and detected by FACS analysis after co-culture with the Treg cells of the invention.
  • PI propidium iodide
  • Propidium iodide is a fluorescent intercalating agent that binds to double- stranded nucleic acids. Propidium iodide cannot pass through an intact cell membrane and thus is not imported into live cells.
  • the B cells are labeled with calcein-AM and their survival is assessed through the detection by FACS analysis of calcein-AM released in the culture medium after co-culture with the monospecific Treg cell population of the invention.
  • the B cells and Treg cells are co-cultured at 37°C and with 5% CO2 in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics.
  • the Treg cells of the monospecific Treg cell population of the invention display a cytotoxic activity when the percentage of dead B cells after co-culture with the monospecific Treg cell population of the invention is at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 50%, preferably at least 60%>, at least 65%o, or at least 70%>, and more preferably at least 75%, at least 80%>, at least 85%, at least 90%, or at least 95% of the total number of B cells in co-culture with the monospecific Treg cell population of the invention.
  • the Treg cells of the monospecific Treg cell population of the invention display a cytotoxic activity when the rate of survival of B cells after co-culture with the monospecific Treg cell population of the invention is at most 85%o, at most 80%>, at most 75%, at most 70%, at most 60% or at most 50%, preferably at most 40%, at most 35%, or at most 30%, and more preferably at most 25%, at most 20%, at most 15%, at most 10%, or at most 5% as compared with the starting number of B cells in the co-culture with the monospecific Treg cell population of the invention.
  • the cells of the monospecific Treg cell population of the invention present low cytotoxic activity for B cells when not expressing the chimeric receptor recognizing a B cell surface marker on a B cell, and their cytotoxicity for B cells is increased by the expression of the chimeric receptor.
  • cells are considered to present a low cytotoxic activity for B cells when the percentage of dead B cells after co-culture with said cells is at most about 30%, preferably at most about 20%, about 15%, about 10% or about 5% of the total number of B cells in co-culture with the monospecific Treg cell population of the invention.
  • the expression of the chimeric receptor by the cells of the invention induces an increase of their cytotoxicity for B cells by a factor of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5 or more.
  • the cytotoxicity of the Treg cells of the monospecific Treg cell population of the invention is determined in the conditions of Test A.
  • Test A is a non-radioactive cell killing assay based on FACS analysis to detect B cells labeled with propidium iodide (PI). Briefly, B cells (5xl0 4 in 100 ⁇ ) are co-cultured with the monospecific Treg cell population of the invention at various Treg cells:B cells ratios in a 96-well U-bottom tissue culture plate (Costar, Cambridge, MA). The 96-well plate is centrifuged at 400g for 5 min prior to cell culture in a 37°C, 5% C02 incubator. After 4 hours of co-culture, propidium iodide is added. The cells are resuspended and transferred to 4mL FACS tubes for FACS analysis.
  • PI propidium iodide
  • the Treg cells of the monospecific Treg cell population of the invention express at least one cytotoxic molecule, preferably express 2, 3, 4, 5, 6 or 7 cytotoxic molecules selected from the group comprising granzyme A, granzyme B, granzyme M, granzyme K, granzyme H, granulysin and perforin.
  • the Treg cells of the monospecific Treg cell population of the invention express granzyme B and preferably further express at least one or at least two or more cytotoxic molecules selected from granzyme A, granzyme M, granzyme K, granzyme H, granulysin and perforin.
  • the Treg cells of the monospecific Treg cell population of the invention express granzyme B and granzyme H, and preferably further express at least one cytotoxic molecule selected from granzyme A, granzyme M, granzyme K, granulysin and perforin. In one embodiment, the Treg cells of the monospecific Treg cell population of the invention express granzyme B and granzyme A.
  • the monospecific Treg cell population of the invention is a mammal monospecific Treg cell population, preferably a human monospecific Treg cell population. In one embodiment, the monospecific Treg cell population of the invention has been frozen and thawed.
  • the monospecific Treg cell population of the invention is specific of an antigen, a fragment of an antigen, a variant of an antigen or a mixture thereof. In one embodiment, the monospecific Treg cell population of the invention is specific of a food antigen from the common human diet.
  • food antigen from common human diet refers to an immunogenic peptide, which comes from foodstuffs common for humans, such as food antigens of the following non-limiting list: bovine antigens such as lipocalin, Ca-binding SI 00, alpha-lactalbumin, lactoglobulins such as beta-lactoglobulin, bovine serum albumin, caseins.
  • Food-antigens may also be atlantic salmon antigens such as parvalbumin; chicken antigens such as ovomucoid, ovalbumin, Ag22, conalbumin, lysozyme or chicken serum albumin; peanut antigens; shrimp antigens such as tropomyosin; wheat antigens such as agglutinin or gliadin; celery antigens such as celery profilin; carrot antigens such as carrot profilin; apple antigens such as thaumatin, apple lipid transfer protein, or apple profilin; pear antigens such as pear profilin, or isoflavone reductase; avocado antigens such as endochitinase; apricot antigens such as apricot lipid transfer protein; peach antigens such as peach lipid transfer protein or peach profilin; soybean antigens such as HPS, soybean profilin or (SAM22) PR-IO prot; fragments, variants and mixtures thereof.
  • the monospecific Treg cell population of the invention is specific of an autoantigen, such as a multiple sclerosis-associated antigen, a joint-associated antigen, an eye-associated antigen, or a human HSP antigen.
  • an autoantigen such as a multiple sclerosis-associated antigen, a joint-associated antigen, an eye-associated antigen, or a human HSP antigen.
  • multiple sclerosis-associated antigen refers to myelin basic protein (MBP).
  • MBP myelin associated glycoprotein
  • MOG myelin oligodendrocyte glycoprotein
  • PGP proteolipid protein
  • OMGP myelin associated oligodendrocyte basic protein
  • MOBP myelin associated oligodendrocyte specific protein
  • OSP/Claudinl 1 heat shock proteins, oligodendrocyte specific proteins (OSP), NOGO A, glycoprotein Po, peripheral myelin protein 22 (PMP22), 2'3'-cyclic nucleotide 3'- phosphodiesterase (CNPase), fragments, variants and mixtures thereof.
  • PMP22 peripheral myelin protein 22
  • CNPase 2'3'-cyclic nucleotide 3'- phosphodiesterase
  • joint-associated antigen refers to citrulline-substituted cyclic and linear filaggrin peptides, type II collagen peptides, human cartilage glycoprotein 39 (HCgp39) peptides, HSP, heterogeneous nuclear ribonucleoprotein (hnRNP) A2 peptides, hnRNP Bl, hnRNP D, Ro60/52, HSP60, HSP65, HSP70 and HSP90, BiP, keratin, vimentin, fibrinogen, type I, III, IV and V collagen peptides, annexin V, Glucose 6 phosphate isomerase (GPI), acetyl-calpastatin, pyruvate dehydrogenase (PDH), aldolase, topoisomerase I, snRNP, PARP, Scl-70, Scl-100, phospholipid antigens including anionic cardiolipin and phosphatidylser
  • eye-associated antigen refers to type II collagen, retinal arrestin, S-arrestin, interphotoreceptor retinoid-binding proteins (IRBPl), beta-crystallin Bl, retinal proteins, choroid proteins and fragments, variants and mixtures thereof.
  • human HSP antigen refers to human HSP60, HSP70, HSP90, fragments, variants and mixtures thereof.
  • the monospecific Treg cell population of the invention is specific of an inhaled allergen, an ingested allergen or a contact allergen.
  • the monospecific Treg cell population of the invention is specific of an antigen selected from the group comprising ovalbumin, MOG, type II collagen fragments, variants and mixtures thereof.
  • the monospecific Treg cell population of the invention is specific of ovalbumin, fragments, variants and mixtures thereof.
  • the monospecific Treg cell population of the invention is specific of MOG, fragments, variants and mixtures thereof.
  • the monospecific Treg cell population of the invention is specific of type II collagen, fragments, variants and mixtures thereof.
  • Examples of cells of the B cell lineage include, but are not limited to, proB cells, preB cells, immature (or transitional) B cells, mature naive B cells, activated B cells, memory B cells, plasma cells, lymphoblasts, marginal zone B cells, germinal center B cells, plasmablasts and regulatory B cells (Breg cells).
  • the monospecific Treg cell population of the invention is cytotoxic for proB cells, preB cells, immature (or transitional) B cells, mature naive B cells, activated B cells, memory B cells, plasma cells, lymphoblasts, marginal zone B cells, germinal center B cells, plasmablasts and/or regulatory B cells (Breg cells).
  • the monospecific Treg cell population of the invention is not cytotoxic for Breg cells.
  • the monospecific Treg cell population of the invention is cytotoxic for B cells expressing and presenting an immunoglobulin at their surface.
  • B cells expressing and presenting an immunoglobulin at their surface include, but are not limited to, preB cells, immature (or transitional) B cells, mature naive B cells, activated B cells, memory B cells, marginal zone B cells, germinal center B cells, and regulatory B cells (Breg cells).
  • the monospecific Treg cell population of the invention is cytotoxic for mature B cells, preferably for mature activated B cells.
  • the monospecific Treg cell population of the invention is cytotoxic for mature B cells (preferably for mature activated B cells) expressing at their surface the autoantibody recognized by the chimeric receptor expressed by the cells of the monospecific Treg cell population.
  • the monospecific Treg cell population of the invention is cytotoxic for at least one cell type selected from proB cells, preB cells, immature (or transitional) B cells, mature na ' ive B cells, activated B cells, memory B cells, plasma cells, lymphoblasts, marginal zone B cells, germinal center B cells, plasmablasts and regulatory B cells (Breg cells); preferably is cytotoxic for at least one cell type selected from preB cells, immature (or transitional) B cells, mature na ' ive B cells, activated B cells, memory B cells, marginal zone B cells, germinal center B cells, and regulatory B cells (Breg cells).
  • the monospecific Treg cell population of the invention is cytotoxic for at least one cell type selected from proB cells, preB cells, immature (or transitional) B cells, mature na ' ive B cells, activated B cells, memory B cells, plasma cells, lymphoblasts, marginal zone B cells, germinal center B cells, and plasmablasts; preferably is cytotoxic for at least one cell type selected from preB cells, immature (or transitional) B cells, mature naive B cells, activated B cells, memory B cells, marginal zone B cells, and germinal center B cells.
  • the chimeric receptor comprises, in one or more polypeptides (i) an extracellular autoantigen, or a variant or fragment of an autoantigen, (ii) optionally an extracellular hinge domain, (iii) optionally a transmembrane domain, and (iv) an intracellular signaling domain.
  • the chimeric receptor further comprises a Tag and/or a leader sequence.
  • the Treg cells of monospecific Treg cell population of the invention comprise at least one nucleic acid sequences encoding the chimeric receptor, wherein said at least one nucleic acid sequences comprises (i) a nucleic acid sequence of an autoantigen, or a variant or fragment of an autoantigen, (ii) optionally a nucleic acid sequence of an extracellular hinge domain, (iii) optionally a nucleic acid sequence of a transmembrane domain, (iv) one or more nucleic acid sequence(s) of an intracellular signaling domain.
  • the nucleic acid sequence further comprises a sequence encoding a Tag and/or a leader sequence.
  • the autoantigen or variant or fragment thereof may be connected to a transmembrane domain by a hinge domain.
  • a short oligo- or polypeptide linker preferably having a length ranging from 2 and 10 amino acids may form the hinge domain.
  • a glycine-serine doublet provides a particularly suitable hinge domain (GS linker).
  • the hinge domain is a Gly/Ser linker. Examples of Gly/Ser linkers include, but are not limited to, GS linkers, G2S linkers, G3S linkers, G 4 S linkers.
  • G2S linkers include, but are not limited to, GGS.
  • GsS linkers include, but are not limited to, GGGSGGGSGGGSGGGS (SEQ ID NO: 6).
  • G 4 S linkers include, but are not limited to, (Gly 4 Ser) corresponding to GGGGS (SEQ ID NO: 5); (Gly 4 Ser) 2 corresponding to GGGGSGGGGS (SEQ ID NO: 4); (Gly 4 Ser) 3 corresponding to GGGGSGGGGSGGGGS (SEQ ID NO: 3); and (Gly 4 Ser) 4 corresponding to GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 2). Also included within the scope of the invention are hinge domains described in WO2012/138475, incorporated herein by reference.
  • the hinge domain comprises the amino acid sequence AGSSSSGGSTTGGSTT (SEQ ID NO: 7), the amino acid sequence GTTAASGSSGGSSSGA (SEQ ID NO: 8), the amino acid sequence SSATATAGTGSSTGST (SEQ ID NO: 9), and/or the amino acid sequence TSGSTGTAASSTSTST (SEQ ID NO: 10).
  • the hinge domain is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 11).
  • the hinge domain is a KIR 2 DS 2 hinge corresponding to KIRRDSS (SEQ ID NO: 12).
  • the hinge domain comprises or consists in the amino acid sequence of a CD8 hinge (SEQ ID NO: 13) or an amino acid sequence with 95-99% identity to SEQ ID NO: 13.
  • the hinge domain is a CD8 hinge encoded by the nucleic acid sequence SEQ ID NO: 14 or a nucleic acid sequence with 95-99%) identity to SEQ ID NO: 14.
  • the hinge domain comprises or consists in the amino acid sequence of a IgG4 hinge (SEQ ID NO: 15), or an amino acid sequence with 95-99%) identity to SEQ ID NO: 15.
  • the hinge domain is an IgG4 hinge encoded by the nucleic acid sequence SEQ ID NO: 16 or a nucleic acid sequence with 95-99% identity to SEQ ID NO: 16.
  • the hinge domain comprises or consists in the amino acid sequence of a IgD hinge (SEQ ID NO: 17) or an amino acid sequence with 95-99%) identity to SEQ ID NO: 17.
  • the hinge domain is an IgD hinge encoded by the nucleic acid sequence SEQ ID NO: 18 or a nucleic acid sequence with 95-99% identity to SEQ ID NO: 18.
  • the hinge region comprises or consists in the amino acid sequence of a CD28 hinge (SEQ ID NO: 19) or an amino acid sequence with 95-99% identity to SEQ ID NO: 19.
  • the hinge domain is a CD28 hinge encoded by the nucleic acid SEQ ID NO: 20 or a nucleic acid sequence with 95-99%) identity to SEQ ID NO: 20.
  • transmembrane domains that may be used in the chimeric receptor of the invention include, but are not limited to, transmembrane domains of an alpha, beta or zeta chain of a T-cell receptor, or of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, IT
  • transmembrane domains that may be used in the chimeric receptor of the invention include, but are not limited to, transmembrane domains of an alpha, beta or zeta chain of a T-cell receptor, or of CD28, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, CD 154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD1 la, CD 18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D,
  • the transmembrane domain comprises or consists in the amino acid sequence of a CD8 transmembrane domain (SEQ ID NO: 21), or an amino acid sequence with 95-99% identity to SEQ ID NO: 21.
  • the transmembrane domain comprises or consists in an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO: 21, or an amino acid sequence with 95-99% identity to SEQ ID NO: 21.
  • the transmembrane domain is encoded by the nucleotide sequence of a CD8 transmembrane domain (SEQ ID NO: 22), or a nucleotide sequence with 95-99% identity to SEQ ID NO: 22.
  • the transmembrane domain comprises or consists in the amino acid sequence of a CD28 transmembrane domain (SEQ ID NO: 23) or an amino acid sequence with 95-99%> identity to SEQ ID NO: 23.
  • the transmembrane domain is a CD28 transmembrane domain encoded by the nucleic acid sequence SEQ ID NO: 24 or a nucleic acid sequence with 95-99%) identity to SEQ ID NO: 24.
  • the transmembrane domain may be recombinant, in which case it will comprise predominantly hydrophobic amino acids such as valine or leucine.
  • the intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment or derivative thereof.
  • the intracellular signaling domain comprises a T cell primary signaling domain (or a sequence derived therefrom) and optionally one or more intracellular domain(s) of a T cell costimulatory molecule (or sequences derived therefrom).
  • the intracellular signaling domain of the chimeric receptor of the invention consists in a primary signaling domain.
  • the intracellular signaling domain comprises one or more intracellular domain(s) of a T cell costimulatory molecule. In one embodiment, the intracellular signaling domain consists in one or more intracellular domain(s) of a T cell costimulatory molecule.
  • the intracellular signaling domain of the chimeric receptor of the invention comprises at least one costimulatory domain and a primary signaling domain.
  • the intracellular signaling domain of the chimeric receptor of the invention comprises at least two costimulatory domains and a primary signaling domain.
  • the T cell primary signaling domain comprises a signaling domain of a protein selected in the group of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP 10, and DAP 12 or sequences derived therefrom.
  • the T cell primary signaling domain comprises or consists in a functional signaling domain of CD3 zeta.
  • the T cell primary signaling domain comprises or consists in the amino acid sequence of the CD3-zeta domain of SEQ ID NO: 25, 26, 46 or 47, or an amino acid sequence with 95-99% identity to SEQ ID NO: 25, 26, 46 or 47.
  • the CD3 zeta primary signaling domain comprises or consists in an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO: 25, 26, 46 or 47, or an amino acid sequence with 95-99% identity to SEQ ID NO: 25, 26, 46 or 47.
  • the nucleic acid sequence encoding the T cell primary signaling domain comprises or consists in the nucleic acid sequence of the CD3-zeta domain of SEQ ID NO: 27 or SEQ ID NO: 28, or a nucleotide sequence with 95-99% identity to SEQ ID NO: 27 or SEQ ID NO: 28.
  • T cell primary signaling domains that act in a stimulatory manner may comprise signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMS).
  • ITAMS immunoreceptor tyrosine-based activation motifs
  • ITAM containing T cell primary intracellular signaling domains examples include, but are not limited to, those of (or derived from) CD3 zeta, common FcR gamma (FCER1G), Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.
  • ITAM containing T cell primary intracellular signaling domains include, but are not limited to, those of (or derived from) CD3 zeta, common FcR gamma (FCER1G), Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD66b, CD79a, CD79b, DAP10, and DAP12.
  • the T cell primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain.
  • a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and/or truncated ITAM-containing primary intracellular signaling domain.
  • a primary signaling domain comprises one, two, three, four or more ITAM motifs.
  • the intracellular signaling domain of the chimeric receptor of the invention comprises a T cell primary signaling domain (such as, for example, a CD3-zeta signaling domain), combined with one or more costimulatory signaling domains.
  • a costimulatory signaling domain(s) refers to a portion of the chimeric receptor comprising at least one intracellular domain of a costimulatory molecule.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen.
  • intracellular domains of a T cell costimulatory molecule include, but are not limited to, the signaling domains of proteins selected in the group of CD27, CD28, 4-1BB (CD 137), an MHC class I molecule, BTLA and a Toll ligand receptor, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160 (BY55), CD 19, CD 19a, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D
  • intracellular domains of a T cell costimulatory molecule include, but are not limited to, the signaling domains of proteins selected in the group of CD27, CD28, 4-1BB (CD137), an MHC class I molecule, BTLA, a Toll ligand receptor, OX40, CD30, CD40, PD-1, ICOS (CD278), lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM- 1, GITR, ARHR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160 (BY55), CD 19, CD 19a, CD4, CD8alpha, CD8beta, IL2ra, IL6Ra, IL2R beta, IL2R gamma, IL7R alpha
  • the T cell costimulatory signaling domain comprises or consists in the amino acid sequence of a 4- IBB intracellular domain (SEQ ID NO: 29) or an amino acid sequence with 95-99% identity to SEQ ID NO: 29. In another embodiment, the T cell costimulatory signaling domain comprises or consists in an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO: 29. In one embodiment, the T cell costimulatory signaling domain comprises or consists in the amino acid sequence of a CD27 intracellular domain (SEQ ID NO: 30) or an amino acid sequence with 95-99% identity to SEQ ID NO: 30. In another embodiment, the T cell costimulatory signaling domain comprises or consists in an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO: 30.
  • the T cell costimulatory signaling domain comprises or consists in the amino acid sequence of a CD28 intracellular domain (SEQ ID NO: 31) or an amino acid sequence with 95-99% identity to SEQ ID NO: 31.
  • the T cell costimulatory signaling domain comprises or consists in an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO: 31.
  • the chimeric receptor comprises a combination of at least two intracellular domains of a T cell costimulatory molecule, preferably selected from an intracellular domain of CD28, an intracellular domain of CD27 and an intracellular domain of 4- IBB.
  • the chimeric receptor comprises the amino acid sequence of a 4- IBB intracellular domain (SEQ ID NO: 29) or an amino acid sequence with 95-99% identity to SEQ ID NO: 29 and the amino acid sequence of a CD27 intracellular domain (SEQ ID NO: 30) or an amino acid sequence with 95-99% identity to SEQ ID NO: 30.
  • the chimeric receptor comprises the amino acid sequence of a 4- 1BB intracellular domain (SEQ ID NO: 29) or an amino acid sequence with 95-99% identity to SEQ ID NO: 29 and the amino acid sequence of a CD28 intracellular domain (SEQ ID NO: 31) or an amino acid sequence with 95-99% identity to SEQ ID NO: 31.
  • the chimeric receptor comprises the amino acid sequence of a CD27 intracellular domain (SEQ ID NO: 30) or an amino acid sequence with 95-99% identity to SEQ ID NO: 30 and the amino acid sequence of a CD28 intracellular domain (SEQ ID NO: 31) or an amino acid sequence with 95-99% identity to SEQ ID NO: 31.
  • the chimeric receptor comprises the amino acid sequence of a 4- IBB intracellular domain (SEQ ID NO: 29) or an amino acid sequence with 95-99% identity to SEQ ID NO: 29 and the amino acid sequence of a CD27 intracellular domain (SEQ ID NO: 30) or an amino acid sequence with 95-99% identity to SEQ ID NO: 30 and the amino acid sequence of a CD28 intracellular domain (SEQ ID NO: 31) or an amino acid sequence with 95-99%) identity to SEQ ID NO: 31.
  • the nucleic acid sequence encoding the T cell costimulatory signaling domain comprises the nucleic acid sequence of a 4- IBB intracellular domain (SEQ ID NO: 32) or a nucleic acid sequence with 95-99% identity to SEQ ID NO: 32, and/or the nucleic acid sequence of a CD27 intracellular domain (SEQ ID NO: 33) or a nucleic acid sequence with 95-99%) identity to SEQ ID NO: 33, and/or the nucleic acid sequence of a CD28 intracellular domain (SEQ ID NO: 34), or a nucleic acid sequence with 95-99% identity to SEQ ID NO: 34.
  • the intracellular signaling domain of the chimeric receptor of the invention comprises:
  • sequences comprised in the intracellular domain are expressed in the same frame and as a single polypeptide chain.
  • the nucleic acid sequence encoding the intracellular signaling domain of the chimeric receptor of the invention comprises:
  • nucleic acid sequence of a 4- IBB intracellular domain of SEQ ID NO: 32 or a nucleic acid sequence with 95-99% identity to SEQ ID NO: 32 and/or the nucleic acid sequence of a CD27 intracellular domain of SEQ ID NO: 33 or a nucleic acid sequence with 95-99%> identity to SEQ ID NO: 33, and/or the nucleic acid sequence of a CD28 intracellular domain of SEQ ID NO: 34 or a nucleic acid sequence with 95-99% identity to SEQ ID NO: 34;
  • the intracellular signaling domain of the chimeric receptor of the invention comprises at least two different domains (e.g. , a primary signaling domain and at least one intracellular domain of a T cell costimulatory molecule) that may be linked to each other in a random or in a specified order.
  • a short oligo- or polypeptide linker for example, between 2 and 10 amino acids (e.g. , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between distinct signaling domains.
  • a glycine-serine doublet (GS) is used as a suitable linker.
  • a single amino acid e.g., an alanine (A), a glycine (G), is used as a suitable linker.
  • the intracellular signaling domain of the chimeric receptor of the invention comprises two or more, e.g. , 2, 3, 4, 5, or more, costimulatory signaling domains.
  • the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains are separated by a linker molecule, e.g., a linker molecule as described hereinabove.
  • the intracellular signaling domain of the chimeric receptor of the invention comprises the primary signaling domain of CD3-zeta (preferably SEQ ID NO: 25, 26, 46 or 47) and the co-stimulatory signaling domain of CD28 (preferably SEQ ID NO: 31).
  • the intracellular signaling domain of the chimeric receptor of the invention comprises the primary signaling domain of CD3-zeta (preferably SEQ ID NO: 25, 26, 46 or 47) and the co-stimulatory signaling domain of 4- IBB (preferably SEQ ID NO: 29).
  • the intracellular signaling domain of the chimeric receptor of the invention comprises the signaling domain of CD3-zeta (preferably SEQ ID NO: 25, 26, 46 or 47) and the signaling domain of CD27 (preferably SEQ ID NO: 30).
  • the chimeric receptor comprises an autoantigen (that may also be referred as self-antigen) or a fragment or variant thereof. Consequently, according to the invention, the chimeric receptor of the invention recognizes antibodies directed to said autoantigen. Therefore, in one embodiment, the Treg cell of the invention is cytotoxic for B cells producing antibodies directed to said autoantigen.
  • autoantigen or self-antigen” refers to an endogenous antigen that stimulates production of autoantibodies.
  • the autoantigen is involved in an autoimmune disease.
  • variant of an antigen refers herein to an autoantigen that is almost identical to the natural antigen and which shares the same biological activity.
  • the minimal difference between the natural antigen and its variants may lie for example in an amino-acid substitution, deletion, and/or addition.
  • variants may contain for example conservative amino acid substitutions in which amino acid residues are replaced with amino acid residues having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. , alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. , threonine, valine, isoleucine) and aromatic side chains (e.g. , tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., as
  • the variant of an antigen presents a sequence identity of at least 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% with the sequence of the natural antigen.
  • autoantigens include but are not limited to, aquaporin water channels (such as, for example, aquaporin-4 water channel (AQP4)), Hu, Ma2, collapsin response- mediator protein 5 (CRMP5), and amphiphysin, voltage-gated potassium channel (VGKC), N-methyl-d-aspartate receptor (NMDAR), a-amino-3-hydroxy-5-methyl-4- isoxazoleproprionic acid (AMPAR), thyroid peroxidase, thyroglobulin, anti-N-methyl- D-aspartate receptor (NRl subunit), Rh blood group antigens, I antigen, desmoglein 1 or 3 (Dsgl/3), BP180, BP230, Acetylcholine nicotinic postsynaptic receptors, thyrotropin receptors, platelet integrin, GpIIb:IIIa, Collagen (such as, for example, Collagen alpha- 3 (IV) chain), rheumatoi
  • autoantigens include but are not limited to, aquaporin-4 water channel (AQP4), Hu, Ma2, collapsin response-mediator protein 5 (CRMP5), and amphiphysin, voltage-gated potassium channel (VGKC), N-methyl-d-aspartate receptor (NMDAR), a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPAR), thyroid peroxidase, thyroglobulin, anti-N-methyl-D-aspartate receptor (NR1 subunit), Rh blood group antigens, I antigen, desmoglein 1 or 3 (Dsgl/3), BP180, BP230, Acetylcholine nicotinic postsynaptic receptors, thyrotropin receptors, platelet integrin, GpIIb:IIIa, Collagen alpha-3(IV) chain, rheumatoid factor, calpastatin, citrullinated proteins, Myelin basic protein (
  • autoantigens include, without limitation, multiple sclerosis-associated antigens (such as, for example, myelin basic protein (MBP), myelin associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), oligodendrocyte myelin oligoprotein (OMGP), myelin associated oligodendrocyte basic protein (MOBP), oligodendrocyte specific protein (OSP/Claudinl 1), heat shock proteins, oligodendrocyte specific proteins (OSP), NOGO A, glycoprotein Po, peripheral myelin protein 22 (PMP22), 2'3'-cyclic nucleotide 3'- phosphodiesterase (CNPase), fragments, variants and mixtures thereof); joint-associated antigens (such as, for example, citrulline-substituted cyclic and linear filaggrin peptides, type II collagen peptides, human carti
  • joint-associated antigens such
  • the autoantigen is desmoglein 1 or desmoglein 3 or a variant or fragment thereof, such as, for example, extracellular domains of desmoglein 1 or 3.
  • the chimeric receptor comprises extracellular domains 1 to 4 of desmoglein 3, such as, for example, a sequence comprising or consisting of SEQ ID NO: 40.
  • the chimeric receptor of the invention further comprises a leader sequence located N-terminally from the B cell surface marker binding domain.
  • leader sequence is a leader sequence of CD8, that may comprise or consists in the sequence SEQ ID NO: 36.
  • the chimeric receptor further comprises a tag, such as, for example, a tag for quality control, enrichment, tracking in vivo and the like. Said Tag may be localized N-terminally, C-terminally and/or internally. Examples of tags that may be used in the chimeric receptor of the invention are well known by the skilled artisan.
  • a tag used in the invention can be a tag selected from the group comprising or consisting of Hemagglutinin Tag, Poly Arginine Tag, Poly Histidine Tag, Myc Tag, Strep Tag, S-Tag, HAT Tag, 3x Flag Tag, Calmodulin-binding peptide Tag, SBP Tag, Chitin binding domain Tag, GST Tag, Maltose-Binding protein Tag, Fluorescent Protein Tag, T7 Tag, V5 Tag and Xpress Tag.
  • Other examples of tag include, without limitation, NWSHPQFEK (SEQ ID NO: 44) or SAWSHPQFEK (SEQ ID NO: 45).
  • the chimeric receptor of the invention comprises an autoantigen or variant or fragment thereof, optionally an extracellular hinge domain, a transmembrane domain, and a T cell primary signaling domain.
  • the chimeric receptor of the invention comprises an autoantigen or variant or fragment thereof; a transmembrane domain of CD8 (preferably SEQ ID NO: 21); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a transmembrane domain of CD28 (preferably SEQ ID NO: 23); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD8 (preferably SEQ ID NO: 13); a transmembrane domain of CD 8 (preferably SEQ ID NO: 21); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD8 (preferably SEQ ID NO: 13); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgG4 (preferably SEQ ID NO: 15); a transmembrane domain of CD 8 (preferably SEQ ID NO: 21); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgG4 (preferably SEQ ID NO: 15); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgD (preferably SEQ ID NO: 17); a transmembrane domain of CD 8 (preferably SEQ ID NO: 21); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention an autoantigen, or a variant or fragment thereof; a hinge domain of IgD (preferably SEQ ID NO: 17); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD28 (preferably SEQ ID NO: 19); a transmembrane domain of CD 8 (preferably SEQ ID NO: 21); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD28 (preferably SEQ ID NO: 19); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof, optionally an extracellular hinge domain, a transmembrane domain, a single intracellular domain of a T cell costimulatory molecule and a T cell primary signaling domain.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a transmembrane domain of CD 8 (preferably SEQ ID NO: 21); an intracellular domain of 4- IBB (preferably SEQ ID NO: 29); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of CD27 (preferably SED ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of CD28 (preferably SED ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of 4- IBB (preferably SED ID NO: 29); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of CD27 (preferably SED ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of CD28 (preferably SED ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD8 (preferably SEQ ID NO: 13); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of 4-1BB (preferably SED ID NO: 29); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD8 preferably SEQ ID NO: 13
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of 4-1BB preferably SED ID NO: 29
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD8 (preferably SEQ ID NO: 13); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of CD27 (preferably SEQ ID NO: 30); and a CD3- zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD8 preferably SEQ ID NO: 13
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of CD27 preferably SEQ ID NO: 30
  • a CD3- zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD8 (preferably SEQ ID NO: 13); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD8 preferably SEQ ID NO: 13
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of CD28 preferably SEQ ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD8 (preferably SEQ ID NO: 13); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of 4-1BB (preferably SEQ ID NO: 29); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD8 preferably SEQ ID NO: 13
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of 4-1BB preferably SEQ ID NO: 29
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD8 (preferably SEQ ID NO: 13); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of CD27 (preferably SEQ ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD8 preferably SEQ ID NO: 13
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of CD27 preferably SEQ ID NO: 30
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD8 (preferably SEQ ID NO: 13); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD8 preferably SEQ ID NO: 13
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of CD28 preferably SEQ ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgG4 (preferably SEQ ID NO: 15); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of 4-1BB (preferably SED ID NO: 29); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgG4 (preferably SEQ ID NO: 15); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of CD27 (preferably SEQ ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgG4 (preferably SEQ ID NO: 15); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of CD27 (preferably SEQ ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgG4 (preferably SEQ ID NO: 15); a transmembran
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgG4 (preferably SEQ ID NO: 15); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of 4-1BB (preferably SED ID NO: 29); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgG4 (preferably SEQ ID NO: 15); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of CD27 (preferably SED ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgG4 (preferably SEQ ID NO: 15); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of CD28 (preferably SED ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgD (preferably SEQ ID NO: 17); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of 4-1BB (preferably SED ID NO: 29); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgD (preferably SEQ ID NO: 17); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of CD27 (preferably SEQ ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgD (preferably SEQ ID NO: 17); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgD (preferably SEQ ID NO: 17); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of 4-1BB (preferably SED ID NO: 29); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgD (preferably SEQ ID NO: 17); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of CD27 (preferably SED ID NO: 30); and a CD3- zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgD (preferably SEQ ID NO: 17); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD28 (preferably SEQ ID NO: 19); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of 4-1BB (preferably SED ID NO: 29); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD28 preferably SEQ ID NO: 19
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of 4-1BB preferably SED ID NO: 29
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD28 (preferably SEQ ID NO: 19); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of CD27 (preferably SEQ ID NO: 30); and a CDS- zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD28 preferably SEQ ID NO: 19
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of CD27 preferably SEQ ID NO: 30
  • a CDS- zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD28 (preferably SEQ ID NO: 19); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD28 preferably SEQ ID NO: 19
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of CD28 preferably SEQ ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD28 (preferably SEQ ID NO: 19); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of 4-1BB (preferably SED ID NO: 29); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD28 preferably SEQ ID NO: 19
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of 4-1BB preferably SED ID NO: 29
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD28 (preferably SEQ ID NO: 19); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of CD27 (preferably SEQ ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD28 preferably SEQ ID NO: 19
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of CD27 preferably SEQ ID NO: 30
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD28 (preferably SEQ ID NO: 19); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of CD28 (preferably SED ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof, optionally an extracellular hinge domain, a transmembrane domain, two intracellular domains of a T cell costimulatory molecule and a T cell primary signaling domain.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a transmembrane domain of CD 8 (preferably SEQ ID NO: 21); an intracellular domain of 4- IBB (preferably SED ID NO: 29); an intracellular domain of CD27 (preferably SEQ ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a transmembrane domain of CD 8 preferably SEQ ID NO: 21
  • an intracellular domain of 4- IBB preferably SED ID NO: 29
  • an intracellular domain of CD27 preferably SEQ ID NO: 30
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of 4- IBB (preferably SEQ ID NO: 29); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of 4- IBB preferably SEQ ID NO: 29
  • an intracellular domain of CD28 preferably SEQ ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of CD27 (preferably SEQ ID NO: 30); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of CD27 preferably SEQ ID NO: 30
  • an intracellular domain of CD28 preferably SEQ ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of 4- IBB (preferably SED ID NO: 29); an intracellular domain of CD27 (preferably SED ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of 4- IBB preferably SED ID NO: 29
  • an intracellular domain of CD27 preferably SED ID NO: 30
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of 4-1BB (preferably SED ID NO: 29); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of 4-1BB preferably SED ID NO: 29
  • an intracellular domain of CD28 preferably SEQ ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of CD27 (preferably SED ID NO: 30); an intracellular domain of CD28 (preferably SED ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of CD27 preferably SED ID NO: 30
  • an intracellular domain of CD28 preferably SED ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD8 (preferably SEQ ID NO: 13); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of 4- IBB (preferably SED ID NO: 29); an intracellular domain of CD27 (preferably SED ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD8 preferably SEQ ID NO: 13
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of 4- IBB preferably SED ID NO: 29
  • an intracellular domain of CD27 preferably SED ID NO: 30
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD 8 (preferably SEQ ID NO: 13); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of 4- IBB (preferably SED ID NO: 29); an intracellular domain of CD28 (preferably SED ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD 8 preferably SEQ ID NO: 13
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of 4- IBB preferably SED ID NO: 29
  • an intracellular domain of CD28 preferably SED ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD8 (preferably SEQ ID NO: 13); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of CD27 (preferably SEQ ID NO: 30); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD8 preferably SEQ ID NO: 13
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of CD27 preferably SEQ ID NO: 30
  • an intracellular domain of CD28 preferably SEQ ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD8 (preferably SEQ ID NO: 13); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of 4- IBB (preferably SED ID NO: 29); an intracellular domain of CD27 (preferably SED ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD8 preferably SEQ ID NO: 13
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of 4- IBB preferably SED ID NO: 29
  • an intracellular domain of CD27 preferably SED ID NO: 30
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD 8 (preferably SEQ ID NO: 13); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of 4- IBB (preferably SEQ ID NO: 29); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD 8 preferably SEQ ID NO: 13
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of 4- IBB preferably SEQ ID NO: 29
  • an intracellular domain of CD28 preferably SEQ ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD8 (preferably SEQ ID NO : 13); a transmembrane domain of CD28 (preferably SEQ ID NO : 23); an intracellular domain of CD27 (preferably SED ID NO: 30); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD8 preferably SEQ ID NO : 13
  • a transmembrane domain of CD28 preferably SEQ ID NO : 23
  • an intracellular domain of CD27 preferably SED ID NO: 30
  • an intracellular domain of CD28 preferably SEQ ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgG4 (preferably SEQ ID NO: 15); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of 4- IBB (preferably SED ID NO: 29); an intracellular domain of CD27 (preferably SEQ ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgG4 (preferably SEQ ID NO: 15); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of 4- IBB (preferably SEQ ID NO: 29); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgG4 (preferably SEQ ID NO: 15); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of CD27 (preferably SEQ ID NO: 30); an intracellular domain of CD28 (preferably SED ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of IgG4 preferably SEQ ID NO: 15
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of CD27 preferably SEQ ID NO: 30
  • an intracellular domain of CD28 preferably SED ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgG4 (preferably SEQ ID NO: 15); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of 4- IBB (preferably SED ID NO: 29); an intracellular domain of CD27 (preferably SED ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • IgG4 preferably SEQ ID NO: 15
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of 4- IBB preferably SED ID NO: 29
  • an intracellular domain of CD27 preferably SED ID NO: 30
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgG4 (preferably SEQ ID NO: 15); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of 4- IBB (preferably SEQ ID NO: 29); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • IgG4 preferably SEQ ID NO: 15
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of 4- IBB preferably SEQ ID NO: 29
  • an intracellular domain of CD28 preferably SEQ ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgG4 (preferably SEQ ID NO: 15); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of CD27 (preferably SED ID NO: 30); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • IgG4 preferably SEQ ID NO: 15
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of CD27 preferably SED ID NO: 30
  • an intracellular domain of CD28 preferably SEQ ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgD, preferably comprising the amino acid sequence SEQ ID NO: 17; a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of 4- IBB (preferably SEQ ID NO: 29); an intracellular domain of CD27 (preferably SEQ ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of IgD preferably comprising the amino acid sequence SEQ ID NO: 17
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of 4- IBB preferably SEQ ID NO: 29
  • an intracellular domain of CD27 preferably SEQ ID NO: 30
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgD (preferably SEQ ID NO: 17); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of 4-1BB (preferably SED ID NO: 29); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3- zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgD (preferably SEQ ID NO: 17); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of CD27 (preferably SEQ ID NO: 30); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgD (preferably SEQ ID NO: 17); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of 4- IBB (preferably SED ID NO: 29); an intracellular domain of CD27 (preferably SEQ ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgD (preferably SEQ ID NO: 17); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of 4- IBB (preferably SED ID NO: 29); an intracellular domain of CD28 (preferably SED ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • IgD preferably SEQ ID NO: 17
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of 4- IBB preferably SED ID NO: 29
  • an intracellular domain of CD28 preferably SED ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of IgD (preferably SEQ ID NO : 17); a transmembrane domain of CD28 (preferably SEQ ID NO : 23); an intracellular domain of CD27 (preferably SED ID NO: 30); an intracellular domain of CD28 (preferably SED ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of IgD preferably SEQ ID NO : 17
  • a transmembrane domain of CD28 preferably SEQ ID NO : 23
  • an intracellular domain of CD27 preferably SED ID NO: 30
  • an intracellular domain of CD28 preferably SED ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD28 (preferably SEQ ID NO: 19); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of 4- IBB (preferably SED ID NO: 29); an intracellular domain of CD27 (preferably SEQ ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD28 preferably SEQ ID NO: 19
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of 4- IBB preferably SED ID NO: 29
  • an intracellular domain of CD27 preferably SEQ ID NO: 30
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD28 (preferably SEQ ID NO: 19); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of 4- IBB (preferably SED ID NO: 29); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD28 preferably SEQ ID NO: 19
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of 4- IBB preferably SED ID NO: 29
  • an intracellular domain of CD28 preferably SEQ ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD28 (preferably SEQ ID NO: 19); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); an intracellular domain of CD27 (preferably SEQ ID NO: 30); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD28 preferably SEQ ID NO: 19
  • a transmembrane domain of CD8 preferably SEQ ID NO: 21
  • an intracellular domain of CD27 preferably SEQ ID NO: 30
  • an intracellular domain of CD28 preferably SEQ ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD28 (preferably SEQ ID NO: 19); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of 4- IBB (preferably SEQ ID NO: 29); an intracellular domain of CD27 (preferably SEQ ID NO: 30); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD28 preferably SEQ ID NO: 19
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of 4- IBB preferably SEQ ID NO: 29
  • an intracellular domain of CD27 preferably SEQ ID NO: 30
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD28 (preferably SEQ ID NO: 19); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of 4- IBB (preferably SEQ ID NO: 29); an intracellular domain of CD28 (preferably SEQ ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD28 preferably SEQ ID NO: 19
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of 4- IBB preferably SEQ ID NO: 29
  • an intracellular domain of CD28 preferably SEQ ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises an autoantigen, or a variant or fragment thereof; a hinge domain of CD28 (preferably SEQ ID NO: 19); a transmembrane domain of CD28 (preferably SEQ ID NO: 23); an intracellular domain of CD27 (preferably SED ID NO: 30); an intracellular domain of CD28 (preferably SED ID NO: 31); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 25, 26, 46 or 47).
  • a hinge domain of CD28 preferably SEQ ID NO: 19
  • a transmembrane domain of CD28 preferably SEQ ID NO: 23
  • an intracellular domain of CD27 preferably SED ID NO: 30
  • an intracellular domain of CD28 preferably SED ID NO: 31
  • a CD3-zeta primary signaling domain preferably SEQ ID NO: 25, 26, 46 or 47.
  • the chimeric receptor of the invention comprises (i) an autoantigen, or a variant or fragment thereof, (ii) a hinge region of human CD28, (iii) a transmembrane domain of human CD28, (iv) an intracellular domain of human CD28 and (v) an intracellular domain of human CD3 ⁇ chain.
  • the part of the chimeric receptor comprising a hinge region of human CD28, a transmembrane domain of human CD28, an intracellular domain of human CD28 and an intracellular domain of human CD3 ⁇ chain corresponds to the amino acid sequence of SEQ ID NO: 35 or 48 or an amino acid sequence with 95-99% identity to SEQ ID NO: 35 or 48.
  • the chimeric receptor of the invention comprises a B cell surface marker binding domain, or an autoantigen, or a variant or fragment thereof linked to an amino acid sequence of SEQ ID NO: 35 or 48 or a sequence or an amino acid sequence with 95-99% identity to SEQ ID NO: 35 or 48.
  • the chimeric receptor of the invention comprises (i) an autoantigen, or a variant or fragment thereof, (ii) a hinge region of human CD8, (iii) a transmembrane domain of human CD8, (iv) an intracellular domain of human 4-lBB and (v) an intracellular domain of human CD3 ⁇ .
  • the part of the chimeric receptor comprising a hinge region of human CD8, a transmembrane domain of human CD8, an intracellular domain of human 4-lBB and an intracellular domain of human CD3 ⁇ comprises or consists in the amino acid sequence SEQ ID NO: 37, 38, 49 or 50, or any amino acid sequence with 95-99% identity with SEQ ID NO: 37, 38, 49 or 50.
  • the chimeric receptor of the invention comprises (i) a B cell surface marker binding domain, (ii) a hinge region of human CD8, (iii) a transmembrane domain of human CD8, (iv) an intracellular domain of human CD28 and (v) an intracellular domain of human CD3 ⁇ .
  • the part of the chimeric receptor comprising a hinge region of human CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28 and an intracellular domain of human CD3 ⁇ comprises or consists in the amino acid sequence SEQ ID NO: 39 or 51, or any amino acid sequence with 95-99% identity with SEQ ID NO: 39 or 51.
  • the chimeric receptor of the invention comprises extracellular domains 1-4 of desmoglein 3, a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human 4-lBB and an intracellular domain of human CD3 ⁇ .
  • said chimeric receptor comprises or consists in SEQ ID NO: 42 or 53 or an amino acid sequence with 95-99% identity to SEQ ID NO: 42 or 53.
  • the chimeric receptor of the invention comprises a leader sequence of CD8, extracellular domains 1-4 of desmoglein 3, a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human 4-lBB and an intracellular domain of human CD3 ⁇ .
  • said chimeric receptor comprises or consists in SEQ ID NO: 41 or 52 or an amino acid sequence with 95-99%) identity to SEQ ID NO: 41 or 52.
  • the cells of the monospecific Treg cell population of the invention express at their surface a chimeric receptor of the invention, and another receptor (herein referred to as "second receptor"), that binds to another ligand than the autoantibody recognized by the chimeric receptor of the invention.
  • this other receptor comprises an extracellular ligand binding domain, optionally a hinge, optionally a transmembrane domain, and an intracellular signaling domain, as previously described.
  • the second receptor is endogenous (such as, for example, the endogenous TCR).
  • the second receptor is exogenous, and its expression is induced in the Treg cells of the monospecific Treg cell population of the invention by transformation or transduction of a nucleic acid encoding it.
  • Said exogenous receptor may be an exogenous TCR or a chimeric receptor. Therefore, in one embodiment, the Treg cells of the monospecific Treg cell population of the invention expresses two chimeric receptors, wherein the first one recognizes an autoantibody on a B cell, and the second one recognizes a distinct ligand.
  • the chimeric receptor of the invention comprises a first intracellular signaling domain, and the second receptor comprises a distinct second intracellular signaling domain.
  • the chimeric receptor of the invention comprises a T cell primary signaling domain (such as, for example, CD3zeta), and the second receptor comprises a costimulatory signaling domain (such as, for example, of 4- IBB, CD28 or a combination of costimulatory signaling domain of 4- IBB and CD28).
  • the chimeric receptor of the invention comprises a costimulatory signaling domain (such as, for example, of 4- IBB, CD28 or a combination of costimulatory signaling domain of 4- IBB and CD28), and the second receptor comprises a T cell primary signaling domain (such as, for example, CD3zeta).
  • a costimulatory signaling domain such as, for example, of 4- IBB, CD28 or a combination of costimulatory signaling domain of 4- IBB and CD28
  • a T cell primary signaling domain such as, for example, CD3zeta
  • the complete activation of the Treg cells of the monospecific Treg cell population of the invention may thus require both the binding of the chimeric receptor of the invention to an autoantibody on B cells, and the binding of the second receptor to the ligand to which it is directed.
  • the ligand recognized by the second receptor is expressed or present at the diseased tissue or organ, or at the site of the autoimmune response. Consequently, cytotoxicity for B cells will be induced only at the diseased tissue or organ or at the site of the autoimmune response, when said ligand will be present and recognized by the second receptor on the Treg cells of the monospecific Treg cell population.
  • the chimeric receptor of the invention further comprises an extracellular ligand binding domain recognizing a ligand distinct from the autoantibody recognized by the autoantigen.
  • said ligand binding domain is an antibody or an antigen binding fragment thereof.
  • the portion of the chimeric receptor of the invention comprising an antibody or antigen binding fragment thereof may exist in a variety of forms where the ligand binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody or bispecific antibody (Harlow et al, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al, 1988, Science 242:423-426).
  • sdAb single domain antibody fragment
  • scFv single chain antibody
  • humanized antibody or bispecific antibody Harlow et al, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989
  • the ligand binding domain of a chimeric receptor composition of the invention comprises an antibody fragment.
  • the chimeric receptor comprises an antibody fragment that comprises a scFv.
  • the precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat” numbering scheme), Al-Lazikani et al, (1997) JMB 273,927-948 ("Chothia” numbering scheme), or a combination thereof.
  • the invention also relates to an isolated and/or substantially purified monospecific Treg cell population as defined hereinabove.
  • one object of the invention is an isolated and/or substantially purified monospecific Treg cell population, wherein the cells of the population comprise a chimeric receptor, wherein said chimeric receptor recognizes an autoantibody on a B cell, and wherein said Treg cells are cytotoxic for B cells, preferably are cytotoxic for B cells expressing at their surface the autoantibody recognized by the chimeric receptor.
  • isolated population refers to a cell population that is removed from its natural environment (such as the peripheral blood) and that is isolated, purified or separated, and is at least about 75% free, 80% free, 85% free and preferably about 90%, 95%, 96%, 97%, 98%, 99% free, from other cells with which it is naturally present, but which lack the cell surface markers based on which the cells were isolated.
  • the isolated monospecific Treg cell population of the invention has been frozen and thawed.
  • Another object of the invention is a vector comprising the nucleic acid sequence encoding a chimeric receptor (CR) as described hereinabove.
  • vectors that may be used in the present invention include, but are not limited to, a DNA vector, a RNA vector, a plasmid, a phagemid, a phage derivative, an animal virus and a cosmid.
  • Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO01/29058; and US6,326, 193 incorporated herein by reference).
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • promoter elements e.g. , enhancers
  • promoters regulate the frequency of transcriptional initiation.
  • these are located in the region 30-1 10 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or independently to activate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • EF-la Elongation Growth Factor - la
  • other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • inducible promoters are also contemplated as part of the invention.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked to when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • bi-directional promoters allowing efficient and coordinate expression of two or more genes may also be of interest in the present invention.
  • bi-directional promoters include but are not limited to the promoters described by Luigi Naldini in US2006200869, incorporated herein by reference, disclosing a bi-directional promoter comprising i) a first minimal promoter sequence derived from cytomegalovirus (CMV) or mouse mammary tumor virus (MMTV) genomes and ii) a full efficient promoter sequence derived from an animal gene.
  • CMV cytomegalovirus
  • MMTV mouse mammary tumor virus
  • the expression vector to be introduced into a T cell can also contain either a selectable marker gene such as CD34 or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • a selectable marker gene such as CD34 or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-trans fection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
  • suicide gene technology may be used. Different suicide gene technologies are described in the art depending on their mechanism of action (Jones et al. Frontiers in Pharmacology, 2014 (5): 254). Examples of gene-directed enzyme prodrug therapy (GDEPT) converting a nontoxic drug to a toxic drug include herpes simplex virus thymidine kinase (HSV-TK) and cytosine deaminase (CD). Other examples are chimeric proteins composed of a drug binding domain linked to apoptotic components such as for example the inducible Fas (iFas) or the inducible Caspase 9 (iCasp9) systems.
  • GDEPT gene-directed enzyme prodrug therapy
  • HSV-TK herpes simplex virus thymidine kinase
  • CD cytosine deaminase
  • Other examples are chimeric proteins composed of a drug binding domain linked to apoptotic components such as for example the inducible Fas (iFas)
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al, 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, US5, 350,674 and 5,585,362.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • an exemplary delivery vehicle is a liposome.
  • lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Lipids suitable for use can be obtained from commercial sources.
  • DMPC dimyristyl phosphatidylcholine
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al, 1991 Glycobiology 5: 505-10).
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine-nucleic acid complexes are also contemplated.
  • assays include, for example, "molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT- PCR and PCR; "biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • moleukin assays well known to those of skill in the art, such as Southern and Northern blotting, RT- PCR and PCR
  • biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • the genetically modified T cells of the invention are modified through the introduction of RNA.
  • an in vitro transcribed RNA CR can be introduced to a cell as a form of transient transfection.
  • the RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template.
  • DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase.
  • the source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.
  • the desired template for in vitro transcription is the CR of the present invention.
  • the DNA to be used for PCR contains an open reading frame.
  • the DNA can be from a naturally occurring DNA sequence from the genome of an organism.
  • the DNA is a full length gene of interest or a portion of a gene.
  • the gene can include some or all of the 5' and/or 3' untranslated regions (UTRs).
  • the gene can include exons and introns.
  • the DNA to be used for PCR is a human gene.
  • the DNA to be used for PCR is a human gene including the 5' and 3' UTRs.
  • the DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism.
  • An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.
  • PCR is used to generate a template for in vitro transcription of mRNA which is used for transfection.
  • Methods for performing PCR are well known in the art.
  • Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR.
  • “Substantially complementary”, as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR.
  • the primers can be designed to be substantially complementary to any portion of the DNA template.
  • the primers can be designed to amplify the portion of a gene that is normally transcribed in cells (the open reading frame), including 5' and 3' UTRs.
  • the primers can also be designed to amplify a portion of a gene that encodes a particular domain of interest.
  • the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5' and 3' UTRs. Primers useful for PCR are generated by synthetic methods that are well known in the art.
  • Forward primers are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified.
  • Upstream is used herein to refer to a location 5', to the DNA sequence to be amplified relative to the coding strand.
  • reverse primers are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified.
  • Downstream is used herein to refer to a location 3' to the DNA sequence to be amplified relative to the coding strand. Any DNA polymerase useful for PCR can be used in the methods disclosed herein. The reagents and polymerase are commercially available from a number of sources.
  • the RNA preferably has 5' and 3' UTRs.
  • the 5' UTR is between zero and 3000 nucleotides in length.
  • the length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.
  • the 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest.
  • UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
  • the use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA.
  • AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
  • the 5' UTR can contain the Kozak sequence of the endogenous gene.
  • a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence.
  • Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art.
  • the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells.
  • various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the mRNA.
  • a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed.
  • the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
  • the promoter is a T7 polymerase promoter, as described elsewhere herein.
  • Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
  • the mRNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
  • RNA polymerase produces a long concatemeric product which is not suitable for expression in eukaryotic cells.
  • the transcription of plasmid DNA linearized at the end of the 3' UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.
  • phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003).
  • the polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100T tail (size can be 50-5000 T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination.
  • Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.
  • Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP).
  • E-PAP E. coli polyA polymerase
  • increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA.
  • the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds.
  • ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
  • RNAs produced by the methods disclosed herein include a 5' cap.
  • the 5' cap is provided using techniques known in the art and described herein (Cougot, et al, Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al, RNA, 7: 1468-95 (2001); Elango, et al, Biochim. Biophys. Res. Commun., 330:958-966 (2005)).
  • RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence.
  • IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
  • RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as "gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).
  • the CR sequences are delivered into cells using a retroviral or lentiviral vector.
  • CR-expressing retroviral and lentiviral vectors can be delivered into different types of eukaryotic cells as well as into tissues and whole organisms using transduced cells as carriers or cell-free local or systemic delivery of encapsulated, bound or naked vectors.
  • the method used can be for any purpose where stable expression is required or sufficient.
  • the CR sequences are delivered into cells using in vitro transcribed mRNA.
  • In vitro transcribed mRNA CR can be delivered into different types of eukaryotic cells as well as into tissues and whole organisms using transfected cells as carriers or cell-free local or systemic delivery of encapsulated, bound or naked mRNA.
  • the method used can be for any purpose where transient expression is required or sufficient.
  • the desired CR can be expressed in the cells by way of transposons.
  • T cells Prior to expansion and genetic modification of the T cells of the invention, a source of T cells is obtained from a subject.
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • any number of T cell lines available in the art may be used.
  • T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FicollTM separation.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations.
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca 2+ -free, Mg 2+ -free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • buffers such as, for example, Ca 2+ -free, Mg 2+ -free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient or by counterflow centrifugal elutriation.
  • a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.
  • T cells are isolated by incubation with anti- CD3/anti-CD28 (i.e., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes.
  • the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In one preferred embodiment, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immune-compromised individuals.
  • TIL tumor infiltrating lymphocytes
  • subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process.
  • subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.
  • the skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention. In another embodiment, it may be desirable to perform the selection procedure and use the "unselected" cells in the activation and expansion process.
  • Unselected cells can also be subjected to further rounds of selection. Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD l ib, CD 16, HLA-DR, and CD8.
  • T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.
  • the concentration of cells and surface can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/ml is used. In a further embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (i.e. , leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain.
  • target antigens of interest such as CD28-negative T cells
  • T cells for stimulation can also be frozen after a washing step.
  • the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
  • the cells may be suspended in a freezing solution.
  • one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.
  • cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation.
  • a blood sample or an apheresis product is taken from a generally healthy subject.
  • a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use.
  • the T cells may be expanded, frozen, and used at a later time.
  • the T cells can be activated and expanded generally using methods as described, for example, in US6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and US20060121005, incorporated herein by reference.
  • the monospecific Treg cell population of the invention is expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the Treg cells of the monospecific Treg cell population.
  • the monospecific Treg cell population populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
  • a protein kinase C activator e.g., bryostatin
  • a ligand that binds the accessory molecule is used for co-stimulation of an accessory molecule on the surface of the T cells.
  • a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
  • an anti-CD3 antibody and an anti- CD28 antibody may be used.
  • an anti-CD28 antibody include, without being limited to, 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France).
  • Other expansion methods commonly known in the art can be used (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al, J. Exp. Med. 190(9): 13191328, 1999; Garland et al, J. Immunol Meth. 227(l-2):53-63, 1999).
  • the primary stimulatory signal and the co-stimulatory signal for the Treg cells of the monospecific Treg cell population may be provided by different protocols.
  • the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in "cis” formation) or to separate surfaces (i.e., in "trans” formation).
  • one agent may be coupled to a surface and the other agent in solution.
  • the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution.
  • the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents.
  • a surface such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents.
  • aAPCs artificial antigen presenting cells
  • the two agents are immobilized on beads, either on the same bead, i.e., "cis," or to separate beads, i.e., "trans.”
  • the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the co-stimulatory signal is an anti-CD28 antibody or antigen- binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts.
  • a 1 : 1 ratio of each antibody bound to the beads for CD4 + T cell expansion and T cell growth is used.
  • a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1 : 1. In one particular embodiment an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1 : 1. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100: 1 to 1 : 100 and all integer values there between. In one aspect of the present invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments of the invention, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2:1.
  • a 1 : 100 CD3:CD28 ratio of antibody bound to beads is used.
  • a 1 :75 CD3:CD28 ratio of antibody bound to beads is used.
  • a 1 :50 CD3:CD28 ratio of antibody bound to beads is used.
  • a 1 :30 CD3:CD28 ratio of antibody bound to beads is used.
  • a 1 :10 CD3:CD28 ratio of antibody bound to beads is used.
  • a 1 :3 CD3:CD28 ratio of antibody bound to the beads is used.
  • a 3: 1 CD3:CD28 ratio of antibody bound to the beads is used.
  • Ratios of particles to cells from 1 :500 to 500: 1 and any integer values in between may be used to stimulate T cells or other target cells.
  • the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many.
  • the ratio of cells to particles ranges from 1 : 100 to 100: 1 and any integer values in-between and in further embodiments the ratio comprises 1 :9 to 9: 1 and any integer values in between, can also be used to stimulate T cells.
  • the ratio of anti-CD3- and anti-CD28-coupled particles to T cells that result in T cell stimulation can vary as noted above, however certain preferred values include 1 : 100, 1 :50, 1 :40, 1 :30, 1 :20, 1 : 10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 : 1, 2: 1, 3:1, 4: 1, 5: 1, 6:1, 7: 1, 8: 1, 9: 1, 10: 1, and 15: 1 with one preferred ratio being at least 1 :1 particles per T cell.
  • a ratio of particles to cells of 1 : 1 or less is used.
  • a preferred particle: cell ratio is 1 :5.
  • the ratio of particles to cells can be varied depending on the day of stimulation.
  • the ratio of particles to cells is from 1 : 1 to 10:1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1 : 1 to 1 : 10 (based on cell counts on the day of addition).
  • the ratio of particles to cells is 1 : 1 on the first day of stimulation and adjusted to 1 :5 on the third and fifth days of stimulation.
  • particles are added on a daily or every other day basis to a final ratio of 1 : 1 on the first day, and 1 :5 on the third and fifth days of stimulation.
  • the ratio of particles to cells is 2: 1 on the first day of stimulation and adjusted to 1 :10 on the third and fifth days of stimulation.
  • particles are added on a daily or every other day basis to a final ratio of 1 : 1 on the first day, and 1 :10 on the third and fifth days of stimulation.
  • ratios will vary depending on particle size and on cell size and type.
  • the Treg cells are combined with agent- coated beads, the beads and the cells are subsequently separated, and then the cells are cultured.
  • the agent-coated beads and cells prior to culture, are not separated but are cultured together.
  • the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.
  • cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3x28 beads) to contact the Treg cells of the monospecific Treg cell population.
  • the cells for example, 10 4 to 10 9 T cells
  • beads for example, DYNABEADS® M-450 CD3/CD28 T paramagnetic beads at a ratio of 1 : 1
  • PBS without divalent cations such as, calcium and magnesium
  • the target cell may be very rare in the sample and comprise only 0.01% of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the context of the present invention.
  • a concentration of about 2 billion cells/ml is used.
  • greater than 100 million cells/ml is used.
  • a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such populations of cells may have therapeutic value and would be desirable to obtain in certain embodiments.
  • the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another embodiment, the mixture may be cultured for 21 days.
  • the beads and the T cells are cultured together for about eight days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired such that culture time of T cells can be 60 days or more.
  • Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN- ⁇ , IL-4, IL-7, GM-CSF, IL- 10, IL-12, IL- 15, ⁇ , and TNF-a or any other additives for the growth of cells known to the skilled artisan.
  • serum e.g., fetal bovine or human serum
  • IL-2 interleukin-2
  • insulin IFN- ⁇
  • IL-4 interleukin-7
  • GM-CSF GM-CSF
  • additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.
  • Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F- 12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum- free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells.
  • Antibiotics e.g.
  • penicillin and streptomycin are included only in experimental cultures, not in cultures of cells that are to be infused into a subject.
  • the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g. , 37° C) and atmosphere (e.g. , air plus 5% CO2).
  • T cells that have been exposed to varied stimulation times may exhibit different characteristics.
  • typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (Th, CD4 + ) that is greater than the cytotoxic or suppressor T cell population (Tc, CD8 + ).
  • Th, CD4 + helper T cell population
  • Tc, CD8 + cytotoxic or suppressor T cell population
  • Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of Th cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of Tc cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of Th cells may be advantageous.
  • the T cells may be cultured in the presence of rapamycin in order to obtain regulatory T cells, as described for example in WO20071 10785 incorporated herein by reference.
  • T cells are cultured with a T cell receptor (TCR)/CD3 activator such as for example TCR/CD3 antibodies, a TCR co-stimulator activator such as for example CD28, CD 137 (4-1 BB), GITR, B7-1/2, CD5, ICOS, OX40, CD40 or CD137 antibodies, and rapamycin.
  • TCR T cell receptor
  • the T cells genetically modified by expression of the CR may also have been genetically modified by expression of at least one intracellular factor such as ROR-C, Foxp3, Foxol , T-bet, or Gata 3, c-Maf, AhR.
  • the genetically modified immune cell of the invention expresses Foxp3.
  • the genetically modified immune cell of the invention express Foxol .
  • the genetically modified Treg cells of the monospecific Treg cell population can be an allogeneic Treg cell.
  • the allogeneic Treg cell can be a T cell lacking expression of a functional human leukocyte antigen (HLA), e.g. , HLA class I and/or HLA class II.
  • HLA human leukocyte antigen
  • a Treg cell described herein can be engineered such that it does not express a functional HLA on its surface.
  • a Treg cell described herein can be engineered such that cell surface expression HLA, e.g. , HLA class 1 and/or HLA class II or non-classical HLA molecules is downregulated.
  • Modified Treg cells that lack expression of a functional HLA can be obtained by any suitable means, including a knock out or knock down of one or more subunit of HLA.
  • the Treg cell can include a knock down of HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR) transcription-activator like effector nuclease (TALEN), zinc finger endonuclease (ZFN), meganuclease (mn, also known as homing endonuclease), or megaTAL (combining a TAL effector with a mn cleavage domain).
  • CRISPR clustered regularly interspaced short palindromic repeats
  • TALEN transcription-activator like effector nuclease
  • ZFN zinc finger endonuclease
  • mn meganuclease
  • megaTAL combining a TAL effector with a mn cleavage domain
  • HLA expression can be inhibited using siRNA or shRNA that target a nucleic acid encoding a HLA in a T cell.
  • siRNA and shRNAs in T cells can be achieved using any conventional expression system, e.g., such as a lentiviral expression system.
  • Exemplary siR A and shRNA that downregulate expression of HLA class I and/or HLA class II genes are described, e.g. , in US2007/0036773.
  • CRISPR or “CRISPR to HLA” or “CRISPR to inhibit HLA” as used herein refers to a set of clustered regularly interspaced short palindromic repeats, or a system comprising such a set of repeats.
  • Cas refers to a CRISPR-associated protein.
  • a “CRISPR/Cas” system refers to a system derived from CRISPR and Cas which can be used to silence or mutate a HLA gene.
  • CRISPR/Cas systems are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. (2007) Science 315 : 1709-1712; Marragini et al. (2008) Science 322: 1843-1845. The CRISPR/Cas system has been modified for use in gene editing (silencing, enhancing or changing specific genes) in eukaryotes such as mice or primates. Wiedenheft et al.
  • the CRISPR sequence sometimes called a CRISPR locus, comprises alternating repeats and spacers.
  • the spacers usually comprise sequences foreign to the bacterium such as a plasmid or phage sequence; in the HLA CRISPR/Cas system, the spacers are derived from the HLA gene sequence.
  • RNA from the CRISPR locus is constitutively expressed and processed by Cas proteins into small RNAs. These comprise a spacer flanked by a repeat sequence.
  • the RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Horvath et al. (2010) Science 327: 167-170; Makarova et al. (2006) Biology Direct 1 : 7. The spacers thus serve as templates for RNA molecules, analogously to siRNAs. Pennisi (2013) Science 341 : 833-836.
  • the CRISPR/Cas system can thus be used to edit a HLA gene (adding or deleting a basepair), or introducing a premature stop which thus decreases expression of a HLA.
  • the CRISPR/Cas system can alternatively be used like RNA interference, turning off HLA gene in a reversible fashion.
  • the RNA can guide the Cas protein to a HLA promoter, sterically blocking RNA polymerases.
  • TALEN or "TALEN to HLA” or “TALEN to inhibit HLA” refers to a transcription activator- like effector nuclease, an artificial nuclease which can be used to edit the HLA gene.
  • TALENs are produced artificially by fusing a TAL effector DNA binding domain to a DNA cleavage domain.
  • Transcription activator-like effectors can be engineered to bind any desired DNA sequence, including a portion of the HLA gene. By combining an engineered TALE with a DNA cleavage domain, a restriction enzyme can be produced which is specific to any desired DNA sequence, including a HLA sequence. These can then be introduced into a cell, wherein they can be used for genome editing.
  • Boch (201 1) Nature Biotech. 29: 135-6; and Boch et al. (2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501.
  • TALEs are proteins secreted by Xanthomonas bacteria.
  • the DNA binding domain contains a repeated, highly conserved 33-34 amino acid sequence, with the exception of the 12th and 13th amino acids. These two positions are highly variable, showing a strong correlation with specific nucleotide recognition. They can thus be engineered to bind to a desired DNA sequence.
  • N nuclease
  • Several mutations to Fokl have been made for its use in TALENs; these, for example, improve cleavage specificity or activity. Cermak et al. (201 1) Nucl. Acids Res.
  • a HLA TALEN can be used inside a cell to produce a double- stranded break (DSB).
  • a mutation can be introduced at the break site if the repair mechanisms improperly repair the break via non-homologous end joining. For example, improper repair may introduce a frame shift mutation.
  • foreign DNA can be introduced into the cell along with the TALEN; depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to correct a defect in the HLA gene or introduce such a defect into a wt HLA gene, thus decreasing expression of HLA.
  • TALENs specific to sequences in HLA can be constructed using any method known in the art, including various schemes using modular components. Zhang et al. (2011) Nature Biotech. 29: 149-53; Geibler et al. (2011) PLoS ONE 6: el9509.
  • ZFN Zinc Finger Nuclease
  • ZFN to HLA or “ZFN to inhibit HLA” refer to a zinc finger nuclease, an artificial nuclease which can be used to edit the HLA gene.
  • a ZFN comprises a Fokl nuclease domain (or derivative thereof) fused to a DNA-binding domain.
  • the DNA-binding domain comprises one or more zinc fingers.
  • a zinc finger is a small protein structural motif stabilized by one or more zinc ions.
  • a zinc finger can comprise, for example, Cys 2 His 2 , and can recognize an approximately 3-bp sequence.
  • Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides which recognize about 6, 9, 12, 15 or 18-bp sequences.
  • selection and modular assembly techniques are available to generate zinc fingers (and combinations thereof) recognizing specific sequences, including phage display, yeast one -hybrid systems, bacterial one - hybrid and two-hybrid systems, and mammalian cells.
  • a ZFN Like a TALEN, a ZFN must dimerize to cleave DNA. Thus, a pair of ZFNs are required to target non-palindromic DNA sites. The two individual ZFNs must bind opposite strands of the DNA with their nucleases properly spaced apart. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10570-5. Also like a TALEN, a ZFN can create a double- stranded break in the DNA, which can create a frame-shift mutation if improperly repaired, leading to a decrease in the expression and amount of HLA in a cell. ZFNs can also be used with homologous recombination to mutate in the HLA gene.
  • ZFNs specific to sequences in HLA can be constructed using any method known in the art. See, e.g., Provasi (2011) Nature Med. 18: 807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al. (2008) Mol. Ther. 16: 1200-7; Quo et al. (2010) /. Mol. Biol. 400: 96; US2011/0158957; and US2012/0060230.
  • Meganuclease or “meganuclease to HLA” or “meganuclease to inhibit HLA” refers to a monomeric endonuclease with large (>14 base pairs) recognition sites, which can be used to edit the HLA gene.
  • Meganucleases mn are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases and engineered for a unique target site.
  • Homing endonucleases are DNA-cleaving enzymes that can generate double strand breaks at individual loci in their host genomes, and thereby drive site-specific gene conversion events. (Stoddard, Structure. 2011 Jan 12;19(1):7-15).
  • homing endonucleases recognize long DNA sequences (typically 20 to 30 base pairs). Homing endonucleases are extremely widespread and are found in microbes, as well as in phages and viruses.
  • the LAGLIDADG and His-Cys box enzymes (which are the most sequence-specific of these enzymes) rely upon antiparallel ⁇ -sheets that dock into the major grooves of their DNA target sites (Flick et al., 1998; Jurica et al., 1998). There they establish a collection of sequence-specific and non-specific contacts that are distributed nonuniformly across multiple consecutive basepairs (Chevalier et al., 2003; Scalley-Kim et al, 2007).
  • LAGLIDADG homing endonuclease (LHE) family is the primary source of the engineered enzymes used for gene targeting applications.
  • the LHE family is primarily encoded within archaea and in the chloroplast and mitochondrial genomes of algae and fungi (Chevalier et al., 2005; Dalgaard et al., 1997; Sethuraman et al., 2009).
  • Meganucleases that possess a single conserved LAGLIDADG motif (SEQ ID NO: 43) per protein chain form homodimeric proteins that cleave palindromic and nearly palindromic DNA target sequences, while those that contain two such motifs per protein chain form larger, pseudo-symmetric monomers that can target completely asymmetric DNA sequences.
  • Meganucleases can be engineered to target HLA and thus create a double-stranded break in the DNA, which can create a frame-shift mutation if improperly repaired, leading to a decrease in the expression and amount of HLA in a cell.
  • MegaTAL or “megaTAL to HLA” or “ megaTAL to inhibit HLA” refers to an artificial nuclease, which can be used to edit the HLA gene.
  • MegaTALs are hybrid monomeric nucleases obtained through the fusion of minimal TAL (transcription activator-like) effector domains to the N-terminus of meganucleases derived from the LAGLIDADG homing endonuclease family (Nucleic Acids Res. 2014 Feb;42(4):2591-601 ; Takeuchi et al, Methods Mol Biol. 2015; 1239: 105-32. doi: 10.1007/978-1-4939-1862-1 6).
  • MegaTALs thus consist of a site-specific meganuclease cleavage head with additional affinity and specificity provided by a TAL effector DNA binding domain. MegaTALs can be engineered to target HLA and thus create a double-stranded break in the DNA, which can create a frame-shift mutation if improperly repaired, leading to a decrease in the expression and amount of HLA in a cell.
  • a therapeutic T cell has short term persistence in a patient, due to shortened telomeres in the T cell; accordingly, transfection with a telomerase gene can lengthen the telomeres of the T cell and improve persistence of the T cell in the patient.
  • the genetically modified T cell ectopically expresses a telomerase subunit, e.g. , the catalytic subunit of telomerase, e.g., TERT, e.g., hTERT.
  • this disclosure provides a method of producing a CR-expressing cell, comprising contacting a cell with a nucleic acid encoding a telomerase subunit, e.g., the catalytic subunit of telomerase, e.g. , TERT, e.g., hTERT.
  • the cell may be contacted with the nucleic acid before, simultaneous with, or after being contacted with a construct encoding a CR.
  • Another object of the invention is a composition comprising, consisting or consisting essentially of at least one monospecific Treg cell population of the invention.
  • said composition comprises, consists or consists essentially of an isolated and/or substantially purified monospecific Treg cell population of the invention. In one embodiment, said composition has been frozen and thawed.
  • Another object of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising, consisting or consisting essentially of at least one monospecific Treg cell population as described hereinabove and at least one pharmaceutically acceptable excipient.
  • Another object of the invention is a medicament comprising, consisting or consisting essentially of at least one monospecific Treg cell population as described hereinabove.
  • the pharmaceutical composition or medicament comprises at least one isolated and/or substantially purified monospecific Treg cell population of the invention.
  • the pharmaceutical composition or medicament comprises a combination of monospecific Treg cell populations as described hereinabove ( . e. , at least two distinct populations of monospecific Treg cell populations of the invention).
  • the term "consisting essentially of, with reference to a pharmaceutical composition or medicament, means that the at least one monospecific Treg cell population of the invention is the only one therapeutic agent or agent with a biologic activity within said pharmaceutical composition or medicament.
  • compositions and medicaments may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins polypeptides or amino acids such as glycine
  • antioxidants e.g., chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • compositions described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection.
  • the at least one monospecific Treg cell population of the present invention are administered by i.v. injection.
  • the compositions of the present invention are thus in one embodiment formulated for intravenous administration.
  • the at least one monospecific Treg cell population of the present invention may be injected directly into the site of the autoimmune disease.
  • Another object of the invention is a method for treating an autoimmune disease in a subject in need thereof, preferably an autoantibody-mediated autoimmune disease, wherein said method comprises administering a therapeutically effective amount of at least one monospecific Treg cell population as described hereinabove.
  • the present invention thus relates to at least one monospecific Treg cell population as described hereinabove (preferably in a composition, pharmaceutical composition or medicament as described hereinabove), for treating or for use in the treatment of an autoimmune disease, preferably an autoantibody-mediated autoimmune disease.
  • the cells of the monospecific Treg cell population of the invention express a chimeric receptor comprising the autoantigen involved in the autoimmune disease, i.e., recognizing the autoantibody involved in the autoantibody-mediated autoimmune disease to be treated.
  • autoantibody-mediated autoimmune diseases include but are not limited to, bullous pemphigoid, lupus (including systemic lupus erythematosus (SLE), lupus nephritis (LN), discoid lupus, lupus erythematosus profundus, Chilbrain lupus erythematosus, tumidus lupus erythematosus, severe systemic lupus erythematosus, acute cutaneous lupus, chronic cutaneous lupus), multiple sclerosis, neuromyelitis optica (NMO), autoimmune limbic encephalitis (LE), Hashimoto's disease, N-methyl-D- aspartate receptor (NMDAR) encephalitis, autoimmune hemolytic anemia, pemphigus vulgaris, myasthenia gravis, Graves' disease, idiopathic thrombocytopenic purpura, Goodpasture's syndrome,
  • said autoantibody-mediated autoimmune disease is selected from the group comprising bullous pemphigoid, lupus (including systemic lupus erythematosus (SLE), lupus nephritis (LN), discoid lupus, lupus erythematosus profundus, Chilbrain lupus erythematosus, tumidus lupus erythematosus, severe systemic lupus erythematosus, acute cutaneous lupus, chronic cutaneous lupus) and multiple sclerosis.
  • lupus including systemic lupus erythematosus (SLE), lupus nephritis (LN), discoid lupus, lupus erythematosus profundus, Chilbrain lupus erythematosus, tumidus lupus erythematosus, severe systemic
  • autoantibody-mediated autoimmune diseases include but are not limited to, uveitis, bullous pemphigoid, lupus (including systemic lupus erythematosus (SLE), lupus nephritis (LN), discoid lupus, lupus erythematosus profundus, Chilbrain lupus erythematosus, tumidus lupus erythematosus, severe systemic lupus erythematosus, acute cutaneous lupus, chronic cutaneous lupus), multiple sclerosis, neuromyelitis optica (NMO), autoimmune limbic encephalitis (LE), Hashimoto's disease, N-methyl-D- aspartate receptor (NMDAR) encephalitis, autoimmune hemolytic anemia, pemphigus vulgaris, myasthenia gravis, Graves' disease, idiopathic thrombocytopenic purpura, Good
  • said autoantibody-mediated autoimmune disease is selected from the group comprising bullous pemphigoid, lupus (including systemic lupus erythematosus (SLE), lupus nephritis (LN), discoid lupus, lupus erythematosus profundus, Chilbrain lupus erythematosus, tumidus lupus erythematosus, severe systemic lupus erythematosus, acute cutaneous lupus, chronic cutaneous lupus) and multiple sclerosis.
  • lupus including systemic lupus erythematosus (SLE), lupus nephritis (LN), discoid lupus, lupus erythematosus profundus, Chilbrain lupus erythematosus, tumidus lupus erythematosus, severe systemic
  • the autoantibody-mediated autoimmune disease may be treated by apheresis.
  • diseases that may be treated by apheresis include but are not limited to, systemic lupus erythematosus (severe, nephritis), multiple sclerosis, Guillain- Barre syndrome, Myasthenia gravis, chronic inflammatory demyelinating polyneuropathy, thrombotic thrombocytopenic purpura, hyperviscosity in monoclonal gammopathies, goodpasture syndrome, hemolytic uremic syndrome (atypical, due to antibody to factor H), Wilson disease, fulminant, Lambert-Eaton myasthenic syndrome, RBC alloimmunization in pregnancy, mushroom poisoning, acute disseminated encephalomyelitis, hemolytic uremic syndrome (atypical, due to complement factor mutations), autoimmune hemolytic anemia (life-threatening cold agglutinin disease), myeloma cast ne
  • the autoantibody-mediated autoimmune disease may be treated by plasmapheresis.
  • diseases that may be treated by plasmapheresis include but are not limited to, systemic lupus erythematosus, ABO-incompatible solid organ transplantation (SOT), thrombotic thrombocytopenic purpura, cryoglobulinemia, heparin-induced thrombocytopenia, thyroid storm, chronic inflammatory demyelinating polyradiculoneuropathy, ANCA associated diseases, focal segmental glomerulosclerosis, fulminant hepatic failure, myasthenia gravis, Goodpasture's syndrome, Guillain-Barre Syndrome, autoimmune hemolytic anemia, IgA nephropathy, hemolytic uremic syndrome.
  • the autoantibody-mediated autoimmune disease may be treated by rituximab.
  • autoantibody-mediated autoimmune diseases include, but are not limited to, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis, Wegener granulomatosis, microscopic polyangiitis, immune thrombocytopenic purpura (ITP), pemphigus vulgaris, sicca syndrome (Sjogren), glomerulonephritis, myasthenia gravis, liver transplant rejection, idiopathic thrombocytopenic purpura (immune thrombocytopenic purpura), kidney transplant rejection, hemophilia A, neuromyelitis optica (Devic's syndrome), pemphigus vulgaris, and thrombotic thrombocytopenic purpura.
  • the autoimmune disease is neuromyelitic optica (NMO)
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with NMO.
  • autoantigens associated with neuromyelitis optica include, but are not limited to, aquaporin-4 water channel (AQP4).
  • the autoimmune disease is LE (lupus erythematosus), and the chimeric receptor comprises an autoantigen associated with LE.
  • autoantigens associated with LE include, but are not limited to, Hu, Ma2, collapsin response-mediator protein 5 (CRMP5), voltage-gated potassium channel (VGKC), N- methyl-d-aspartate receptor (NMDAR), and a-amino-3-hydroxy-5-methyl-4- isoxazoleproprionic acid (AMPAR).
  • the autoimmune disease is SLE (systemic lupus erythematosus)/ LN (lupus nephritis), and the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with SLE / LN.
  • autoantigens associated with SLE / LN include, but are not limited to, DNA, histone, ribosomes, and RNP.
  • the autoimmune disease is acute cutaneous lupus erythematosus (ACLE)
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with acute cutaneous lupus erythematosus.
  • autoantigens associated with acute cutaneous lupus erythematosus include, but are not limited to, DNA, and RNP.
  • the autoimmune disease is chronic cutaneous lupus erythematosus
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with chronic cutaneous lupus erythematosus.
  • autoantigens associated with chronic cutaneous lupus erythematosus include, but are not limited to, RNP.
  • the autoimmune disease is discoid lupus erythematosus/lupus erythematosus profundus/ chilblain lupus erythematosus/tumidus lupus erythematosus nephropathy
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with discoid lupus erythematosus/lupus erythematosus profundus/ chilblain lupus erythematosus/tumidus lupus erythematosus nephropathy.
  • Examples of autoantigens associated with discoid lupus erythematosus/lupus erythematosus profundus/ chilblain lupus erythematosus/tumidus lupus erythematosus nephropathy include, but are not limited to, ANA.
  • the autoimmune disease is Hashimoto's disease
  • the chimeric receptor comprises an autoantigen associated with Hashimoto's disease.
  • autoantigens associated with Hashimoto's disease include, but are not limited to, thyroid peroxidase, and thyroglobulin.
  • the autoimmune disease is NMDAR encephalitis
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with NMDAR encephalitis.
  • autoantigens associated with NMDAR encephalitis include, but are not limited to, anti-N-methyl-D-aspartate receptor (NR1 subunit).
  • the autoimmune disease is autoimmune hemolytic anemia
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with autoimmune hemolytic anemia.
  • autoantigens associated with autoimmune hemolytic anemia include, but are not limited to, Rh blood group antigens, and I antigen.
  • the autoimmune disease is pemphigus vulgaris
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with pemphigus vulgaris.
  • autoantigens associated with pemphigus vulgaris include, but are not limited to, Dsgl/3.
  • the autoimmune disease is bullous pemphigoid
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with bullous pemphigoid.
  • autoantigens associated with bullous pemphigoid include, but are not limited to, BP 180, and BP230.
  • the autoimmune disease is Myasthenia Gravis
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with Myasthenia Gravis.
  • autoantigens associated with myasthenia gravis include, but are not limited to, acetylcholine nicotinic postsynaptic receptors.
  • the autoimmune disease is Graves' disease
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with Graves' disease.
  • autoantigens associated with Graves' disease include but are not limited to, thyrotropin receptors.
  • the autoimmune disease is idiopathic thrombocytopenic purpura (ITP)
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with idiopathic thrombocytopenic purpura (ITP).
  • autoantigens associated with idiopathic thrombocytopenic purpura include, but are not limited to, Platelet integrin, and GpIIb:IIIa.
  • the autoimmune disease is Goodpasture's syndrome
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with Goodpasture's syndrome.
  • autoantigens associated with Goodpasture's syndrome include, but are not limited to, Collagen alpha-3(IV) chain.
  • the autoimmune disease is rheumatoid arthritis
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with rheumatoid arthritis.
  • autoantigens associated with rheumatoid arthritis include, but are not limited to, Rheumatoid factor, and calpastatin.
  • the autoimmune disease is juvenile idiopathic arthritis
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with juvenile idiopathic arthritis.
  • autoantigens associated with juvenile idiopathic arthritis include, but are not limited to, RF, citrullinated proteins.
  • the autoimmune disease is multiple sclerosis
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with multiple sclerosis.
  • autoantigens associated with multiple sclerosis include, but are not limited to, Myelin basic protein (MBP), Myelin oligodendrocyte glycoprotein (MOG) peptides, and alpha-beta-crystallin.
  • the autoimmune disease is celiac disease
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with celiac disease.
  • autoantigens associated with celiac disease include, but are not limited to, tissue transglutaminase (TG2).
  • the autoimmune disease is pernicious anemia
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with pernicious anemia.
  • autoantigens associated with pernicious anemia include, but are not limited to, intrinsic factor of gastric parietal cells.
  • the autoimmune disease is vitiligo
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with vitiligo.
  • autoantigens associated with vitiligo include, but are not limited to, 65-kDa antigen.
  • the autoimmune disease is Behcet's disease
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with Behcet's disease.
  • autoantigens associated with Behcet's disease include, but are not limited to, phosphatidylserine, ribosomal phosphoproteins, and anti-neutrophil cytoplasmic antibody.
  • the autoimmune disease is scleroderma
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with scleroderma.
  • autoantigens associated with scleroderma include, but are not limited to, Scl- 70, U1-RNP.
  • the autoimmune disease is psoriasis
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with psoriasis.
  • autoantigens associated with psoriasis include, but are not limited to, calpastatin.
  • the autoimmune disease is ulcerative colitis (UC) and Crohn's disease
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with UC and Crohn's disease.
  • autoantigens associated with UC and Crohn's disease include, but are not limited to, ANA.
  • the autoimmune disease is Sjogren's syndrome
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with Sjogren's syndrome.
  • autoantigens associated with Sjogren's syndrome include, but are not limited to, SSA and anti-SSB.
  • the autoimmune disease is Wegener's granulomatosis
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with Wegener's granulomatosis.
  • autoantigens associated with Wegener's granulomatosis include, but are not limited to, ANA, and ANCA.
  • the autoimmune disease is polymyositis or dermatomyositis
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with polymyositis or dermatomyositis.
  • autoantigens associated with polymyositis or dermatomyositis include, but are not limited to, Jo-1.
  • the autoimmune disease is primary biliary cirrhosis
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with primary biliary cirrhosis.
  • autoantigens associated with primary biliary cirrhosis include, but are not limited to, anti-mitochondrial antibodies, gp210, p62, sp 100.
  • the autoimmune disease is antiphospholipid syndrome (APS), and the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with antiphospholipid syndrome.
  • autoantigens associated with antiphospholipid syndrome include, but are not limited to, anti-phospholipid antibodies.
  • the autoimmune disease is mixed connective tissue disease (MCTD), and the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with mixed connective tissue disease. Examples of autoantigens associated with mixed connective tissue disease include, but are not limited to, Ul-RNP, Ul-70 kd snRNP.
  • the autoimmune disease is Miller Fisher syndrome
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with Miller Fisher syndrome.
  • autoantigens associated with Miller Fisher syndrome include, but are not limited to, GQlb ganglioside.
  • the autoimmune disease is Guillain-Barre syndrome
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with Guillain-Barre syndrome.
  • autoantigens associated with Guillain-Barre syndrome include, but are not limited to, GM1, asialo GM1, and GDlb.
  • the autoimmune disease is acute motor axonal neuropathy
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with acute motor axonal neuropathy.
  • autoantigens associated with acute motor axonal neuropathy include, but are not limited to, GM1.
  • the autoimmune disease is autoimmune hepatitis
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with autoimmune hepatitis.
  • autoantigens associated with autoimmune hepatitis include, but are not limited to, antinuclear antibodies (ANA) and anti-smooth muscle antibodies (ASMA), anti-liver-kidney microsome- 1 antibodies (ALKM-1) and anti-liver cytosol antibody- 1 (ALC-1).
  • the autoimmune disease is dermatitis herpetiformis
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with dermatitis herpetiformis.
  • autoantigens associated with dermatitis herpetiformis include, but are not limited to, IgA anti-endomysial antibodies.
  • the autoimmune disease is Churg-Strauss syndrome
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with Churg-Strauss syndrome.
  • autoantigens associated with Churg-Strauss syndrome include, but are not limited to, anti-neutrophil cytoplasm antibodies (ANCAs).
  • the autoimmune disease is microscopic polyangiitis
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with microscopic polyangiitis.
  • autoantigens associated with microscopic polyangiitis include, but are not limited to, ANCAs.
  • the autoimmune disease is ANCA vasculitis
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with ANCA vasculitis.
  • autoantigens associated with ANCA vasculitis include, but are not limited to, neutrophil granule proteins.
  • the autoimmune disease is acute rheumatic fever
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with acute rheumatic fever.
  • autoantigens associated with acute rheumatic fever include, but are not limited to, streptococcal cell wall antigen.
  • the autoimmune disease is type 1 Diabetes (TID)
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with TID.
  • autoantigens associated with TID include, but are not limited to, insulin (IAA), glutamic acid decarboxylase (GAA or GAD) and protein tyrosine phosphatase (IA2 or ICA512).
  • the autoimmune disease is membranous nephropathy
  • the chimeric receptor comprises an autoantigen (or a variant or fragment thereof) associated with membranous nephropathy.
  • autoantigens associated with membranous nephropathy include, but are not limited to, PLA2R1 and THSD7A1.
  • the subject receives an initial administration of the monospecific Treg cell population of the invention, and one or more subsequent administrations, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration.
  • the amount of Treg cells of the at least one monospecific Treg cell population of the invention administered to the subject ranges from about 10 2 to about 10 9 , from about 10 3 to about 10 8 , from about 10 4 to about 10 7 , or from about 10 5 to about
  • the amount of Treg cells of the at least one monospecific Treg cell population of the invention administrated to the subject ranges from about 10 6 to about 10 9 , from about 10 6 to 10 7 , from about 10 6 to 10 8 , from about 10 7 to 10 9 , from about
  • the amount of Treg cells of the at least one monospecific Treg cell population of the invention administrated to the subject is about 10 6 , about 10 7 , about 10 8 , or is about 10 9 .
  • the amount of Treg cells of the at least one monospecific Treg cell population of the invention administered to the subject ranges from about 10 4 to 10 9 cells/kg body weight or 10 5 to 10 8 cells/kg body weight, including all integer values within those ranges.
  • more than one administration of the at least one monospecific Treg cell population of the invention are administered to the subject (e.g., human) per week, e.g. , 2, 3, or 4 administrations of the genetically modified CR Treg cells of the invention are administered per week.
  • Another object of the present invention is an article of manufacture containing materials useful for the treatment of an autoimmune disease.
  • the article of manufacture may comprise a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, pouch, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for treating the autoimmune disease, such as an autoantibody-mediated autoimmune disease, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is a monospecific Treg cell population of the invention.
  • the label or package insert may indicate that the composition is used for treating an autoimmune disease.
  • the article of manufacture, label or package insert may further comprise instructional material for administering the monospecific Treg cell population of the invention to the patient.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as, for example, bacteriostatic water for injection (BWFI), phosphate -buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • kit comprising at least one monospecific Treg cell population of the invention.
  • kit any manufacture (e.g., a package or a container) comprising at least one monospecific Treg cell population of the invention.
  • the kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention.
  • any or all of the kit reagents may be provided within containers that protect them from the external environment, such as in sealed containers.
  • kits may also contain a package insert describing the kit and methods for its use. Kits are also provided that are useful for various purposes (e.g., for treating an autoimmune disease). Kits can be provided which contain the monospecific Treg cell population of the invention. As with the article of manufacture, the kit may comprise a container and a label or package insert on or associated with the container. The container holds a composition comprising at least one monospecific Treg cell population of the invention. Additional containers may be included that contain, e.g. , diluents and buffers. The label or package insert may provide a description of the composition as well as instructions for the intended use.
  • Figure 1 is a scheme depicting the structure and nucleotide sequence of the chimeric receptor.
  • A Schematic of native desmoglein 3 and the desmoglein 3 CR.
  • B The chimeric receptor is composed of the sequence leader of the CD8 (written in small letter) followed by domain 1 to 4 of the extracellular part of the native desmoglein and the STII tag (NWSHPQFEK). This extracellular domain is linked to the signaling sequence via the hinge region (outlined) and the transmembrane region (in framed) of the huCD8.
  • the signaling sequence is composed to the intracellular domain of 4- IBB (written in italics) followed by intracellular 1 ⁇ _ ⁇ )3 ⁇ chain (written in small case letters).
  • Figure 2 is a histogram showing the contact independent suppression of CD4 + effector T cells by monospecific Treg cells expressing a chimeric receptor of the invention (CAAR Treg) or a chimeric receptor directed to CD 19 (CD19-CAR Treg).
  • CAAR Treg monospecific Treg cells expressing a chimeric receptor of the invention
  • CD19-CAR Treg a chimeric receptor directed to CD 19
  • No Act no activation.
  • Beads activation of Treg cells with beads coated with anti-CD3 and anti-CD28
  • NALM6- aDSG mito activation of Treg cells with mitomycin-treated NALM6 cells expressing aDSG.
  • Figure 3 is a combination of graphs showing the specific induction of NALM6-aDSG cells cytolysis by monospecific Treg cells expressing a chimeric receptor of the invention (NALM6 aDSG + CAAR, grey line).
  • NALM6 + CAAR Untransduced (UT) monospecific Treg cells were further used as control (NALM6 + UT, dotted black line and NALM6 aDSG + UT dotted grey line). Results obtained with 3 different clones are shown.
  • NALM6, clone G5 (ATCC® CRL-3273TM) were obtained from the American Type Culture Collection (ATCC) and cultivated in RPMI -1640 supplemented with 10% fetal bovine serum according to the supplier's instructions.
  • Nalm6 cells were modified by lentiviral vectors to stably express a b-cell receptor (BCR) specifically recognizing Desmoglin.
  • BCR b-cell receptor
  • Nalm6 cells were transduced with 2 vectors bearing different selection markers, one transferring CD79a+b, and one transferring a membrane bound anti-Desmoglin antibody derived from the previously described F779 clone (Cho et al 2014; Nat Commun. 2014; 5: 4167).
  • the vectors were generated by gene synthesis. All coding sequences were codon optimized for human gene expression by a proprietary algorithm from Geneart (Life Technologies).
  • SFFV Spleen Focus Forming Virus
  • T2A-site a ribosomal cleavage site "T2A-site” was added, which was followed by a sequence coding for human CD79a.
  • IRS internal ribosomal entry site
  • EMCV encephalomyocarditis virus
  • a 3 rd generation lentiviral transfer vector was similarly equipped with a Spleen Focus Forming Virus (SFFV) promoter to drive the expression of the transgene cassette.
  • the transgene consisted of the F779 heavy chain variable region grafted to the human IgG constant chain, which was anchored in the cell membrane using an IgG2 C-terminus.
  • the variable region of the F779 light chain was consecutively fused via a T2A-site to the C-terminus of the heavy chain and grafted to a human IgG kappa light chain constant domain.
  • an internal ribosomal entry site (IRES) from the encephalomyocarditis virus (EMCV) was added, followed by a Phleomycin resistance gene to allow chemical selection of the transduced cells.
  • Lentiviral vectors were produced using an 293T-based, 3 rd generation packaging system with VSV- G env (http://tronolab.epfl.ch).
  • Nalm6 cells were transduced at an MOI of -0.5 with the CD79a/b-transferring vector followed by a selection using puromycin at 2 ⁇ g/ml. Selected cells were further transduced with the anti-Desmoglin transferring vector at an MOI of -0.5 and selected using Zeocin TM (Life technologies) at 200 ⁇ g/ml obtain Nalm6-aDSG. OVA -specific Treg obtention
  • PBMCs Peripheral blood mononuclear cells
  • Ficoll GE Healthcare, Uppsala, Sweden
  • OVA-specific Treg cells PBMCs were cultured at 37°C and 5% CO2 in the presence of ovalbumin in X-Vivol5 supplemented with recombinant human IL-2 and recombinant human IL-4.
  • a monospecific Treg cell population comprising 0.5xl0 6 human Treg in 1 ml PL24 well was activated with anti-CD3/anti-CD28 coated beads for two days before transduction with lentiviral supernatant expressing a chimeric receptor construct according to the present invention.
  • Tregs were transduced with a chimeric receptor composed of the sequence leader of the CD 8 followed by domain 1 to 4 of the extracellular part of the native desmoglein. This extracellular domain is linked to the signaling sequence via the hinge and the transmembrane region of the huCD8.
  • the signaling sequence is composed to the intracellular domain of 4- IBB followed by intracellular 1 ⁇ 0 ⁇ 3 ⁇ chain ( Figure 1).
  • transduction was carried out by loading 0.5 to 1 xlO 7 Transduction unit (TU) per ml to each well. After 6 hours at 37°C, viral particles were removed by washout. The plates were then incubated at 37°C with 5% CO2. The efficiency of transduction (assessed by GFP expression) and the level of chimeric receptor (CAAR) at cell surface (assessed by AK23 staining) were checked 5 days after transduction.
  • TU Transduction unit
  • Cytolysis was assessed by a simple non-radioactive cell killing assay that is based on FACS analysis combined with dye450-labeled target cells. Briefly, dye450 pre-labeled target cells (5xl0 4 cells in ⁇ ) were co-cultured with Treg cells at various E : T ratios in 96-well U-bottom tissue culture plate (Costar, Cambridge, MA). We used as target cell Nalm6 or Nalm6-aDSG cell lines. After 4h-coincubation, the percentage of dying target cells was assessed by propidium iodide (PI) staining. In vitro suppression contact independent assay.
  • PI propidium iodide
  • the contact dependent mediated suppression was assessed using Transwell culture of CD4 + T cells pre-labeled with dye 450 and (i) unlabeled CD19-CAR transduced monospecific Treg cells or (ii) unlabeled CAAR transduced monospecific Treg cells at various ratios of monospecific Tregs to responder cells.
  • CD4 + T cells were first stimulated with antiCD3/anti-CD28 coated beads at 1 :3 ratio.
  • Monospecific Tregs were unstimulated (no Act) or stimulated with antiCD3/anti-CD28 coated beads (beads) or with NALM6- aDSG. Because NALM6-aDSG is a cell line with a high proliferative capacity, it was treated with mitomycin.
  • CD4 + T cells in the lower chamber and monospecific Treg cells in the upper chamber were then incubated for 3 days at 37°C in a 5% CO2 incubator. At day 3, cells were harvested, and proliferation of CD4 T cells was assessed by flow cytometry (Miltenyi, MACSQUANT) through the determination of dye 450 dilution.
  • Antibodies listed in Table 1 were used for immuno-phenotypic characterization of Tregs. Briefly, 2.10 5 Treg were washed in PBS and incubated at 4°C for 20 minutes with the specified mAb conjugates. After incubation and washing, samples were analyzed on a MACQUANT (Miltenyi) using MacsQuantify software (Miltenyi). When necessary cells were permeabilized and fixed for intracellular staining using the BD kit.
  • Monospecific Treg cells were obtained, expressing a chimeric receptor composed of the sequence leader of the CD 8 followed by domain 1 to 4 of the extracellular part of the native desmoglein. This extracellular domain is linked to the signaling sequence via the hinge and the transmembrane region of the huCD8.
  • the signaling sequence is composed to the intracellular domain of 4- IBB followed by intracellular 1 ⁇ _ ⁇ )3 ⁇ chain. This chimeric receptor is shown in Figure 1.
  • the cytolytic activity of the Treg cells of the invention was assessed.
  • the monospecific Treg cells of the present invention display a lytic potential toward a cell which express the ligand (the BCR specific for DSG) for their chimeric receptor (CAAR-DSG).
  • Treg of the invention is capable to (i) induce cytolysis of autoreactive B cells and after activation through their CAAR to (ii) mediate suppression of CD4 T effector cells.

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Abstract

La présente invention concerne une population monospécifique de lymphocytes Treg, les lymphocytes Treg comprenant un récepteur chimérique, ledit récepteur chimérique reconnaissant un auto-anticorps sur une cellule B, et lesdits lymphocytes Treg étant cytotoxiques pour les lymphocytes B. La présente invention concerne également des procédés d'obtention de ladite population de lymphocytes Treg monospécifiques et l'utilisation de ladite population de lymphocytes Treg monospécifiques, en particulier pour le traitement d'une maladie auto-immune médiée par les auto-anticorps.
PCT/EP2018/050347 2017-01-06 2018-01-08 Population de lymphocytes t régulateurs monospécifiques avec cytotoxicité pour les lymphocytes b WO2018127585A1 (fr)

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