WO2024008274A1 - T regulatory cell modification - Google Patents

T regulatory cell modification Download PDF

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Publication number
WO2024008274A1
WO2024008274A1 PCT/EP2022/068488 EP2022068488W WO2024008274A1 WO 2024008274 A1 WO2024008274 A1 WO 2024008274A1 EP 2022068488 W EP2022068488 W EP 2022068488W WO 2024008274 A1 WO2024008274 A1 WO 2024008274A1
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cells
treg
cell
polynucleotide
antigen
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PCT/EP2022/068488
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French (fr)
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Ibo JANSSENS
Nathalie COOLS
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Universiteit Antwerpen
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Priority to PCT/EP2022/068488 priority Critical patent/WO2024008274A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N2510/00—Genetically modified cells

Definitions

  • the invention is broadly applicable in the cell biology and medical fields, and more particularly concerns methods for introducing exogenous polynucleotides into regulatory T cells (Tregs), and further pertains to the modified Treg cells and their compositions and uses.
  • Tregs regulatory T cells
  • T regulatory cells T regulatory cells
  • Tregs can suppress not only CD4 + T cells, but also CD8 + T cells and many other immune cells when recruited to an identical antigen-presenting cell.
  • Tregs are the most potent immunosuppressive cells in the human body and play a pivotal role in the delicate but crucial balance between immunity and tolerance. For instance, Tregs can directly interact with and downmodulate self-reactive T cells, thereby regulating self-tolerance and ultimately preventing the development of autoimmunity. Tregs have been described as CD4 + T cells or CD8 + T cells which display the expression of high levels of the interleukin (IL)-2 receptor a chain (IL-2Ra, also called CD25) and the expression of the master regulator forkhead box P3 (FOXP3) transcription factor.
  • IL interleukin
  • IL-2Ra also called CD25
  • FOXP3 master regulator forkhead box P3
  • FOXP3 orchestrates the transcriptional machinery of Tregs by binding >1400 genes and acting as both a transcriptional repressor and an activator of the expression of genes associated with the function of Tregs, including IL2RA and cytotoxic T lymphocyte associated protein 4 (CTLA-4).
  • CTLA-4 cytotoxic T lymphocyte associated protein 4
  • Tregs are heterogeneous mixture of cellular sub-phenotypes with a high degree of phenotypic complexity reflecting distinct developmental states, methods of suppression, homing properties and suppression targets.
  • CD4 + CD127 CD25 hl Tregs are characterized by high expression of the Treg master regulator FOXP3, whereas CD4 CD I 27 CD25 + CD45RA + Tregs (also denoted CD45RA + Treg cells), expressing the naivety marker CD45RA, are superior for expansion purposes.
  • Tregs exploit a broad spectrum of suppressive mechanisms, including cell contact dependent mechanisms involving CTLA-4 and the secretion of immune regulatory cytokines such as IL-10 and transforming growth factor (TGF- ). These mechanisms are influenced by the surrounding microenvironment, the type of immune reaction and the target cell. Furthermore, Tregs can transfer suppressor activity to conventional CD4 + T cells. This process, termed “infectious tolerance,” creates a local tolerogenic environment in which naive T cells convert into an induced Treg phenotype. In addition, Tregs are responsible for “bystander suppression” by inducing tolerance to cells without direct interaction.
  • Tregs are present throughout the body, and can be conveniently sourced from peripheral blood. Treg frequencies have been reported to be about 5% to 7% of CD4 + T cells in the periphery. A broad range of Treg isolation and expansion protocols have been developed (MacDonald et al. Clinical and Experimental Immunology 2019, vol. 197, 52-63), to arrive at satisfactory Treg numbers while preserving the desired Treg characteristics. A suitable activation reagent for ex vivo expansion of Tregs partially mimics the interaction with antigen-presenting cells, using anti-CD3 and anti-CD28 monoclonal antibodies covalently linked to magnetic beads (Trickett and Kwan. Journal of Immunological Methods 2003, vol. 275, 251-255), while many other clinical-grade Tregs isolation and expansion methods have been described.
  • Tregs While safe and feasible, only modest clinical efficacy was observed with unmodified Tregs. This could be, at least in part, because polyclonal Tregs were used, collectively targeting a broad mix of antigens, not all disease-related, and therefore potentially weakening their clinical effect. This prompted the field to move into a more antigen-specific approach to generate Tregs, aiming for a durable patient-tailored cell therapy without the risk for general immunosuppression. Preclinical studies demonstrated increased potency of antigen-specific Tregs compared with polyclonal Tregs in models of type 1 diabetes and transplantation.
  • Tregs can also be modulated for increased stability of their phenotype and function by introducing key regulators for Treg function, such as Helios, after introduction of cytokines, such as IL-10 and TGF-P involved in Tregs’ mechanism of action, or by creating Tregs that gained new functions, e.g. regenerative capacity by introducing neurotrophic factors such as brain-derived neurotrophic factor (BDNF) or amphiregulin.
  • BDNF brain-derived neurotrophic factor
  • Different genetic engineering technologies, including retro- and lentiviral transduction as well as nonviral transfection methods have been explored to introduce the expression of TCRs or chimeric antigen receptors into Tregs. Transduction of cells using viral vectors is the most used method resulting in high transfection efficiency.
  • RNA such as messenger RNA (mRNA)
  • mRNA messenger RNA
  • the present invention is at least in part based on the discovery that T regulatory cells (Treg cells or Tregs) are amenable to genetic engineering by means of RNA electroporation, and allow for highly efficient delivery of RNA molecules into the cells. Moreover, while the reaction of various immune cell types to non-physiological external treatments and to the introduction of exogenous materials tends to be unpredictable, the present inventors have discovered that the delivered RNA is not recognised as a danger signal by Treg cells and that the modified Treg cells retain their immunosuppressive functions, such as their capacity to inhibit the proliferation of T effector cells.
  • RNA is safer without the risk of insertional mutagenesis.
  • messenger RNA mRNA
  • mRNA messenger RNA
  • expression levels can be adjusted with the amount of supplied mRNA, expression is almost instant, and it does not rely on promoter strength.
  • introduced mRNA results in transient gene expression, being subjected to the natural decay of mRNA, providing an accurate system to control the synthesis of exogenous proteins.
  • electroporation is safe, versatile and robust, which can simplify its adoption in the clinic.
  • an aspect of the invention provides a method for introducing an unmodified or modified RNA polynucleotide into a Treg cell, comprising electroporation of a suspension comprising the polynucleotide and Treg cells.
  • a further aspect provides Treg cells comprising an unmodified or modified RNA polynucleotide, wherein the cells are obtainable or obtained by a method comprising electroporation of a suspension comprising the polynucleotide and Treg cells.
  • a pharmaceutical composition comprising Treg cells comprising an unmodified or modified RNA polynucleotide, wherein the cells are obtainable or obtained by a method comprising electroporation of a suspension comprising the polynucleotide and Treg cells.
  • a further aspect provides said Treg cells comprising the unmodified or modified RNA polynucleotide, or said pharmaceutical composition comprising said Treg cells, for use in medicine.
  • An aspect provides said Treg cells comprising the unmodified or modified RNA polynucleotide, or said pharmaceutical composition comprising said Treg cells, wherein the polynucleotide encodes a polypeptide which endows the Treg cell with specificity to an antigen or an antigenic peptide thereof, for use in a method of treating a disease caused by or associated with an increased activity of the immune system against said antigen.
  • a method for treating, in a subject in need thereof, a disease caused by or associated with an increased activity of the subject’s immune system against an antigen comprising administering to the subject an effective amount of said Treg cells comprising the unmodified or modified RNA polynucleotide, or said pharmaceutical composition comprising said Treg cells, wherein the polynucleotide encodes a polypeptide which endows the Treg cell with specificity to an antigen or an antigenic peptide thereof.
  • Fig. 1 illustrates multiparametric flow cytometry-based sorting of CD45RA + and CD25 hl Tregs and confirmation of their phenotype and functionality.
  • A Gating strategy for FACS sorting of CD4 + CD127"CD25 CD45RA + and CD4 + CD127 CD25 hi Tregs after CD4 + magnetic bead enrichment from PBMCs Tregs were sorted, from left to right, as lymphocytes (FSCA/SSC-A), single cells (FSC-A/FSC-H), living cells (LIVE/DEAD Fixable Aqua Dead Cell Stain negative population) and CD4 + T cells (CD3 + CD4 + ), and further elimination of irrelevant cell subsets based on dump channel (CD8 CD14 CD16 CD19 ) and CD127"CD25 + cells, whereas effector CD4 + T cells were sorted as CD127 + CD25" cells.
  • naive Tregs were based on expression of CD45RA, and CD25 hl Tregs were sorted as CD45RA CD25 hl .
  • B Representative overlay of dot plot and histogram of FOXP3 expression in CD127 + CD25" effector T cells (a), CD45RA + Tregs (b) and CD25 hl Tregs (c). FMO was used as control (d).
  • FOXP3 expression was analyzed by flow cytometry in CD45RA + and CD25 hl Tregs after sorting and compared to control CD I 27 CD25 effector T cells.
  • Fig. 2 illustrates gating strategy used for in vitro T-cell suppression assay. Effector T cells are stained with a CellTrace Violet Cell Proliferation kit, which allows tracking of cell division. Gating was conducted in following steps: (A) lymphocytes (FSC-A/SSC-A), (B) single cells (FSC-A/FSC- H), (C) living cells (SSC-A/7-AAD ), (D) CD4 + T cells (SSC-A/CD4 + ), (E) gating out the added Tregs, which were not stained with CellTrace Violet (CellTrace Violet/count) and (F) setting the gate for the undivided CD I 27 CD25 T cell using the unstimulated condition.
  • A lymphocytes
  • FSC-A/FSC-H single cells
  • C living cells
  • SSC-A/7-AAD living cells
  • E CD4 + T cells
  • E gating out the added Tregs, which were not stained with CellTrace Violet (CellTrace
  • Fig. 3 illustrates a mean 186.5 ⁇ 123.8-fold expansion of CD45RA + Tregs and 71.4 ⁇ 50.3-fold expansion of CD25 hl Tregs upon 19 days of ex vivo expansion and a >80% expression of a transgenic TCR by means of mRNA electroporation was achieved by using a novel GMP- compatible engineering protocol.
  • Tregs were FACS sorted, cultured in IMDM supplemented with 500 lU/mL IL-2 and 5% hAB serum, and activated using a soluble polymer conjugated with antibodies to CD3 and CD28 (1: 100 dilution) on days 0, 7 and 14.
  • Fig. 4 illustrates a representative dot plot and histogram overlay of eGFP expression in Tregs 24h following eGFP-encoding mRNA electroporation. Mock electroporation is indicated with ‘b’ and eGFP -encoding mRNA-electroporation is indicated with ‘a’.
  • Fig. 5 illustrates high FOXP3 expression and low methylation levels in engineered CD45RA + and CD25 hl Tregs, which confirm conservation of stable Treg phenotype.
  • FOXP3 expression in CD45RA + Tregs (A) and CD25 hl Tregs (B) after sorting, expansion, and MBPss-gg-specific TCR- encoding mRNA electroporation was compared with control CD I 27 CD25 T cells.
  • Fig. 6 illustrates Helios, CTLA-4 and CCR4 expression in engineered CD45RA + and CD25 hl Tregs, confirming conservation of stable Treg phenotype.
  • Helios (left), CTLA-4 (middle) and CCR4 (right) expression in CD45RA + Tregs and CD25 hl Tregs after expansion and MBPss-gg-specific TCR-encoding mRNA electroporation (EP) were compared to control CD4 + T cells. Results are shown as representative data graphs, representing median percentage ⁇ interquartile range for three independent donors.
  • Statistical analysis was performed using the nonparametric Friedman test with Dunn’s multiple comparisons test: *P ⁇ 0.05, **P ⁇ 0.01.
  • Fig. 7 illustrates that ex vivo expanded and mRNA-electroporated (EP) CD45RA + and CD25 hl Tregs induce the suppression of effector T cell proliferation in vitro and produce antiinflammatory, but not pro-inflammatory, cytokines when activated.
  • Autologous CD I 27 CD25 T cells were stained using CellTrace Violet and activated with human Treg suppression inspector beads, leading to cell proliferation.
  • Inhibition of CD 127 CD25 T cell proliferation was obtained by different ratios of expanded CD45RA + Tregs (A) and expanded CD25 hl Tregs (B). MBP85-99- specific TCR-encoding mRNA-electroporated cells are indicated with an asterisk (*).
  • Graphs represent median percentage of suppression ⁇ interquartile range for four independent donors. Each donor was measured in duplicate, and the mean division index was used to calculate the suppression percentage: 100 - (DI CO ndition of interest/DIi : o) * 100.
  • Statistical analysis was performed using the nonparametric Friedman test with Dunn’s multiple comparisons test. Pro-inflammatory (C) and anti-inflammatory (D) cytokine production of expanded CD45RA + Tregs and expanded CD25 hl Tregs, which are MBP 8 5-99-specific TCR-encoding mRNA-electroporated (EP) or not, compared with control CD4 + T cells.
  • Statistical analysis was performed using the nonparametric Friedman test with Dunn’s multiple comparisons test: *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****p ⁇ 0.0001.
  • EP mRNA electroporation.
  • Fig. 8 illustrates representative dot plot and histogram overlay of the l ⁇ BPs'-99-spccific TCR and CD3 expression in TCR-deficient cell lines 2D3 and SKW-3 6h following MBP 8 5-99-specific TCR- encoding mRNA-electroporation. Mock electroporation is indicated by ‘b’ and MBP 8 5-99-specific TCR-encoding mRNA-electroporation, leading to surface expression of CD3 and the transgenic TCR, is indicated by ‘a’ in both 2D3 (A) and SKW-3 (B) cells, which are TCR-deficient cell lines.
  • Fig. 9 illustrates TCR-dependent activation following TransAct stimulation of MBP 8 5-99-specific TCR-encoding mRNA-electroporated 2D3 and SKW-3 cells.
  • 2D3 (A) and SKW-3 (B) cells were MBP 85 -99-specific TCR-encoding mRNA-electroporated and unstimulated (grey) or TCR-specific stimulated using TransAct (black) 6 h after mRNA electroporation.
  • NFAT-dependent GFP expression in 2D3 cells and expression of activation markers CD69 and CD 137 in SKW-3 cells were analyzed 12 h after activation. Results are represented as data plot and histogram overlay for unstimulated and stimulated electroporated cells.
  • one or more or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.
  • “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.
  • the term “and/or” when used in a list of two or more items means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a list is described as comprising group A, B, and/or C, the list can comprise A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.
  • T regulatory cells T regulatory cells or Tregs
  • Treg cells can be highly efficiently engineered by means of RNA electroporation, even while they retain their immunosuppressive functions.
  • an aspect of the invention provides a method for introducing an unmodified or modified RNA polynucleotide into a Treg cell, comprising electroporation of a suspension comprising the polynucleotide and Treg cells.
  • the terms “nucleic acid”, “nucleic acid molecule”, and “polynucleotide” as used herein refer to a polymer (preferably a linear polymer) of any length composed essentially of nucleoside units.
  • a nucleoside unit commonly includes a heterocyclic base and a sugar group.
  • Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine), as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases.
  • A adenine
  • G guanine
  • C cytosine
  • T thymine
  • U uracil
  • other naturally-occurring bases e.g., xanthine, inosine, hypoxanthine
  • chemically or biochemically modified e.g., methylated
  • modified nucleobases include, without limitation, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, 5 -methylcytosine, and 5- propynylcytosine.
  • modified nucleobases include N6- isopentenyladenine, 1 -methyladenine, 2-methyladenine, N6-methyladenine, 2-methylthio-N6- isopentenyladenine, 4-acetylcytosine, 3 -methylcytosine, 5 -methylcytosine, 2-thiocytosine, 1- methylguanine, 2,2-dimethylguanine, 2-methylguanine, 7-methylguanine, 5- (carboxyhydroxymethyl)uracil, 5 -(carboxymethylaminomethyl)-2 -thiouracil, 5 - carboxymethylaminomethyluracil, dihydrouracil, 1 -methyluracil, 5 -methylaminomethyluracil, 5- methoxyaminomethyl -2 -thiouracil, 5 -methoxy carbonylmethyl -2 -thiouracil, 5 - methoxy carbonylmethyluracil, 5 -methoxyuracil, 5
  • Sugar groups may include inter alia pentose (pentofuranose) groups such as preferably ribose and/or 2-deoxyribose common in naturally-occurring nucleic acids (RNA and DNA, respectively), or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups (such as, without limitation, 2’-0-alkylated, e.g., 2’-O-methylated or 2’-0-ethylated sugars such as ribose; 2’-O-alkyloxyalkylated, e.g., 2’-O-methoxyethylated sugars such as ribose; or 2’-O,4’-C-alkylene-linked, e.g., 2’-O,4’-C-methylene-linked or 2’-O,4’-C-ethylene-linked sugars such as ribose; 2 ’-fluoro-
  • Naturally-occurring ribonucleosides include in particular adenosine, guanosine, uridine, and cytidine.
  • Naturally-occurring deoxyribonucleosides include in particular deoxyadenosine, deoxyguanosine, thymidine, and deoxy cytidine.
  • modified nucleosides include, without limitation, 4-acetylcytidine, 5- (carboxyhydroxymethyl)uridine, 2 ’ -O-methylcytidine, 5 -carboxymethylaminomethyl -2 -thiouridine, 5 -carboxymethylaminomethyluridine, dihydrouridine, 2’-O-methylpseudouridine, D- galactosylqueuosine, 2’-O-methylguanosine, inosine, N6-isopentenyladenosine, 1- methyladenosine, 1 -methylpseudouridine, 1 -methylguanosine, 1 -methylinosine, 2,2- dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3 -methylcytidine, 5 -methylcytidine, N6-methyladenosine, 7-methylguanosine
  • Nucleoside units may be linked to one another by any one of numerous known inter-nucleoside linkages, including inter alia phosphodiester linkages common in naturally-occurring nucleic acids, and further modified phosphate- or phosphonate-based linkages such as phosphorothioate, alkyl phosphorothioate such as methyl phosphorothioate, phosphorodithioate, alkylphosphonate such as methylphosphonate, alkylphosphonothioate, phosphotriester such as alkylphosphotriester, phosphoramidate, phosphoropiperazidate, phosphoromorpholidate, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate; and further siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate such as 3’-N-carbamate, morpholino, borano, thioether, 3 ’-
  • inter-nucleoside linkages may be phosphate-based linkages including modified phosphate-based linkages, such as more preferably phosphodiester, phosphorothioate or phosphorodithioate linkages or combinations thereof.
  • the term “nucleic acid” may also encompass any other nucleobase containing polymers such as nucleic acid mimetics, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino phosphorodiamidate-backbone nucleic acids (PMO), cyclohexene nucleic acids (CeNA), tricyclo- DNA (tcDNA), and nucleic acids having backbone sections with alkyl linkers or amino linkers (see, e.g., Kurreck 2003 (Eur J Biochem 270: 1628-1644)).
  • Alkyl as used herein particularly encompasses lower hydrocarbon moieties, e.g., C1-C4 linear or branched, saturated or unsaturated hydrocarbon, such as methyl, ethyl, ethenyl, propyl, 1 -propenyl, 2-propenyl, and isopropyl.
  • Nucleic acids as intended herein may include naturally occurring nucleosides, modified nucleosides, or mixtures thereof.
  • Reference to an “unmodified” polynucleotide may conveniently denote a polynucleotide composed of nucleosides which also constitute the corresponding type or kind of polynucleotide in nature, such as adenosine, guanosine, uridine, and/or cytidine for ribonucleic acid (RNA) polynucleotides, or deoxyadenosine, deoxyguanosine, thymidine, and/or deoxycytidine for deoxyribonucleic acid (DNA) polynucleotides, and connected by inter- nucleoside phosphodiester linkages.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • Reference to a “modified” polynucleotide may conveniently denote a polynucleotide which comprises one or more modified nucleoside, one or more modified inter-nucleoside linkage, or a combination thereof.
  • the one or more modified nucleoside may each independently comprise a modified heterocyclic base, a modified sugar moiety, a modified connection between the base and the sugar moiety, or a combination thereof.
  • an RNA polynucleotide may be denoted as “modified” when it comprises one or more nucleoside other than adenosine, guanosine, uridine, and cytidine, one or more inter-nucleoside linkage other than a phosphodiester bond, or a combination thereof.
  • a modified RNA polynucleotide may be primarily composed of adenosine, guanosine, uridine, and/or cytidine nucleosides connected by phosphodiester linkages, and may comprise a minority of nucleosides other than adenosine, guanosine, uridine, and cytidine, and/or a minority of inter-nucleoside linkages other than phosphodiester bond.
  • a modified RNA polynucleotide may be primarily or exclusively composed of nucleosides other than adenosine, guanosine, uridine, and cytidine, and/or may comprise a majority of or may exclusively comprise inter-nucleoside linkages other than phosphodiester bond.
  • nucleic acid further preferably encompasses DNA, RNA and DNA/RNA hybrid molecules, specifically including heteronuclear RNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesized) DNA, RNA (such as but not limited to Dicer-substrate small interfering RNAs (DsiRNA)) or DNA/RNA hybrids.
  • DNA RNA and DNA/RNA hybrid molecules, specifically including heteronuclear RNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesized) DNA, RNA (such as but not limited to Dicer-substrate small interfering RNAs (DsiRNA)) or DNA/RNA hybrids.
  • DsiRNA Dicer-substrate small interfering RNAs
  • a nucleic acid can be naturally occurring, e.g., present in or isolated from nature (and may be of any origin, e.g., prokaryotic, eukaryotic, archaeal, or viral), can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesized.
  • a “nucleic acid” can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand.
  • RNA polynucleotides are typically single-stranded molecules, can, however, also be provided in a double-stranded form by partial complementary base pairing. RNA polynucleotides typically do not form long double helical stretches.
  • nucleic acid can be circular or linear. Nucleic acids may also, in certain embodiments, comprise artificial additions such as tags or labels.
  • ribonucleic acid polynucleotide or “RNA polynucleotide” more particularly denotes nucleic acids which comprise two or more ribonucleosides, i.e., nucleosides in which the sugar group to which the heterocyclic base is linked is a ribose or a modified ribose, preferably ribose, but not deoxyribose.
  • ribose include, without limitation, 2’-O-alkylation, e.g., 2’-O-methylation or 2’-O-ethylation, 2’-O-alkyloxyalkylation, e.g., 2’-O-methoxyethylation; or 2’-O,4’-C-alkylene-linkage, e.g., 2’-O,4’-C-methylene-linked or 2’-O,4’-C-ethylene-linked ribose.
  • 2’-O-alkylation e.g., 2’-O-methylation or 2’-O-ethylation
  • 2’-O-alkyloxyalkylation e.g., 2’-O-methoxyethylation
  • 2’-O,4’-C-alkylene-linkage e.g., 2’-O,4’-C-methylene-linked or 2’-O,4’-C-ethylene-linked ribos
  • a ribonucleoside may comprise one of the prevalent nucleobases found in naturally- occurring RNA molecules, i.e., adenine, guanine, uracil, or cytosine, or may comprise a modified (whether naturally-occurring or non-naturally-occurring) nucleobase, such as one of the modified nucleobases described above.
  • the two or more ribonucleosides may be connected by a phosphodiester bond, or by an alternative inter-nucleoside linkage such as described above, preferably by a phosphodiester bond.
  • RNA polynucleotide comprises one or more nucleoside other than a ribonucleoside
  • the RNA polynucleotide may be primarily composed of ribonucleosides, for example, in an increasing order of preference, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99% of the RNA polynucleotide’s nucleosides are ribonucleosides.
  • the RNA polynucleotide may comprise only nucleosides which are ribonucleosides.
  • the RNA polynucleotide may be primarily composed of nucleosides selected from the group consisting of adenosine, guanosine, uridine, cytidine, and combinations thereof, for example, in an increasing order of preference, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or 100% of the RNA polynucleotide’s nucleosides are selected from the group consisting of adenosine, guanosine, uridine, cytidine, and combinations thereof.
  • the RNA polynucleotide’s nucleosides may be primarily connected by phosphodiester bonds, for example, in an increasing order of preference, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or 100% of the RNA polynucleotide’s nucleosides are connected by phosphodiester bonds.
  • the RNA polynucleotide is primarily composed of nucleosides selected from the group consisting of adenosine, guanosine, uridine, cytidine, and combinations thereof, connected by phosphodiester bonds, for example, in an increasing order of preference, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or 100% of the RNA polynucleotide’s nucleosides are nucleosides selected from the group consisting of adenosine, guanosine, uridine, cytidine, and combinations thereof, connected by phosphodiester bonds.
  • the RNA polynucleotide is a linear polynucleotide.
  • the modified RNA polynucleotide is nucleobase-modified, or backbone-modified, or nucleobase- modified and backbone-modified (wherein the modifications may be on the same or on different nucleosides).
  • the RNA polynucleotide is a naked polynucleotide, i.e., a polynucleotide free from any delivery vehicle that can act to facilitate entry into the cell, for example, the polynucleotide sequences are free of viral sequences, particularly any viral particles that may carry genetic information. They are similarly free from, or “naked” with respect to, any material that promotes transfection, such as liposomal formulations, charged lipids, or precipitating agents such as calcium phosphate.
  • any polynucleotides as discussed herein may be purified.
  • the term “purified” does not require absolute purity. Instead, it denotes that such nucleic acids are in a discrete environment in which their abundance (conveniently expressed in terms of mass or weight or concentration) relative to other analytes is greater than in a source material they have been purified from (e.g., from an in vitro transcription reaction, from a cell recombinantly producing them, etc.)
  • a discrete environment denotes a single medium, such as for example a single solution, gel, precipitate, lyophilisate, etc.
  • Purified nucleic acids may preferably constitute by weight > 10%, more preferably > 50%, such as > 60%, yet more preferably > 70%, such as > 80%, and still more preferably > 90%, such as > 95%, > 96%, > 97%, > 98%, > 99% or even 100%, of the nucleic acid content of the discrete environment.
  • Quantity of nucleic acids may be determined by measuring absorbance A260.
  • Purity of nucleic acids may be determined by measuring absorbance A260/A280, or by agarose- or polyacrylamide-gel electrophoresis and ethidium bromide or similar staining.
  • Tregs are potent immunosuppressive cells, which among others can down-modulate the functions of T effector cells.
  • Treg markers include LAG-3, CD25, cytotoxic T lymphocyte-associated antigen 4 (CD152), FoxP3, GITR, IFN-y, and neuropilin-1 but often only a subset of these are expressed.
  • CD4 + Tregs have been phenotypically described as CD4 positive, CD25 positive, and CD 127 low or negative.
  • CD8 + Tregs have been described which are CD8 positive, LAG3 positive, CD25 positive and CD 127 negative.
  • positive cells may on average generate a signal that is significantly different from the control, e.g., but without limitation, at least 1.5-fold higher than such signal generated by control cells, e.g., at least 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold higher or even higher.
  • a cell not positive for a given marker can be denoted as being negative for said marker.
  • the expression of cell-specific markers can be detected using any suitable immunological technique known in the art, such as immunohistochemistry or affinity adsorption, Western blot analysis, flow cytometry, ELISA, etc., or by any suitable biochemical assay of enzyme activity, or by any suitable technique of measuring the quantity of the marker mRNA, e.g., Northern blot, semi-quantitative or quantitative RT-PCR, etc. Sequence data for markers listed in this disclosure are known and can be obtained from public databases such as GenBank (http://www.ncbi.nlm.nih.gov/).
  • the cell surface marker phenotype of Treg cells allows their isolation from heterogeneous immune cell populations by immunological techniques which preserve the viability of the sorted cells, such as by fluorescence activated cell sorting (FACS) or by magnetic-activated cell sorting, in which distinct cell phenotypes within the cell populations can be separated.
  • FACS fluorescence activated cell sorting
  • a CD8 + Treg subset can be isolated based on their CD4 CD25 CD127" phenotype
  • a CD8 + subset could be isolated based on a CD8 + LAG3 + CD25 + FoxP3 + CCL4 + phenotype.
  • FACS is particularly convenient, since it allows to visualise cells in a sample according to their physical properties and their surface marker expression, and allows the user to define gates around the cell populations with the desired properties, and separate out the cells sorted into any gates of interest.
  • Treg cells have also been phenotypically described as expressing the master regulator forkhead box P3 (F0XP3) transcription factor (even while F0XP3 -independent maintenance of the human Treg identity has been shown in F0XP3-ablated Tregs) and as harbouring a substantially demethylated Treg-specific demethylated region (TSDR). While such intracellular characteristics may be less suited for isolation or sorting of viable Treg cells, they allow to classify cells as Treg cells, for example by analysing a sample of a larger cell population.
  • F0XP3 master regulator forkhead box P3
  • TSDR substantially demethylated Treg-specific demethylated region
  • the terms further include any and all Treg subtypes, subpopulations, and differentiation stages, in isolation as well as their combinations or mixtures. These will typically display the aforementioned defining Treg characteristics, and will be further distinguished from one another on the basis of other properties.
  • the terms encompass naive Treg cells (nTregs), central memory Treg cells (cmTregs), effector memory Treg cells (emTregs), and effector Treg (eTreg) lymphocytes.
  • isolated need not be expressly recited with relation to the cells as intended herein, it may conveniently be included, as the present disclosure pertains to manipulation of cells outside of the body, in vitro or ex vivo, and subsequent uses, such as therapeutic uses, of so- manipulated cells.
  • isolated with reference to a particular component generally denotes that such component exists in separation from - for example, has been separated from or prepared and/or maintained in separation from - one or more other components of its natural environment.
  • isolated as used herein in relation to a cell or cell population denotes that such cell or cell population does not form part of an animal or human body, for example the cell may be cultured, sorted or stored in vitro or ex vivo.
  • the Treg cell as disclosed herein is a vertebrate cell, preferably a warmblooded animal cell, even more preferably a mammalian cell, and most preferably a human cell.
  • the Treg cells are CD4 + CD45RA + Treg cells.
  • the Treg cells are a population of CD4 + CD45RA + Treg cells with purity, in increasing order of preference, of at least 50% (i.e., at least 50% of the cells of the cell population are CD4 + CD45RA + Treg cells), at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or 100%.
  • the Treg cells are CD4 + CD25 hl Treg cells.
  • the Treg cells are a population of CD4 + CD45RA + Treg cells with purity, in increasing order of preference, of at least 50% (i.e., at least 50% of the cells of the cell population are CD4 + CD25 hl Treg cells), at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or 100%.
  • the Treg cells are CD8 + LAG3 + CD25 FoxP3 CCL4 + Treg cells.
  • the Treg cells are a population of CD8 + LAG3 + CD25 + FoxP3 + CCL4 + Treg cells with purity, in increasing order of preference, of at least 50% (i.e., at least 50% of the cells of the cell population are CD8 + LAG3 + CD25 + FoxP3 + CCL4 + Treg cells), at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or 100%.
  • the Treg cells to be electroporated are freshly isolated, such as freshly isolated from peripheral blood mononuclear cells (PBMC) or from an internal organ, such as lung, liver, or spleen.
  • PBMC peripheral blood mononuclear cells
  • the Treg cells are not expanded in culture prior to electroporation.
  • the Treg cells to be electroporated have been obtained by in vitro or ex vivo expansion of isolated Treg cells, such as Treg cells isolated from PBMC or from an internal organ, such as lung, liver, or spleen.
  • isolated Treg cells such as Treg cells isolated from PBMC or from an internal organ, such as lung, liver, or spleen.
  • in vitro as used herein is to denote outside, or external to, animal or human body.
  • in vitro as used herein should be understood to include “ex vivo”.
  • ex vivo typically refers to tissues or cells removed from an animal or human body and maintained or propagated outside the body, e.g., in a culture vessel.
  • peripheral blood mononuclear cells are nucleated peripheral blood cells, in particular cells having a round nucleus.
  • PBMC in particular include lymphocytes, monocytes, and dendritic cells.
  • the lymphocyte population of PBMC typically consists of T-cells, B-cells and NK cells.
  • PBMC may be isolated from whole blood samples by methods well known in the art, such as for example density gradient centrifugation (e.g., Ficoll gradient).
  • the Treg isolation comprises isolating T cells from PBMC or from the internal organ, thereby obtaining a population of T cells; and isolating Treg cells from the population of T cells. Since Treg cells are a subset of CD4 + immune cells and/or CD8 + immune cells, in certain embodiments, the Treg isolation comprises isolating CD4 + cells, in particular CD4 + T cells from PBMC or from the internal organ and/or the Treg isolation comprises isolating CD8 + cells, in particular CD8 + T cells from PBMC or from the internal organ, thereby obtaining a population of CD4 + cells and/or a population of CD8 + cells, in particular CD4 + T cells and/or CD8 + T cells; and isolating Treg cells from the population of CD4 + cells and/or CD8 + cells in particular CD4 + T cells and/or CD8 + T cells.
  • the Treg cell can be directly isolated from PBMC or from the internal organ, i.e., without the
  • the isolation of Treg cells from the population of T cells, CD4 + cells, or in particular CD4 + T cells may involve isolating CD 127 negative (CD 127 ) cells therefrom.
  • the isolation comprises isolating CD4 + cells from PBMC or from the internal organ, thereby obtaining a population of CD4 + cells; and isolating CD 127’ cells from the population of CD4 + cells, thereby obtaining Treg cells.
  • the CD4 CD I27 cells can be directly isolated from PBMC or from the internal organ, i.e., without the interposed step of CD4 + cell enrichment.
  • the method may comprise isolating specifically CD45RA + Treg cells from the population of T cells, CD4 + cells, or in particular CD4 + T cells, or directly from PBMC or from the internal organ; or isolating specifically CD25 hl Treg cells from the population of T cells, CD4 + cells, or in particular CD4 + T cells, or directly from PBMC or from the internal organ; or isolating specifically a mixture of CD45RA + Treg cells and CD25 hl Treg cells from the population of T cells, CD4 + cells, or in particular CD4 + T cells, or directly from PBMC or from the internal organ.
  • the isolation of Treg cells from the population of T cells, CD8 + cells, or in particular CD8 + T cells may involve isolating CD 127 negative (CD 127 ) cells therefrom and/or further selecting for expression of LAG3 + , CD25 + , FoxP3 + , and/or CCL4 + .
  • the isolation comprises isolating CD8 + cells from PBMC or from the internal organ, thereby obtaining a population of CD8 + cells; and isolating CD 127’ cells (or cells enriched based on one or more of the markers specified above) from the population of CD8 + cells, thereby obtaining Treg cells.
  • Treg cells can be isolated from the T cell population based on the expression of specific cell-surface markers, using any suitable cell separation method which substantially preserves the viability of the cells, in particular an immunological methods, such as flow cytometry (FACS) and/or affinity separation.
  • FACS flow cytometry
  • Flow cytometry encompasses methods by which individual cells of a cell population are analyzed by their optical properties (e.g., light absorbance, light scattering and fluorescence properties, etc.) as they pass in a narrow stream in single file through a laser beam.
  • Flow cytometry methods include fluorescence activated cell sorting (FACS) methods by which a population of cells having particular optical properties are separated from other cells.
  • FACS fluorescence activated cell sorting
  • Affinity separation also referred to as affinity chromatography broadly encompasses techniques involving specific interactions of cells present in a mobile phase, such as a suitable liquid phase (e.g., cell population in an aqueous suspension) with, and thereby adsorption of the cells to, a stationary phase, such as a suitable solid phase; followed by separation of the stationary phase from the remainder of the mobile phase; and recovery (e.g., elution) of the adsorbed cells from the stationary phase.
  • Affinity separation may be columnar, or alternatively, may entail batch treatment, wherein the stationary phase is collected / separated from the liquid phases by suitable techniques, such as centrifugation or application of magnetic field (e.g., where the stationary phase comprises magnetic substrate, such as magnetic particles or beads).
  • suitable techniques such as centrifugation or application of magnetic field (e.g., where the stationary phase comprises magnetic substrate, such as magnetic particles or beads).
  • magnetic cell separation or magnetic-activated cell sorting is also envisaged herein.
  • the T cells or CD4 + cells, or in particular CD4 + T cells are isolated from PBMC or from the internal organ using MACS or FACS, preferably by MACS.
  • the Treg cells are isolated from the population of T cells or CD4 + cells and/or CD8 + cells, or in particular CD4 + T and/or CD8 + cells, using MACS or FACS, preferably by FACS.
  • the T cells or CD4 + cells, or in particular CD4 + T cells and/or CD8 + T cells are isolated from PBMC or from the internal organ using MACS or FACS, and the Treg cells are isolated from the population of T cells or CD4 + cells, or in particular CD4 + T cells and/or CD8 + T cells, using MACS or FACS.
  • the T cells or CD4 + cells and/or CD8 + cells, or in particular CD4 + T cells and/or CD8 + T cells are isolated from PBMC or from the internal organ using MACS, and the Treg cells are isolated from the population of T cells or CD4 + cells and/or CD8 + cells, or in particular CD4 + T cells, using FACS.
  • the Treg cells are directly isolated from PBMC or from the internal organ using FACS.
  • FACS Fluorescence Activated Cell Sorting
  • the method may advantageously include a step of expanding the isolated Treg cells in cell culture.
  • T cell cultivation may typically occur in cell cultures, in suitable liquid cell culture media.
  • the medium will comprise a basal medium formulation as known in the art.
  • basal media formulations can be used to culture the cells herein, including but not limited Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), BGJb, F-12 Nutrient Mixture (Ham), or Iscove's Modified Dulbecco's Medium (IMDM), and modifications and/or combinations thereof.
  • Compositions of the above basal media are generally known in the art and it is within the skill of one in the art to modify or modulate concentrations of media and/or media supplements as necessary for the cells cultured.
  • basal media formulations contain ingredients necessary for mammal cell development, which are known per se.
  • these ingredients may include inorganic salts (in particular salts containing Na, K, Mg, Ca, Cl, P and possibly Cu, Fe, Se and Zn), physiological buffers (e.g., HEPES, bicarbonate), nucleotides, nucleosides and/or nucleic acid bases, ribose, deoxyribose, amino acids, vitamins, antioxidants (e.g., glutathione) and sources of carbon (e.g., glucose, sodium pyruvate, sodium acetate), etc.
  • inorganic salts in particular salts containing Na, K, Mg, Ca, Cl, P and possibly Cu, Fe, Se and Zn
  • physiological buffers e.g., HEPES, bicarbonate
  • nucleotides e.g., nucleosides and/or nucleic acid bases
  • ribose e.g., deoxyribose
  • basal media can be supplied with one or more further components.
  • additional supplements can be used to supply the cells with the necessary trace elements and substances for optimal growth and expansion.
  • Such supplements include insulin, transferrin, selenium salts, and combinations thereof.
  • These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution.
  • Further antioxidant supplements may be added, e.g., P-mercaptoethanol. While many basal media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution.
  • a medium may be further supplied with antibiotic and/or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and/or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin.
  • antibiotic and/or antimycotic compounds such as, typically, mixtures of penicillin and streptomycin, and/or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neo
  • Such lipids and carriers can include, but are not limited to cyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic-oleic- arachidonic acid conjugated to albumin, oleic acid unconjugated and conjugated to albumin, among others.
  • Albumin can similarly be used in fatty-acid free formulations.
  • Plasma, serum or a substitute thereof such as bovine serum, fetal calf serum, or preferably human serum, may also be comprised in said media at a proportion (volume of one or more of plasma, serum, or a substitute thereof / volume of medium) between about 0.5% and about 30% v/v, preferably between about 5% and about 10%.
  • v/v. Standard cell culture can be performed in humidified 5% CO2 incubator at 37°C.
  • the culture medium can be replenished or refreshed, completely or partly, at with suitable regularity, such as twice daily, once daily, once in two days, or similar, depending on factors such as cell density, medium pH etc.
  • suitable regularity such as twice daily, once daily, once in two days, or similar, depending on factors such as cell density, medium pH etc.
  • cells may be passaged once they have reached a certain degree of confluency.
  • Treg cell expansion comprises a step of culturing the isolated Treg cells, such as Treg cells isolated from PBMC or from the internal organ, in the presence of interleukin-2 (IL-2).
  • IL-2 may be included in the media in which the Treg cells are cultured, in a quantity sufficient to promote expansion of the Treg cells.
  • IL-2 can be included in the media at a concentration of between 50 lU/mL and 2000 lU/mL, preferably between 100 lU/mL and 1000 lU/mL, more preferably between 250 lU/mL and 750 lU/mL, such as at about 500 lU/ml.
  • the Treg cells are expanded in a complete medium comprising or consisting of Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 5% v/v human AB serum and 500 lU/mL IL-2.
  • Iscove’s modified Dulbecco’s medium IMDM
  • the Treg cells may be activated prior to electroporation.
  • Methods for Treg cell activation are generally known and may comprise a step of contacting the isolated and optionally expanded Treg cells, such as the Treg cells isolated and optionally and preferably expanded from PBMC or from the internal organ, with an anti-CD3 antibody and an anti-CD28 antibody.
  • the antibodies may be covalently linked to a polymer carrier.
  • T cell TransActTM commercially available from Miltenyi Biotec, which is a clinical-grade colloidal reagent comprising iron oxide crystals embedded into a biocompatible polysaccharide matrix with an overall diameter of -100 nm.
  • Agonistic humanized anti-CD3 and anti-CD28 antibodies are coated onto the nanomatrix.
  • the matrix can be produced under GMP conditions, sterilized by fdtration and unbound reagent can easily be removed from the cell suspension by centrifugation of the cells.
  • the Treg cells may be activated two or more times during the Treg expansion and activation protocol. The later activation steps may also be denoted as reactivation steps.
  • the frequency of the activation / reactivation steps may be for example twice weekly, weekly, every 1.5 week, or every two weeks, such as for example every 3, 4, 5, 6, 7, 8, 9, 10, or 11 days, preferably every 6, 7, or 8 days, and more preferably every 7 days.
  • the activation is repeated on days 0, 7, 14, and 19 of the Treg expansion and activation.
  • Each activation step may be followed by washing away the activation reagent, such as between about 24 hours and about 72 hours after its addition to the cells / medium, preferably between about 36 hours and about 60 hours, and more preferably at about 48 hours its addition to the cells / medium.
  • the present methods comprise electroporation of a suspension comprising the polynucleotide and Treg cells.
  • the polynucleotide and the Treg cells may be suspended in any suitable electroporation medium or buffer.
  • suitable electroporation medium or buffer Such compositions conducive to cell viability and to the electroporation process are well-known, and may include for example serum-free media such as IMDM, RPMI, or a serum reduced medium (e.g., Opti-MEM I®, Gibco Invitrogen).
  • the suspension for electroporation may be kept at room temperature or may be kept at less than ambient temperature, such as on ice, i.e., about 4°C.
  • the cells can be replenished in a serum-containing medium, such as in IMDM supplemented with 10% v/v/ human serum.
  • an electroporation cuvette typically dimensions of 1 mm to 4 mm, such as 1-mm, 2 -mm or 4-mm cuvettes, and preferably 4-mm cuvettes.
  • One or more settings of the electroporation process may be adjusted depending on the cuvette size.
  • a 4-mm cuvette and about 200 pl of the cell suspension may be used in the electroporation.
  • the electroporation may be performed with any suitable device available from a variety of vendors, such as BTX ECM 830 square wave electroporator, Gene Pulser Xcell (BioRad), Gene Pulser II® (Bio-Rad), or Easyject Plus® (Equibio) exponential decay pulse electroporator, etc.
  • a conventional electroporation apparatus which provides for an exponential decay pulse; the electroporation may be performed at a voltage from 100 to 500 V, more preferably from 200 to 350 V, most preferably from 250 to 300 V; the capacitance is preferably 100 pF to below 300 pF, more preferably 150 pF to 250 pF; the pulsing time can depend from the type of the tray (cuvette) and the amount of the cell suspension in the cuvette and is preferably below 50 ms, more preferably below 40 ms (for example, for a 4 mm cuvette and 200 pl cell suspension, the pulsing time may be from 5 to 40 ms, preferably 1 to 25 ms, and most preferably 7 to 10 ms).
  • so-called “soft pulse” electroporation device is utilized; a voltage of 300 to 600 V and a time of 100 ps to 1 ms may be used which are believed to correspond to a capacitance of below 300 pF (although, due to the use of eukaryotic cell suspensions, a correct conversion is not possible); the pulse form provided by commercially available soft pulse electroporation devices may be a square wave pulse or an exponential decay pulse; preferred settings for the soft pulse devices may be 350 to 450 V for 300 to 600 ps.
  • any one or any combination of two or more, or all of the following may apply:
  • the concentration of the Treg cells in the suspension is 100 cells per ml to 1x10 9 cells per ml, such as IxlO 3 cells per ml to IxlO 9 cells per ml, or IxlO 4 cells per ml to IxlO 9 cells per ml, or 1x10 s cells per ml to IxlO 9 cells per ml, or IxlO 6 cells per ml to IxlO 9 cells per ml, preferably IxlO 7 to IxlO 8 cells per ml, such as about IxlO 7 cells per ml, about 2.5xl0 7 cells per ml, about 5xl0 7 cells per ml, about 7.5xl0 7 cells per ml, or about IxlO 8 cells per ml;
  • the pulse is a square wave pulse or an exponential decay pulse, preferably a square wave pulse;
  • the dimension of the cuvette is from 1 mm to 4 mm, preferably 1-mm, 2 -mm, or 4-mm, more preferably 4-mm;
  • the voltage is from 100 V to 700 V, such as from 350 V to 650 V, preferably from 400 V to 600 V, such as from 450 V to 550 V, such as particularly preferably about 500 V;
  • the pulse is a square wave pulse and the voltage is from 300 V to 700 V, such as from 350 V to 650 V, preferably from 400 V to 600 V, such as from 450 V to 550 V, such as particularly preferably about 500 V;
  • the pulsing time is from 1 ms to 40 ms, such as from 1 ms to 30 ms, or from 1 ms to 20 ms, preferably from 1 to 10 ms, such as about 2 ms, or about 3 ms, or about 4 ms, or about 5 ms, or about 6 ms, or about 7 ms, or about 8 ms, or about 9 ms, particularly preferably about 5 ms;
  • the concentration of the polynucleotide in the suspension is from 100 ng / IxlO 6 cells to 10 pg / IxlO 6 cells, such as from 250 ng / IxlO 6 cells to 7.5 pg / IxlO 6 cells, preferably from 500 ng / IxlO 6 cells to 5 pg / IxlO 6 cells, such as from 750 ng / IxlO 6 cells to 2.5 pg / IxlO 6 cells, more preferably about 1 pg / IxlO 6 cells.
  • RNA polynucleotide to be introduced into the Treg cells is synthetic, or in vitro transcribed, or isolated from a host cell or a non-human host organism genetically engineered to produce the polynucleotide.
  • Methods for nucleic acid synthesis, in vitro transcription, or recombinant expression in host cells and host organisms are well-known in the art, and need not be discussed in detail.
  • RNA can be readily prepared from the corresponding DNA in vitro.
  • conventional techniques utilize phage RNA polymerases SP6, T3, or T7 to prepare RNA from DNA templates in the presence of the individual ribonucleoside triphosphates.
  • An appropriate phage promoter, such as a T7 origin of replication site is placed in the template DNA immediately upstream of the sequence to be transcribed.
  • the RNA polynucleotide is coding, i.e., its sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question to a particular amino acid sequence, e.g., the amino acid sequence of one or more desired proteins or polypeptides. In certain other embodiments, the RNA polynucleotide is non-coding.
  • the RNA polynucleotide is selected from the group consisting of messenger RNA (mRNA), guide RNA (gRNA), single guide RNA (sgRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), long non-coding RNA (IncRNA), ribozyme, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, apt
  • the polynucleotide is mRNA.
  • the mRNA preferably encodes one or more polypeptide, preferably one or more biologically active polypeptide.
  • biologically active or “functionally active” or “functional” particularly conveys that the polypeptide displays some activity or function, such as a biochemical activity, an enzymatic activity, a signalling activity, an interaction activity, a ligand activity, and/or structural activity, particularly preferably wherein such activity can control, impact, or modulate the function or phenotype of Treg cells.
  • RNA polynucleotides may advantageously include structural and sequence elements for efficient and correct translation, together with those elements which will enhance the stability of the introduced mRNA.
  • translational efficiency has been found to be regulated by specific sequence elements in the 5 ’-non-coding or untranslated region (5’-UTR) of mRNA.
  • Positive sequence motifs include the Kozak translational initiation consensus sequence and the 5 G 7-methyl GpppG cap structure.
  • Negative elements include stable intramolecular 5’ UTR stem -loop structures and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5’ UTR.
  • mRNA-based polynucleotides suitable for use herein ideally include appropriate 5’ UTR translational elements flanking the coding sequence for the protein of interest. Further, capping and 3’ polyadenylation are major positive determinants of eukaryotic mRNA stability and function to protect the 5’ and 3’ ends of the mRNA from degradation. Other regulatory elements that affect the stability of eukaryotic mRNAs may also need to be considered in the development of mRNA-based polynucleotides.
  • One example includes uridine rich 3’ untranslated region (3’ UTR) destabilizer sequences found in many short half-life mRNAs.
  • the RNA polynucleotide may be chemically modified or blocked at the 5’ and/or 3’ end to prevent access by RNase.
  • the polynucleotide produces a loss-of-function phenotype when introduced into the Treg cell.
  • the expression of one or more RNA products or one or more proteins endogenously produced by the Treg cell may be downregulated or abolished by the introduction of the polynucleotide.
  • antisense oligonucleotides, RNA interference agents such as siRNA or shRNA, or gene editing systems (such as CRISPR/Cas) systems may be utilised to this aim.
  • one or more polynucleotide comprising or encoding components of a gene editing system may be introduced into the Treg cell in order to effect a gene editing event in the Treg cell’s genomic material.
  • a DNA molecule homologous to the target locus may be co-introduced to induce a recombination / swapping of the native genetic information for the exogenously provided one at that locus.
  • one or more antisense or RNA interference polynucleotide may be introduced into the Treg cell in order to reduce the amount or the translation of an RNA molecule, such as an mRNA molecule, produced by the Treg cell.
  • the polynucleotide produces a gain-of-function phenotype when introduced into the Treg cell.
  • the polynucleotide when the polynucleotide is mRNA encoding one or more polypeptide, preferably one or more biologically active polypeptide, the polypeptide produces a gain-of-function phenotype when expressed by the Treg cell.
  • This may broadly encompass situations in which the polynucleotide endows the Treg cell with a new function, or in which a certain existing function of the Treg cell is increased or enhanced. In particular, this may refer to the expression of a protein normally not expressed by Treg cells, or an increase in expression of a protein normally expressed by Treg cells above the endogenous level.
  • proteins or peptides encoded by the RNA polynucleotides may include, but are not limited to, tumor antigens, microbial antigens, viral antigens, immunostimulatory or tolerogenic molecules, cytokines, interleukins, anti-apoptotic molecules, adhesion and homing molecules and antigen processing molecules, differentiation-regulating proteins, differentiation-associated proteins, tissue specific proteins, etc.
  • proteins or peptides encoded by the RNA polynucleotides may further include, but are not limited to, B-cell antibody receptors (BARs), growth factors, neurotrophic factors (e.g., BDNF, CCN3, amphiregulin), cytokines (e.g., TGF-beta, IL-10), regulators of Treg function, such as Helios, etc. and any combinations thereof.
  • BARs B-cell antibody receptors
  • growth factors e.g., BDNF, CCN3, amphiregulin
  • cytokines e.g., TGF-beta, IL-10
  • regulators of Treg function such as Helios, etc. and any combinations thereof.
  • Tregs can be modulated for increased stability of their phenotype and function by introducing key regulators for Treg function, such as Helios, after introduction of cytokines, such as IL-10 and/or TGF-P involved in Tregs’ mechanism of action, or by creating Tregs that have gained new functions, e.g. regenerative capacity, such as by introducing neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and/or amphiregulin.
  • key regulators for Treg function such as Helios
  • cytokines such as IL-10 and/or TGF-P involved in Tregs’ mechanism of action
  • Tregs that have gained new functions, e.g. regenerative capacity, such as by introducing neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and/or amphiregulin.
  • BDNF brain-derived neurotrophic factor
  • the one or more polypeptide encoded by the RNA polynucleotide endows the Treg cell with specificity to an antigen or an antigenic peptide thereof.
  • the one or more polypeptide is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
  • T cell receptor refers to a protein complex found on the surface of T cells that recognizes fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules.
  • Naturally occurring T cell receptors comprise two subunits, an a-subunit and a P-subunit or a y-subunit and a 5-subunit each of which is a unique protein produced by recombination event in each T cell's genome.
  • Each a-, P-, y- or 5- subunit contains variable (V) and constant (C) region domains, and the latter is followed by a transmembrane region and a short cytoplasmic tail.
  • Each V domain contains three loops (i.e.
  • CDR1, CDR2, and CDR3 complementarity-determining regions CDR1, CDR2, and CDR3, which interact with the antigen.
  • the CDR loops project from each TCR chain and physically contact portions of the MHC molecule alone or in complex with a peptide.
  • the centrally located CDR3 loops are most hypervariable by virtue of somatic rearrangement, dominate the interactions with the peptide, and therefore often contribute to the fine specificity of a TCR for a specific peptide.
  • the outward-facing and germline-encoded CDR1 and CDR2 loops provide a basal level of TCR affinity for generic MHC molecules through relatively conserved interactions, although CDR1 can contact and contribute to peptide specificity.
  • the constant domain of the TCR consists of short connecting sequences in which a cysteine residue forms disulfide bonds, which form a link between the two chains.
  • the polynucleotide or protein sequence of an aP or y5 TCR can be cloned with standard techniques from one or a plurality of a or y5 T-cell(s) that have engaged with an antigen of interest, such as a tumor antigen or a viral antigen.
  • an antigen of interest such as a tumor antigen or a viral antigen.
  • the polynucleotide sequence of an aP or y5 TCR can be designed in silico.
  • a nucleic acid comprising the engineered aP or y5 TCR can be synthesized with, for example, oligonucleotide synthesis techniques. High throughput screening techniques can be used to characterize the binding of the engineered TCR to the antigen of interest.
  • the one or more nucleic acids encoding the alpha- and beta-chain of a TCR are in a multicistronic construct.
  • a linker sequence may be inserted between the nucleic acid sequence encoding the alpha-chain of TCR and the beta-chain of TCR.
  • the linker sequences may be any linker sequence known in the art.
  • the linker sequence is a 2A self-cleaving peptide, as described elsewhere in this specification. The cleavage observed is not a proteolytic event but is rather the result of cis-acting hydrolase activity, which causes ribosomal skipping during translation.
  • the alpha-, beta-, delta- and/or gamma- subunits may have one or more amino acid substitutions, deletions, insertions, or modifications compared to the naturally occurring subunit, as long as the subunits retain the ability to form TCRs conferring upon transfected immune effector cells the ability to home to target cells.
  • one or more additional polypeptides are attached to the TCR.
  • Such one or more polypeptides may be attached to the TCR so long as the attached additional polypeptide does not interfere with the ability of the a-chain or P-chain to form a functional T cell receptor and the MHC dependent antigen recognition.
  • T cell activation upon TCR binding to the antigen involves several other cell surface molecules, also known as costimulatory molecules, that collectively initiate and amplify the signal.
  • the a and y5 heterodimer lack their own intracellular signaling domains and, thus, must associate with cluster of differentiation 3 (CD3).
  • CD3 is a six-subunit complex comprising three dimers: CD3sy. CD3so. and CD3 .
  • the cytoplasmic domains of CD3y, 5, and 8 each contain one immunoreceptor tyrosine-rich activation motif (ITAM) and each CD3 ⁇ contains three ITAMs, which serve as substrates for the Src-family kinase lymphocyte-specific protein tyrosine kinase (Lek).
  • ITAM immunoreceptor tyrosine-rich activation motif
  • each chain of the TCR comprises a CD3 ⁇ chain fused to its C-terminus.
  • the structure of the TCR may be modified to enhance the safety, efficacy and scalability of TCR- based immunotherapies, by any methods known in the art.
  • the nucleic acid encoding the TCR may comprise substitutions of all or selected murine residues in place of the human sequence in the TCR constant regions (murinization) for obtaining a higher exogenous TCR surface expression, increased functional avidity, and enhanced antigen-specific effector functions compared with a fully human TCR, at least one additional cysteine residue to promote a second disulfide bond (cysteine -modification), modification of the hydrophobicity of the TCR, such as the TCRa, transmembrane region (transmembrane-modification), swapping constant domains or fragments thereof between the a and P chains of the TCR (domain-swapping), mutagenesis of the CDR loops (affinity-enhancement), and/or consolidation of a normal TCR heterodimer into a single-chain format by covalent
  • Mutagenesis of the CDR loops to increase affinity of the TCR to an antigen may be achieved by inserting amino acid substitutions in one or more of the CDR loops of the TCR either empirically or through directed evolution using phage-display libraries.
  • various single-chain TCR chimeras have been used in atempts to limit the problems associated with pairing of endogenous TCRs in a cell.
  • Using genetic engineering such constructs can be freely designed, and ensure a covalent l: l-stoichiometry of the heterodimeric, variable domains.
  • the variable domains are typically covalently connected by a linker (e.g. a short peptide), whereby one of both constant domains is omited.
  • three-domain TCRs can comprise the variable region of the a-chain and the variable and constant region of the P-chain of the TCR.
  • the TCR is a TCR exogenous to the Treg cell, meaning that the cell does not normally express the particular TCR.
  • the TCR is a human TCR, i.e., a TCR wherein at least the variable regions of the TCR chains are human.
  • the qualifier “human” in this connection relates to the primary sequence of the respective peptides, polypeptides, proteins, or nucleic acids, rather than to its origin or source.
  • such peptides, polypeptides, proteins, or nucleic acids may be present in or isolated from samples of human subjects or may be obtained by other means (e.g., by recombinant expression, cell-free transcription or translation, or non-biological nucleic acid or peptide synthesis).
  • a murinized (mu) human TCR i.e. wherein the constant regions are replaced by corresponding murine counterparts
  • the nucleic acid may encode one or more CARs.
  • the nucleic acid may encode at least two, such as two, three, four, five, six, seven or eight, different CARs.
  • CAR Chimeric Antigen Receptor
  • a CAR refers to a recombinant polypeptide or a set of polypeptides, which ,when expressed by an immune effector cell, endows the cell with specificity for a target molecule on the surface of a target cell, and with intracellular signal transduction.
  • a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signalling domain (also referred to herein as “an intracellular signalling domain” or “an intracellular activation domain”) comprising a functional signalling domain derived from a stimulatory molecule and/or a costimulatory molecule.
  • a CAR refers to the functional portion of a protein which acts by transmiting information within the cell to regulate cellular activity via defined signalling pathways by generating second messengers or functioning as effectors by responding to such messengers.
  • a CAR may comprise a chimeric fusion protein, such that for example an antigen binding domain and an intracellular signalling domain are comprised within the same polypeptide chain.
  • a CAR may be formed by a set of polypeptides not contiguous with each other, such that for example an antigen binding domain and an intracellular signalling domain may be provided in separate polypeptide chains, configured to heterodimerise to form the CAR.
  • the antigen binding domain and the intracellular signalling domain may each be provided with a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides containing said domains to one another.
  • first-generation CARs the intracellular signalling domain contains or consists essentially of the zeta chain associated with the T cell receptor complex (CD3Q or the y subunit of the immunoglobulin Fc receptor (FcRy).
  • the cytoplasmic signalling domain of second generation CARs further comprises an intracellular costimulatory domain, i.e., a functional signalling domain derived from at least one costimulatory molecule, such as CD28, 4-1BB (CD137), DAP10, ICOS, or 0X40 (CD 134), and third-generation CARs include a combination of two or more such costimulatory endodomains.
  • the CAR comprises an ectodomain, a transmembrane domain and an intracellular portion.
  • the ectodomain comprises an extracellular antigen recognition domain.
  • antigen or “Ag” as used herein is defined as a molecule capable of being bound by an antigen recognition domain, such as capable being bound to an antibody or receptor (e.g. T-cell receptor).
  • binding domain or “antigen-specific binding domain” as used herein refers to the domain of the CAR that binds to a specific target molecule.
  • a binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a target molecule.
  • the extracellular antigen recognition domain is derived from an antibody or an antibody fragment.
  • antibody is used herein in its broadest sense and generally refers to any immunologic binding agent, such as a whole antibody, including without limitation a chimeric, humanized, human, recombinant, transgenic, grafted and single chain antibody, and the like, or any fusion proteins, conjugates, fragments, or derivatives thereof that contain one or more domains that selectively bind to an antigen of interest.
  • the term antibody thereby includes a whole immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or an immunologically effective fragment of any of these.
  • the term thus specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g., 2-, 3- or more-valent) and/or multi-specific antibodies (e.g., bi- or more-specific antibodies) formed from at least two intact antibodies, and antibody fragments insofar they exhibit the desired biological activity (particularly, ability to specifically bind an antigen of interest), as well as multivalent and/or multi-specific composites of such fragments.
  • multivalent e.g., 2-, 3- or more-valent
  • multi-specific antibodies e.g., bi- or more-specific antibodies
  • antibody is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide, e.g., a recombinantly expressed polypeptide, which is made to encompass at least one complementarity-determining region (CDR) capable of specifically binding to an epitope on an antigen of interest. Hence, the term applies to such molecules regardless whether they are produced in vitro, in cell culture, or in vivo.
  • CDR complementarity-determining region
  • antibody fragment or “antigen -binding moiety” comprises a portion or region of a full length antibody, generally the antigen binding or variable domain thereof.
  • antibody fragments include Fab, Fab', F(ab)2, Fv, scFv fragments, single domain (sd)Fv, such as VH domains , VL domains and VHH domains, diabodies, linear antibodies, single-chain antibody molecules, in particular heavy-chain antibodies; and multivalent and/or multispecific antibodies formed from antibody fragment(s), e.g., dibodies, tribodies, and multibodies.
  • Fab, Fab', F(ab')2, Fv, scFv etc. are intended to have their art- established meaning.
  • a full-length antibody as it exists naturally is an immunoglobulin molecule comprising 2 heavy (H) chains and 2 light (L) chains interconnected by disulfide bonds.
  • the amino terminal portion of each chain includes a variable region of about 100-110 amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein.
  • the carboxyterminal portion of each chain defines a constant region primarily responsible for effector function.
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDRs of the light chain are referred to as “LCDR1, LCDR2, and LCDR3” and the 3 CDRs of the heavy chain are referred to as “HCDR1, HCDR2, and HCDR3.”
  • the CDRs contain most of the residues which form specific interactions with the antigen.
  • the numbering and positioning of CDR amino acid residues within the LCVR and HCVR regions is in accordance with the well-known Kabat numbering convention, which refers to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain regions of an antibody (Kabat, et al., Ann. NYAcad. Sci. 190:382-93 (1971 ); Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242 (1991 )).
  • Kabat numbering convention refers to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain regions of an antibody (Kabat, et al., Ann. NYAcad. Sci. 190:382-93 (1971 ); Kabat, et al., Sequences
  • Light chains are classified as kappa or lambda, and are characterized by a particular constant region as known in the art.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the isotype of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively.
  • IgG antibodies can be further divided into subclasses, e.g., IgGl, IgG2, IgG3, IgG4.
  • Each heavy chain type is characterized by a particular constant region with a sequence well known in the art.
  • an antibody may be any of IgA, IgD, IgE, IgG and IgM classes, and preferably IgG class antibody.
  • the extracellular antigen recognition domain comprises, consists essentially of, or consists of the antigen-binding region of an antibody or an antibody fragment.
  • antigen-binding portion or “antigen-binding region” refers to one or more fragments of an antibody, such as a particular site, part, domain or stretch of amino acid residues, that retain the ability to specifically bind to an antigen of interest. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. These may be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens.
  • binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature, 341 : 544-546 (1989); PCT publication WO 90/05144), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
  • a F(ab')2 fragment a bivalent fragment compris
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) (Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci., 85: 5879-5883 (1988)).
  • scFv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody.
  • Other forms of single chain antibodies, such as diabodies are also encompassed.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (Holliger, et al., Proc. Natl. Acad. Sci., 90: 6444-6448 (1993); Poljak, et al., Structure 2: 1121-1123 (1994)).
  • Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer- Verlag. New York. 790 pp. (ISBN 3-540-41354-5).
  • sequence as used herein (for example in terms like “variable domain sequence”, “VHH sequence” or “protein sequence”), should generally be understood to include both the relevant amino acid sequence as well as nucleic acid sequences or nucleotide sequences encoding the same, unless the context requires a more limited interpretation.
  • sequence as used herein (for example in terms like “variable domain sequence”, “VHH sequence” or “protein sequence”), should generally be understood to include both the relevant amino acid sequence as well as nucleic acid sequences or nucleotide sequences encoding the same, unless the context requires a more limited interpretation.
  • the antigen-binding region of an antibody or an antibody fragment specifically binds to an antigen of interest.
  • specifically bind means that an agent (denoted herein also as “binding agent” or “specific-binding agent”) binds to one or more desired targets (e.g., peptides, polypeptides, proteins, nucleic acids, or cells) substantially to the exclusion of other entities which are random or unrelated, and optionally substantially to the exclusion of other molecules that are structurally related.
  • desired targets e.g., peptides, polypeptides, proteins, nucleic acids, or cells
  • an agent may be said to specifically bind to target(s) of interest if its affinity for such intended target(s) under the conditions of binding is at least about 2-fold greater, preferably at least about 5 -fold greater, more preferably at least about 10-fold greater, yet more preferably at least about 25 -fold greater, still more preferably at least about 50-fold greater, and even more preferably at least about 100-fold, or at least about 1000-fold, or at least about 10 4 - fold, or at least about 10 5 -fold, or at least about 10 6 -fold or more greater, than its affinity for a nontarget.
  • the binding or interaction between the agent and its intended target(s) may be covalent (i.e., mediated by one or more chemical bonds that involve the sharing of electron pairs between atoms) or, more typically, non-covalent (i.e., mediated by non-covalent forces, such as for example, hydrogen bridges, dipolar interactions, van der Waals interactions, and the like).
  • the extracellular antigen recognition domain comprises, consists essentially of, or consists of a single chain variable fragment of an antibody (scFv).
  • the extracellular antigen recognition domain may comprise, consist essentially of, or consist of divalent scFv.
  • CARs comprising di-scFvs
  • two scFvs specific for each antigen are linked together by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs, such as described in Xiong, C.Y. et al., 2006, Protein Engineering Design and Selection 19 (8): 359-367; Kufer, P. et al., 2004, Trends in Biotechnology 22 (5): 238- 244).
  • scFvs may be obtained using standard recombinant DNA techniques.
  • scFvs may be prepared by the isolation of the coding sequence from a hybridoma producing antibodies, identification of V-chain types and design of a nucleic acid encoding the scFv, as described in Koksal H. et al., 2019, Antibody Therapeutics, 2(2):56-63.
  • the scFv may comprise, consist essentially of, or consist of, the VL sequence, the linker peptide, and the VH sequence, wherein the VL sequence is located N-terminally of the linker peptide, and the linker peptide is located N- terminally of the VH sequence.
  • Different scFv designs are possible, such as wherein the position of the VL and VH sequence is swapped.
  • the extracellular antigen recognition domain comprises, consists essentially of, or consists of a single domain variable fragment of a heavy chain antibody (VHH) specific for the antigen or a Nanobody®.
  • VHH heavy chain antibody
  • Nanobody® and “Nanobodies®” are trademarks of Ablynx NV (Belgium).
  • the term “Nanobody” is well-known in the art and as used herein in its broadest sense encompasses an immunological binding agent obtained (1) by isolating the VHH domain of a naturally occurring heavy-chain antibody, preferably a heavy-chain antibody derived from camelids; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanization” of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized VHH domain; (4) by "camelization” of a naturally occurring VH domain from any animal species, and in particular from a mammalian species, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by "camelisation” of a "domain antibody” or “dAb” as described in the art, or by expression
  • single domain antibodies or single variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", see for example WO 05/18629); (6) by using synthetic or semisynthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (7) by preparing a nucleic acid encoding a Nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and/or (8) by any combination of one or more of the foregoing.
  • “Camelids” as used herein comprise old world camelids ⁇ Camelus bactrianus and Camelus dromade rius) and new world camelids (for example Lama paccos, Lama glama and Lama vicugna).
  • the amino acid sequence and structure of a Nanobody can be considered - without however being limited thereto - to be comprised of four framework regions or "FR's", which are referred to in the art and herein as “Framework region 1" or “FR1”; as “Framework region 2" or “FR2”; as “Framework region 3” or “FR3”; and as “Framework region 4" or “FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CDR's”, which are referred to in the art as “Complementarity Determining Region l”or “CDR1”; as “Complementarity Determining Region 2" or “CDR2”; and as “Complementarity Determining Region 3" or “CDR3", respectively.
  • CDR's complementary determining regions
  • the total number of amino acid residues in a Nanobody can be in the region of 110-120, and preferably 112-115. It should however be noted that parts, fragments, analogs or derivatives of a Nanobody are not particularly limited as to their length and/or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are preferably suitable for the purposes described herein.
  • variable domains present in naturally occurring heavy chain antibodies are also be referred to as “VHH domains”, in order to distinguish them from the heavy chain variable domains that are present in conventional 4-chain antibodies (which will be referred to herein as “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to herein as “VL domains”).
  • VHH domains have a number of unique structural characteristics and functional properties which make isolated VHH domains (as well as Nanobodies based thereon, which share these structural characteristics and functional properties with the naturally occurring VHH domains) and proteins containing the same highly advantageous for use as functional antigen-binding domains or proteins.
  • VHH domains (which have been “designed” by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain) and Nanobodies can function as a single, relatively small, functional antigen-binding structural unit, domain or protein.
  • VHH domains from the VH and VL domains of conventional 4-chain antibodies, which by themselves are generally not suited for practical application as single antigen-binding proteins or domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in ScFv's fragments, which consist of a VH domain covalently linked to a VL domain, as described elsewhere in this specification).
  • the antigen-binding region is obtained from a multispecific antibody or antibody fragment (such as a bispecific, trispecific, etc. antibody) comprising at least two (such as two, three, etc.) binding sites, each directed against a different antigen or antigenic determinant.
  • the antigen-binding region may be obtained from antibodies or antibody fragments originating from or comprising one or more portions derived from any animal species, preferably vertebrate species, including, e.g., birds and mammals.
  • the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant.
  • the antibodies may be human, murine (e.g., mouse, rat, etc.), porcine, donkey, rabbit, goat, sheep, guinea pig, monkey (e.g., cynomolus monkeys), camel (e.g., Camelus bactrianus and Camelus dromade ruts) also including camel heavy-chain antibodies, llama (e.g., Lama paccos, Lama glama or Lama vicugna) also including llama heavy-chain antibodies, or horse.
  • the antigen-binding region may be obtained from a chimeric antibody or chimeric antibody fragment, such as a chimeric antibody or chimeric antibody fragment originating from at least two animal species.
  • a chimeric antibody or chimeric antibodies refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as for example antibodies having murine heavy and light chain variable regions linked to human, nonhuman primate, canine, equine, or feline constant regions.
  • Chimeric antibodies comprise a portion of the heavy and/or light chain that is identical to or homologous with corresponding sequences from antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous with corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, exhibiting the desired biological activity (See e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)). Chimeric antibodies are made through merging DNA encoding a portion, such as the Fv region, of a monoclonal antibody from one species, e.g. mouse or monkey, with the antibody-producing DNA from another species, e.g. human.
  • the antigen-binding region may be obtained from a fully human antibody or antibody fragment.
  • the term “fully human antibody” refers to an antibody of which the encoding genetic information is of human origin. Accordingly, the term “fully human antibody” refers to antibodies having variable and constant regions derived only from human germline immunoglobulin sequences. The term “fully human antibody” is thus not to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the antigen-binding region may be obtained from a humanized antibody or antibody fragment.
  • humanized antibody refers to antibodies which comprise heavy and light chain variable region sequences from a non -human species (e.g., a mouse) but in which at least a portion of the VH and/or VU sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences.
  • a CDR-grafted antibody in which non-human CDR sequences are introduced into human VH and VU sequences to replace the corresponding human CDR sequences.
  • antigen-binding region obtained from an antibody or antibody fragment can include one or more amino acid deletions, additions and/or substitutions (e.g., conservative substitutions), insofar such alterations preserve its binding of the respective antigen.
  • the CAR ectodomain or CAR antigen recognition domain comprises, consists essentially of or consists of an antibody-like scaffold, a cognate receptor or ligand for the antigen or an antigen-binding portion of said receptor or ligand, or a synthetic receptor.
  • the CAR ectodomain comprises one or more antibody-like scaffolds.
  • antibody-like scaffold refers to a synthetic or natural binding molecules having a stable scaffold holding the molecule together and a variable arm binding to specific targets thereby mimicking the general structure and function of an antibody.
  • Non-limiting examples of antibody-like scaffolds include designed ankyrin repeat proteins (DARPins), affimers and monobodies.
  • DARPins ankyrin repeat proteins
  • the CAR ectodomain comprises a cognate receptor or ligand for the antigen or an antigen-binding portion of said receptor or ligand.
  • cognate refers to the receptor or ligand with which the target molecule preferentially interacts under physiological conditions, or under in vitro conditions substantially approximating physiological conditions.
  • preferentially interacts is synonymous with “preferentially binding” and refers to an interaction that is statistically significantly greater in degree relative to a control.
  • antigen-binding portion or “antigen-binding region” refers to one or more fragments of a receptor or ligand that retain the ability to specifically bind to an antigen.
  • the CAR ectodomain comprises a synthetic receptor.
  • synthetic receptor or “recombinant receptor” refers to a receptor that cannot be found in nature as such and is being artificially produced by man.
  • a polypeptide sequence can be intentionally modified by man in the laboratory.
  • the CAR includes a single chain variable fragment of an antibody (scFv) or a single domain variable fragment of a heavy chain antibody (VHH) specific for the antigen, an antibody-like scaffold, a cognate receptor or ligand for the antigen or an antigen-binding portion of said receptor or ligand, or a synthetic receptor known to specifically bind to an antigen of interest, such as a tumor antigen or viral antigen
  • the capacity of such CAR to bind to the antigen of interest is meaningfully similar or comparable to the ability of the scFv or VHH specific for the antigen, the antibody-like scaffold, the cognate receptor or ligand for the antigen or the antigen-binding portion of said receptor or ligand, or the synthetic receptor.
  • the “transmembrane domain” is the portion of the CAR that fuses the extracellular binding portion and intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell.
  • the transmembrane domain may be derived either from a natural, synthetic, semisynthetic, or recombinant source.
  • the transmembrane domain may be derived from, such as may comprise, consist essentially of, or consist of, at least the transmembrane region(s) of, the alpha or beta chain of the T-cell receptor, CD3 epsilon, CD3 zeta, CD4, CD5, CD8 (e.g. CD8 alpha), CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, or CD 154, but is not limited thereto.
  • the transmembrane domain comprises, consists essentially of, or consists of the CD28 transmembrane domain or the CD8 alpha transmembrane domain.
  • the CD28 transmembrane domain may be a human CD28 transmembrane domain or the CD8 alpha transmembrane domain may be a human CD8 alpha transmembrane domain.
  • the intracellular portion of the CAR comprises, consists essentially of, or consists of, an intracellular activation domain.
  • intracellular signaling domain or “intracellular activation domain” as used herein refers to intracellular part of a CAR that participates in transducing the message of effective CAR binding to a target molecule into the interior of the immune effector cell to elicit effector cell function (i.e. to perform a specialized function).
  • Functions of the effector cell may encompass activation, cytokine production, proliferation and cytotoxic activity, such as releasing cytotoxic factors to the target cell bound by the CAR.
  • effector cell function refers to a specialized function of the immune effector cell.
  • the intracellular activation domain comprises an immunoreceptor tyrosine-based activation motif or a signaling motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • the primary intracellular activation domain comprises, consists essentially of, or consists of a CD3 zeta activation domain, a FcR gamma activation domain, a FcRbeta activation domain, a CD3 gamma activation domain, a CD3 delta activation domain, a CD3 epsilon activation domain, a CD5 activation domain, a CD22 activation domain, a CD79a activation domain, a CD79b activation domain, a FcsRI activation domain, a CD32 activation domain, a DAP 10 activation domain, a DAP 12 activation domain, and/or a multiple EGF-like domains 10 (MEGF10) activation domain, preferably a CD3 zeta activation domain or a FcR gamma activation domain.
  • the intracellular portion of the CAR further comprises, essentially consists of, or consists of, at least one, such as at least two, costimulatory
  • costimulatory intracellular domain refers to the intracellular domain of a costimulatory molecule of an immune effector cell that specifically binds with a cognate stimulatory ligand, wherein the ligand is present on an antigen presenting cell (e.g. antigen presenting cell (APC), B-cell or dendritic cell).
  • APC antigen presenting cell
  • B-cell B-cell or dendritic cell
  • the costimulatory intracellular domain is able to mediate or enhance the primary response by the immune effector cell in the presence of the primary antigen receptor, such as activation, initiation of an immune response and/or proliferation.
  • the intracellular signaling domain may comprise the entire intracellular portion of the molecule from which it is derived, or the entire intrinsic intracellular signaling domain, or a functional fragment thereof.
  • Non-limiting examples of costimulatory molecules are the costimulatory domain of MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, (CDla / CD18), 4-1BB (CD137), B7-H3, ICAM-1, ICOS (CD278), GITR, CD4, CD27, CD28, CD30, CD40, ICAM- CD8 beta, IL2Rbeta, IL2R gamma, IL7Ralpha, ITGA4, VLA1, CD49a, IT49A, NKp80, NKp80, NKp44, NKp30, NKp46, NKp30, NKp46, CD19, CD4, CD8 alpha, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, IA4, CD49D, ITGA6, VLA-6, CD49f
  • the costimulatory domain most commonly used is that of CD28. This supplies the most potent costimulatory signal - namely immunological signal 2, which triggers proliferation of the immune effector cell, such as T-cell proliferation. Furthermore, TNF receptor family endodomains, such as the 0X40 and 4- IBB, transmit survival signals.
  • the at least one costimulatory intracellular domain comprises, consists essentially of, or consists of the CD28 costimulatory domain, the 4-1BB costimulatory domain, the DNAX-activation protein 10 (DAP10) costimulatory domain, the 0X40 (CD134) costimulatory domain and/or the ICOS (CD278) costimulatory domain.
  • DAP10 DNAX-activation protein 10
  • CD134 0X40
  • CD278 costimulatory domain
  • the CAR comprises two complementary costimulatory intracellular domains, preferably selected from the group consisting of the CD28 costimulatory domain, the 4-1BB costimulatory domain, the OX-40 costimulatory domain, the ICOS costimulatory domain and the CD27 costimulatory domain.
  • the intracellular portion of the CAR comprises, consists essentially of or consists of, at least one intracellular activation domain selected from the group consisting of the CD3 ⁇ and FcRy intracellular activation domain and at least one intracellular costimulatory domain selected from the group consisting of the CD28 costimulatory domain, the 4-1BB costimulatory domain, the DAP 10 costimulatory domain, the 0X40 costimulatory domain and the ICOS costimulatory domain.
  • the CAR may comprise one or more linkers between the ectodomain and the transmembrane domain and/or between the transmembrane domain and the intracellular portion.
  • linkers include flexible linkers such as glycine polymers (G) n , glycine-serine polymers (Gi-5Si-5)n, where n is an integer of at least one, two, three, four, or five, glycine-alanine polymers, alanine-serine polymers, or the like.
  • the CAR ectodomain further comprises one or more hinge regions.
  • hinge region refers to an amino acid sequence located between the antigen recognition domain and the transmembrane domain of the CAR, which is able to position the antigen recognition domain away from the immune effector cell surface to enable proper cellcell contact, antigen binding and activation.
  • the hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • the hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
  • Illustrative hinge domains suitable for use in the CARs include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a, CD4, CD28 and CD7.
  • a TCR or a CAR molecule as discussed here is a transmembrane protein and will typically require the inclusion of a suitable signal or leader sequence when expressed to effect the cellular membrane localisation of the protein.
  • signal sequences are typically short (3-60 amino acids long) N-terminally located peptide chains, which are optionally and advantageously cleaved off or processed away by signal peptidase after the proteins are transported, such as to yield the mature protein.
  • Signal sequences are widely known in the art and they may be applied for the expression of the TCR or CAR as taught herein.
  • the signal sequence comprise, consist essentially of or consist essentially of the leader sequence of CD8 alpha, preferably human CD8 alpha.
  • such signal sequence can be N-terminally fused to any one of the TCR or CAR molecules individualised above.
  • the CAR ectodomain comprises a single chain variable fragment of an antibody (scFv) or a single domain variable fragment of a heavy chain antibody (VHH) specific for the antigen, an antibody-like scaffold, a cognate receptor or ligand for the antigen or an antigen-binding portion of said receptor or ligand, or a synthetic receptor, and/or
  • the intracellular portion of the CAR comprises at least one intracellular activation domain, such as a CD3 ⁇ or FcRy intracellular activation domain, and optionally and preferably at least one intracellular costimulatory domain, such as a CD28, 4-1BB, DAP10, 0X40 and/or ICOS intracellular costimulatory domain.
  • intracellular activation domain such as a CD3 ⁇ or FcRy intracellular activation domain
  • intracellular costimulatory domain such as a CD28, 4-1BB, DAP10, 0X40 and/or ICOS intracellular costimulatory domain.
  • the antigen is an autoantigen, alloantigen, or an allergen.
  • auto-antigen self-antigen
  • a substance e.g., a cell or tissue or a component thereof, that is normally present in the body, but that provokes an immune response.
  • said auto-antigen is involved in the induction and/or progression of an autoimmune disease.
  • the autoantigen is involved in the induction and/or progression of multiple sclerosis, rheumatoid arthritis, type I diabetes, autoimmune uveitis, autoimmune myasthenia gravis, psoriasis, celiac disease, systemic lupus erythematosus, inflammatory bowel disease, Addison’s disease, Graves’ disease, Sjogren’s syndrome, Hashimoto’s thyroiditis, autoimmune vasculitis, pernicious anemia, or idiopathic thrombocytopenic purpura (ITP), or wherein the alloantigen is involved in the induction and/or progression of graft-versus-host disease or in transplant rejection.
  • multiple sclerosis rheumatoid arthritis
  • type I diabetes autoimmune uveitis
  • autoimmune myasthenia gravis psoriasis
  • celiac disease systemic lupus erythematosus
  • inflammatory bowel disease Addison
  • auto-antigens are generally known in the art and it shall be appreciated that the skilled person is capable of selecting an auto-antigen that may be suitable target for the Treg cells according to certain embodiments of the invention.
  • auto-antigens involved in type I diabetes may include one or more of insulin, pro-insulin, glutamic acid decarboxylase 65 (GAD65), GAD67, insulinoma-associated antigen 2 (IAA2), heat shock protein 65 (hsp65), islet cell antigen 69 (ICA69), zinc transporter 8 (ZnT8), and immunodominant peptides thereof;
  • auto-antigens involved in multiple sclerosis may include one or more of myelin-binding protein (MBP), alphaB -crystallin, SlOObeta, proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG)-alpha and MOG-beta isoforms, enolase and arrestin, and immunodominant peptides
  • alloantigen is known in the art and encompasses a genetically determined antigen present in some but not all individuals of a species (as those of a particular blood group) and capable of inducing the production of an alloantibody by individuals which lack it. Alloantigens may for example be involved in the induction and/or progression of graft-versus-host disease or in transplant rejection.
  • allergen is known in the art and encompasses any substance, chemical, particle or composition which is capable of stimulating an allergic response in a susceptible individual. Allergens may be contained within or derived from a food item such as, for example, dairy products (e.g., cow’s milk), egg, celery, sesame, wheat, soy, fish, shellfish, sugars (e.g., sugars present on meat such as alpha-galactose), peanuts, other legumes (e.g., beans, peas, soybeans, etc.), and tree nuts.
  • dairy products e.g., cow’s milk
  • egg celery
  • sesame e.g., sugars present on meat such as alpha-galactose
  • peanuts e.g., peanuts, other legumes (e.g., beans, peas, soybeans, etc.), and tree nuts.
  • an allergen may be contained within or derived from a non-food item such as, for example , dust (e.g., containing dust mite), pollen, insect venom (e.g., venom of bees, wasps, mosquitos, fire ants, etc.), mold, animal fur, animal dander, wool, latex, metals (e.g., nickel), household cleaners, detergents, medication, cosmetics (e.g., perfumes, etc.), drugs (e.g., penicillin, sulfonamides, salicylate, etc.), therapeutic monoclonal antibodies (e.g., cetuximab), ragweed, grass and birch.
  • a non-food item such as, for example , dust (e.g., containing dust mite), pollen, insect venom (e.g., venom of bees, wasps, mosquitos, fire ants, etc.), mold, animal fur, animal dander, wool, latex, metals (
  • pollen allergens include tree pollens such as birch pollen, cedar pollen, oak pollen, alder pollen, hornbeam pollen, aesculus pollen, willow pollen, poplar pollen, plantanus pollen, tilia pollen, olea pollen, Ashe juniper pollen, and Alstonia scholaris pollen.
  • tree pollens such as birch pollen, cedar pollen, oak pollen, alder pollen, hornbeam pollen, aesculus pollen, willow pollen, poplar pollen, plantanus pollen, tilia pollen, olea pollen, Ashe juniper pollen, and Alstonia scholaris pollen.
  • Allergens are involved in the induction and/or progression of allergic reactions, which may include one or more signs or symptoms selected from the group consisting of urticaria (e.g., hives), angioedema, rhinitis, asthma, vomiting, sneezing, runny nose, sinus inflammation, watery eyes, wheezing, bronchospasm, reduced peak expiratory flow (PEF), gastrointestinal distress, flushing, swollen lips, swollen tongue, reduced blood pressure, anaphylaxis, and organ dysfimction/failure.
  • urticaria e.g., hives
  • angioedema e.g., rhinitis
  • rhinitis e.g., asthma, vomiting, sneezing, runny nose, sinus inflammation, watery eyes, wheezing, bronchospasm
  • PEF reduced peak expiratory flow
  • gastrointestinal distress flushing, swollen lips, swollen tongue
  • the endogenous T cell receptor (TCR) of the Treg cells may have been knocked-out or knocked-down. Any method may be used to this end, such as antisense oligonucleotides, RNA interference agents, or genetically engineering the Treg cell’s genome, for example using a gene editing system, such as CRISPR/Cas. In certain embodiments, the present methods are good manufacturing practice (GMP) compliant.
  • TCR T cell receptor
  • method further comprises cryopreservation of the Treg cells comprising the polynucleotide.
  • Cryopreservation media are well known, and may include, for example, a liquid medium comprising 10% v/v DMSO, such as a liquid medium consisting of 90% serum (such as human serum) and 10% DMSO.
  • Cryopreservation temperature may be typically about -80°C, such as in a -80°C freezer or in a liquid nitrogen container.
  • the method may further comprise formulating the Treg cells comprising the polynucleotide into a pharmaceutical composition or a kit-of-parts suitable for medicinal use.
  • the pharmaceutical composition will typically also comprise one or more pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant.
  • a further aspect thus provides the Treg cells comprising the polynucleotide, obtainable or obtained by the methods disclosed herein.
  • a further aspects provides a pharmaceutical composition comprising the Treg cells comprising the polynucleotide, obtainable or obtained by the methods disclosed herein.
  • pharmaceutically acceptable as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.
  • Carriers, diluents, excipients and/or adjuvants include any and all solvents, diluents, buffers (e.g., neutral buffered saline or phosphate buffered saline), solubilizers, colloids, dispersion media, vehicles, fdlers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, stabilisers, antioxidants, tonicity controlling agents, absorption delaying agents, and the like.
  • buffers e.g., neutral buffered saline or phosphate buffered saline
  • solubilizers colloids
  • dispersion media e.
  • the composition may be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds., Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.
  • Liquid pharmaceutical compositions may generally include a liquid carrier such as water or a pharmaceutically acceptable aqueous solution.
  • a liquid carrier such as water or a pharmaceutically acceptable aqueous solution.
  • physiological saline solution, tissue or cell culture media, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the composition may include one or more cell protective molecules, cell regenerative molecules, growth factors, anti-apoptotic factors or factors that regulate gene expression in the cells. Such substances may render the cells independent of its environment.
  • compositions may contain further components ensuring the viability of the cells therein.
  • the compositions may comprise a suitable buffer system (e.g., phosphate or carbonate buffer system) to achieve desirable pH, more usually near neutral pH, and may comprise sufficient salt to ensure isosmotic conditions for the cells to prevent osmotic stress.
  • suitable solution for these purposes may be phosphate-buffered saline (PBS), sodium chloride solution, Ringer's Injection or Lactated Ringer's Injection, as known in the art.
  • the composition may comprise a carrier protein, e.g., albumin (e.g., bovine or human albumin), which may increase the viability of the cells.
  • albumin e.g., bovine or human albumin
  • suitably pharmaceutically acceptable carriers or additives are well known to those skilled in the art and for instance may be selected from proteins such as collagen or gelatine, carbohydrates such as starch, polysaccharides, sugars (dextrose, glucose and sucrose), cellulose derivatives like sodium or calcium carboxymethylcellulose, hydroxypropyl cellulose or hydroxypropylmethyl cellulose, pregeletanized starches, pectin agar, carrageenan, clays, hydrophilic gums (acacia gum, guar gum, arabic gum and xanthan gum), alginic acid, alginates, hyaluronic acid, polyglycolic and polylactic acid, dextran, pectins, synthetic polymers such as water-soluble acrylic polymer or polyvinylpyrrolidone, proteoglycans, calcium phosphate and the like.
  • proteins such as collagen or gelatine
  • carbohydrates such as starch, polysaccharides, sugars (dextrose, glucose and sucrose), cellulose derivatives like
  • the pharmaceutical cell preparation as defined above may be administered in a form of liquid composition.
  • the cells or pharmaceutical composition comprising such can be administered systemically, topically, within an organ, at a site of organ dysfunction or lesion or at a site of tissue lesion.
  • the pharmaceutical compositions may comprise a therapeutically effective amount of the desired cells.
  • therapeutically effective amount refers to an amount which can elicit a biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, and in particular can prevent or alleviate one or more of the local or systemic symptoms or features of a disease or condition being treated. Appropriate therapeutically effective amounts may be determined by a qualified physician with due regard to the nature of the desired cells, the disease condition and severity, and the age, size and condition of the subject.
  • kits of parts comprising a surgical instrument or device for administration of the cells as taught herein or the pharmaceutical compositions as defined herein to a subject, such as for example systemically, for example, by injection, and further comprising the cells as taught herein or the pharmaceutical compositions as defined herein.
  • the pharmaceutical composition as define above may be administered in a form of a liquid composition.
  • the quantity of cells to be administered will vary for the subject being treated.
  • the quantity of cells to be administered is between 10 2 to IO 10 or between 10 2 to 10 9 , or between 10 3 to IO 10 or between 10 3 to 10 9 , or between 10 4 to IO 10 or between 10 4 to 10 9 , such as between 10 4 and 10 8 , or between 10 5 and 10 7 , e.g., about IxlO 5 , about 5xl0 5 , about IxlO 6 , about 5xl0 6 , about IxlO 7 , about 5xl0 7 , about IxlO 8 , about 5xl0 8 , about IxlO 9 , about 5xl0 9 , or about IxlO 10 cells can be administered to a human subject.
  • such administration may be suitably distributed over one or more doses (e.g., distributed over 2, 3, 4, 5, 6, 7, 8 9 or 10 or more doses) administered over one or more days (e.g., over 1, 2, 3, 4 or 5 or more days).
  • doses e.g., distributed over 2, 3, 4, 5, 6, 7, 8 9 or 10 or more doses
  • days e.g., over 1, 2, 3, 4 or 5 or more days.
  • the precise determination of a therapeutically effective dose may be based on factors individual to each patient, including their size, age, tissue damage, and can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.
  • cells in a composition to be administered, may be present at a concentration between about 10 4 /ml to about 10 9 /ml, preferably between about 10 5 /ml and about 10 8 /ml, yet more preferably between about lxl0 6 /ml and about lxl0 8 /ml.
  • a further aspect provides the T reg cells or the pharmaceutical compositions as taught herein, for use in medicine, i.e., for use in therapy.
  • Reference to “therapy” or “treatment” broadly encompasses both curative and preventative treatments, and the terms may particularly refer to the alleviation or measurable lessening of one or more symptoms or measurable markers of a pathological condition such as a disease or disorder.
  • the terms encompass primary treatments as well as neo-adjuvant treatments, adjuvant treatments and adjunctive therapies. Measurable lessening includes any statistically significant decline in a measurable marker or symptom.
  • the terms encompass both curative treatments and treatments directed to reduce symptoms and/or slow progression of the disease.
  • the terms encompass both the therapeutic treatment of an already developed pathological condition, as well as prophylactic or preventative measures, wherein the aim is to prevent or lessen the chances of incidence of a pathological condition.
  • the terms may relate to therapeutic treatments. In certain other embodiments, the terms may relate to preventative treatments. Treatment of a chronic pathological condition during the period of remission may also be deemed to constitute a therapeutic treatment.
  • the term may encompass ex vivo or in vivo treatments as appropriate in the context of the present invention.
  • subject typically and preferably denote humans, but may also encompass reference to non-human animals, preferably warm-blooded animals, even more preferably non-human mammals. Particularly preferred are human subjects including both genders and all age categories thereof. In other embodiments, the subject is an experimental animal or animal substitute as a disease model. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. The term subject is further intended to include transgenic non-human species.
  • subject in need of treatment refers to subjects diagnosed with or having a disease as recited herein and/or those in whom said disease is to be prevented.
  • a further aspect provides T reg cells or the pharmaceutical composition as taught herein, wherein the polynucleotide encodes a polypeptide which endows the Treg cell with specificity to an antigen or an antigenic peptide thereof, for use in a method of treating a disease caused by or associated with an increased activity of the immune system against said antigen.
  • antigen may for example be an auto-antigen (such as an auto-antigen causative of or associated with an autoimmune disease, such as an autoimmune disease discussed elsewhere in this specification), or may be an allergen, or an allo-antigen.
  • a related aspect provides a method for treating, in a subject in need thereof, a disease caused by or associated with an increased activity of the subject’s immune system against an antigen, comprising administering to the subject an effective amount of the T reg cells or the pharmaceutical composition as taught herein, wherein the polynucleotide encodes a polypeptide which endows the Treg cell with specificity to the antigen or an antigenic peptide thereof.
  • the Treg cells are CD45RA + Treg cells, preferably an at least 90% pure population of CD45RA + Treg cells; or
  • the Treg cells are CD25 hl Treg cells, preferably an at least 90% pure population of CD25 hl Treg cells.
  • Treg cells have been isolated from peripheral blood mononuclear cells (PBMC) or from an internal organ, such as lung, liver, or spleen.
  • PBMC peripheral blood mononuclear cells
  • T cells preferably CD4 + T cells and/or CD8 + cells
  • PBMC PBMC or from an internal organ, thereby obtaining a population of T cells, preferably CD4 + T cells and/or CD8 + cells
  • Treg cells from the population of T cells; or b) wherein the isolation comprises directly isolating Treg cells from PBMC or from an internal organ.
  • CD4 + cells and/or CD8 + cells from PBMC or from an internal organ, thereby obtaining a population of CD4 + cells and/or CD8 + cells;
  • the T cells or CD4 + or CD8 + cells are isolated from PBMC or from the internal organ using magnetic-activated cell sorting, and/or wherein the Treg cells are isolated from the population of T cells or CD4 + or CD8 + cells using fluorescence-activated cell sorting (FACS); or
  • Treg cells are directly isolated from PBMC or from the internal organ using FACS.
  • Statement 9 The method according to any one of Statements 3 to 7, wherein the Treg expansion comprises a step of culturing the isolated Treg cells in the presence of interleukin-2 (IL-2).
  • IL-2 interleukin-2
  • Statement 10 The method according to any one of Statements 1 to 9, wherein the Treg cells are activated prior to electroporation, preferably wherein the Treg activation comprises a step of contacting the isolated and optionally expanded Treg cells with an anti-CD3 antibody and an anti- CD28 antibody.
  • Statement 11 The method according to Statement 10, wherein the antibodies are covalently linked to a polymer carrier.
  • Statement 12 The method according to Statement 10 or 11, wherein the contacting step is repeated two or more times during the Treg expansion and activation.
  • Statement 13 The method according to Statement 12, wherein the contacting step is repeated on days 0, 7, 14, and 19 of the Treg expansion and activation.
  • Statement 14 The method according to any one of Statements 1 to 13, wherein the concentration of the Treg cells in the suspension is 100 cells per ml to IxlO 9 cells per ml, preferably IxlO 7 to IxlO 8 cells per ml, such as about 2.5xl0 7 cells per ml.
  • Statement 15 The method according to any one of Statements 1 to 13, wherein the voltage is from 100 V to 700 V, preferably from 400 V to 600 V, such as about 500 V.
  • Statement 16 The method according to any one of Statements 1 to 15, wherein the pulsing time is from 1 to 40 ms, preferably from 1 to 10 ms, such as about 5 ms.
  • Statement 17 The method according to any one of Statements 1 to 16, wherein the pulse is a square wave pulse.
  • Statement 18 The method according to any one of Statements 1 to 17, wherein the concentration of the polynucleotide in the suspension is from 100 ng / IxlO 6 cells to 10 pg / IxlO 6 cells, preferably from 500 ng / IxlO 6 cells to 5 pg / IxlO 6 cells, more preferably about 1 pg / IxlO 6 cells.
  • Statement 19 The method according to any one of Statements 1 to 18, wherein the polynucleotide is synthetic, or in vitro transcribed, or isolated from a host cell or a non-human host organism genetically engineered to produce the polynucleotide.
  • Statement 20 The method according to any one of Statements 1 to 19, wherein the polynucleotide is a naked polynucleotide.
  • Statement 21 The method according to any one of Statements 1 to 20, wherein the polynucleotide is a linear polynucleotide.
  • Statement 22 The method according to any one of Statements 1 to 21, wherein the modified RNA polynucleotide is nucleobase and/or backbone-modified.
  • Statement 23 The method according to any one of Statements 1 to 22, wherein the polynucleotide is coding or non-coding.
  • Statement 24 The method according to any one of Statements 1 to 23, wherein the polynucleotide is selected from the group consisting of messenger RNA (mRNA), guide RNA (gRNA), single guide RNA (sgRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), long non-coding RNA (IncRNA), ribozyme, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer, aptamer,
  • Statement 25 The method according to any one of Statements 1 to 24, wherein the polynucleotide is mRNA encoding one or more polypeptide, preferably one or more biologically active polypeptide.
  • Statement 26 The method according to any one of Statements 1 to 25, wherein the polynucleotide produces a gain-of-fimction phenotype when introduced into the Treg cell, such as wherein the polynucleotide is mRNA encoding one or more polypeptide, preferably one or more biologically active polypeptide, and the polypeptide produces a gain-of-fimction phenotype when expressed by the Treg cell.
  • Statement 27 The method according to Statement 26, wherein the one or more polypeptide endows the Treg cell with specificity to an antigen or an antigenic peptide thereof.
  • Statement 28 The method according to Statement 27, wherein the one or more polypeptide is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • the CAR ectodomain comprises a single chain variable fragment of an antibody (scFv) or a single domain variable fragment of a heavy chain antibody (VHH) specific for the antigen, an antibody-like scaffold, a cognate receptor or ligand for the antigen or an antigen-binding portion of said receptor or ligand, or a synthetic receptor, and/or
  • the intracellular portion of the CAR comprises at least one intracellular activation domain, such as a CD3 ⁇ or FcRy intracellular activation domain, and optionally and preferably at least one intracellular costimulatory domain, such as a CD28, 4-1BB, DAP10, 0X40 and/or ICOS intracellular costimulatory domain.
  • intracellular activation domain such as a CD3 ⁇ or FcRy intracellular activation domain
  • intracellular costimulatory domain such as a CD28, 4-1BB, DAP10, 0X40 and/or ICOS intracellular costimulatory domain.
  • Statement 30 The method according to any one of Statements 27 to 29, wherein the antigen is an autoantigen, alloantigen, or an allergen.
  • Statement 31 The method according to Statement 28, wherein the autoantigen is involved in the induction and/or progression of multiple sclerosis, rheumatoid arthritis, type I diabetes, autoimmune uveitis, autoimmune myasthenia gravis, psoriasis, celiac disease, systemic lupus erythematosus, inflammatory bowel disease, Addison’s disease, Graves’ disease, Sjogren’s syndrome, Hashimoto’s thyroiditis, autoimmune vasculitis, pernicious anemia, or idiopathic thrombocytopenic purpura (ITP), or wherein the alloantigen is involved in the induction and/or progression of graft-versus-host disease or in transplant rejection.
  • the autoantigen is involved in the induction and/or progression of multiple sclerosis, rheumatoid arthritis, type I diabetes, autoimmune uveitis, autoimmune myasthenia gravis, psoriasis, celia
  • Statement 32 The method according to any one of Statements 1 to 31, wherein the endogenous T cell receptor (TCR) of the Treg cells has been knocked-out or knocked-down.
  • TCR T cell receptor
  • Statement 33 The method according to any one of Statements 1 to 32, wherein the method is good manufacturing practice (GMP) compliant.
  • GMP good manufacturing practice
  • Statement 34 The method according to any one of Statements 1 to 33, wherein the method further comprises cryopreservation of the Treg cells comprising the polynucleotide.
  • Statement 35 The method according to any one of Statements 1 to 34, wherein the method further comprises formulating the Treg cells comprising the polynucleotide into a pharmaceutical composition or a kit-of-parts suitable for medicinal use.
  • Treg cells comprising the polynucleotide, obtainable or obtained by the method of any one of Statements 1 to 35.
  • Statement 37 A pharmaceutical composition comprising the Treg cells comprising the polynucleotide, obtainable or obtained by the method of any one of Statements 1 to 35.
  • T reg cells according to Statement 36 or the pharmaceutical composition according to Statement 37 for use in medicine.
  • Statement 39. The T reg cells according to Statement 36 or the pharmaceutical composition according to Statement 37, wherein the polynucleotide encodes a polypeptide which endows the Treg cell with specificity to an antigen or an antigenic peptide thereof, for use in a method of treating a disease caused by or associated with an increased activity of the immune system against said antigen.
  • a method for treating, in a subject in need thereof, a disease caused by or associated with an increased activity of the subject’s immune system against an antigen comprising administering to the subject an effective amount of the T reg cells according to Statement 36 or the pharmaceutical composition according to Statement 37, wherein the polynucleotide encodes a polypeptide which endows the Treg cell with specificity to the antigen or an antigenic peptide thereof.
  • Buffy coats from anonymous healthy donors were provided by the Blood Service of the Flemish Red Cross (Mechelen, Belgium). This study was approved by the Ethics Committee of the University of Antwerp and the Antwerp University Hospital (Belgium) under reference number EC 18/18/236. Information, if known, of the healthy donors used in this study is depicted in Table 1.
  • Table 1 Known information of the anonymous healthy donors used in this study. For each donor used in this study, year of birth and sex is given.
  • PBMCs Human peripheral blood mononuclear cells
  • PBMCs Human peripheral blood mononuclear cells
  • CD4 + cells were positively isolated from 400 x 10 6 PBMCs using human CD4 MicroBeads for magnetic- activated cell sorting (MACS, Miltenyi Biotec, Leiden, Netherlands), according to the manufacturer’s instructions.
  • Isolated CD4 + cells were stained with fluorochrome-conjugated monoclonal antibodies (mAbs) (Table 2).
  • CD25 hl Tregs were sorted as CD3 + , CD4 + , CD127" and CD25hi; naive Tregs were sorted as CD3 + , CD4 + , CD 127’, CD25 + and CD45RA + ; and effector CD4 + T cells were sorted as CD3 + , CD4 + , CD127 + and CD25" ( Figure 1A). Remaining CD8 + T cells, monocytes, natural killer (NK) cells and B cells were excluded from the sorting gate using a dump channel consisting of anti-CD8, -CD14, -CD16 and -CD19. Sorting was performed by flow cytometry using a FACSAria II device (BD Biosciences). After flow cytometric cell sorting, an aliquot of the sorted cells was used to confirm purity.
  • Tregs were expanded ex vivo in complete medium, consisting of Iscove’s modified Dulbecco’s medium (IMDM; Life Technologies) supplemented with 5% human AB serum (hAB; Life Technologies) and 500 lU/mL IL-2 (ImmunoTools GmbH, Friesoythe, Germany). Treg activation was achieved by using T cell TransAct (1: 100 dilution, Miltenyi Biotec) on day 0. TransAct is a clinical-grade colloidal reagent comprising iron oxide crystals embedded into a biocompatible polysaccharide matrix with a diameter of ⁇ 100 nm. Agonistic anti-CD3 and anti-CD28 antibodies are coated onto the nanomatrix. Reactivation was performed on days 7 and 14.
  • TCR recognizing HLA-DR2_restricted myelin basic protein (MBP)s5-99 peptide was kindly provided by Prof. Dr. David W. Scott of the Uniformed Services School of Health Sciences (USUHS) in Bethesda, MD (Kim et al. Journal of Autoimmunity 2018, vol. 92, 77-86). Sequences encoding the TCR a- and P-chains were linked with the 2A sequence from porcine teschovirus-1 (P2A) (Szymczak et al. Nature Biotechnology 2004, vol. 22, 589-594). The sequences were cloned into the Spel-Xhol site of the pSTl plasmid (kindly provided by Dr.
  • P2A porcine teschovirus-1
  • plasmids were propagated in Escherichia coli SoloPack Golden supercompetent cells (Agilent Technologic, Machelen, Belgium), and plasmid DNA was purified using a NucleoBond Xtra Midi EF kit (Macherey-Nagel, Duren, Germany). Next, purified plasmid DNA was linearized by SapI digestion (Thermo Fisher Scientific) for the MBPgj-gg-specific TCR plasmid and by Spel digestion (Thermo Fisher Scientific) for the eGFP plasmid.
  • Tregs were electroporated with mRNA encoding the MBP85-99- specific TCR. Tregs were washed twice and resuspended in cold serum-free Opti-MEM I medium (Gibco Invitrogen) at a concentration of 25xl0 6 cells/mL. 200 pL of the cell suspension was transferred to a 4.0-mm electroporation cuvette (Cell Projects, Kent, United Kingdom), and 1 pg/10 6 cells of in vitro transcribed mRNA was added to the cuvette. Electroporations were performed with a Gene Pulser Xcell device (Bio-Rad, Temse, Belgium) using a square wave pulse of 500 V for 5 ms.
  • Opti-MEM I medium Gibco Invitrogen
  • PBMCs Remaining PBMCs, not used for CD4+ isolation using MACS, and transfected Tregs, used for post-cryopreservation kinetics of the transgenic TCR, were washed and resuspended in cryopreservation medium consisting of fetal bovine serum (FBS; Life Technologies) supplemented with 10% DMSO (Sigma-Aldrich, Diegem, Belgium). Aliquots were stored in a -80°C freezer. When needed, cells were thawed in prewarmed IMDM supplemented with 10% hAB serum.
  • FBS fetal bovine serum
  • DMSO Sigma-Aldrich, Diegem, Belgium
  • Dry pelleted cell samples (0.5 x 10 6 cells; centrifuged at 480g for 5 min) of CD127 + CD25" effector CD4 + T cells and CD45RA + or CD25 hl Tregs were collected before and after ex vivo expansion and mRNA electroporation, stored at -80°C and shipped on dry ice to EpigenDx (Hopkinton, MA) to perform TSDR methylation analysis (assay ID ADS783-FS2). The analysis covered nine CpG sites spanning positions -2263 to -2330 (upstream from the ATG start codon) of FOXP3.
  • pelleted cell samples were lysed using ZymoResearch Mdigestion buffer and 20 mg/mL protease K (ZymoResearch, Irvine, CA), and incubated at 65°C for a minimum of 2 h.
  • supernatants from the sample lysate were bisulfite modified using EZ-96 DNA Methylation-Direct kit (ZymoResearch) as per the manufacturer’s protocol with minor modifications.
  • Polymerase chain reactions PCRs were performed using 1 pL of the bisulfite-treated DNA and 0.2 pM of each primer (EpigenDx’s proprietary information).
  • One primer was biotin labeled and HPLC purified to purify the final PCR product using Sepharose beads.
  • PCR product was bound to Streptavidin Sepharose HP (GE Healthcare Life Sciences), after which the immobilized PCR products were purified, washed, denatured with a 0.2-pM NaOH solution, and washed again using the Pyrosequencing Vacuum Prep Tool (Pyrosequencing, Qiagen), as per the manufacturer’s protocol.
  • Pyrosequencing Vacuum Prep Tool
  • 0.5 pM of sequencing primer was annealed to the purified single-stranded PCR products, and 10 pL of the PCR products were sequenced by Pyrosequencing on the PSQ96 HS System (Pyrosequencing, Qiagen) following the manufacturer’s instructions.
  • the methylation status of each CpG site was determined individually as an artificial C/T singlenucleotide polymorphism (SNP) using QCpG software (Pyrosequencing, Qiagen).
  • the methylation level at each CpG site was calculated as the percentage of the methylated alleles divided by the sum of all methylated and unmethylated alleles.
  • the mean methylation level was calculated using methylation levels of all measured CpG sites within the targeted region of each gene.
  • Each experiment included non-CpG cytosines as internal controls to detect incomplete bisulfite conversion of the input DNA.
  • a series of unmethylated and methylated DNA were included as controls in each PCR.
  • PCR bias testing was performed by mixing unmethylated control DNA with in vitro methylated DNA at different ratios (0%, no methylated DNA; 5%; 10%; 25%; 50%; 75%; and 100%, only methylated DNA), followed by bisulfite modification, PCR, and Pyrosequencing analysis. For female donors, this ratio was corrected with a factor 2 since one of the two TSDR alleles is methylated because of X-inactivation.
  • Effector CD4 + T cells were stained using a CellTrace Violet Cell Proliferation kit (Thermo Fisher Scientific), which allows tracking of cell division, according to the manufacturer’s instructions. Subsequently, stained effector CD4 + T cells were stimulated with Treg Suppression Inspector beads (Miltenyi Biotec) in a 1 : 1 ratio, providing optimal polyclonal stimulation of effector CD4 + T cells based on anti-CD2, anti-CD3 and anti-CD28, while incremental numbers of Tregs were added to the cell culture. More specifically, the different effector CD4 + T cell/Treg ratios were 2:0, 1:0, 1: 1, 2: 1, 4: 1, 8: 1 and 16: 1.
  • Expanded Tregs were cultured (0.5 x 10 6 cells/mL) in IMDM supplemented with 5% hAB serum (i.e., control, not activated) or in complete medium with TransAct (1: 100 dilution), mimicking TCR activation. Subsequently, these cell cultures were incubated in a humidified 5% CO2 incubator at 37°C. As a positive control, thawed cryopreserved autologous effector CD4 + cells were cultured in the same conditions. As a negative control, cell-free IMDM supplemented with 5% hAB serum and complete medium was used.
  • a volume of 500 pl culture medium of each condition was extracted for the simultaneous quantitative determination of both natural and recombinant human interferon-y (IFN-y), IL-2, IL-4, IL-5, IL-10, IL-13 and tumor necrosis factor a (TNF-a) using a chemiluminescence-based assay from Meso Scale Discovery (Human TH1/TH2 10-Plex Tissue Culture Kit, MSD, Gaithersburg, MD), according to the manufacturer’s instructions. The plate was washed and read with MSD reading buffer on the QuickPlex SQ 120 (MSD). All conditions were measured in duplicate and run at the same time. Background measurements of nonactivated cells were deducted from the measurements of corresponding activated cells.
  • 2D3 cells were generated from TCR-deficient Jurkat 76 cells (human acute T cell leukemia), as described before (Versteven et al. Oncotarget 2018, vol. 9, 27797-808).
  • SKW-3 cells were purchased from cell bank of German Collection of Microorganisms and Cell Cultures. Exponential growth was maintained by culturing the cells in RPMI 1640 (Gibco Invitrogen) supplemented with 10% FBS.
  • FACS data were analyzed using FlowJo software version 10.5.3 (TreeStar, Ashland, OR), and multiplex data were analyzed using Discovery Workbench 4.0 software. Results were analyzed using Prism software version 8 (GraphPad, San Diego, CA), and given as mean values ⁇ standard deviation (SD). Statistical analysis was performed using nonparametric Kruskal -Wallis test or Friedman test, followed by a post hoc Dunn’s multiple comparison test where applicable. For transgenic TCR expression over time, mixed-models test with the Geisser-Greenhouse correction, followed by a post hoc Dunnett’s multiple comparisons test, was used. Any P value ⁇ 0.05 is considered statistically significant.
  • Treg-mediated suppressive capacity of the FACSsorted Treg populations we evaluated their suppression on CD4 CD 127 CD25 effector T cell proliferation, induced by in vitro stimulation with beads coated with anti-CD2, anti-CD3 and anti-CD28 ( Figure 1C and D).
  • Tregs were able to suppress effector T cell proliferation at different TeffTreg ratios compared with effector T cells alone (no suppression of T cells).
  • No significant difference in suppression of proliferation rate (P 0.7434) was observed between the 2:0 ratio (-21.7% ⁇ 26.8% suppression of T cells) and the 1:0 ratio, excluding the possibility that proliferation differences occur based on T cell numbers in the cell culture instead of Treg presence.
  • Example 4 Electroporation of expanded Tregs with TCR-encoding mRNA resulted in significant amounts of TCR-expressing Tregs
  • Tregs were electroporated with eGFP-encoding mRNA. On average, we observed 98.5% ⁇ 0.8% eGFP- expressing CD45RA + Tregs and 98.1% ⁇ 0.9% eGFP -expressing CD25 hl Tregs 24 h after electroporation ( Figure 4).
  • Example 6 GMP-compliant activation, expansion and transfection of CD45RA + and CD25 hl Tregs did not affect CTLA-4 and CCR4 expression but showed a slight decrease in Helios expression by CD25 hl Tregs
  • Example 7 GMP-compliant expanded and transfected Tregs were capable of inducing in vitro immunosuppression and produced anti-inflammatory, but not pro-inflammatory, cytokines
  • Example 8 TCR-dependent stimulation of cells electroporated with MBPss-99-specific TCR- encoding mRNA led to cell activation
  • the present Examples provide an illustration of a GMP -compliant and easy-to-use protocol for the expansion and genetic engineering of two different Treg subtypes according to certain embodiments of the invention.
  • the expansion and mRNA-based genetic engineering of both CD45RA + and CD25 hl Tregs did not negatively affect the Treg characteristics of both subtypes.
  • Our data indicate no significant difference in the expression levels of the Treg master regulator FOXP3, expression after activation, expansion and transfection of both CD45RA + Tregs and CD25 hl Tregs.
  • Tregs were still capable of producing anti-inflammatory cytokines after anti-CD3 and anti-CD28 activation of expanded and mRNA-electroporated Tregs, indicative of the stability of the Treg phenotype and function.
  • Tregs remained functional after in vitro expansion and mRNA electroporation, as indicated by their capacity to inhibit the proliferation of CD127 + CD25" effector T cells in vitro in a ratio-dependent manner.
  • the illustrated protocols provide GMP -compliant approach for ex vivo expansion and RNA-based engineering of Tregs, which is convenient and robust and allows for transient genetic engineering of different subtypes of Tregs, without affecting Treg phenotype and function.
  • Our findings offer new opportunities for RNA engineering of Tregs for future clinical applicability, in which this approach can be used for the induction of antigen specificity or evaluating possible gain of function, after introduction of proteins or cytokines involved in Tregs’ mechanism of action.

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Abstract

The invention relates to methods for modifying T regulatory cells by introducing RNApolynucleotides thereto by electroporation, and further pertains to the modified Treg cells and their compositions and uses.

Description

T REGULATORY CELL MODIFICATION
FIELD
The invention is broadly applicable in the cell biology and medical fields, and more particularly concerns methods for introducing exogenous polynucleotides into regulatory T cells (Tregs), and further pertains to the modified Treg cells and their compositions and uses.
BACKGROUND
The therapeutic landscape for autoimmune diseases and transplant rejection is constantly advancing. However, despite progress in targeted biologic and pharmacologic interventions, none of the currently available treatments results in permanent stabilization of disease, and most of the current treatments indiscriminately suppress the immune system. Moreover, general immune modulation may be accompanied by undesired adverse events, such as opportunistic infections and secondary autoimmunity. Breakthroughs in cell and molecular biology have enabled the development of cell-based vaccines. The potential of T regulatory cells (Tregs) as an adoptive cell therapy for the treatment of autoimmune diseases and transplant rejection seems undisputed. Indeed, Tregs can suppress not only CD4+ T cells, but also CD8+ T cells and many other immune cells when recruited to an identical antigen-presenting cell.
Tregs are the most potent immunosuppressive cells in the human body and play a pivotal role in the delicate but crucial balance between immunity and tolerance. For instance, Tregs can directly interact with and downmodulate self-reactive T cells, thereby regulating self-tolerance and ultimately preventing the development of autoimmunity. Tregs have been described as CD4+ T cells or CD8+ T cells which display the expression of high levels of the interleukin (IL)-2 receptor a chain (IL-2Ra, also called CD25) and the expression of the master regulator forkhead box P3 (FOXP3) transcription factor. FOXP3 orchestrates the transcriptional machinery of Tregs by binding >1400 genes and acting as both a transcriptional repressor and an activator of the expression of genes associated with the function of Tregs, including IL2RA and cytotoxic T lymphocyte associated protein 4 (CTLA-4). Although the importance of FOXP3 in Tregs has long been undisputed, activated conventional T cells can also temporarily express FOXP3 as a negative feedback mechanism, and FOXP3 -independent maintenance of the human Treg identity has been shown in FOXP3-ablated Tregs.
Because stable FOXP3 expression is inversely correlated with the expression of IL-7R (CD 127), the combination of positive expression of CD25 and negative expression of CD 127 is a commonly used strategy to flow cytometrically isolate viable Tregs. Current insights define Tregs as a heterogeneous mixture of cellular sub-phenotypes with a high degree of phenotypic complexity reflecting distinct developmental states, methods of suppression, homing properties and suppression targets. For instance, CD4+CD127 CD25hl Tregs (also denoted CD25hl Treg cells) are characterized by high expression of the Treg master regulator FOXP3, whereas CD4 CD I 27 CD25+CD45RA+ Tregs (also denoted CD45RA+ Treg cells), expressing the naivety marker CD45RA, are superior for expansion purposes.
To induce tolerance, Tregs exploit a broad spectrum of suppressive mechanisms, including cell contact dependent mechanisms involving CTLA-4 and the secretion of immune regulatory cytokines such as IL-10 and transforming growth factor (TGF- ). These mechanisms are influenced by the surrounding microenvironment, the type of immune reaction and the target cell. Furthermore, Tregs can transfer suppressor activity to conventional CD4+ T cells. This process, termed “infectious tolerance,” creates a local tolerogenic environment in which naive T cells convert into an induced Treg phenotype. In addition, Tregs are responsible for “bystander suppression” by inducing tolerance to cells without direct interaction.
Tregs are present throughout the body, and can be conveniently sourced from peripheral blood. Treg frequencies have been reported to be about 5% to 7% of CD4+ T cells in the periphery. A broad range of Treg isolation and expansion protocols have been developed (MacDonald et al. Clinical and Experimental Immunology 2019, vol. 197, 52-63), to arrive at satisfactory Treg numbers while preserving the desired Treg characteristics. A suitable activation reagent for ex vivo expansion of Tregs partially mimics the interaction with antigen-presenting cells, using anti-CD3 and anti-CD28 monoclonal antibodies covalently linked to magnetic beads (Trickett and Kwan. Journal of Immunological Methods 2003, vol. 275, 251-255), while many other clinical-grade Tregs isolation and expansion methods have been described.
While safe and feasible, only modest clinical efficacy was observed with unmodified Tregs. This could be, at least in part, because polyclonal Tregs were used, collectively targeting a broad mix of antigens, not all disease-related, and therefore potentially weakening their clinical effect. This prompted the field to move into a more antigen-specific approach to generate Tregs, aiming for a durable patient-tailored cell therapy without the risk for general immunosuppression. Preclinical studies demonstrated increased potency of antigen-specific Tregs compared with polyclonal Tregs in models of type 1 diabetes and transplantation. Mutatis mutandis Tregs can also be modulated for increased stability of their phenotype and function by introducing key regulators for Treg function, such as Helios, after introduction of cytokines, such as IL-10 and TGF-P involved in Tregs’ mechanism of action, or by creating Tregs that gained new functions, e.g. regenerative capacity by introducing neurotrophic factors such as brain-derived neurotrophic factor (BDNF) or amphiregulin. Different genetic engineering technologies, including retro- and lentiviral transduction as well as nonviral transfection methods have been explored to introduce the expression of TCRs or chimeric antigen receptors into Tregs. Transduction of cells using viral vectors is the most used method resulting in high transfection efficiency. However, a number of limitations have been attributed to the use of viral vectors, including host immune responses against the viral vector, potential insertional mutagenesis, the maximum insert size, variability of infection potencies, the laborious viral vector production and introduction, and the elevated laboratory costs because of the requirement for a higher biosafety level.
Previous studies have demonstrated the possibility to introduce RNA such as messenger RNA (mRNA) into certain cell types such as human hematopoietic cells by means of electroporation (W02003000907; Van Tendeloo et al. Blood 2001, vol. 98, 49-56).
SUMMARY
The present invention is at least in part based on the discovery that T regulatory cells (Treg cells or Tregs) are amenable to genetic engineering by means of RNA electroporation, and allow for highly efficient delivery of RNA molecules into the cells. Moreover, while the reaction of various immune cell types to non-physiological external treatments and to the introduction of exogenous materials tends to be unpredictable, the present inventors have discovered that the delivered RNA is not recognised as a danger signal by Treg cells and that the modified Treg cells retain their immunosuppressive functions, such as their capacity to inhibit the proliferation of T effector cells.
Advantageously, compared to viral delivery of genes, RNA is safer without the risk of insertional mutagenesis. Additionally, messenger RNA (mRNA) has some advantages over DNA, mostly because no translocation to the nucleus is needed, with no risk of integration into the genome, while expression levels can be adjusted with the amount of supplied mRNA, expression is almost instant, and it does not rely on promoter strength. Moreover, introduced mRNA results in transient gene expression, being subjected to the natural decay of mRNA, providing an accurate system to control the synthesis of exogenous proteins. Also, electroporation is safe, versatile and robust, which can simplify its adoption in the clinic.
In view of the inventors’ unexpected findings, an aspect of the invention provides a method for introducing an unmodified or modified RNA polynucleotide into a Treg cell, comprising electroporation of a suspension comprising the polynucleotide and Treg cells.
A further aspect provides Treg cells comprising an unmodified or modified RNA polynucleotide, wherein the cells are obtainable or obtained by a method comprising electroporation of a suspension comprising the polynucleotide and Treg cells. Also provided is a pharmaceutical composition comprising Treg cells comprising an unmodified or modified RNA polynucleotide, wherein the cells are obtainable or obtained by a method comprising electroporation of a suspension comprising the polynucleotide and Treg cells.
A further aspect provides said Treg cells comprising the unmodified or modified RNA polynucleotide, or said pharmaceutical composition comprising said Treg cells, for use in medicine.
An aspect provides said Treg cells comprising the unmodified or modified RNA polynucleotide, or said pharmaceutical composition comprising said Treg cells, wherein the polynucleotide encodes a polypeptide which endows the Treg cell with specificity to an antigen or an antigenic peptide thereof, for use in a method of treating a disease caused by or associated with an increased activity of the immune system against said antigen.
Further provided is a method for treating, in a subject in need thereof, a disease caused by or associated with an increased activity of the subject’s immune system against an antigen, comprising administering to the subject an effective amount of said Treg cells comprising the unmodified or modified RNA polynucleotide, or said pharmaceutical composition comprising said Treg cells, wherein the polynucleotide encodes a polypeptide which endows the Treg cell with specificity to an antigen or an antigenic peptide thereof.
These and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject-matter of the appended claims is hereby specifically incorporated in this specification.
BRIEF DESCRIPTION OF DRAWINGS
The following description of the figures of specific embodiments of the invention is merely exemplary in nature and is not intended to limit the present teachings, their application or uses.
Fig. 1 illustrates multiparametric flow cytometry-based sorting of CD45RA+ and CD25hl Tregs and confirmation of their phenotype and functionality. (A) Gating strategy for FACS sorting of CD4+CD127"CD25 CD45RA+ and CD4+CD127 CD25hi Tregs after CD4+ magnetic bead enrichment from PBMCs Tregs were sorted, from left to right, as lymphocytes (FSCA/SSC-A), single cells (FSC-A/FSC-H), living cells (LIVE/DEAD Fixable Aqua Dead Cell Stain negative population) and CD4+ T cells (CD3+CD4+), and further elimination of irrelevant cell subsets based on dump channel (CD8 CD14 CD16 CD19 ) and CD127"CD25+ cells, whereas effector CD4+ T cells were sorted as CD127+CD25" cells. Selection of the naive Tregs was based on expression of CD45RA, and CD25hl Tregs were sorted as CD45RA CD25hl. (B) Representative overlay of dot plot and histogram of FOXP3 expression in CD127+CD25" effector T cells (a), CD45RA+ Tregs (b) and CD25hl Tregs (c). FMO was used as control (d). FOXP3 expression was analyzed by flow cytometry in CD45RA+ and CD25hl Tregs after sorting and compared to control CD I 27 CD25 effector T cells. (C and D) In vitro analysis of suppression of autologous effector T cell proliferation was performed using FACS-sorted CD45RA+ (C) and CD25hl (D) Tregs at different TeffTreg ratios. All ratios were performed in duplicate, and the mean division index was used to calculate the suppression percentage: 100 - (DICOndition of interest/DIi:o) * 100. Results are shown as median ± interquartile range for four independent donors. Statistical analysis was performed using the nonparametric Friedman test with Dunn’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 2 illustrates gating strategy used for in vitro T-cell suppression assay. Effector T cells are stained with a CellTrace Violet Cell Proliferation kit, which allows tracking of cell division. Gating was conducted in following steps: (A) lymphocytes (FSC-A/SSC-A), (B) single cells (FSC-A/FSC- H), (C) living cells (SSC-A/7-AAD ), (D) CD4+ T cells (SSC-A/CD4+), (E) gating out the added Tregs, which were not stained with CellTrace Violet (CellTrace Violet/count) and (F) setting the gate for the undivided CD I 27 CD25 T cell using the unstimulated condition. Next, the division index for the effector T cell only condition (G: ratio 1:0) is obtained and used to calculated the suppression percentage for each condition by the division index method: 100-(DICOndition of interest/DIi;o)* 100. For all samples 10,000 events were recorded in Pl (= Lymphocytes).
Fig. 3 illustrates a mean 186.5 ± 123.8-fold expansion of CD45RA+ Tregs and 71.4 ± 50.3-fold expansion of CD25hl Tregs upon 19 days of ex vivo expansion and a >80% expression of a transgenic TCR by means of mRNA electroporation was achieved by using a novel GMP- compatible engineering protocol. Tregs were FACS sorted, cultured in IMDM supplemented with 500 lU/mL IL-2 and 5% hAB serum, and activated using a soluble polymer conjugated with antibodies to CD3 and CD28 (1: 100 dilution) on days 0, 7 and 14. Expansion in total cell numbers (top) and fold expansion (bottom) of CD45RA+ Tregs (A) and CD25hl Tregs (B) was assessed for 19 days after the initiation of the expansion protocol. Graphs represent the median cell count ± interquartile range for seven independent donors. Statistical analysis was performed using the nonparametric Friedman test with Dunn’s multiple comparisons test. (C) Representative plots of Treg purity assessed by expression of CD127 and CD25 on days 7, 14 and 19 during Treg expansion. (D) Representative histograms of the MBPgj-gg-specific TCR expression measured for 8 days after mRNA electroporation using anti-TCR Vb2-PE antibody in expanded and transfected CD45RA+ Tregs (E) and CD25hl Tregs (F). Transgenic TCR kinetics after cryopreservation 4 h after mRNA electroporation (EP) are represented by striped bars. Baseline TCR Vb2 expression per day is also indicated. Graphs represent median percentage of Vp2+ Tregs ± interquartile range for three independent donors. Statistical analysis was performed using mixed-models test with the Geisser-Greenhouse correction; matched values are stacked into a subcolumn, with Dunnett’s multiple comparisons test, with individual variances computed for each comparison. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 4 illustrates a representative dot plot and histogram overlay of eGFP expression in Tregs 24h following eGFP-encoding mRNA electroporation. Mock electroporation is indicated with ‘b’ and eGFP -encoding mRNA-electroporation is indicated with ‘a’.
Fig. 5 illustrates high FOXP3 expression and low methylation levels in engineered CD45RA+ and CD25hl Tregs, which confirm conservation of stable Treg phenotype. FOXP3 expression in CD45RA+ Tregs (A) and CD25hl Tregs (B) after sorting, expansion, and MBPss-gg-specific TCR- encoding mRNA electroporation was compared with control CD I 27 CD25 T cells. Percentage of TSDR DNA methylation in FACS-sorted, expanded and MBPss-gg-specific TCR-encoding mRNA- electroporated CD45RA+ (C) and CD25hl (D) Tregs, compared with that of freshly FACS-sorted CD I 27 CD25 T cells. Heatmaps represent the methylation percentages of nine CpG sites located in intron 1 of human FOXP3 gene. Data plots show the summarized data of median percentage FOXP3 (A, B) or median percentage methylation (C, D) ± interquartile range for four (freshly sorted cells, two male and two female donors) or seven independent donors. Statistical analysis was performed using the nonparametric Kruskal-Wallis test with Dunn’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 6 illustrates Helios, CTLA-4 and CCR4 expression in engineered CD45RA+ and CD25hl Tregs, confirming conservation of stable Treg phenotype. Helios (left), CTLA-4 (middle) and CCR4 (right) expression in CD45RA+ Tregs and CD25hl Tregs after expansion and MBPss-gg-specific TCR-encoding mRNA electroporation (EP) were compared to control CD4+ T cells. Results are shown as representative data graphs, representing median percentage ± interquartile range for three independent donors. Statistical analysis was performed using the nonparametric Friedman test with Dunn’s multiple comparisons test: *P < 0.05, **P < 0.01.
Fig. 7 illustrates that ex vivo expanded and mRNA-electroporated (EP) CD45RA+ and CD25hl Tregs induce the suppression of effector T cell proliferation in vitro and produce antiinflammatory, but not pro-inflammatory, cytokines when activated. Autologous CD I 27 CD25 T cells were stained using CellTrace Violet and activated with human Treg suppression inspector beads, leading to cell proliferation. Inhibition of CD 127 CD25 T cell proliferation was obtained by different ratios of expanded CD45RA+ Tregs (A) and expanded CD25hl Tregs (B). MBP85-99- specific TCR-encoding mRNA-electroporated cells are indicated with an asterisk (*). Graphs represent median percentage of suppression ± interquartile range for four independent donors. Each donor was measured in duplicate, and the mean division index was used to calculate the suppression percentage: 100 - (DICOndition of interest/DIi:o) * 100. Statistical analysis was performed using the nonparametric Friedman test with Dunn’s multiple comparisons test. Pro-inflammatory (C) and anti-inflammatory (D) cytokine production of expanded CD45RA+ Tregs and expanded CD25hl Tregs, which are MBP85-99-specific TCR-encoding mRNA-electroporated (EP) or not, compared with control CD4+ T cells. Statistical analysis was performed using the nonparametric Friedman test with Dunn’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001. EP, mRNA electroporation.
Fig. 8 illustrates representative dot plot and histogram overlay of the l\ BPs'-99-spccific TCR and CD3 expression in TCR-deficient cell lines 2D3 and SKW-3 6h following MBP85-99-specific TCR- encoding mRNA-electroporation. Mock electroporation is indicated by ‘b’ and MBP85-99-specific TCR-encoding mRNA-electroporation, leading to surface expression of CD3 and the transgenic TCR, is indicated by ‘a’ in both 2D3 (A) and SKW-3 (B) cells, which are TCR-deficient cell lines.
Fig. 9 illustrates TCR-dependent activation following TransAct stimulation of MBP85-99-specific TCR-encoding mRNA-electroporated 2D3 and SKW-3 cells. 2D3 (A) and SKW-3 (B) cells were MBP85 -99-specific TCR-encoding mRNA-electroporated and unstimulated (grey) or TCR-specific stimulated using TransAct (black) 6 h after mRNA electroporation. NFAT-dependent GFP expression in 2D3 cells and expression of activation markers CD69 and CD 137 in SKW-3 cells were analyzed 12 h after activation. Results are represented as data plot and histogram overlay for unstimulated and stimulated electroporated cells. (C) The data graph represents median percentage ± interquartile range for eight independent experiments, representing unstimulated and stimulated, mock and electroporated cells. Statistical analysis was performed using the nonparametric Friedman test with Dunn’s multiple comparisons test: **P < 0.01, ***P < 0.001, ****P < 0.0001.
DESCRIPTION OF EMBODIMENTS
As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.
The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes”, “containing”, or “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “constituted of’, “consists in”, “consisting of’, and “consists of’, and also the terms “consisting essentially of’, “consisting essentially in” and “consists essentially of’, which enjoy well- established meanings in patent terminology.
The recitation of numerical ranges by endpoints includes all integer numbers and, where appropriate, fractions subsumed within the respective ranges, as well as the recited endpoints. This applies to numerical ranges irrespective of whether they are introduced by the expression “from... to... ” or the expression “between... and... ” or another expression. Any numerical range recited herein is intended to include all sub-ranges subsumed therein.
The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, preferably +/-5% or less, more preferably +/-1% or less, and still more preferably +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.
Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.
As used herein, the term “and/or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a list is described as comprising group A, B, and/or C, the list can comprise A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.
The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.
Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference. Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation or meaning is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined.
In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.
Similarly, it should be appreciated that in the description of illustrative embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects.
As corroborated by the experimental section, which illustrates certain representative embodiments of the present invention, the inventors have unexpectedly demonstrated that T regulatory cells (Treg cells or Tregs) can be highly efficiently engineered by means of RNA electroporation, even while they retain their immunosuppressive functions.
Accordingly, an aspect of the invention provides a method for introducing an unmodified or modified RNA polynucleotide into a Treg cell, comprising electroporation of a suspension comprising the polynucleotide and Treg cells. The terms “nucleic acid”, “nucleic acid molecule”, and “polynucleotide” as used herein refer to a polymer (preferably a linear polymer) of any length composed essentially of nucleoside units. A nucleoside unit commonly includes a heterocyclic base and a sugar group.
Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine), as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases. Examples of modified nucleobases (whether naturally-occurring or non-naturally-occurring) include, without limitation, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, 5 -methylcytosine, and 5- propynylcytosine. Further non-limiting examples of modified nucleobases include N6- isopentenyladenine, 1 -methyladenine, 2-methyladenine, N6-methyladenine, 2-methylthio-N6- isopentenyladenine, 4-acetylcytosine, 3 -methylcytosine, 5 -methylcytosine, 2-thiocytosine, 1- methylguanine, 2,2-dimethylguanine, 2-methylguanine, 7-methylguanine, 5- (carboxyhydroxymethyl)uracil, 5 -(carboxymethylaminomethyl)-2 -thiouracil, 5 - carboxymethylaminomethyluracil, dihydrouracil, 1 -methyluracil, 5 -methylaminomethyluracil, 5- methoxyaminomethyl -2 -thiouracil, 5 -methoxy carbonylmethyl -2 -thiouracil, 5 - methoxy carbonylmethyluracil, 5 -methoxyuracil, 5 -methyl -2 -thiouracil, 2-thiouracil, 4-thiouracil, 5- methyluracil, and 3-(3-amino-3-carboxy-propyl)uracil.
Sugar groups may include inter alia pentose (pentofuranose) groups such as preferably ribose and/or 2-deoxyribose common in naturally-occurring nucleic acids (RNA and DNA, respectively), or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups (such as, without limitation, 2’-0-alkylated, e.g., 2’-O-methylated or 2’-0-ethylated sugars such as ribose; 2’-O-alkyloxyalkylated, e.g., 2’-O-methoxyethylated sugars such as ribose; or 2’-O,4’-C-alkylene-linked, e.g., 2’-O,4’-C-methylene-linked or 2’-O,4’-C-ethylene-linked sugars such as ribose; 2 ’-fluoro-arabinose, etc.).
Naturally-occurring ribonucleosides include in particular adenosine, guanosine, uridine, and cytidine. Naturally-occurring deoxyribonucleosides include in particular deoxyadenosine, deoxyguanosine, thymidine, and deoxy cytidine. Examples of modified nucleosides (whether naturally-occurring or non-naturally-occurring) include, without limitation, 4-acetylcytidine, 5- (carboxyhydroxymethyl)uridine, 2 ’ -O-methylcytidine, 5 -carboxymethylaminomethyl -2 -thiouridine, 5 -carboxymethylaminomethyluridine, dihydrouridine, 2’-O-methylpseudouridine, D- galactosylqueuosine, 2’-O-methylguanosine, inosine, N6-isopentenyladenosine, 1- methyladenosine, 1 -methylpseudouridine, 1 -methylguanosine, 1 -methylinosine, 2,2- dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3 -methylcytidine, 5 -methylcytidine, N6-methyladenosine, 7-methylguanosine, 5 -methylaminomethyluridine, 5 -methoxyaminomethyl - 2 -thiouridine, D-mannosylqueuosine, 5 -methoxycarbonylmethyl -2 -thiouridine, 5- methoxycarbonylmethyluridine, 5 -methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N-((9- beta-D-ribofiiranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine, mt6a N-((9-beta-D- ribofiiranosylpurine-6-yl)N-methylcarbamoyl)threonine, uridine-5 -oxyacetic acid-methylester, uridine-5 -oxyacetic acid, wybutoxosine, pseudouridine, queuosine, 2-thiocytidine, 5 -methyl -2- thiouridine, 2-thiouridine, 4-thiouridine, 5 -methyluridine, N-((9-beta-D-ribofiiranosylpurine-6-yl)- carbamoyl)threonine, 2 ’-O-methyl-5 -methyluridine, 2’-O-methyluridine, wybutosine, and 3-(3- amino-3 -carboxy-propyl)uridine .
Nucleoside units may be linked to one another by any one of numerous known inter-nucleoside linkages, including inter alia phosphodiester linkages common in naturally-occurring nucleic acids, and further modified phosphate- or phosphonate-based linkages such as phosphorothioate, alkyl phosphorothioate such as methyl phosphorothioate, phosphorodithioate, alkylphosphonate such as methylphosphonate, alkylphosphonothioate, phosphotriester such as alkylphosphotriester, phosphoramidate, phosphoropiperazidate, phosphoromorpholidate, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate; and further siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate such as 3’-N-carbamate, morpholino, borano, thioether, 3 ’-thioacetal, and sulfone intemucleoside linkages. Preferably, inter-nucleoside linkages may be phosphate-based linkages including modified phosphate-based linkages, such as more preferably phosphodiester, phosphorothioate or phosphorodithioate linkages or combinations thereof. The term “nucleic acid” may also encompass any other nucleobase containing polymers such as nucleic acid mimetics, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino phosphorodiamidate-backbone nucleic acids (PMO), cyclohexene nucleic acids (CeNA), tricyclo- DNA (tcDNA), and nucleic acids having backbone sections with alkyl linkers or amino linkers (see, e.g., Kurreck 2003 (Eur J Biochem 270: 1628-1644)). “Alkyl” as used herein particularly encompasses lower hydrocarbon moieties, e.g., C1-C4 linear or branched, saturated or unsaturated hydrocarbon, such as methyl, ethyl, ethenyl, propyl, 1 -propenyl, 2-propenyl, and isopropyl.
Nucleic acids as intended herein may include naturally occurring nucleosides, modified nucleosides, or mixtures thereof. Reference to an “unmodified” polynucleotide may conveniently denote a polynucleotide composed of nucleosides which also constitute the corresponding type or kind of polynucleotide in nature, such as adenosine, guanosine, uridine, and/or cytidine for ribonucleic acid (RNA) polynucleotides, or deoxyadenosine, deoxyguanosine, thymidine, and/or deoxycytidine for deoxyribonucleic acid (DNA) polynucleotides, and connected by inter- nucleoside phosphodiester linkages. Reference to a “modified” polynucleotide may conveniently denote a polynucleotide which comprises one or more modified nucleoside, one or more modified inter-nucleoside linkage, or a combination thereof. The one or more modified nucleoside may each independently comprise a modified heterocyclic base, a modified sugar moiety, a modified connection between the base and the sugar moiety, or a combination thereof. By means of an example, an RNA polynucleotide may be denoted as “modified” when it comprises one or more nucleoside other than adenosine, guanosine, uridine, and cytidine, one or more inter-nucleoside linkage other than a phosphodiester bond, or a combination thereof.
By means of an example, a modified RNA polynucleotide may be primarily composed of adenosine, guanosine, uridine, and/or cytidine nucleosides connected by phosphodiester linkages, and may comprise a minority of nucleosides other than adenosine, guanosine, uridine, and cytidine, and/or a minority of inter-nucleoside linkages other than phosphodiester bond. In another example, a modified RNA polynucleotide may be primarily or exclusively composed of nucleosides other than adenosine, guanosine, uridine, and cytidine, and/or may comprise a majority of or may exclusively comprise inter-nucleoside linkages other than phosphodiester bond.
The term “nucleic acid” further preferably encompasses DNA, RNA and DNA/RNA hybrid molecules, specifically including heteronuclear RNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesized) DNA, RNA (such as but not limited to Dicer-substrate small interfering RNAs (DsiRNA)) or DNA/RNA hybrids. A nucleic acid can be naturally occurring, e.g., present in or isolated from nature (and may be of any origin, e.g., prokaryotic, eukaryotic, archaeal, or viral), can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesized. A “nucleic acid” can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. RNA polynucleotides are typically single-stranded molecules, can, however, also be provided in a double-stranded form by partial complementary base pairing. RNA polynucleotides typically do not form long double helical stretches. In addition, nucleic acid can be circular or linear. Nucleic acids may also, in certain embodiments, comprise artificial additions such as tags or labels.
The term “ribonucleic acid polynucleotide” or “RNA polynucleotide” more particularly denotes nucleic acids which comprise two or more ribonucleosides, i.e., nucleosides in which the sugar group to which the heterocyclic base is linked is a ribose or a modified ribose, preferably ribose, but not deoxyribose. Available modifications of ribose include, without limitation, 2’-O-alkylation, e.g., 2’-O-methylation or 2’-O-ethylation, 2’-O-alkyloxyalkylation, e.g., 2’-O-methoxyethylation; or 2’-O,4’-C-alkylene-linkage, e.g., 2’-O,4’-C-methylene-linked or 2’-O,4’-C-ethylene-linked ribose. A ribonucleoside may comprise one of the prevalent nucleobases found in naturally- occurring RNA molecules, i.e., adenine, guanine, uracil, or cytosine, or may comprise a modified (whether naturally-occurring or non-naturally-occurring) nucleobase, such as one of the modified nucleobases described above. The two or more ribonucleosides may be connected by a phosphodiester bond, or by an alternative inter-nucleoside linkage such as described above, preferably by a phosphodiester bond.
Where an RNA polynucleotide comprises one or more nucleoside other than a ribonucleoside, the RNA polynucleotide may be primarily composed of ribonucleosides, for example, in an increasing order of preference, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99% of the RNA polynucleotide’s nucleosides are ribonucleosides. In certain embodiments, the RNA polynucleotide may comprise only nucleosides which are ribonucleosides. In certain preferred embodiments, the RNA polynucleotide may be primarily composed of nucleosides selected from the group consisting of adenosine, guanosine, uridine, cytidine, and combinations thereof, for example, in an increasing order of preference, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or 100% of the RNA polynucleotide’s nucleosides are selected from the group consisting of adenosine, guanosine, uridine, cytidine, and combinations thereof. In certain preferred embodiments, the RNA polynucleotide’s nucleosides may be primarily connected by phosphodiester bonds, for example, in an increasing order of preference, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or 100% of the RNA polynucleotide’s nucleosides are connected by phosphodiester bonds. In certain particularly preferred embodiments, the RNA polynucleotide is primarily composed of nucleosides selected from the group consisting of adenosine, guanosine, uridine, cytidine, and combinations thereof, connected by phosphodiester bonds, for example, in an increasing order of preference, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or 100% of the RNA polynucleotide’s nucleosides are nucleosides selected from the group consisting of adenosine, guanosine, uridine, cytidine, and combinations thereof, connected by phosphodiester bonds.
In accordance with the above explanations, in certain embodiments the RNA polynucleotide is a linear polynucleotide. In accordance with the above explanations, in certain embodiments the modified RNA polynucleotide is nucleobase-modified, or backbone-modified, or nucleobase- modified and backbone-modified (wherein the modifications may be on the same or on different nucleosides).
In certain embodiments, the RNA polynucleotide is a naked polynucleotide, i.e., a polynucleotide free from any delivery vehicle that can act to facilitate entry into the cell, for example, the polynucleotide sequences are free of viral sequences, particularly any viral particles that may carry genetic information. They are similarly free from, or “naked” with respect to, any material that promotes transfection, such as liposomal formulations, charged lipids, or precipitating agents such as calcium phosphate.
Any polynucleotides as discussed herein may be purified. As used herein, the term “purified” does not require absolute purity. Instead, it denotes that such nucleic acids are in a discrete environment in which their abundance (conveniently expressed in terms of mass or weight or concentration) relative to other analytes is greater than in a source material they have been purified from (e.g., from an in vitro transcription reaction, from a cell recombinantly producing them, etc.) A discrete environment denotes a single medium, such as for example a single solution, gel, precipitate, lyophilisate, etc. Purified nucleic acids may preferably constitute by weight > 10%, more preferably > 50%, such as > 60%, yet more preferably > 70%, such as > 80%, and still more preferably > 90%, such as > 95%, > 96%, > 97%, > 98%, > 99% or even 100%, of the nucleic acid content of the discrete environment. Quantity of nucleic acids may be determined by measuring absorbance A260. Purity of nucleic acids may be determined by measuring absorbance A260/A280, or by agarose- or polyacrylamide-gel electrophoresis and ethidium bromide or similar staining.
The terms “T regulatory cell” and “Treg” encompass any and all cell types, sub-types, and phenotypes known under these designations in the art. By means of further guidance, Tregs are potent immunosuppressive cells, which among others can down-modulate the functions of T effector cells. Treg markers include LAG-3, CD25, cytotoxic T lymphocyte-associated antigen 4 (CD152), FoxP3, GITR, IFN-y, and neuropilin-1 but often only a subset of these are expressed. CD4+ Tregs have been phenotypically described as CD4 positive, CD25 positive, and CD 127 low or negative. CD8+ Tregs have been described which are CD8 positive, LAG3 positive, CD25 positive and CD 127 negative.
When a cell is said to be positive for (or to express or comprise expression of) a particular marker, this means that a skilled person will conclude the presence or evidence of a distinct signal, e.g., antibody-detectable or detection by reverse transcription polymerase chain reaction, for that marker when carrying out the appropriate measurement, compared to suitable controls. Where the method allows for quantitative assessment of the marker, positive cells may on average generate a signal that is significantly different from the control, e.g., but without limitation, at least 1.5-fold higher than such signal generated by control cells, e.g., at least 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold higher or even higher. Conversely, a cell not positive for a given marker can be denoted as being negative for said marker. The expression of cell-specific markers can be detected using any suitable immunological technique known in the art, such as immunohistochemistry or affinity adsorption, Western blot analysis, flow cytometry, ELISA, etc., or by any suitable biochemical assay of enzyme activity, or by any suitable technique of measuring the quantity of the marker mRNA, e.g., Northern blot, semi-quantitative or quantitative RT-PCR, etc. Sequence data for markers listed in this disclosure are known and can be obtained from public databases such as GenBank (http://www.ncbi.nlm.nih.gov/).
The cell surface marker phenotype of Treg cells allows their isolation from heterogeneous immune cell populations by immunological techniques which preserve the viability of the sorted cells, such as by fluorescence activated cell sorting (FACS) or by magnetic-activated cell sorting, in which distinct cell phenotypes within the cell populations can be separated. For instance, a CD4+ Treg subset can be isolated based on their CD4 CD25 CD127" phenotype, or a CD8+ subset could be isolated based on a CD8+LAG3+CD25+FoxP3+CCL4+ phenotype. FACS is particularly convenient, since it allows to visualise cells in a sample according to their physical properties and their surface marker expression, and allows the user to define gates around the cell populations with the desired properties, and separate out the cells sorted into any gates of interest.
Treg cells have also been phenotypically described as expressing the master regulator forkhead box P3 (F0XP3) transcription factor (even while F0XP3 -independent maintenance of the human Treg identity has been shown in F0XP3-ablated Tregs) and as harbouring a substantially demethylated Treg-specific demethylated region (TSDR). While such intracellular characteristics may be less suited for isolation or sorting of viable Treg cells, they allow to classify cells as Treg cells, for example by analysing a sample of a larger cell population.
The terms further include any and all Treg subtypes, subpopulations, and differentiation stages, in isolation as well as their combinations or mixtures. These will typically display the aforementioned defining Treg characteristics, and will be further distinguished from one another on the basis of other properties. Hence, for example, the terms encompass naive Treg cells (nTregs), central memory Treg cells (cmTregs), effector memory Treg cells (emTregs), and effector Treg (eTreg) lymphocytes. The terms further encompass CD4 CD127"CD25+, CD4+CD127"CD25 CD45RA+ Tregs (CD4+CD45RA+ Tregs or CD45RA+ Tregs, for reasons of brevity), CD4 CD127"CD25hl Tregs (CD4+CD25hl or CD25hl Tregs, for reasons of brevity), CD8+LAG3+CD25+FoxP3+CCL4+ Tregs (CD8+LAG3+ Tregs, for reasons of brevity) ICOS+ Tregs, CD49d+ Tregs, and CD62L+ Tregs, and their combinations or mixtures.
Whereas the qualifier “isolated” need not be expressly recited with relation to the cells as intended herein, it may conveniently be included, as the present disclosure pertains to manipulation of cells outside of the body, in vitro or ex vivo, and subsequent uses, such as therapeutic uses, of so- manipulated cells. The term “isolated” with reference to a particular component generally denotes that such component exists in separation from - for example, has been separated from or prepared and/or maintained in separation from - one or more other components of its natural environment. More particularly, the term “isolated” as used herein in relation to a cell or cell population denotes that such cell or cell population does not form part of an animal or human body, for example the cell may be cultured, sorted or stored in vitro or ex vivo.
In some embodiments, the Treg cell as disclosed herein is a vertebrate cell, preferably a warmblooded animal cell, even more preferably a mammalian cell, and most preferably a human cell.
In certain embodiments, the Treg cells are CD4+CD45RA+ Treg cells. In certain preferred embodiments, the Treg cells are a population of CD4+CD45RA+ Treg cells with purity, in increasing order of preference, of at least 50% (i.e., at least 50% of the cells of the cell population are CD4+CD45RA+ Treg cells), at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or 100%. In certain embodiments, the Treg cells are CD4+CD25hl Treg cells. In certain preferred embodiments, the Treg cells are a population of CD4+CD45RA+ Treg cells with purity, in increasing order of preference, of at least 50% (i.e., at least 50% of the cells of the cell population are CD4+CD25hl Treg cells), at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or 100%.
In certain embodiments, the Treg cells are CD8+LAG3+CD25 FoxP3 CCL4+ Treg cells. In certain preferred embodiments, the Treg cells are a population of CD8+LAG3+CD25+FoxP3+CCL4+ Treg cells with purity, in increasing order of preference, of at least 50% (i.e., at least 50% of the cells of the cell population are CD8+LAG3+CD25+FoxP3+CCL4+ Treg cells), at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or 100%.
In certain embodiments, the Treg cells to be electroporated are freshly isolated, such as freshly isolated from peripheral blood mononuclear cells (PBMC) or from an internal organ, such as lung, liver, or spleen. In these embodiments, the Treg cells are not expanded in culture prior to electroporation.
In certain other embodiments, the Treg cells to be electroporated have been obtained by in vitro or ex vivo expansion of isolated Treg cells, such as Treg cells isolated from PBMC or from an internal organ, such as lung, liver, or spleen. The term “in vitro” as used herein is to denote outside, or external to, animal or human body. The term “in vitro” as used herein should be understood to include “ex vivo”. The term “ex vivo” typically refers to tissues or cells removed from an animal or human body and maintained or propagated outside the body, e.g., in a culture vessel.
By means of explanation and without limitation, peripheral blood mononuclear cells (PBMC) are nucleated peripheral blood cells, in particular cells having a round nucleus. PBMC in particular include lymphocytes, monocytes, and dendritic cells. The lymphocyte population of PBMC typically consists of T-cells, B-cells and NK cells. PBMC may be isolated from whole blood samples by methods well known in the art, such as for example density gradient centrifugation (e.g., Ficoll gradient).
In certain embodiments, the Treg isolation comprises isolating T cells from PBMC or from the internal organ, thereby obtaining a population of T cells; and isolating Treg cells from the population of T cells. Since Treg cells are a subset of CD4+ immune cells and/or CD8+ immune cells, in certain embodiments, the Treg isolation comprises isolating CD4+ cells, in particular CD4+ T cells from PBMC or from the internal organ and/or the Treg isolation comprises isolating CD8+ cells, in particular CD8+ T cells from PBMC or from the internal organ, thereby obtaining a population of CD4+ cells and/or a population of CD8+ cells, in particular CD4+ T cells and/or CD8+ T cells; and isolating Treg cells from the population of CD4+ cells and/or CD8+ cells in particular CD4+ T cells and/or CD8+ T cells. In certain embodiments, the Treg cell can be directly isolated from PBMC or from the internal organ, i.e., without the interposed step of T cell or CD4+ and/or CD8+ cell enrichment.
In certain embodiments, the isolation of Treg cells from the population of T cells, CD4+ cells, or in particular CD4+ T cells, may involve isolating CD 127 negative (CD 127 ) cells therefrom. Hence, in certain embodiments, the isolation comprises isolating CD4+ cells from PBMC or from the internal organ, thereby obtaining a population of CD4+ cells; and isolating CD 127’ cells from the population of CD4+ cells, thereby obtaining Treg cells. In certain embodiments, the CD4 CD I27 cells (Treg cells) can be directly isolated from PBMC or from the internal organ, i.e., without the interposed step of CD4+ cell enrichment.
In further embodiments, the method may comprise isolating specifically CD45RA+ Treg cells from the population of T cells, CD4+ cells, or in particular CD4+ T cells, or directly from PBMC or from the internal organ; or isolating specifically CD25hl Treg cells from the population of T cells, CD4+ cells, or in particular CD4+ T cells, or directly from PBMC or from the internal organ; or isolating specifically a mixture of CD45RA+ Treg cells and CD25hl Treg cells from the population of T cells, CD4+ cells, or in particular CD4+ T cells, or directly from PBMC or from the internal organ.
In certain embodiments, the isolation of Treg cells from the population of T cells, CD8+ cells, or in particular CD8+ T cells, may involve isolating CD 127 negative (CD 127 ) cells therefrom and/or further selecting for expression of LAG3+, CD25+, FoxP3+, and/or CCL4+. Hence, in certain embodiments, the isolation comprises isolating CD8+ cells from PBMC or from the internal organ, thereby obtaining a population of CD8+ cells; and isolating CD 127’ cells (or cells enriched based on one or more of the markers specified above) from the population of CD8+ cells, thereby obtaining Treg cells.
As explained above, Treg cells can be isolated from the T cell population based on the expression of specific cell-surface markers, using any suitable cell separation method which substantially preserves the viability of the cells, in particular an immunological methods, such as flow cytometry (FACS) and/or affinity separation.
Flow cytometry encompasses methods by which individual cells of a cell population are analyzed by their optical properties (e.g., light absorbance, light scattering and fluorescence properties, etc.) as they pass in a narrow stream in single file through a laser beam. Flow cytometry methods include fluorescence activated cell sorting (FACS) methods by which a population of cells having particular optical properties are separated from other cells.
Affinity separation also referred to as affinity chromatography broadly encompasses techniques involving specific interactions of cells present in a mobile phase, such as a suitable liquid phase (e.g., cell population in an aqueous suspension) with, and thereby adsorption of the cells to, a stationary phase, such as a suitable solid phase; followed by separation of the stationary phase from the remainder of the mobile phase; and recovery (e.g., elution) of the adsorbed cells from the stationary phase. Affinity separation may be columnar, or alternatively, may entail batch treatment, wherein the stationary phase is collected / separated from the liquid phases by suitable techniques, such as centrifugation or application of magnetic field (e.g., where the stationary phase comprises magnetic substrate, such as magnetic particles or beads). Accordingly, magnetic cell separation or magnetic-activated cell sorting (MACS) is also envisaged herein.
In certain embodiments, the T cells or CD4+ cells, or in particular CD4+ T cells, are isolated from PBMC or from the internal organ using MACS or FACS, preferably by MACS. In certain embodiments, the Treg cells are isolated from the population of T cells or CD4+ cells and/or CD8+ cells, or in particular CD4+ T and/or CD8+ cells, using MACS or FACS, preferably by FACS. In certain preferred embodiments, the T cells or CD4+ cells, or in particular CD4+ T cells and/or CD8+ T cells, are isolated from PBMC or from the internal organ using MACS or FACS, and the Treg cells are isolated from the population of T cells or CD4+ cells, or in particular CD4+ T cells and/or CD8+ T cells, using MACS or FACS. In certain particularly preferred embodiments, the T cells or CD4+ cells and/or CD8+ cells, or in particular CD4+ T cells and/or CD8+ T cells, are isolated from PBMC or from the internal organ using MACS, and the Treg cells are isolated from the population of T cells or CD4+ cells and/or CD8+ cells, or in particular CD4+ T cells, using FACS. In certain embodiments, the Treg cells are directly isolated from PBMC or from the internal organ using FACS. Whereas the above describes certain preferred ways to isolate Treg cells, other approaches and strategies have been developed in the art and can also be employed in the present context, such as other gating strategies using FACS, other magnetic selection methods, and/or enrichment methods using pharmaceuticals, such as for example, rapamycin.
Because Treg cells typically constitute a relatively small fraction of PBMC or of the cells of the internal organ, the method may advantageously include a step of expanding the isolated Treg cells in cell culture. T cell cultivation may typically occur in cell cultures, in suitable liquid cell culture media. Typically, the medium will comprise a basal medium formulation as known in the art. Many basal media formulations (available, e.g., from the American Type Culture Collection, ATCC; or from Invitrogen, Carlsbad, California) can be used to culture the cells herein, including but not limited Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), BGJb, F-12 Nutrient Mixture (Ham), or Iscove's Modified Dulbecco's Medium (IMDM), and modifications and/or combinations thereof. Compositions of the above basal media are generally known in the art and it is within the skill of one in the art to modify or modulate concentrations of media and/or media supplements as necessary for the cells cultured. Such basal media formulations contain ingredients necessary for mammal cell development, which are known per se. By means of illustration and not limitation, these ingredients may include inorganic salts (in particular salts containing Na, K, Mg, Ca, Cl, P and possibly Cu, Fe, Se and Zn), physiological buffers (e.g., HEPES, bicarbonate), nucleotides, nucleosides and/or nucleic acid bases, ribose, deoxyribose, amino acids, vitamins, antioxidants (e.g., glutathione) and sources of carbon (e.g., glucose, sodium pyruvate, sodium acetate), etc.
For use in culture, basal media can be supplied with one or more further components. For example, additional supplements can be used to supply the cells with the necessary trace elements and substances for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution. Further antioxidant supplements may be added, e.g., P-mercaptoethanol. While many basal media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution. A medium may be further supplied with antibiotic and/or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and/or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin. Lipids and lipid carriers can also be used to supplement cell culture media. Such lipids and carriers can include, but are not limited to cyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic-oleic- arachidonic acid conjugated to albumin, oleic acid unconjugated and conjugated to albumin, among others. Albumin can similarly be used in fatty-acid free formulations. Plasma, serum or a substitute thereof, such as bovine serum, fetal calf serum, or preferably human serum, may also be comprised in said media at a proportion (volume of one or more of plasma, serum, or a substitute thereof / volume of medium) between about 0.5% and about 30% v/v, preferably between about 5% and about 10%. v/v. Standard cell culture can be performed in humidified 5% CO2 incubator at 37°C.
During cell culture, the culture medium can be replenished or refreshed, completely or partly, at with suitable regularity, such as twice daily, once daily, once in two days, or similar, depending on factors such as cell density, medium pH etc. During cell culture, cells may be passaged once they have reached a certain degree of confluency.
In certain embodiments, Treg cell expansion comprises a step of culturing the isolated Treg cells, such as Treg cells isolated from PBMC or from the internal organ, in the presence of interleukin-2 (IL-2). Conveniently, IL-2 may be included in the media in which the Treg cells are cultured, in a quantity sufficient to promote expansion of the Treg cells. Typically, IL-2 can be included in the media at a concentration of between 50 lU/mL and 2000 lU/mL, preferably between 100 lU/mL and 1000 lU/mL, more preferably between 250 lU/mL and 750 lU/mL, such as at about 500 lU/ml.
In certain particularly preferred embodiments, the Treg cells are expanded in a complete medium comprising or consisting of Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 5% v/v human AB serum and 500 lU/mL IL-2.
In certain embodiments, such as in particular when immunomodulatory properties of the electroporated Treg cells are to be exploited, the Treg cells may be activated prior to electroporation. Methods for Treg cell activation are generally known and may comprise a step of contacting the isolated and optionally expanded Treg cells, such as the Treg cells isolated and optionally and preferably expanded from PBMC or from the internal organ, with an anti-CD3 antibody and an anti-CD28 antibody. In certain embodiments, the antibodies may be covalently linked to a polymer carrier. One example of such reagent is T cell TransAct™, commercially available from Miltenyi Biotec, which is a clinical-grade colloidal reagent comprising iron oxide crystals embedded into a biocompatible polysaccharide matrix with an overall diameter of -100 nm. Agonistic humanized anti-CD3 and anti-CD28 antibodies are coated onto the nanomatrix. The matrix can be produced under GMP conditions, sterilized by fdtration and unbound reagent can easily be removed from the cell suspension by centrifugation of the cells. In certain embodiments, the Treg cells may be activated two or more times during the Treg expansion and activation protocol. The later activation steps may also be denoted as reactivation steps. The frequency of the activation / reactivation steps may be for example twice weekly, weekly, every 1.5 week, or every two weeks, such as for example every 3, 4, 5, 6, 7, 8, 9, 10, or 11 days, preferably every 6, 7, or 8 days, and more preferably every 7 days. In certain preferred embodiments, the activation is repeated on days 0, 7, 14, and 19 of the Treg expansion and activation. Each activation step may be followed by washing away the activation reagent, such as between about 24 hours and about 72 hours after its addition to the cells / medium, preferably between about 36 hours and about 60 hours, and more preferably at about 48 hours its addition to the cells / medium.
As mentioned, the present methods comprise electroporation of a suspension comprising the polynucleotide and Treg cells. The polynucleotide and the Treg cells may be suspended in any suitable electroporation medium or buffer. Such compositions conducive to cell viability and to the electroporation process are well-known, and may include for example serum-free media such as IMDM, RPMI, or a serum reduced medium (e.g., Opti-MEM I®, Gibco Invitrogen). The suspension for electroporation may be kept at room temperature or may be kept at less than ambient temperature, such as on ice, i.e., about 4°C. Following electroporation, preferably immediately after electroporation, the cells can be replenished in a serum-containing medium, such as in IMDM supplemented with 10% v/v/ human serum.
Various dimensions of an electroporation cuvette may be utilised, typically dimensions of 1 mm to 4 mm, such as 1-mm, 2 -mm or 4-mm cuvettes, and preferably 4-mm cuvettes. One or more settings of the electroporation process, for example voltage settings, may be adjusted depending on the cuvette size.
Optimally, a 4-mm cuvette and about 200 pl of the cell suspension may be used in the electroporation. The electroporation may be performed with any suitable device available from a variety of vendors, such as BTX ECM 830 square wave electroporator, Gene Pulser Xcell (BioRad), Gene Pulser II® (Bio-Rad), or Easyject Plus® (Equibio) exponential decay pulse electroporator, etc.
In certain embodiments, a conventional electroporation apparatus is utilized which provides for an exponential decay pulse; the electroporation may be performed at a voltage from 100 to 500 V, more preferably from 200 to 350 V, most preferably from 250 to 300 V; the capacitance is preferably 100 pF to below 300 pF, more preferably 150 pF to 250 pF; the pulsing time can depend from the type of the tray (cuvette) and the amount of the cell suspension in the cuvette and is preferably below 50 ms, more preferably below 40 ms (for example, for a 4 mm cuvette and 200 pl cell suspension, the pulsing time may be from 5 to 40 ms, preferably 1 to 25 ms, and most preferably 7 to 10 ms).
In certain other embodiments, so-called “soft pulse” electroporation device is utilized; a voltage of 300 to 600 V and a time of 100 ps to 1 ms may be used which are believed to correspond to a capacitance of below 300 pF (although, due to the use of eukaryotic cell suspensions, a correct conversion is not possible); the pulse form provided by commercially available soft pulse electroporation devices may be a square wave pulse or an exponential decay pulse; preferred settings for the soft pulse devices may be 350 to 450 V for 300 to 600 ps.
In certain preferred embodiments, any one or any combination of two or more, or all of the following may apply:
- the concentration of the Treg cells in the suspension is 100 cells per ml to 1x109 cells per ml, such as IxlO3 cells per ml to IxlO9 cells per ml, or IxlO4 cells per ml to IxlO9 cells per ml, or 1x10s cells per ml to IxlO9 cells per ml, or IxlO6 cells per ml to IxlO9 cells per ml, preferably IxlO7 to IxlO8 cells per ml, such as about IxlO7 cells per ml, about 2.5xl07 cells per ml, about 5xl07 cells per ml, about 7.5xl07 cells per ml, or about IxlO8 cells per ml;
- the pulse is a square wave pulse or an exponential decay pulse, preferably a square wave pulse;
- the dimension of the cuvette is from 1 mm to 4 mm, preferably 1-mm, 2 -mm, or 4-mm, more preferably 4-mm;
- the voltage is from 100 V to 700 V, such as from 350 V to 650 V, preferably from 400 V to 600 V, such as from 450 V to 550 V, such as particularly preferably about 500 V;
- the pulse is a square wave pulse and the voltage is from 300 V to 700 V, such as from 350 V to 650 V, preferably from 400 V to 600 V, such as from 450 V to 550 V, such as particularly preferably about 500 V;
- the pulsing time is from 1 ms to 40 ms, such as from 1 ms to 30 ms, or from 1 ms to 20 ms, preferably from 1 to 10 ms, such as about 2 ms, or about 3 ms, or about 4 ms, or about 5 ms, or about 6 ms, or about 7 ms, or about 8 ms, or about 9 ms, particularly preferably about 5 ms;
- the concentration of the polynucleotide in the suspension is from 100 ng / IxlO6 cells to 10 pg / IxlO6 cells, such as from 250 ng / IxlO6 cells to 7.5 pg / IxlO6 cells, preferably from 500 ng / IxlO6 cells to 5 pg / IxlO6 cells, such as from 750 ng / IxlO6 cells to 2.5 pg / IxlO6 cells, more preferably about 1 pg / IxlO6 cells.
In certain embodiments, RNA polynucleotide to be introduced into the Treg cells is synthetic, or in vitro transcribed, or isolated from a host cell or a non-human host organism genetically engineered to produce the polynucleotide. Methods for nucleic acid synthesis, in vitro transcription, or recombinant expression in host cells and host organisms are well-known in the art, and need not be discussed in detail. Conveniently, RNA can be readily prepared from the corresponding DNA in vitro. For example, conventional techniques utilize phage RNA polymerases SP6, T3, or T7 to prepare RNA from DNA templates in the presence of the individual ribonucleoside triphosphates. An appropriate phage promoter, such as a T7 origin of replication site is placed in the template DNA immediately upstream of the sequence to be transcribed.
In certain embodiments, the RNA polynucleotide is coding, i.e., its sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question to a particular amino acid sequence, e.g., the amino acid sequence of one or more desired proteins or polypeptides. In certain other embodiments, the RNA polynucleotide is non-coding.
In certain embodiments, the RNA polynucleotide is selected from the group consisting of messenger RNA (mRNA), guide RNA (gRNA), single guide RNA (sgRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), long non-coding RNA (IncRNA), ribozyme, aptamer, spiegelmer, and combinations thereof.
In particularly preferred embodiments, the polynucleotide is mRNA. The mRNA preferably encodes one or more polypeptide, preferably one or more biologically active polypeptide. The reference to “biologically active” or “functionally active” or “functional” particularly conveys that the polypeptide displays some activity or function, such as a biochemical activity, an enzymatic activity, a signalling activity, an interaction activity, a ligand activity, and/or structural activity, particularly preferably wherein such activity can control, impact, or modulate the function or phenotype of Treg cells.
RNA polynucleotides may advantageously include structural and sequence elements for efficient and correct translation, together with those elements which will enhance the stability of the introduced mRNA. In general, translational efficiency has been found to be regulated by specific sequence elements in the 5 ’-non-coding or untranslated region (5’-UTR) of mRNA. Positive sequence motifs include the Kozak translational initiation consensus sequence and the 5G 7-methyl GpppG cap structure. Negative elements include stable intramolecular 5’ UTR stem -loop structures and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5’ UTR. mRNA-based polynucleotides suitable for use herein ideally include appropriate 5’ UTR translational elements flanking the coding sequence for the protein of interest. Further, capping and 3’ polyadenylation are major positive determinants of eukaryotic mRNA stability and function to protect the 5’ and 3’ ends of the mRNA from degradation. Other regulatory elements that affect the stability of eukaryotic mRNAs may also need to be considered in the development of mRNA-based polynucleotides. One example includes uridine rich 3’ untranslated region (3’ UTR) destabilizer sequences found in many short half-life mRNAs. Further, the RNA polynucleotide may be chemically modified or blocked at the 5’ and/or 3’ end to prevent access by RNase.
In certain embodiments, the polynucleotide produces a loss-of-function phenotype when introduced into the Treg cell. For example, the expression of one or more RNA products or one or more proteins endogenously produced by the Treg cell may be downregulated or abolished by the introduction of the polynucleotide. By means of an example, antisense oligonucleotides, RNA interference agents such as siRNA or shRNA, or gene editing systems (such as CRISPR/Cas) systems, may be utilised to this aim. For example, one or more polynucleotide comprising or encoding components of a gene editing system may be introduced into the Treg cell in order to effect a gene editing event in the Treg cell’s genomic material. Optionally, a DNA molecule homologous to the target locus may be co-introduced to induce a recombination / swapping of the native genetic information for the exogenously provided one at that locus. In another example, one or more antisense or RNA interference polynucleotide may be introduced into the Treg cell in order to reduce the amount or the translation of an RNA molecule, such as an mRNA molecule, produced by the Treg cell.
In certain embodiments, the polynucleotide produces a gain-of-function phenotype when introduced into the Treg cell. In certain embodiments, when the polynucleotide is mRNA encoding one or more polypeptide, preferably one or more biologically active polypeptide, the polypeptide produces a gain-of-function phenotype when expressed by the Treg cell. This may broadly encompass situations in which the polynucleotide endows the Treg cell with a new function, or in which a certain existing function of the Treg cell is increased or enhanced. In particular, this may refer to the expression of a protein normally not expressed by Treg cells, or an increase in expression of a protein normally expressed by Treg cells above the endogenous level.
Without limitation, proteins or peptides encoded by the RNA polynucleotides may include, but are not limited to, tumor antigens, microbial antigens, viral antigens, immunostimulatory or tolerogenic molecules, cytokines, interleukins, anti-apoptotic molecules, adhesion and homing molecules and antigen processing molecules, differentiation-regulating proteins, differentiation-associated proteins, tissue specific proteins, etc.
Without limitation, proteins or peptides encoded by the RNA polynucleotides may further include, but are not limited to, B-cell antibody receptors (BARs), growth factors, neurotrophic factors (e.g., BDNF, CCN3, amphiregulin), cytokines (e.g., TGF-beta, IL-10), regulators of Treg function, such as Helios, etc. and any combinations thereof. In certain embodiments, Tregs can be modulated for increased stability of their phenotype and function by introducing key regulators for Treg function, such as Helios, after introduction of cytokines, such as IL-10 and/or TGF-P involved in Tregs’ mechanism of action, or by creating Tregs that have gained new functions, e.g. regenerative capacity, such as by introducing neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and/or amphiregulin.
In certain embodiments, the one or more polypeptide encoded by the RNA polynucleotide endows the Treg cell with specificity to an antigen or an antigenic peptide thereof. In certain embodiments, the one or more polypeptide is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
The term “T cell receptor” or “TCR” as used herein refers to a protein complex found on the surface of T cells that recognizes fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. Naturally occurring T cell receptors comprise two subunits, an a-subunit and a P-subunit or a y-subunit and a 5-subunit each of which is a unique protein produced by recombination event in each T cell's genome. Each a-, P-, y- or 5- subunit contains variable (V) and constant (C) region domains, and the latter is followed by a transmembrane region and a short cytoplasmic tail. Each V domain contains three loops (i.e. complementarity-determining regions CDR1, CDR2, and CDR3), which interact with the antigen. The CDR loops project from each TCR chain and physically contact portions of the MHC molecule alone or in complex with a peptide. The centrally located CDR3 loops are most hypervariable by virtue of somatic rearrangement, dominate the interactions with the peptide, and therefore often contribute to the fine specificity of a TCR for a specific peptide. By contrast, the outward-facing and germline-encoded CDR1 and CDR2 loops provide a basal level of TCR affinity for generic MHC molecules through relatively conserved interactions, although CDR1 can contact and contribute to peptide specificity. The constant domain of the TCR consists of short connecting sequences in which a cysteine residue forms disulfide bonds, which form a link between the two chains.
The polynucleotide or protein sequence of an aP or y5 TCR can be cloned with standard techniques from one or a plurality of a or y5 T-cell(s) that have engaged with an antigen of interest, such as a tumor antigen or a viral antigen. Alternatively, the polynucleotide sequence of an aP or y5 TCR can be designed in silico. A nucleic acid comprising the engineered aP or y5 TCR can be synthesized with, for example, oligonucleotide synthesis techniques. High throughput screening techniques can be used to characterize the binding of the engineered TCR to the antigen of interest.
In particular embodiments, the one or more nucleic acids encoding the alpha- and beta-chain of a TCR are in a multicistronic construct. A linker sequence may be inserted between the nucleic acid sequence encoding the alpha-chain of TCR and the beta-chain of TCR. The linker sequences may be any linker sequence known in the art. Preferably, the linker sequence is a 2A self-cleaving peptide, as described elsewhere in this specification. The cleavage observed is not a proteolytic event but is rather the result of cis-acting hydrolase activity, which causes ribosomal skipping during translation.
The alpha-, beta-, delta- and/or gamma- subunits may have one or more amino acid substitutions, deletions, insertions, or modifications compared to the naturally occurring subunit, as long as the subunits retain the ability to form TCRs conferring upon transfected immune effector cells the ability to home to target cells.
In particular embodiments, one or more additional polypeptides are attached to the TCR. Such one or more polypeptides may be attached to the TCR so long as the attached additional polypeptide does not interfere with the ability of the a-chain or P-chain to form a functional T cell receptor and the MHC dependent antigen recognition.
T cell activation upon TCR binding to the antigen involves several other cell surface molecules, also known as costimulatory molecules, that collectively initiate and amplify the signal. In fact, the a and y5 heterodimer lack their own intracellular signaling domains and, thus, must associate with cluster of differentiation 3 (CD3). CD3 is a six-subunit complex comprising three dimers: CD3sy. CD3so. and CD3 . The cytoplasmic domains of CD3y, 5, and 8 each contain one immunoreceptor tyrosine-rich activation motif (ITAM) and each CD3^ contains three ITAMs, which serve as substrates for the Src-family kinase lymphocyte-specific protein tyrosine kinase (Lek).
In particular embodiments, each chain of the TCR comprises a CD3^ chain fused to its C-terminus.
The structure of the TCR may be modified to enhance the safety, efficacy and scalability of TCR- based immunotherapies, by any methods known in the art. For example, the nucleic acid encoding the TCR may comprise substitutions of all or selected murine residues in place of the human sequence in the TCR constant regions (murinization) for obtaining a higher exogenous TCR surface expression, increased functional avidity, and enhanced antigen-specific effector functions compared with a fully human TCR, at least one additional cysteine residue to promote a second disulfide bond (cysteine -modification), modification of the hydrophobicity of the TCR, such as the TCRa, transmembrane region (transmembrane-modification), swapping constant domains or fragments thereof between the a and P chains of the TCR (domain-swapping), mutagenesis of the CDR loops (affinity-enhancement), and/or consolidation of a normal TCR heterodimer into a single-chain format by covalently linking the variable domains of the TCR chains (single-chain TCR or scTCR). Mutagenesis of the CDR loops to increase affinity of the TCR to an antigen may be achieved by inserting amino acid substitutions in one or more of the CDR loops of the TCR either empirically or through directed evolution using phage-display libraries. Furthermore, various single-chain TCR chimeras have been used in atempts to limit the problems associated with pairing of endogenous TCRs in a cell. Using genetic engineering such constructs can be freely designed, and ensure a covalent l: l-stoichiometry of the heterodimeric, variable domains. In scTCR, the variable domains are typically covalently connected by a linker (e.g. a short peptide), whereby one of both constant domains is omited. For example, three-domain TCRs can comprise the variable region of the a-chain and the variable and constant region of the P-chain of the TCR.
In particular embodiments, the TCR is a TCR exogenous to the Treg cell, meaning that the cell does not normally express the particular TCR.
In particular embodiments, the TCR is a human TCR, i.e., a TCR wherein at least the variable regions of the TCR chains are human. Hence, the qualifier “human” in this connection relates to the primary sequence of the respective peptides, polypeptides, proteins, or nucleic acids, rather than to its origin or source. For example, such peptides, polypeptides, proteins, or nucleic acids may be present in or isolated from samples of human subjects or may be obtained by other means (e.g., by recombinant expression, cell-free transcription or translation, or non-biological nucleic acid or peptide synthesis). For example, a murinized (mu) human TCR (i.e. wherein the constant regions are replaced by corresponding murine counterparts) are also considered herein as a human TCR.
In particular embodiments, the nucleic acid may encode one or more CARs. In particular embodiments, the nucleic acid may encode at least two, such as two, three, four, five, six, seven or eight, different CARs.
The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers to a recombinant polypeptide or a set of polypeptides, which ,when expressed by an immune effector cell, endows the cell with specificity for a target molecule on the surface of a target cell, and with intracellular signal transduction. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signalling domain (also referred to herein as “an intracellular signalling domain” or “an intracellular activation domain”) comprising a functional signalling domain derived from a stimulatory molecule and/or a costimulatory molecule. The term “signalling domain” refers to the functional portion of a protein which acts by transmiting information within the cell to regulate cellular activity via defined signalling pathways by generating second messengers or functioning as effectors by responding to such messengers. Typically, a CAR may comprise a chimeric fusion protein, such that for example an antigen binding domain and an intracellular signalling domain are comprised within the same polypeptide chain. In alternative embodiments, a CAR may be formed by a set of polypeptides not contiguous with each other, such that for example an antigen binding domain and an intracellular signalling domain may be provided in separate polypeptide chains, configured to heterodimerise to form the CAR. By means of illustration, the antigen binding domain and the intracellular signalling domain may each be provided with a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides containing said domains to one another.
Alternative CAR constructs may typically be characterised as belonging to successive generations. In first-generation CARs the intracellular signalling domain contains or consists essentially of the zeta chain associated with the T cell receptor complex (CD3Q or the y subunit of the immunoglobulin Fc receptor (FcRy). The cytoplasmic signalling domain of second generation CARs further comprises an intracellular costimulatory domain, i.e., a functional signalling domain derived from at least one costimulatory molecule, such as CD28, 4-1BB (CD137), DAP10, ICOS, or 0X40 (CD 134), and third-generation CARs include a combination of two or more such costimulatory endodomains.
In particular embodiments, the CAR comprises an ectodomain, a transmembrane domain and an intracellular portion.
In particular embodiments, the ectodomain comprises an extracellular antigen recognition domain.
The term “antigen” or “Ag” as used herein is defined as a molecule capable of being bound by an antigen recognition domain, such as capable being bound to an antibody or receptor (e.g. T-cell receptor).
The term “antigen recognition domain” or “binding domain” or “antigen-specific binding domain” as used herein refers to the domain of the CAR that binds to a specific target molecule. A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a target molecule.
In particular embodiments, the extracellular antigen recognition domain is derived from an antibody or an antibody fragment. The term “antibody” is used herein in its broadest sense and generally refers to any immunologic binding agent, such as a whole antibody, including without limitation a chimeric, humanized, human, recombinant, transgenic, grafted and single chain antibody, and the like, or any fusion proteins, conjugates, fragments, or derivatives thereof that contain one or more domains that selectively bind to an antigen of interest. The term antibody thereby includes a whole immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or an immunologically effective fragment of any of these. The term thus specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g., 2-, 3- or more-valent) and/or multi-specific antibodies (e.g., bi- or more-specific antibodies) formed from at least two intact antibodies, and antibody fragments insofar they exhibit the desired biological activity (particularly, ability to specifically bind an antigen of interest), as well as multivalent and/or multi-specific composites of such fragments. The term “antibody” is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide, e.g., a recombinantly expressed polypeptide, which is made to encompass at least one complementarity-determining region (CDR) capable of specifically binding to an epitope on an antigen of interest. Hence, the term applies to such molecules regardless whether they are produced in vitro, in cell culture, or in vivo. The term “antibody fragment” or “antigen -binding moiety” comprises a portion or region of a full length antibody, generally the antigen binding or variable domain thereof. Examples of antibody fragments include Fab, Fab', F(ab)2, Fv, scFv fragments, single domain (sd)Fv, such as VH domains , VL domains and VHH domains, diabodies, linear antibodies, single-chain antibody molecules, in particular heavy-chain antibodies; and multivalent and/or multispecific antibodies formed from antibody fragment(s), e.g., dibodies, tribodies, and multibodies. The above designations Fab, Fab', F(ab')2, Fv, scFv etc. are intended to have their art- established meaning.
A full-length antibody as it exists naturally is an immunoglobulin molecule comprising 2 heavy (H) chains and 2 light (L) chains interconnected by disulfide bonds. The amino terminal portion of each chain includes a variable region of about 100-110 amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein. The carboxyterminal portion of each chain defines a constant region primarily responsible for effector function.
The CDRs are interspersed with regions that are more conserved, termed framework regions (FR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the light chain are referred to as “LCDR1, LCDR2, and LCDR3” and the 3 CDRs of the heavy chain are referred to as “HCDR1, HCDR2, and HCDR3.” The CDRs contain most of the residues which form specific interactions with the antigen. The numbering and positioning of CDR amino acid residues within the LCVR and HCVR regions is in accordance with the well-known Kabat numbering convention, which refers to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain regions of an antibody (Kabat, et al., Ann. NYAcad. Sci. 190:382-93 (1971 ); Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242 (1991 )). The positioning of CDRs in the variable region of an antibody follows Kabat numbering or simply, “Kabat.”
Light chains are classified as kappa or lambda, and are characterized by a particular constant region as known in the art. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the isotype of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively. IgG antibodies can be further divided into subclasses, e.g., IgGl, IgG2, IgG3, IgG4. Each heavy chain type is characterized by a particular constant region with a sequence well known in the art.
In certain embodiments, an antibody may be any of IgA, IgD, IgE, IgG and IgM classes, and preferably IgG class antibody.
In particular embodiments, the extracellular antigen recognition domain comprises, consists essentially of, or consists of the antigen-binding region of an antibody or an antibody fragment.
The term “antigen-binding portion” or “antigen-binding region” refers to one or more fragments of an antibody, such as a particular site, part, domain or stretch of amino acid residues, that retain the ability to specifically bind to an antigen of interest. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. These may be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature, 341 : 544-546 (1989); PCT publication WO 90/05144), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) (Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci., 85: 5879-5883 (1988)). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (Holliger, et al., Proc. Natl. Acad. Sci., 90: 6444-6448 (1993); Poljak, et al., Structure 2: 1121-1123 (1994)). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer- Verlag. New York. 790 pp. (ISBN 3-540-41354-5). In addition, the term “sequence” as used herein (for example in terms like “variable domain sequence”, “VHH sequence” or “protein sequence”), should generally be understood to include both the relevant amino acid sequence as well as nucleic acid sequences or nucleotide sequences encoding the same, unless the context requires a more limited interpretation. In particular embodiments, the antigen-binding region of an antibody or an antibody fragment specifically binds to an antigen of interest.
The term “specifically bind” means that an agent (denoted herein also as “binding agent” or “specific-binding agent”) binds to one or more desired targets (e.g., peptides, polypeptides, proteins, nucleic acids, or cells) substantially to the exclusion of other entities which are random or unrelated, and optionally substantially to the exclusion of other molecules that are structurally related. The term “specifically bind” does not necessarily require that an agent binds exclusively to its intended target(s). For example, an agent may be said to specifically bind to target(s) of interest if its affinity for such intended target(s) under the conditions of binding is at least about 2-fold greater, preferably at least about 5 -fold greater, more preferably at least about 10-fold greater, yet more preferably at least about 25 -fold greater, still more preferably at least about 50-fold greater, and even more preferably at least about 100-fold, or at least about 1000-fold, or at least about 104- fold, or at least about 105-fold, or at least about 106-fold or more greater, than its affinity for a nontarget.
The binding or interaction between the agent and its intended target(s) may be covalent (i.e., mediated by one or more chemical bonds that involve the sharing of electron pairs between atoms) or, more typically, non-covalent (i.e., mediated by non-covalent forces, such as for example, hydrogen bridges, dipolar interactions, van der Waals interactions, and the like). Preferably, the agent may bind to or interact with its intended target(s) with affinity constant (KA) of such binding KA > IxlO6 M 1, more preferably KA > IxlO7 M 1, yet more preferably KA > IxlO8 M 1, even more preferably KA > IxlO9 M 1, and still more preferably KA > IxlO10 M 1 or KA > IxlO11 M 1, wherein KA = [A_T]/[A][T], A denotes the agent, T denotes the intended target. Determination of KA can be carried out by methods known in the art, such as for example, using equilibrium dialysis and Scatchard plot analysis.
In preferred embodiments, the extracellular antigen recognition domain comprises, consists essentially of, or consists of a single chain variable fragment of an antibody (scFv).
In further embodiments, the extracellular antigen recognition domain may comprise, consist essentially of, or consist of divalent scFv. In CARs comprising di-scFvs, two scFvs specific for each antigen are linked together by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs, such as described in Xiong, C.Y. et al., 2006, Protein Engineering Design and Selection 19 (8): 359-367; Kufer, P. et al., 2004, Trends in Biotechnology 22 (5): 238- 244). scFvs may be obtained using standard recombinant DNA techniques. For example, scFvs may be prepared by the isolation of the coding sequence from a hybridoma producing antibodies, identification of V-chain types and design of a nucleic acid encoding the scFv, as described in Koksal H. et al., 2019, Antibody Therapeutics, 2(2):56-63. The scFv may comprise, consist essentially of, or consist of, the VL sequence, the linker peptide, and the VH sequence, wherein the VL sequence is located N-terminally of the linker peptide, and the linker peptide is located N- terminally of the VH sequence. Different scFv designs are possible, such as wherein the position of the VL and VH sequence is swapped.
In preferred embodiments, the extracellular antigen recognition domain comprises, consists essentially of, or consists of a single domain variable fragment of a heavy chain antibody (VHH) specific for the antigen or a Nanobody®.
The terms “Nanobody®” and “Nanobodies®” are trademarks of Ablynx NV (Belgium). The term “Nanobody” is well-known in the art and as used herein in its broadest sense encompasses an immunological binding agent obtained (1) by isolating the VHH domain of a naturally occurring heavy-chain antibody, preferably a heavy-chain antibody derived from camelids; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanization" of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized VHH domain; (4) by "camelization" of a naturally occurring VH domain from any animal species, and in particular from a mammalian species, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by "camelisation" of a "domain antibody" or "dAb" as described in the art, or by expression of a nucleic acid encoding such a camelized dAb, for the term "dAb", reference is for example made to Ward et al. (Nature 1989 Oct. 12; 341 (6242): 544- 6), to Holt et al., Trends Biotechnol., 2003, 21(11):484-490; as well as to for example WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd, single domain antibodies or single variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", see for example WO 05/18629); (6) by using synthetic or semisynthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (7) by preparing a nucleic acid encoding a Nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and/or (8) by any combination of one or more of the foregoing. “Camelids" as used herein comprise old world camelids {Camelus bactrianus and Camelus dromade rius) and new world camelids (for example Lama paccos, Lama glama and Lama vicugna).
The amino acid sequence and structure of a Nanobody can be considered - without however being limited thereto - to be comprised of four framework regions or "FR's", which are referred to in the art and herein as "Framework region 1" or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CDR's", which are referred to in the art as "Complementarity Determining Region l"or "CDR1"; as "Complementarity Determining Region 2" or "CDR2"; and as "Complementarity Determining Region 3" or "CDR3", respectively. The total number of amino acid residues in a Nanobody can be in the region of 110-120, and preferably 112-115. It should however be noted that parts, fragments, analogs or derivatives of a Nanobody are not particularly limited as to their length and/or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are preferably suitable for the purposes described herein.
The variable domains present in naturally occurring heavy chain antibodies are also be referred to as “VHH domains”, in order to distinguish them from the heavy chain variable domains that are present in conventional 4-chain antibodies (which will be referred to herein as “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to herein as “VL domains”). VHH domains have a number of unique structural characteristics and functional properties which make isolated VHH domains (as well as Nanobodies based thereon, which share these structural characteristics and functional properties with the naturally occurring VHH domains) and proteins containing the same highly advantageous for use as functional antigen-binding domains or proteins. In particular, and without being limited thereto, VHH domains (which have been “designed” by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain) and Nanobodies can function as a single, relatively small, functional antigen-binding structural unit, domain or protein. This distinguishes the VHH domains from the VH and VL domains of conventional 4-chain antibodies, which by themselves are generally not suited for practical application as single antigen-binding proteins or domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in ScFv's fragments, which consist of a VH domain covalently linked to a VL domain, as described elsewhere in this specification).
In further embodiments, the antigen-binding region is obtained from a multispecific antibody or antibody fragment (such as a bispecific, trispecific, etc. antibody) comprising at least two (such as two, three, etc.) binding sites, each directed against a different antigen or antigenic determinant.
The antigen-binding region may be obtained from antibodies or antibody fragments originating from or comprising one or more portions derived from any animal species, preferably vertebrate species, including, e.g., birds and mammals. Without limitation, the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant. Also without limitation, the antibodies may be human, murine (e.g., mouse, rat, etc.), porcine, donkey, rabbit, goat, sheep, guinea pig, monkey (e.g., cynomolus monkeys), camel (e.g., Camelus bactrianus and Camelus dromade ruts) also including camel heavy-chain antibodies, llama (e.g., Lama paccos, Lama glama or Lama vicugna) also including llama heavy-chain antibodies, or horse.
In particular embodiments, the antigen-binding region may be obtained from a chimeric antibody or chimeric antibody fragment, such as a chimeric antibody or chimeric antibody fragment originating from at least two animal species. More specifically, the term “chimeric antibody” or “chimeric antibodies” refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as for example antibodies having murine heavy and light chain variable regions linked to human, nonhuman primate, canine, equine, or feline constant regions. Chimeric antibodies comprise a portion of the heavy and/or light chain that is identical to or homologous with corresponding sequences from antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous with corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, exhibiting the desired biological activity (See e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)). Chimeric antibodies are made through merging DNA encoding a portion, such as the Fv region, of a monoclonal antibody from one species, e.g. mouse or monkey, with the antibody-producing DNA from another species, e.g. human.
In particular embodiments, the antigen-binding region may be obtained from a fully human antibody or antibody fragment. As used herein, the term “fully human antibody” refers to an antibody of which the encoding genetic information is of human origin. Accordingly, the term “fully human antibody” refers to antibodies having variable and constant regions derived only from human germline immunoglobulin sequences. The term “fully human antibody” is thus not to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, have been grafted onto human framework sequences.
In particular embodiments, the antigen-binding region may be obtained from a humanized antibody or antibody fragment. More particularly, the term “humanized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non -human species (e.g., a mouse) but in which at least a portion of the VH and/or VU sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which non-human CDR sequences are introduced into human VH and VU sequences to replace the corresponding human CDR sequences.
A skilled person will understand that the antigen-binding region obtained from an antibody or antibody fragment can include one or more amino acid deletions, additions and/or substitutions (e.g., conservative substitutions), insofar such alterations preserve its binding of the respective antigen.
In particular embodiments, the CAR ectodomain or CAR antigen recognition domain comprises, consists essentially of or consists of an antibody-like scaffold, a cognate receptor or ligand for the antigen or an antigen-binding portion of said receptor or ligand, or a synthetic receptor.
In particular embodiments, the CAR ectodomain comprises one or more antibody-like scaffolds.
The term “antibody-like scaffold” as used herein refers to a synthetic or natural binding molecules having a stable scaffold holding the molecule together and a variable arm binding to specific targets thereby mimicking the general structure and function of an antibody. Non-limiting examples of antibody-like scaffolds include designed ankyrin repeat proteins (DARPins), affimers and monobodies.
In particular embodiments, the CAR ectodomain comprises a cognate receptor or ligand for the antigen or an antigen-binding portion of said receptor or ligand.
The term “cognate” as used herein with regard to the receptor or ligand refers to the receptor or ligand with which the target molecule preferentially interacts under physiological conditions, or under in vitro conditions substantially approximating physiological conditions. As used herein, the term “preferentially interacts” is synonymous with “preferentially binding” and refers to an interaction that is statistically significantly greater in degree relative to a control.
As described elsewhere in this specification for antibodies, the term “antigen-binding portion” or “antigen-binding region” refers to one or more fragments of a receptor or ligand that retain the ability to specifically bind to an antigen.
In particular embodiments, the CAR ectodomain comprises a synthetic receptor. The term “synthetic receptor” or “recombinant receptor” refers to a receptor that cannot be found in nature as such and is being artificially produced by man. For example, a polypeptide sequence can be intentionally modified by man in the laboratory.
When the CAR includes a single chain variable fragment of an antibody (scFv) or a single domain variable fragment of a heavy chain antibody (VHH) specific for the antigen, an antibody-like scaffold, a cognate receptor or ligand for the antigen or an antigen-binding portion of said receptor or ligand, or a synthetic receptor known to specifically bind to an antigen of interest, such as a tumor antigen or viral antigen, the capacity of such CAR to bind to the antigen of interest is meaningfully similar or comparable to the ability of the scFv or VHH specific for the antigen, the antibody-like scaffold, the cognate receptor or ligand for the antigen or the antigen-binding portion of said receptor or ligand, or the synthetic receptor. The “transmembrane domain” is the portion of the CAR that fuses the extracellular binding portion and intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell. The transmembrane domain may be derived either from a natural, synthetic, semisynthetic, or recombinant source.
For example, the transmembrane domain may be derived from, such as may comprise, consist essentially of, or consist of, at least the transmembrane region(s) of, the alpha or beta chain of the T-cell receptor, CD3 epsilon, CD3 zeta, CD4, CD5, CD8 (e.g. CD8 alpha), CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, or CD 154, but is not limited thereto.
In particular embodiments, the transmembrane domain comprises, consists essentially of, or consists of the CD28 transmembrane domain or the CD8 alpha transmembrane domain. For example, the CD28 transmembrane domain may be a human CD28 transmembrane domain or the CD8 alpha transmembrane domain may be a human CD8 alpha transmembrane domain.
In one embodiment, the intracellular portion of the CAR comprises, consists essentially of, or consists of, an intracellular activation domain.
The term “intracellular signaling domain” or “intracellular activation domain” as used herein refers to intracellular part of a CAR that participates in transducing the message of effective CAR binding to a target molecule into the interior of the immune effector cell to elicit effector cell function (i.e. to perform a specialized function). Functions of the effector cell may encompass activation, cytokine production, proliferation and cytotoxic activity, such as releasing cytotoxic factors to the target cell bound by the CAR. The term “effector cell function” as used herein refers to a specialized function of the immune effector cell.
In particular embodiments, the intracellular activation domain comprises an immunoreceptor tyrosine-based activation motif or a signaling motif (ITAM).
In particular embodiments, the primary intracellular activation domain comprises, consists essentially of, or consists of a CD3 zeta activation domain, a FcR gamma activation domain, a FcRbeta activation domain, a CD3 gamma activation domain, a CD3 delta activation domain, a CD3 epsilon activation domain, a CD5 activation domain, a CD22 activation domain, a CD79a activation domain, a CD79b activation domain, a FcsRI activation domain, a CD32 activation domain, a DAP 10 activation domain, a DAP 12 activation domain, and/or a multiple EGF-like domains 10 (MEGF10) activation domain, preferably a CD3 zeta activation domain or a FcR gamma activation domain. In particular embodiments, the intracellular portion of the CAR further comprises, essentially consists of, or consists of, at least one, such as at least two, costimulatory intracellular domain.
The term “costimulatory intracellular domain” as used herein refers to the intracellular domain of a costimulatory molecule of an immune effector cell that specifically binds with a cognate stimulatory ligand, wherein the ligand is present on an antigen presenting cell (e.g. antigen presenting cell (APC), B-cell or dendritic cell). The costimulatory intracellular domain is able to mediate or enhance the primary response by the immune effector cell in the presence of the primary antigen receptor, such as activation, initiation of an immune response and/or proliferation. The intracellular signaling domain may comprise the entire intracellular portion of the molecule from which it is derived, or the entire intrinsic intracellular signaling domain, or a functional fragment thereof.
Non-limiting examples of costimulatory molecules are the costimulatory domain of MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, (CDla / CD18), 4-1BB (CD137), B7-H3, ICAM-1, ICOS (CD278), GITR, CD4, CD27, CD28, CD30, CD40, ICAM- CD8 beta, IL2Rbeta, IL2R gamma, IL7Ralpha, ITGA4, VLA1, CD49a, IT49A, NKp80, NKp80, NKp44, NKp30, NKp46, NKp30, NKp46, CD19, CD4, CD8 alpha, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD 18, LFA-2 (CD2), NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 , CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME SLAMF8, SELPLG CD162, LTBR, LAT, GADS, PAG / Cbp, CD19a, and ligands that specifically bind to CD83.
The costimulatory domain most commonly used is that of CD28. This supplies the most potent costimulatory signal - namely immunological signal 2, which triggers proliferation of the immune effector cell, such as T-cell proliferation. Furthermore, TNF receptor family endodomains, such as the 0X40 and 4- IBB, transmit survival signals.
In particular embodiments, the at least one costimulatory intracellular domain comprises, consists essentially of, or consists of the CD28 costimulatory domain, the 4-1BB costimulatory domain, the DNAX-activation protein 10 (DAP10) costimulatory domain, the 0X40 (CD134) costimulatory domain and/or the ICOS (CD278) costimulatory domain.
Even more potent third generation CARs have also been described which have multiple costimulatory endodomains, capable of transmitting activation, proliferation and survival signals, such as a combination of CD28 and 4-1BB (TLR2), as described in Weinkove R. et al., 2019, Clin Transl Immunology, 8(5): el049.
Accordingly, in particular embodiments, the CAR comprises two complementary costimulatory intracellular domains, preferably selected from the group consisting of the CD28 costimulatory domain, the 4-1BB costimulatory domain, the OX-40 costimulatory domain, the ICOS costimulatory domain and the CD27 costimulatory domain.
In particular embodiments, the intracellular portion of the CAR comprises, consists essentially of or consists of, at least one intracellular activation domain selected from the group consisting of the CD3^ and FcRy intracellular activation domain and at least one intracellular costimulatory domain selected from the group consisting of the CD28 costimulatory domain, the 4-1BB costimulatory domain, the DAP 10 costimulatory domain, the 0X40 costimulatory domain and the ICOS costimulatory domain.
In particular embodiments, the CAR may comprise one or more linkers between the ectodomain and the transmembrane domain and/or between the transmembrane domain and the intracellular portion. Non-limiting examples of linkers include flexible linkers such as glycine polymers (G)n, glycine-serine polymers (Gi-5Si-5)n, where n is an integer of at least one, two, three, four, or five, glycine-alanine polymers, alanine-serine polymers, or the like.
In particular embodiments, the CAR ectodomain further comprises one or more hinge regions.
The term “hinge region” as used herein refers to an amino acid sequence located between the antigen recognition domain and the transmembrane domain of the CAR, which is able to position the antigen recognition domain away from the immune effector cell surface to enable proper cellcell contact, antigen binding and activation. The hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a, CD4, CD28 and CD7.
The skilled person shall appreciate that a TCR or a CAR molecule as discussed here is a transmembrane protein and will typically require the inclusion of a suitable signal or leader sequence when expressed to effect the cellular membrane localisation of the protein. Such signal sequences are typically short (3-60 amino acids long) N-terminally located peptide chains, which are optionally and advantageously cleaved off or processed away by signal peptidase after the proteins are transported, such as to yield the mature protein. Signal sequences are widely known in the art and they may be applied for the expression of the TCR or CAR as taught herein. In certain embodiments, the signal sequence comprise, consist essentially of or consist essentially of the leader sequence of CD8 alpha, preferably human CD8 alpha.
In certain embodiments, such signal sequence can be N-terminally fused to any one of the TCR or CAR molecules individualised above.
In certain embodiments,
- the CAR ectodomain comprises a single chain variable fragment of an antibody (scFv) or a single domain variable fragment of a heavy chain antibody (VHH) specific for the antigen, an antibody-like scaffold, a cognate receptor or ligand for the antigen or an antigen-binding portion of said receptor or ligand, or a synthetic receptor, and/or
- the intracellular portion of the CAR comprises at least one intracellular activation domain, such as a CD3^ or FcRy intracellular activation domain, and optionally and preferably at least one intracellular costimulatory domain, such as a CD28, 4-1BB, DAP10, 0X40 and/or ICOS intracellular costimulatory domain.
In certain embodiments, the antigen is an autoantigen, alloantigen, or an allergen.
With an “auto-antigen” (self-antigen) as used herein is meant a substance, e.g., a cell or tissue or a component thereof, that is normally present in the body, but that provokes an immune response. Preferably, said auto-antigen is involved in the induction and/or progression of an autoimmune disease. In certain embodiments, the autoantigen is involved in the induction and/or progression of multiple sclerosis, rheumatoid arthritis, type I diabetes, autoimmune uveitis, autoimmune myasthenia gravis, psoriasis, celiac disease, systemic lupus erythematosus, inflammatory bowel disease, Addison’s disease, Graves’ disease, Sjogren’s syndrome, Hashimoto’s thyroiditis, autoimmune vasculitis, pernicious anemia, or idiopathic thrombocytopenic purpura (ITP), or wherein the alloantigen is involved in the induction and/or progression of graft-versus-host disease or in transplant rejection.
Auto-antigens are generally known in the art and it shall be appreciated that the skilled person is capable of selecting an auto-antigen that may be suitable target for the Treg cells according to certain embodiments of the invention. By means of examples and without limitation, auto-antigens involved in type I diabetes may include one or more of insulin, pro-insulin, glutamic acid decarboxylase 65 (GAD65), GAD67, insulinoma-associated antigen 2 (IAA2), heat shock protein 65 (hsp65), islet cell antigen 69 (ICA69), zinc transporter 8 (ZnT8), and immunodominant peptides thereof; auto-antigens involved in multiple sclerosis may include one or more of myelin-binding protein (MBP), alphaB -crystallin, SlOObeta, proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG)-alpha and MOG-beta isoforms, enolase and arrestin, and immunodominant peptides thereof; auto-antigens involved in rheumatoid arthritis may include one or more of citrullinated proteins against which anti-citrullinated protein antibodies (ACPA) are formed, such as, fibrinogen, vimentin, enolase, collagen type I, fibronectin, a translational initiation factor and viral protein EBNA-1, and immunodominant peptides thereof; auto-antigens involved in autoimmune uveitis may include one or more of Retinal S antigen (S-Ag), tyrosinase-related proteins, interphotoreceptor retinoid-binding protein (IRBP), and immunodominant peptides thereof; auto-antigens involved in psoriasis may include one or more of psoriasis-associated antigen pso p27, and immunodominant peptides thereof; etc.
The term “alloantigen” is known in the art and encompasses a genetically determined antigen present in some but not all individuals of a species (as those of a particular blood group) and capable of inducing the production of an alloantibody by individuals which lack it. Alloantigens may for example be involved in the induction and/or progression of graft-versus-host disease or in transplant rejection.
The term “allergen” is known in the art and encompasses any substance, chemical, particle or composition which is capable of stimulating an allergic response in a susceptible individual. Allergens may be contained within or derived from a food item such as, for example, dairy products (e.g., cow’s milk), egg, celery, sesame, wheat, soy, fish, shellfish, sugars (e.g., sugars present on meat such as alpha-galactose), peanuts, other legumes (e.g., beans, peas, soybeans, etc.), and tree nuts. Alternatively, an allergen may be contained within or derived from a non-food item such as, for example , dust (e.g., containing dust mite), pollen, insect venom (e.g., venom of bees, wasps, mosquitos, fire ants, etc.), mold, animal fur, animal dander, wool, latex, metals (e.g., nickel), household cleaners, detergents, medication, cosmetics (e.g., perfumes, etc.), drugs (e.g., penicillin, sulfonamides, salicylate, etc.), therapeutic monoclonal antibodies (e.g., cetuximab), ragweed, grass and birch. Examples of pollen allergens include tree pollens such as birch pollen, cedar pollen, oak pollen, alder pollen, hornbeam pollen, aesculus pollen, willow pollen, poplar pollen, plantanus pollen, tilia pollen, olea pollen, Ashe juniper pollen, and Alstonia scholaris pollen. Allergens are involved in the induction and/or progression of allergic reactions, which may include one or more signs or symptoms selected from the group consisting of urticaria (e.g., hives), angioedema, rhinitis, asthma, vomiting, sneezing, runny nose, sinus inflammation, watery eyes, wheezing, bronchospasm, reduced peak expiratory flow (PEF), gastrointestinal distress, flushing, swollen lips, swollen tongue, reduced blood pressure, anaphylaxis, and organ dysfimction/failure.
In certain embodiments, the endogenous T cell receptor (TCR) of the Treg cells may have been knocked-out or knocked-down. Any method may be used to this end, such as antisense oligonucleotides, RNA interference agents, or genetically engineering the Treg cell’s genome, for example using a gene editing system, such as CRISPR/Cas. In certain embodiments, the present methods are good manufacturing practice (GMP) compliant.
In certain embodiments, method further comprises cryopreservation of the Treg cells comprising the polynucleotide. Cryopreservation media are well known, and may include, for example, a liquid medium comprising 10% v/v DMSO, such as a liquid medium consisting of 90% serum (such as human serum) and 10% DMSO. Cryopreservation temperature may be typically about -80°C, such as in a -80°C freezer or in a liquid nitrogen container.
The method may further comprise formulating the Treg cells comprising the polynucleotide into a pharmaceutical composition or a kit-of-parts suitable for medicinal use. The pharmaceutical composition will typically also comprise one or more pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant.
A further aspect thus provides the Treg cells comprising the polynucleotide, obtainable or obtained by the methods disclosed herein.
A further aspects provides a pharmaceutical composition comprising the Treg cells comprising the polynucleotide, obtainable or obtained by the methods disclosed herein.
The term “pharmaceutically acceptable” as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.
Carriers, diluents, excipients and/or adjuvants include any and all solvents, diluents, buffers (e.g., neutral buffered saline or phosphate buffered saline), solubilizers, colloids, dispersion media, vehicles, fdlers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, stabilisers, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Such materials should be non-toxic and should not interfere with the activity of the cells.
The precise nature of the carriers, diluents, excipients and/or adjuvants or other material will depend on the route of administration. For example, the composition may be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability. For general principles in medicinal formulation, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds., Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000. Liquid pharmaceutical compositions may generally include a liquid carrier such as water or a pharmaceutically acceptable aqueous solution. For example, physiological saline solution, tissue or cell culture media, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
The composition may include one or more cell protective molecules, cell regenerative molecules, growth factors, anti-apoptotic factors or factors that regulate gene expression in the cells. Such substances may render the cells independent of its environment.
Such pharmaceutical compositions may contain further components ensuring the viability of the cells therein. For example, the compositions may comprise a suitable buffer system (e.g., phosphate or carbonate buffer system) to achieve desirable pH, more usually near neutral pH, and may comprise sufficient salt to ensure isosmotic conditions for the cells to prevent osmotic stress. For example, suitable solution for these purposes may be phosphate-buffered saline (PBS), sodium chloride solution, Ringer's Injection or Lactated Ringer's Injection, as known in the art. Further, the composition may comprise a carrier protein, e.g., albumin (e.g., bovine or human albumin), which may increase the viability of the cells.
Further suitably pharmaceutically acceptable carriers or additives are well known to those skilled in the art and for instance may be selected from proteins such as collagen or gelatine, carbohydrates such as starch, polysaccharides, sugars (dextrose, glucose and sucrose), cellulose derivatives like sodium or calcium carboxymethylcellulose, hydroxypropyl cellulose or hydroxypropylmethyl cellulose, pregeletanized starches, pectin agar, carrageenan, clays, hydrophilic gums (acacia gum, guar gum, arabic gum and xanthan gum), alginic acid, alginates, hyaluronic acid, polyglycolic and polylactic acid, dextran, pectins, synthetic polymers such as water-soluble acrylic polymer or polyvinylpyrrolidone, proteoglycans, calcium phosphate and the like.
In an embodiment, the pharmaceutical cell preparation as defined above may be administered in a form of liquid composition. In embodiments, the cells or pharmaceutical composition comprising such can be administered systemically, topically, within an organ, at a site of organ dysfunction or lesion or at a site of tissue lesion.
Preferably, the pharmaceutical compositions may comprise a therapeutically effective amount of the desired cells. The term “therapeutically effective amount” refers to an amount which can elicit a biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, and in particular can prevent or alleviate one or more of the local or systemic symptoms or features of a disease or condition being treated. Appropriate therapeutically effective amounts may be determined by a qualified physician with due regard to the nature of the desired cells, the disease condition and severity, and the age, size and condition of the subject.
Also provided are methods of producing said pharmaceutical compositions by admixing the cells of the invention with one or more additional components as described above as well as with one or more pharmaceutical carriers, diluents, excipients and/or adjuvants as described above.
Also disclosed is an arrangement or kit of parts comprising a surgical instrument or device for administration of the cells as taught herein or the pharmaceutical compositions as defined herein to a subject, such as for example systemically, for example, by injection, and further comprising the cells as taught herein or the pharmaceutical compositions as defined herein.
In an embodiment, the pharmaceutical composition as define above may be administered in a form of a liquid composition.
The quantity of cells to be administered will vary for the subject being treated. In certain embodiments, the quantity of cells to be administered is between 102 to IO10 or between 102 to 109, or between 103 to IO10 or between 103 to 109, or between 104 to IO10 or between 104 to 109, such as between 104 and 108, or between 105 and 107, e.g., about IxlO5, about 5xl05, about IxlO6, about 5xl06, about IxlO7, about 5xl07, about IxlO8, about 5xl08, about IxlO9, about 5xl09, or about IxlO10 cells can be administered to a human subject. For example, such administration may be suitably distributed over one or more doses (e.g., distributed over 2, 3, 4, 5, 6, 7, 8 9 or 10 or more doses) administered over one or more days (e.g., over 1, 2, 3, 4 or 5 or more days). However, the precise determination of a therapeutically effective dose may be based on factors individual to each patient, including their size, age, tissue damage, and can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art. Suitably but without limitation, in a composition to be administered, cells may be present at a concentration between about 104/ml to about 109/ml, preferably between about 105/ml and about 108/ml, yet more preferably between about lxl06/ml and about lxl08/ml.
A further aspect provides the T reg cells or the pharmaceutical compositions as taught herein, for use in medicine, i.e., for use in therapy.
Reference to “therapy” or “treatment” broadly encompasses both curative and preventative treatments, and the terms may particularly refer to the alleviation or measurable lessening of one or more symptoms or measurable markers of a pathological condition such as a disease or disorder. The terms encompass primary treatments as well as neo-adjuvant treatments, adjuvant treatments and adjunctive therapies. Measurable lessening includes any statistically significant decline in a measurable marker or symptom. Generally, the terms encompass both curative treatments and treatments directed to reduce symptoms and/or slow progression of the disease. The terms encompass both the therapeutic treatment of an already developed pathological condition, as well as prophylactic or preventative measures, wherein the aim is to prevent or lessen the chances of incidence of a pathological condition. In certain embodiments, the terms may relate to therapeutic treatments. In certain other embodiments, the terms may relate to preventative treatments. Treatment of a chronic pathological condition during the period of remission may also be deemed to constitute a therapeutic treatment. The term may encompass ex vivo or in vivo treatments as appropriate in the context of the present invention.
The terms “subject”, “individual” or “patient” are used interchangeably throughout this specification, and typically and preferably denote humans, but may also encompass reference to non-human animals, preferably warm-blooded animals, even more preferably non-human mammals. Particularly preferred are human subjects including both genders and all age categories thereof. In other embodiments, the subject is an experimental animal or animal substitute as a disease model. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. The term subject is further intended to include transgenic non-human species.
The term “subject in need of treatment” or similar as used herein refers to subjects diagnosed with or having a disease as recited herein and/or those in whom said disease is to be prevented.
A further aspect provides T reg cells or the pharmaceutical composition as taught herein, wherein the polynucleotide encodes a polypeptide which endows the Treg cell with specificity to an antigen or an antigenic peptide thereof, for use in a method of treating a disease caused by or associated with an increased activity of the immune system against said antigen. As discussed above, such antigen may for example be an auto-antigen (such as an auto-antigen causative of or associated with an autoimmune disease, such as an autoimmune disease discussed elsewhere in this specification), or may be an allergen, or an allo-antigen.
A related aspect provides a method for treating, in a subject in need thereof, a disease caused by or associated with an increased activity of the subject’s immune system against an antigen, comprising administering to the subject an effective amount of the T reg cells or the pharmaceutical composition as taught herein, wherein the polynucleotide encodes a polypeptide which endows the Treg cell with specificity to the antigen or an antigenic peptide thereof.
The present application also provides aspects and embodiments as set forth in the following Statements. In these statements, the wording “The [subject] according to Statement [number], wherein... ” or “The [subject] according to any one of Statements [numbers], wherein... ” also discloses and may be replaced by the simple wording “In certain embodiments. . . ”. Statement 1. A method for introducing an unmodified or modified RNA polynucleotide into a T regulatory (Treg) cell, comprising electroporation of a suspension comprising the polynucleotide and Treg cells.
Statement 2. The method according to Statement 1, wherein:
- the Treg cells are CD45RA+ Treg cells, preferably an at least 90% pure population of CD45RA+ Treg cells; or
- the Treg cells are CD25hl Treg cells, preferably an at least 90% pure population of CD25hl Treg cells.
Statement 3. The method according to Statement 1 or 2, wherein the Treg cells are freshly-isolated, or wherein the Treg cells have been obtained by in vitro or ex vivo expansion of isolated Treg cells.
Statement 4. The method according to Statement 3, wherein the Treg cells have been isolated from peripheral blood mononuclear cells (PBMC) or from an internal organ, such as lung, liver, or spleen.
Statement 5. The method according to Statement 3 or 4, a) wherein the isolation comprises:
- isolating T cells, preferably CD4+ T cells and/or CD8+ cells, from PBMC or from an internal organ, thereby obtaining a population of T cells, preferably CD4+ T cells and/or CD8+ cells; and
- isolating Treg cells from the population of T cells; or b) wherein the isolation comprises directly isolating Treg cells from PBMC or from an internal organ.
Statement 6. The method according to any one of Statements 3 to 5, a) wherein the isolation comprises:
- isolating CD4+ cells and/or CD8+ cells from PBMC or from an internal organ, thereby obtaining a population of CD4+ cells and/or CD8+ cells; and
- isolating CD 127’ cells from the population of CD4+ cells and/or CD8+ cells, thereby obtaining Treg cells; or b) wherein the isolation comprises directly isolating CD4 CD127" cells and/or CD8+CD 127" cells from PBMC or from an internal organ. Statement 7. The method according to Statement 4 or 5, comprising isolating CD45RA+ Treg cells or CD25hl Treg cells or a mixture thereof from the population of T cells or CD4+ cells or comprising isolating CD8+LAG3+ Treg cells or CD8+CD25+FoxP3+CCL4+ Treg cells or a mixture thereof.
Statement 8. The method according to any one of Statements 5 to 7, wherein
- the T cells or CD4+ or CD8+ cells are isolated from PBMC or from the internal organ using magnetic-activated cell sorting, and/or wherein the Treg cells are isolated from the population of T cells or CD4+ or CD8+ cells using fluorescence-activated cell sorting (FACS); or
- the Treg cells are directly isolated from PBMC or from the internal organ using FACS.
Statement 9. The method according to any one of Statements 3 to 7, wherein the Treg expansion comprises a step of culturing the isolated Treg cells in the presence of interleukin-2 (IL-2).
Statement 10. The method according to any one of Statements 1 to 9, wherein the Treg cells are activated prior to electroporation, preferably wherein the Treg activation comprises a step of contacting the isolated and optionally expanded Treg cells with an anti-CD3 antibody and an anti- CD28 antibody.
Statement 11. The method according to Statement 10, wherein the antibodies are covalently linked to a polymer carrier.
Statement 12. The method according to Statement 10 or 11, wherein the contacting step is repeated two or more times during the Treg expansion and activation.
Statement 13. The method according to Statement 12, wherein the contacting step is repeated on days 0, 7, 14, and 19 of the Treg expansion and activation.
Statement 14. The method according to any one of Statements 1 to 13, wherein the concentration of the Treg cells in the suspension is 100 cells per ml to IxlO9 cells per ml, preferably IxlO7 to IxlO8 cells per ml, such as about 2.5xl07 cells per ml.
Statement 15. The method according to any one of Statements 1 to 13, wherein the voltage is from 100 V to 700 V, preferably from 400 V to 600 V, such as about 500 V.
Statement 16. The method according to any one of Statements 1 to 15, wherein the pulsing time is from 1 to 40 ms, preferably from 1 to 10 ms, such as about 5 ms.
Statement 17. The method according to any one of Statements 1 to 16, wherein the pulse is a square wave pulse. Statement 18. The method according to any one of Statements 1 to 17, wherein the concentration of the polynucleotide in the suspension is from 100 ng / IxlO6 cells to 10 pg / IxlO6 cells, preferably from 500 ng / IxlO6 cells to 5 pg / IxlO6 cells, more preferably about 1 pg / IxlO6 cells.
Statement 19. The method according to any one of Statements 1 to 18, wherein the polynucleotide is synthetic, or in vitro transcribed, or isolated from a host cell or a non-human host organism genetically engineered to produce the polynucleotide.
Statement 20. The method according to any one of Statements 1 to 19, wherein the polynucleotide is a naked polynucleotide.
Statement 21. The method according to any one of Statements 1 to 20, wherein the polynucleotide is a linear polynucleotide.
Statement 22. The method according to any one of Statements 1 to 21, wherein the modified RNA polynucleotide is nucleobase and/or backbone-modified.
Statement 23. The method according to any one of Statements 1 to 22, wherein the polynucleotide is coding or non-coding.
Statement 24. The method according to any one of Statements 1 to 23, wherein the polynucleotide is selected from the group consisting of messenger RNA (mRNA), guide RNA (gRNA), single guide RNA (sgRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), long non-coding RNA (IncRNA), ribozyme, aptamer, spiegelmer, and combinations thereof.
Statement 25. The method according to any one of Statements 1 to 24, wherein the polynucleotide is mRNA encoding one or more polypeptide, preferably one or more biologically active polypeptide.
Statement 26. The method according to any one of Statements 1 to 25, wherein the polynucleotide produces a gain-of-fimction phenotype when introduced into the Treg cell, such as wherein the polynucleotide is mRNA encoding one or more polypeptide, preferably one or more biologically active polypeptide, and the polypeptide produces a gain-of-fimction phenotype when expressed by the Treg cell.
Statement 27. The method according to Statement 26, wherein the one or more polypeptide endows the Treg cell with specificity to an antigen or an antigenic peptide thereof.
Statement 28. The method according to Statement 27, wherein the one or more polypeptide is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
Statement 29. The method according to Statement 28, wherein: - the CAR ectodomain comprises a single chain variable fragment of an antibody (scFv) or a single domain variable fragment of a heavy chain antibody (VHH) specific for the antigen, an antibody-like scaffold, a cognate receptor or ligand for the antigen or an antigen-binding portion of said receptor or ligand, or a synthetic receptor, and/or
- the intracellular portion of the CAR comprises at least one intracellular activation domain, such as a CD3^ or FcRy intracellular activation domain, and optionally and preferably at least one intracellular costimulatory domain, such as a CD28, 4-1BB, DAP10, 0X40 and/or ICOS intracellular costimulatory domain.
Statement 30. The method according to any one of Statements 27 to 29, wherein the antigen is an autoantigen, alloantigen, or an allergen.
Statement 31. The method according to Statement 28, wherein the autoantigen is involved in the induction and/or progression of multiple sclerosis, rheumatoid arthritis, type I diabetes, autoimmune uveitis, autoimmune myasthenia gravis, psoriasis, celiac disease, systemic lupus erythematosus, inflammatory bowel disease, Addison’s disease, Graves’ disease, Sjogren’s syndrome, Hashimoto’s thyroiditis, autoimmune vasculitis, pernicious anemia, or idiopathic thrombocytopenic purpura (ITP), or wherein the alloantigen is involved in the induction and/or progression of graft-versus-host disease or in transplant rejection.
Statement 32. The method according to any one of Statements 1 to 31, wherein the endogenous T cell receptor (TCR) of the Treg cells has been knocked-out or knocked-down.
Statement 33. The method according to any one of Statements 1 to 32, wherein the method is good manufacturing practice (GMP) compliant.
Statement 34. The method according to any one of Statements 1 to 33, wherein the method further comprises cryopreservation of the Treg cells comprising the polynucleotide.
Statement 35. The method according to any one of Statements 1 to 34, wherein the method further comprises formulating the Treg cells comprising the polynucleotide into a pharmaceutical composition or a kit-of-parts suitable for medicinal use.
Statement 36. The Treg cells comprising the polynucleotide, obtainable or obtained by the method of any one of Statements 1 to 35.
Statement 37. A pharmaceutical composition comprising the Treg cells comprising the polynucleotide, obtainable or obtained by the method of any one of Statements 1 to 35.
Statement 38. The T reg cells according to Statement 36 or the pharmaceutical composition according to Statement 37, for use in medicine. Statement 39. The T reg cells according to Statement 36 or the pharmaceutical composition according to Statement 37, wherein the polynucleotide encodes a polypeptide which endows the Treg cell with specificity to an antigen or an antigenic peptide thereof, for use in a method of treating a disease caused by or associated with an increased activity of the immune system against said antigen.
Statement 40. A method for treating, in a subject in need thereof, a disease caused by or associated with an increased activity of the subject’s immune system against an antigen, comprising administering to the subject an effective amount of the T reg cells according to Statement 36 or the pharmaceutical composition according to Statement 37, wherein the polynucleotide encodes a polypeptide which endows the Treg cell with specificity to the antigen or an antigenic peptide thereof.
While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and scope of the appended claims.
The herein disclosed aspects and embodiments of the invention are further supported by the following non-limiting examples.
EXAMPLES
Example 1 - Experimental procedures used in Examples 2-8
Human blood samples
Buffy coats from anonymous healthy donors were provided by the Blood Service of the Flemish Red Cross (Mechelen, Belgium). This study was approved by the Ethics Committee of the University of Antwerp and the Antwerp University Hospital (Belgium) under reference number EC 18/18/236. Information, if known, of the healthy donors used in this study is depicted in Table 1.
Table 1. Known information of the anonymous healthy donors used in this study. For each donor used in this study, year of birth and sex is given.
Figure imgf000050_0001
Figure imgf000051_0001
T cell isolation
Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare, Diegem, Belgium). Subsequently, CD4+ cells were positively isolated from 400 x 106 PBMCs using human CD4 MicroBeads for magnetic- activated cell sorting (MACS, Miltenyi Biotec, Leiden, Netherlands), according to the manufacturer’s instructions. Isolated CD4+ cells were stained with fluorochrome-conjugated monoclonal antibodies (mAbs) (Table 2). CD25hl Tregs were sorted as CD3+, CD4+, CD127" and CD25hi; naive Tregs were sorted as CD3+, CD4+, CD 127’, CD25+ and CD45RA+; and effector CD4+ T cells were sorted as CD3+, CD4+, CD127+ and CD25" (Figure 1A). Remaining CD8+ T cells, monocytes, natural killer (NK) cells and B cells were excluded from the sorting gate using a dump channel consisting of anti-CD8, -CD14, -CD16 and -CD19. Sorting was performed by flow cytometry using a FACSAria II device (BD Biosciences). After flow cytometric cell sorting, an aliquot of the sorted cells was used to confirm purity.
Table 2. Information of antibodies and dyes used in flow cytometry experiments shown in the Examples. For each antibody or dye, the specificity, fluorochrome, clone, reference number and company name is given, when applicable.
Figure imgf000051_0002
Figure imgf000052_0001
Ex vivo expansion of Trees
Tregs were expanded ex vivo in complete medium, consisting of Iscove’s modified Dulbecco’s medium (IMDM; Life Technologies) supplemented with 5% human AB serum (hAB; Life Technologies) and 500 lU/mL IL-2 (ImmunoTools GmbH, Friesoythe, Germany). Treg activation was achieved by using T cell TransAct (1: 100 dilution, Miltenyi Biotec) on day 0. TransAct is a clinical-grade colloidal reagent comprising iron oxide crystals embedded into a biocompatible polysaccharide matrix with a diameter of ~ 100 nm. Agonistic anti-CD3 and anti-CD28 antibodies are coated onto the nanomatrix. Reactivation was performed on days 7 and 14. Complete medium was replenished twice daily, and cell counts were monitored routinely using an automated hemocytometer (ABX Micros 60; Horiba, Diegem, Belgium). On days 7, 14 and 19, purity was assessed using fluorochrome-conjugated mAbs (Table 2) and measured using a CytoFLEX flow cytometer (Beckman Coulter, Analis, Suarlee, Belgium). A fluorescent-minus-one (FMO) control was used as a negative control. The proportion of viable cells was assessed by means of propidium iodide (PI, Thermo Fisher Scientific, Life Technologies, Merelbeke, Belgium) staining. For analytical flow cytometry, 10,000 events were recorded per sample.
Vector construction and in vitro mRNA transcription
The sequence of the TCR recognizing HLA-DR2_restricted myelin basic protein (MBP)s5-99 peptide was kindly provided by Prof. Dr. David W. Scott of the Uniformed Services School of Health Sciences (USUHS) in Bethesda, MD (Kim et al. Journal of Autoimmunity 2018, vol. 92, 77-86). Sequences encoding the TCR a- and P-chains were linked with the 2A sequence from porcine teschovirus-1 (P2A) (Szymczak et al. Nature Biotechnology 2004, vol. 22, 589-594). The sequences were cloned into the Spel-Xhol site of the pSTl plasmid (kindly provided by Dr. Ugur Sahin, Johannes-Gutenberg University, Mainz, Germany) (Holtkamp et al. Blood 2006, vol. 108, 4009-4017) under the control of a T7 promotor and with the addition of a poly(A) tail, and subjected to codon-optimization (GeneArt, Thermo Fisher Scientific). Additionally, a DNA plasmid encoding enhanced green fluorescent protein (eGFP; pGEM4Z/EGFP/A64 vector (Nair et al. Nature Biotechnology 1998, vol. 16, 364-9)) was kindly provided by Dr. Eh Gilboa (Duke University Medical Center, Durham, NC) (Ponsaerts et al. Leukemia 2002, vol. 16, 1324-30). All plasmids were propagated in Escherichia coli SoloPack Golden supercompetent cells (Agilent Technologic, Machelen, Belgium), and plasmid DNA was purified using a NucleoBond Xtra Midi EF kit (Macherey-Nagel, Duren, Germany). Next, purified plasmid DNA was linearized by SapI digestion (Thermo Fisher Scientific) for the MBPgj-gg-specific TCR plasmid and by Spel digestion (Thermo Fisher Scientific) for the eGFP plasmid. Subsequently, linearized plasmid DNA was used as a DNA template for in vitro transcription with a mMessage mMachineT7 in vitro transcription kit (Ambion, Life Technologies), according to the manufacturer’s protocol. mRNA quality was assessed by agarose gel electrophoresis and Nanodrop (Thermo Fisher Scientific). All mRNA constructs were stored at -20°C at a concentration of 1 pg/pL. mRNA electroporation
On day 19 of Treg expansion, Tregs were electroporated with mRNA encoding the MBP85-99- specific TCR. Tregs were washed twice and resuspended in cold serum-free Opti-MEM I medium (Gibco Invitrogen) at a concentration of 25xl06 cells/mL. 200 pL of the cell suspension was transferred to a 4.0-mm electroporation cuvette (Cell Projects, Kent, United Kingdom), and 1 pg/106 cells of in vitro transcribed mRNA was added to the cuvette. Electroporations were performed with a Gene Pulser Xcell device (Bio-Rad, Temse, Belgium) using a square wave pulse of 500 V for 5 ms. As a positive control, cells were electroporated under the same conditions using eGFP-encoding mRNA, while for the negative control no mRNA was added (mock electroporation). Immediately after electroporation, cells were replenished in IMDM supplemented with 10% hAB serum and rested for a minimum of 20 min in a humidified 5% CO2 incubator at 37°C before further analysis.
For the evaluation of transfection efficiency, kinetics of the expression of eGFP and of the MBP^. 99-specific TCR using an anti-TCR Vb2-phycoerythrin (PE) antibody (Beckman Coulter) was evaluated 0, 4, 24, 48, 72, 96, 120, 144, 168 and 192 h after electroporation using a CytoFLEX flow cytometer. The proportion of viable cells was assessed by means of PI staining (Thermo Fisher Scientific). For analytical flow cytometry, 10,000 events were recorded per sample.
Cryopreservation
Remaining PBMCs, not used for CD4+ isolation using MACS, and transfected Tregs, used for post-cryopreservation kinetics of the transgenic TCR, were washed and resuspended in cryopreservation medium consisting of fetal bovine serum (FBS; Life Technologies) supplemented with 10% DMSO (Sigma-Aldrich, Diegem, Belgium). Aliquots were stored in a -80°C freezer. When needed, cells were thawed in prewarmed IMDM supplemented with 10% hAB serum.
Surface and intracellular staining
To assess intracellular expression of FOXP3, Helios and CTLA-4, membrane markers on effector CD4+ T cells and Tregs were stained first (Table 2). Next, cells were fixed and permeabilized using the eBioscience FOXP3/Transcription Factor Staining Buffer Set, according to the manufacturer’s instructions (Thermo Fisher Scientific). Subsequently, cells were intracellularly stained with anti- FOXP3-Alexa Fluor 488 mAb (BD Biosciences), anti-Helios-Alexa Fluor 647 (BioLegend) and anti-CD152 (=CTLA-4)-BV421 (BioLegend) (Table 2). A FMO and CD4+ T cells were used as a negative control. For analytical flow cytometry, 50,000 lymphocytes, gated on light scatter characteristics, were measured using a Novocyte Quanteon (Agilent).
DNA methylation analysis of human Treg-specific demethylation regions
Dry pelleted cell samples (0.5 x 106 cells; centrifuged at 480g for 5 min) of CD127+CD25" effector CD4+ T cells and CD45RA+ or CD25hl Tregs were collected before and after ex vivo expansion and mRNA electroporation, stored at -80°C and shipped on dry ice to EpigenDx (Hopkinton, MA) to perform TSDR methylation analysis (assay ID ADS783-FS2). The analysis covered nine CpG sites spanning positions -2263 to -2330 (upstream from the ATG start codon) of FOXP3.
Briefly, pelleted cell samples were lysed using ZymoResearch Mdigestion buffer and 20 mg/mL protease K (ZymoResearch, Irvine, CA), and incubated at 65°C for a minimum of 2 h. Next, supernatants from the sample lysate were bisulfite modified using EZ-96 DNA Methylation-Direct kit (ZymoResearch) as per the manufacturer’s protocol with minor modifications. Polymerase chain reactions (PCRs) were performed using 1 pL of the bisulfite-treated DNA and 0.2 pM of each primer (EpigenDx’s proprietary information). One primer was biotin labeled and HPLC purified to purify the final PCR product using Sepharose beads. PCR product was bound to Streptavidin Sepharose HP (GE Healthcare Life Sciences), after which the immobilized PCR products were purified, washed, denatured with a 0.2-pM NaOH solution, and washed again using the Pyrosequencing Vacuum Prep Tool (Pyrosequencing, Qiagen), as per the manufacturer’s protocol. Next, 0.5 pM of sequencing primer was annealed to the purified single-stranded PCR products, and 10 pL of the PCR products were sequenced by Pyrosequencing on the PSQ96 HS System (Pyrosequencing, Qiagen) following the manufacturer’s instructions.
The methylation status of each CpG site was determined individually as an artificial C/T singlenucleotide polymorphism (SNP) using QCpG software (Pyrosequencing, Qiagen). The methylation level at each CpG site was calculated as the percentage of the methylated alleles divided by the sum of all methylated and unmethylated alleles. The mean methylation level was calculated using methylation levels of all measured CpG sites within the targeted region of each gene. Each experiment included non-CpG cytosines as internal controls to detect incomplete bisulfite conversion of the input DNA. In addition, a series of unmethylated and methylated DNA were included as controls in each PCR. Furthermore, PCR bias testing was performed by mixing unmethylated control DNA with in vitro methylated DNA at different ratios (0%, no methylated DNA; 5%; 10%; 25%; 50%; 75%; and 100%, only methylated DNA), followed by bisulfite modification, PCR, and Pyrosequencing analysis. For female donors, this ratio was corrected with a factor 2 since one of the two TSDR alleles is methylated because of X-inactivation.
Suppression assay
Effector CD4+ T cells were stained using a CellTrace Violet Cell Proliferation kit (Thermo Fisher Scientific), which allows tracking of cell division, according to the manufacturer’s instructions. Subsequently, stained effector CD4+ T cells were stimulated with Treg Suppression Inspector beads (Miltenyi Biotec) in a 1 : 1 ratio, providing optimal polyclonal stimulation of effector CD4+ T cells based on anti-CD2, anti-CD3 and anti-CD28, while incremental numbers of Tregs were added to the cell culture. More specifically, the different effector CD4+ T cell/Treg ratios were 2:0, 1:0, 1: 1, 2: 1, 4: 1, 8: 1 and 16: 1. Cells were co-cultured in a 96-well U-bottom plate in IMDM (Life Technologies) supplemented with 5% FBS (Life Technologies) for 5 days in a humidified 5% CO2 incubator at 37°C. After 5 days, 10,000 lymphocytes, based on light scatter characteristics, were measured using a FACSAria II flow cytometer (BD Biosciences) (Figure 2 and Table 2). The unstimulated responder T cell condition, was used as a reference, after which the division indices of the stimulated responder T cells alone condition (1:0 ratio) and the conditions containing different ratios of Tregs were obtained. The division index (DI), given by FlowJo software, represents the average number of divisions a cell in the starting population has undergone. Subsequently, the percentage suppression for each condition was calculated by the division index method: 100 - (DICondition of interest/DIi;o) * 100 (McMurchy and Levings. Eur J Immunol 2012, vol. 42, 27-34).
Multiplex cytokine analysis
Expanded Tregs were cultured (0.5 x 106 cells/mL) in IMDM supplemented with 5% hAB serum (i.e., control, not activated) or in complete medium with TransAct (1: 100 dilution), mimicking TCR activation. Subsequently, these cell cultures were incubated in a humidified 5% CO2 incubator at 37°C. As a positive control, thawed cryopreserved autologous effector CD4+ cells were cultured in the same conditions. As a negative control, cell-free IMDM supplemented with 5% hAB serum and complete medium was used. After 4 days, a volume of 500 pl culture medium of each condition was extracted for the simultaneous quantitative determination of both natural and recombinant human interferon-y (IFN-y), IL-2, IL-4, IL-5, IL-10, IL-13 and tumor necrosis factor a (TNF-a) using a chemiluminescence-based assay from Meso Scale Discovery (Human TH1/TH2 10-Plex Tissue Culture Kit, MSD, Gaithersburg, MD), according to the manufacturer’s instructions. The plate was washed and read with MSD reading buffer on the QuickPlex SQ 120 (MSD). All conditions were measured in duplicate and run at the same time. Background measurements of nonactivated cells were deducted from the measurements of corresponding activated cells.
TCR functionality
The functionality of the MBPgj-gg-specific TCR was assessed using two TCR-deficient cell lines, 2D3 and SKW-3. 2D3 cells were generated from TCR-deficient Jurkat 76 cells (human acute T cell leukemia), as described before (Versteven et al. Oncotarget 2018, vol. 9, 27797-808). SKW-3 cells were purchased from cell bank of German Collection of Microorganisms and Cell Cultures. Exponential growth was maintained by culturing the cells in RPMI 1640 (Gibco Invitrogen) supplemented with 10% FBS. After electroporation of the cells with mRNA encoding the MBPss- 99-specific TCR (5 x 106 cells, 1 pg mRNA/106 cells) as described above, cells were stimulated using TransAct, mimicking TCR activation, 6 h after electroporation. Mock electroporation and non-stimulated cells were used as a control. For 2D3 cells, containing a plasmid vector with the eGFP gene under the control of a nuclear factor of activated T cell (NFAT)-dependent promoter, TCR signaling can be measured directly by eGFP expression. For SKW-3, TCR-dependent cell activation was assessedby measuring the expression of activation markers CD69 and CD 137 (Table 2). For analytical flow cytometry, 10,000 lymphocytes, gated on light scatter characteristics, were measured using a CytoFLEX flow cytometer. Data analysis
FACS data were analyzed using FlowJo software version 10.5.3 (TreeStar, Ashland, OR), and multiplex data were analyzed using Discovery Workbench 4.0 software. Results were analyzed using Prism software version 8 (GraphPad, San Diego, CA), and given as mean values ± standard deviation (SD). Statistical analysis was performed using nonparametric Kruskal -Wallis test or Friedman test, followed by a post hoc Dunn’s multiple comparison test where applicable. For transgenic TCR expression over time, mixed-models test with the Geisser-Greenhouse correction, followed by a post hoc Dunnett’s multiple comparisons test, was used. Any P value <0.05 is considered statistically significant.
Example 2 - FACS sorting resulted in pure and functional CD45RA+ and CD25hl Tregs
First, we evaluated whether a sufficient number for ex vivo expansion of highly pure and functional CD4+CD127"CD25 CD45RA+ (CD45RA+) and CD4+CD127 CD25hi (CD25hi) Treg populations could be isolated using FACS sorting. For this, samples were enriched for CD4+ T cells, and CD45RA+ and CD25hl Tregs were sorted with an average purity of 94.9 ± 4.3% and 95.2 ± 4.2%, respectively (Figure 1A). To confirm Treg identity, FOXP3 expression was analyzed (Figure IB). Both CD45RA+ Tregs (70.6 ± 2.8%; P = 0.1573) and CD25hi Tregs (95.8 ± 1.5%; P = 0.0047) showed higher expression of FOXP3 compared with biological control CD I 27 CD25 effector T cells (6.1 ± 3.0%).
To validate the Treg-mediated suppressive capacity of the FACSsorted Treg populations, we evaluated their suppression on CD4 CD 127 CD25 effector T cell proliferation, induced by in vitro stimulation with beads coated with anti-CD2, anti-CD3 and anti-CD28 (Figure 1C and D). Tregs were able to suppress effector T cell proliferation at different TeffTreg ratios compared with effector T cells alone (no suppression of T cells). Indeed, significant inhibition of effector T cell proliferation occurred by CD45RA+ Tregs in a TeffTreg ratio of 1: 1 (81.1% ± 11.2% suppression of T cells; P = 0.0014) and 2: 1 (76.1% ± 13.8% suppression of T cells; P = 0.0025). Similarly, CD25hl Tregs significantly suppressed T cell proliferation at a TeffTreg ratio of 1: 1 (90.7% ± 4.7% suppression of T cells; P = 0.0008), 2: 1 (84.3% ± 13.0% suppression of T cells; P = 0.0112) and 4: 1 (80.7% ± 14.3% suppression of T cells; P = 0.0271). No significant difference in suppression of proliferation rate (P = 0.7434) was observed between the 2:0 ratio (-21.7% ± 26.8% suppression of T cells) and the 1:0 ratio, excluding the possibility that proliferation differences occur based on T cell numbers in the cell culture instead of Treg presence. Altogether, our findings confirm that FACS-sorted CD45RA+ and CD25hl Tregs display a genuine Treg phenotype and are capable of inducing immunosuppression. Example 3 - Good manufacturing practice (GMP)-compliant expansion of Tregs resulted in a >70-fold and a >185-fold increase of CD25hl and CD45RA+ Tregs, respectively
Given the low frequencies of Tregs in the human body, i.e., 5% to 7% of CD4+ T cells, large-scale expansion is advantageous to obtain sufficient Treg numbers for clinical application. Therefore, we developed an expansion protocol compliant with GMP using a combination of soluble colloidal polymeric reagent, with covalently attached anti-CD3/CD28-antibodies (T Cell TransAct), and 500 lU/mL IL-2 to activate and expand CD4+CD127 CD25hi and CD4 CD127 CD25 CD45RA+ Tregs. As depicted in Figure 3, an incremental increase of Treg numbers was observed. Indeed, the number of CD45RA+ Tregs (Figure 3A) and CD25hl Tregs (Figure 3B) was significantly higher from day 12 (A, 14.31 x 106 ± 9.28 x 106; P = 0.0143; B, 4.30 x 106 ± 1.56 x 106; P = 0.0233), compared with the initial value of 0.28 x 106 ± 0.06 x 106 cells and 0.27 x 106 ± 0.05 x 106 cells on day 0, respectively. Ultimately, we observed a 186.5 ± 123.8-fold expansion of CD45RA+ Tregs and a 71.4 ± 50.3-fold expansion of CD25hl Tregs over the course of a 19-day expansion protocol. Assessment of Treg purity at different time points during expansion indicated no outgrowth of contaminating CD127+ effector T cells (Figure 3C). Viability assessment indicated 94.9% ± 1.8% and 93.3% ± 4.0% viable cells over the course of the 19-day ex vivo expansion procedure for CD45RA+ and CD25hl Tregs, respectively.
Example 4 - Electroporation of expanded Tregs with TCR-encoding mRNA resulted in significant amounts of TCR-expressing Tregs
Next, we tested the feasibility of genetically and transiently modifying expanded Tregs using mRNA electroporation. To assess the transfection efficiency of mRNA electroporation, Tregs were electroporated with eGFP-encoding mRNA. On average, we observed 98.5% ± 0.8% eGFP- expressing CD45RA+ Tregs and 98.1% ± 0.9% eGFP -expressing CD25hl Tregs 24 h after electroporation (Figure 4). Subsequently, we evaluated whether electroporation with mRNA encoding an HLA-DR2-restricted MBPgj-gg-specific TCR, which comprises a variable P2 (Vp2) TCR chain, would result in effective TCR surface expression in expanded CD45RA+ and CD25hl Tregs. The kinetics of MBPgj-gg-specific TCR surface expression levels were evaluated using a TCR Vp2-specific mAb, targeting the TRBV20-1 variable segment, for 8 consecutive days (Figure 3D) The highest TCR Vp2 expression was observed 24 h after mRNA electroporation in both CD45RA+ (85.5% ± 6.2% TCR Vp2+ cells; P = 0.0035; Figure 3E) and CD25hi (83.0% ± 10.0% TCR Vp2+ cells; P = 0.0055; Figure 3F) Tregs, compared with mock electroporated cells, indicating successful transfection of Tregs with MBPss-gg-specific TCR-encoding mRNA. TCR Vp2+ expression levels gradually decreased in both Treg subtypes over the evaluated time course. Basal TCR Vp2+ levels in mock-electroporated CD45RA+ and CD25hl Tregs were 11.5% ± 0.7% TCR VP2+ and 7.9% ± 2.6% TCR Vp2+, respectively.
Next, CD45RA+ and CD25hl Tregs were cryopreserved 4 h after transfection. Upon thawing, the kinetics of TCR Vp2 expression was assessed. Importantly, no difference in TCR Vp2 expression could be detected between fresh or cryopreserved Tregs, as demonstrated by similar high expression of the transgenic TCR in cryopreserved CD45RA+ Tregs (81.4% ± 6.8% TCR Vp2+; P = 0.0047; Figure 3E) and in cryopreserved CD25hi Tregs (77.3% ± 7.2% TCR Vp2+; P = 0.0020; Figure 3F) 24 h after mRNA electroporation, and thus 20 h after cryopreservation, compared with mock electroporated cells. In addition, neither mRNA electroporation (94.0% ± 1.5% and 90.5% ± 4.2% viable cells) nor cryopreservation (94.7% ± 0.9% and 96.3 ± 0.4% viable cells) negatively influenced the viability of CD45RA+ and CD25hl Tregs, respectively. At day 8, no significant expression of the Vp2 TCR chain could be detected in all conditions, compared with the basal TCR Vp2+ levels in mock-electroporated CD45RA+ and CD25hl Tregs.
Example 5 - GMP-compliant activation, expansion and transfection of CD45RA+ and CD25hl Tregs did not affect the stable expression of the Treg master regulator FOXP3
Given the importance of the transcription factor FOXP3 in the phenotype and function of Tregs, we assessed whether GMP-compliantactivation, expansion and transfection affected its expression. No difference in FOXP3 expression levels could be found after activation, expansion and transfection of both CD45RA+ Tregs (93.9% ± 4.1% FOXP3+ cells after expansion and 92.0% ± 6.7% FOXP3+ cells after subsequent mRNA electroporation) (Figure 5A) and CD25hl Tregs (94.9% ± 3.0% FOXP3+ cells after expansion and 89.2% ± 7.7% FOXP3+ cells after subsequent mRNA electroporation) (Figure 5B) compared with FOXP3 levels in freshly isolated CD45RA+ Tregs (70.6% ± 2.8% FOXP3+ cells) and freshly isolated CD25hi Tregs (95.8% ± 1.5% FOXP3+ cells). Nonetheless, compared with CD127+CD25" effector T cells (7.0% ± 2.7% FOXP3+ cells), only expanded (P = 0.0001) and transfected (P = 0.0004) CD45RA+ Tregs demonstrated significantly higher expression levels of FOXP3, but not freshly isolated CD45RA+ Tregs (P = 0.2332), whereas freshly isolated (P = 0.0026), expanded (P = 0.0003) and transfected (P = 0.0099) CD25hl Tregs displayed significantly higher expression levels of FOXP3 compared with effector T cells.
Moreover, stable FOXP3 expression is directly linked to high demethylation of TSDR. Therefore, we analyzed the methylation status of FOXP3, as indicated by the percentage of methylation in nine CpG motifs located in intron 1 of human FOXP3 after GMP-compliant isolation, expansion and transfection of Tregs (Figure 5C and D). Significantly lower methylation percentages were measured in freshly isolated (24.3% ± 4.9%; P = 0.0180 and 13.4% ± 8.5%; P = 0.0110), expanded (31.1% ± 3.7%; P = 0.0313 and 23.4% ± 8.2%; P = 0.0370) and mRNA electroporated (30.0% ± 6.3%; P = 0.0376 and 24.1% ± 7.2%; P = 0.0450) CD45RA+ (Figure 5C) and CD25hi (Figure 3D) Tregs, respectively, compared with CD127+CD25" effector T cells (90.0% ± 1.5%). No significant differences in methylation status of the CpG motifs were observed between expanded and transfected Tregs and freshly sorted Tregs.
Example 6 - GMP-compliant activation, expansion and transfection of CD45RA+ and CD25hl Tregs did not affect CTLA-4 and CCR4 expression but showed a slight decrease in Helios expression by CD25hl Tregs
As FOXP3 -independent maintenance of the human Treg identity has been shown in FOXP3- ablated Tregs, we assessed the expression of additional Treg-defming markers, including Helios, CTLA-4 and CCR4. We demonstrated high expression of Helios in expanded CD45RA+ Tregs (66.5% ± 17.1% Helios+; P = 0.0019) and transfected CD45RA+ Tregs (65.5% ± 17.6% Helios+; P = 0.0201), but slightly lower expression in expanded CD25hl Tregs (31.6% ± 12.0% Helios+; P = 0.1967) and transfected CD25hl Tregs (31.8% ± 10.3% Helios+; P = 0.3017), compared with autologous CD4+ T cells (4.7% ± 2.5% Helios+) (Figure 6). It should be noted only Helios+, and not Heliosmid, was considered in the analysis. Nonetheless, high expression of CTLA-4 and CCR4 (Figure 6) was measured in expanded CD45RA+ Tregs (95.7% ± 0.8% CTLA-4+, P = 0.1213; 92.6% ± 5.8% CCR4+, P = 0.0444), transfected CD45RA+ Tregs (92.6% ± 1.4% CTLA-4+, P = 0.4386; 92.3% ± 6.4% CCR4+, P = 0.0550), expanded CD25hi Tregs (97.7% ± 0.1% CTLA-4+, P = 0.0019; 94.4% ± 4.7% CCR4+, P = 0.0495) and transfected CD25hi Tregs (96.7% ± 1.4% CTLA-4+, P = 0.0201; 95.0% ± 4.3% CCR4+, P = 0.0198), compared with autologous CD4+ T cells (7.7% ± 2.9% CTLA-4+ and 22.7% ± 5.3% CCR4+). In addition, our data indicated no differences in expression of Helios, CTLA-4 and CCR4 between expanded and transfected CD45RA+ and CD25hl Tregs, respectively.
Example 7 - GMP-compliant expanded and transfected Tregs were capable of inducing in vitro immunosuppression and produced anti-inflammatory, but not pro-inflammatory, cytokines
Inhibition of Teff proliferation was examined as a measure for Treg functionality after ex vivo expansion and genetic engineering via mRNA electroporation (Figure 7A and B). Significant inhibition of Teff proliferation was detected at a Teff: Treg ratio of 1: 1 compared with Teffs without the addition of Tregs (1:0 ratio; 0.0% ± 0.0% suppression of T cells), as indicated by 70.5% ± 13.3% (P = 0.0006) suppression of T cells by expanded CD45RA+ Tregs and 79.6% ± 10.3% (P = 0.0006) suppression of T cells by expanded and mRNA-engineered CD45RA+ Tregs; 62.6% ± 12.4% (P = 0.0006) suppression of T cells by expanded CD25hl Tregs and 67.8% ± 6.8% (P = 0.0006) suppression of T cells by expanded and engineered CD25hl Tregs. Also at a 2: 1 TeffTreg ratio, significant inhibition of Teff proliferation was observed, as indicated by 60.7% ± 19.2% (P = 0.0054) suppression of T cells by expanded CD45RA+ Tregs and 67.5% ± 13.0% (P = 0.0054) suppression of T cells by expanded and mRNA-engineered CD45RA+ Tregs; 55.9% ± 15.1% (P = 0.0054) suppression of T cells by expanded CD25hl Tregs and 59.1% ± 11.4% (P = 0.0054) suppression of T cells by expanded and mRNA-engineered CD25hl Tregs. Additionally, at a 4: 1 TeffTreg ratio, significant inhibition of Teff proliferation was observed, as indicated by 45.6% ± 18.7% (P = 0.0496) suppression of T cells by expanded CD45RA+ Tregs and 54.0% ± 12.6% (P = 0.0334) suppression of T cells by expanded and mRNA-engineered CD45RA+ Tregs; 47.0% ± 21.6% (P = 0.0334) suppression of T cells by expanded CD25hl Tregs and 46.4% ± 23.3% (P = 0.0334) suppression of T cells by expanded and mRNA-engineered CD25hl Tregs. No significant differences (P > 0.9999) were found between expanded and mRNA-electroporated Tregs, or between the two different Treg subsets (data not shown). No significant difference (P > 0.9999) in proliferation rate was observed between the 2:0 ratio (-11.8% ± 12.1% suppression of T cells) and the 1:0 ratio (no suppression of T cells), excluding the possibility that proliferation differences occur based on T cell numbers in the cell culture instead of Treg presence.
Quantitative analysis of secreted cytokines by expanded and mRNA-electroporated Tregs after TCR activation indicates high production of anti-inflammatory, but not pro-inflammatory, cytokines (Figure 7C and D). Indeed, cell-free supernatant of activated expanded CD45RA+ Tregs (1564 ± 1090 pg/mL IFN-y [P > 0.001]; 658 ± 166 pg/mL TNF-a [P = 0.1441]; 3697 ± 4570 pg/mL IL-2 [P = 0.0106]), expanded and mRNA-electroporated CD45RA+ Tregs (1649 ± 1259 pg/mL IFN-y [P = 0.0003]; 653 ± 423 pg/mL TNF-a [P = 0.0679]; 4222 ± 5503 pg/mL IL-2 [P = 0.0285]), expanded CD25hi Tregs (3333 ± 2051 pg/mL IFN-y [P = 0.0446]; 423 ± 64 pg/mL TNF-a [P = 0.0003]; 639 ± 237 pg/mL IL-2 [P = 0.0035]) and expanded and mRNA-electroporated CD25hl Tregs (3658 ± 2505 pg/mL IFN-y [P = 0.2012]; 436 ± 193 pg/mL TNF-a [P < 0.0001]; 599 ± 311 pg/mL IL-2 [P = 0.0010]) contained lower concentrations of pro-inflammatory cytokines, compared with activated CD4+ T cells (50,881 ± 23,741 pg/mL IFN-y; 11,762 ± 638 pg/mL TNF- a; 22,734 ± 3081 pg/mL IL-2) (Figure 7D). It should be noted that complete medium used for activation contained high concentrations of exogenous IL-2, resulting in 19,437 ± 2857 pg/mL IL-2 when measured with the multiplex. On the other hand, cell-free supernatant of activated expanded CD45RA+ Tregs (5200 ± 4576 pg/mL IL-4 [P = 0.446]; 3893 ± 1414 pg/mL IL-5 [P = 0.4652]; 14,565 ± 974 pg/mL IL-10 [P = 0.0176]; 6371 ± 2287 pg/mL IL-13 [P = 0.0019]), expanded and mRNA-electroporated CD45RA+ Tregs (4430 ± 4143 pg/mL IL-4 [P = 0.2012]; 4115 ± 963 pg/mL IL-5 [P = 0.4652]; 14,799 ± 1021 pg/mL IL-10 [P = 0.0176; 6621 ± 3066 pg/mL IL-13 [P = 0.0019), expanded CD25hi Tregs (11,389 ± 3304 pg/mL IL-4 [P < 0.0001]; 4025 ± 1375 pg/mL IL- 5 [P = 0.3613]; 15,223 ± 3168 pg/mL IL-10 [P = 0.0003]; 4640 ± 1308 pg/mL IL-13 (P = 0.0446]), mRNA-electroporated CD25hi Tregs (10,018 ± 4518 pg/mL IL-4 [P = 0.0010]; 4239 ± 651 pg/mL IL-5 [P = 0.2012]; 14,412 ± 3731 pg/mL IL-10 [P = 0.0106]; 4426 ± 495 pg/mL IL-13 [P = 0.3613]) contained higher concentrations of anti-inflammatory cytokines, compared with activated CD4+ T cells (284 ± 219 pg/mL IL-4; 2630 ± 2411 pg/mL IL-5; 1212 ± 277 pg/mL IL-10; 3116 ± 602 pg/mL IL-13) (Figure 7C). Hence, a genuine Treg phenotype and functionality can be assigned to both expanded and mRNA-engineered Treg subtypes.
Example 8 - TCR-dependent stimulation of cells electroporated with MBPss-99-specific TCR- encoding mRNA led to cell activation
Finally, two TCR-deficient cell lines were electroporated with MBPgj-gg-specific TCR-encoding mRNA, which resulted in TCR and CD3 surface expression (Figure 8). Subsequently, 6 h after electroporation, both cell types were stimulated with TransAct, mimicking TCR activation, to assess TCR functionality. As a negative control, unstimulated and stimulated mock-electroporated cells and unstimulated mRNA-electroporated cells were used. Flow cytometric analyses from both cell types, by either eGFP production (Figure 9A) or expression of activation markers CD69 and CD137 (Figure 9B), indicated TCR-dependent activation. Indeed, only stimulated mRNA- electroporated 2D3 cells expressed eGFP (60.9% ± 1.4% eGFP+), compared with unstimulated mock 2D3 (0.02% ± 0.02% eGFP+; P < 0.0001), stimulated mock 2D3 (0.04% ± 0.03% eGFP+; P < 0.0001) and unstimulated mRNA-electroporated 2D3 cells (3.3% ± 0.1% eGFP+; P = 0.0880). Similarly, only stimulated mRNA-electroporated SKW-3 cells expressed activation markers (91.1% ± 2.0% CD69+; 20.1% ± 2.5% CD137+) compared with unstimulated mock 2D3 (2.8% ± 1.4% CD69+ [P < 0.0001); 0.3% ± 0.2% CD137+ [P = 0.0005]), stimulated mock 2D3 (2.8% ± 0.6% CD69+ [P = 0.0003]; 0.3% ± 0.1% CD137+ [P = 0.0009]) and unstimulated mRNA- electroporated 2D3 cells (3.7% ± 1.4% CD69+ [P = 0.0077]; 0.3% ± 0.2% CD137+ [P = 0.0006]) (Figure 9C). In conclusion, TCR-encoding mRNA-electroporation led to expression of a functional TCR.
The present Examples provide an illustration of a GMP -compliant and easy-to-use protocol for the expansion and genetic engineering of two different Treg subtypes according to certain embodiments of the invention. The expansion and mRNA-based genetic engineering of both CD45RA+ and CD25hl Tregs did not negatively affect the Treg characteristics of both subtypes. Our data indicate no significant difference in the expression levels of the Treg master regulator FOXP3, expression after activation, expansion and transfection of both CD45RA+ Tregs and CD25hl Tregs. We demonstrated that Tregs were still capable of producing anti-inflammatory cytokines after anti-CD3 and anti-CD28 activation of expanded and mRNA-electroporated Tregs, indicative of the stability of the Treg phenotype and function. Tregs remained functional after in vitro expansion and mRNA electroporation, as indicated by their capacity to inhibit the proliferation of CD127+CD25" effector T cells in vitro in a ratio-dependent manner. The illustrated protocols provide GMP -compliant approach for ex vivo expansion and RNA-based engineering of Tregs, which is convenient and robust and allows for transient genetic engineering of different subtypes of Tregs, without affecting Treg phenotype and function. Our findings offer new opportunities for RNA engineering of Tregs for future clinical applicability, in which this approach can be used for the induction of antigen specificity or evaluating possible gain of function, after introduction of proteins or cytokines involved in Tregs’ mechanism of action.

Claims

1. A method for introducing an unmodified or modified RNA polynucleotide into a T regulatory (Treg) cell, comprising electroporation of a suspension comprising the polynucleotide and Treg cells.
2. The method according to claim 1, wherein:
- the Treg cells are CD45RA+ Treg cells, preferably an at least 90% pure population of CD45RA+ Treg cells; or
- the Treg cells are CD25hl Treg cells, preferably an at least 90% pure population of CD25hl Treg cells.
3. The method according to claim 1 or 2, wherein the Treg cells are freshly-isolated, or wherein the Treg cells have been obtained by in vitro or ex vivo expansion of isolated Treg cells.
4. The method according to claim 3, wherein the Treg cells have been isolated from peripheral blood mononuclear cells (PBMC) or from an internal organ, such as lung, liver, or spleen.
5. The method according to claim 3 or 4, a) wherein the isolation comprises:
- isolating T cells, preferably CD4+ T cells and/or CD8+ T cells, from PBMC or from an internal organ, thereby obtaining a population of T cells, preferably CD4+ T cells and/or CD8+ T cells ; and
- isolating Treg cells from the population of T cells; or b) wherein the isolation comprises directly isolating Treg cells from PBMC or from an internal organ.
6. The method according to any one of claims 3 to 5, a) wherein the isolation comprises:
- isolating CD4+ cells and/or CD8+ cells from PBMC or from an internal organ, thereby obtaining a population of CD4+ cells and/or CD8+ cells; and
- isolating CD 127’ cells from the population of CD4+ cells and/or CD8+ cells, thereby obtaining Treg cells; or b) wherein the isolation comprises directly isolating CD4 CD127" cells and/or CD8+ cells from PBMC or from an internal organ.
7. The method according to claim 5 or 6, comprising isolating CD45RA+ Treg cells or CD25hl Treg cells or a mixture thereof from the population of T cells or CD4+ cells.
8. The method according to any one of claims 5 to 7, wherein
- the T cells, CD4+ cells or CD8+ cells are isolated from PBMC or from the internal organ using magnetic-activated cell sorting, and/or wherein the Treg cells are isolated from the population of T cells, CD4+ cells or CD8+ cells using fluorescence-activated cell sorting (FACS); or
- the Treg cells are directly isolated from PBMC or from the internal organ using FACS.
9. The method according to any one of claims 3 to 8, wherein the Treg expansion comprises a step of culturing the isolated Treg cells in the presence of interleukin-2 (IL-2).
10. The method according to any one of claims 1 to 9, wherein the Treg cells are activated prior to electroporation, preferably wherein the Treg activation comprises a step of contacting the isolated and optionally expanded Treg cells with an anti-CD3 antibody and an anti-CD28 antibody.
11. The method according to claim 10, wherein the antibodies are covalently linked to a polymer carrier.
12. The method according to claim 10 or 11, wherein the contacting step is repeated two or more times during the Treg expansion and activation.
13. The method according to claim 12, wherein the contacting step is repeated on days 0, 7, 14, and 19 of the Treg expansion and activation.
14. The method according to any one of claims 1 to 13, wherein the concentration of the Treg cells in the suspension is 100 cells per ml to IxlO9 cells per ml, preferably IxlO7 to IxlO8 cells per ml, such as about 2.5xl07 cells per ml.
15. The method according to any one of claims 1 to 14, wherein the voltage is from 100 V to 700 V, preferably from 400 V to 600 V, such as about 500 V.
16. The method according to any one of claims 1 to 15, wherein the pulsing time is from 1 to 40 ms, preferably from 1 to 10 ms, such as about 5 ms.
17. The method according to any one of claims 1 to 16, wherein the pulse is a square wave pulse.
18. The method according to any one of claims 1 to 17, wherein the concentration of the polynucleotide in the suspension is from 100 ng / IxlO6 cells to 10 pg / IxlO6 cells, preferably from 500 ng / IxlO6 cells to 5 pg / IxlO6 cells, more preferably about 1 pg / IxlO6 cells.
19. The method according to any one of claims 1 to 18, wherein the polynucleotide is synthetic, or in vitro transcribed, or isolated from a host cell or a non-human host organism genetically engineered to produce the polynucleotide.
20. The method according to any one of claims 1 to 19, wherein the polynucleotide is a naked polynucleotide.
21. The method according to any one of claims 1 to 20, wherein the polynucleotide is a linear polynucleotide.
22. The method according to any one of claims 1 to 21, wherein the modified RNA polynucleotide is nucleobase and/or backbone-modified.
23. The method according to any one of claims 1 to 22, wherein the polynucleotide is coding or non-coding.
24. The method according to any one of claims 1 to 23, wherein the polynucleotide is selected from the group consisting of messenger RNA (mRNA), guide RNA (gRNA), single guide RNA (sgRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), long non-coding RNA (IncRNA), ribozyme, aptamer, spiegelmer, and combinations thereof.
25. The method according to any one of claims 1 to 24, wherein the polynucleotide is mRNA encoding one or more polypeptide, preferably one or more biologically active polypeptide.
26. The method according to any one of claims 1 to 25, wherein the polynucleotide produces a gain-of-fimction phenotype when introduced into the Treg cell, such as wherein the polynucleotide is mRNA encoding one or more polypeptide, preferably one or more biologically active polypeptide, and the polypeptide produces a gain-of-fimction phenotype when expressed by the Treg cell.
27. The method according to claim 26, wherein the one or more polypeptide endows the Treg cell with specificity to an antigen or an antigenic peptide thereof.
28. The method according to claim 27, wherein the one or more polypeptide is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
29. The method according to claim 28, wherein:
- the CAR ectodomain comprises a single chain variable fragment of an antibody (scFv) or a single domain variable fragment of a heavy chain antibody (VHH) specific for the antigen, an antibody-like scaffold, a cognate receptor or ligand for the antigen or an antigen-binding portion of said receptor or ligand, or a synthetic receptor, and/or - the intracellular portion of the CAR comprises at least one intracellular activation domain, such as a CD3^ or FcRy intracellular activation domain, and optionally and preferably at least one intracellular costimulatory domain, such as a CD28, 4-1BB, DAP10, 0X40 and/or ICOS intracellular costimulatory domain.
30. The method according to any one of claims 27 to 29, wherein the antigen is an autoantigen, alloantigen, or an allergen.
31. The method according to claim 28, wherein the autoantigen is involved in the induction and/or progression of multiple sclerosis, rheumatoid arthritis, type I diabetes, autoimmune uveitis, autoimmune myasthenia gravis, psoriasis, celiac disease, systemic lupus erythematosus, inflammatory bowel disease, Addison’s disease, Graves’ disease, Sjogren’s syndrome, Hashimoto’s thyroiditis, autoimmune vasculitis, pernicious anemia, or idiopathic thrombocytopenic purpura (ITP), or wherein the alloantigen is involved in the induction and/or progression of graft-versus- host disease or in transplant rejection.
32. The method according to any one of claims 1 to 31, wherein the endogenous T cell receptor (TCR) of the Treg cells has been knocked-out or knocked-down.
33. The method according to any one of claims 1 to 32, wherein the method is good manufacturing practice (GMP) compliant.
34. The method according to any one of claims 1 to 33, wherein the method further comprises cryopreservation of the Treg cells comprising the polynucleotide.
35. The method according to any one of claims 1 to 34, wherein the method further comprises formulating the Treg cells comprising the polynucleotide into a pharmaceutical composition or a kit-of-parts suitable for medicinal use.
36. The Treg cells comprising the polynucleotide, obtainable or obtained by the method of any one of claims 1 to 35.
37. A pharmaceutical composition comprising the Treg cells comprising the polynucleotide, obtainable or obtained by the method of any one of claims 1 to 35.
38. The T reg cells according to claim 36 or the pharmaceutical composition according to claim 37, for use in medicine.
39. The T reg cells according to claim 36 or the pharmaceutical composition according to claim 37, wherein the polynucleotide encodes a polypeptide which endows the Treg cell with specificity to an antigen or an antigenic peptide thereof, for use in a method of treating a disease caused by or associated with an increased activity of the immune system against said antigen.
40. A method for treating, in a subject in need thereof, a disease caused by or associated with an increased activity of the subject’s immune system against an antigen, comprising administering to the subject an effective amount of the T reg cells according to claim 36 or the pharmaceutical composition according to claim 37, wherein the polynucleotide encodes a polypeptide which endows the Treg cell with specificity to the antigen or an antigenic peptide thereof.
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