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WO2025096685A1 - Methods of making and using regulatory t cells for treatment of autoimmune disorders and cancers - Google Patents

Methods of making and using regulatory t cells for treatment of autoimmune disorders and cancers Download PDF

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Publication number
WO2025096685A1
WO2025096685A1 PCT/US2024/053754 US2024053754W WO2025096685A1 WO 2025096685 A1 WO2025096685 A1 WO 2025096685A1 US 2024053754 W US2024053754 W US 2024053754W WO 2025096685 A1 WO2025096685 A1 WO 2025096685A1
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Prior art keywords
cells
car
treg
population
seq
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French (fr)
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Christine E. BROWN
Jemily JUAN
Jamie Wagner
Jose Enrique Montero Casimiro
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City of Hope
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City of Hope
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4231—Cytokines
    • A61K40/4234—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/53—CD2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • Tregs Regulatory T cells
  • GVHD graft versus host disease
  • Tregs that express a chimeric antign receptor (‘'CAR”) targeted to an antigen expressed on a human cell (“CAR Tregs”).
  • the methods can be used to produce both Tregs and CAR Tregs that have a number of desirable characterisics and produce them in therapeutically useful quantities.
  • the Tregs and CAR Tregs can be cryopreserved and retain many desirable characteristics once thawed. This has imporant advantages.
  • Tregs or CAR Tregs can be produced from cells isolated from a patient and the Tregs or CAR Tregs can be preserved in a manner that allows aliquots of the stored cells to be expanded so that they can be used to treat the patient on multiple occassions over a period of many months without the need for isolating additional cells from the patient for the preparation of additional Tregs or CAR Tregs.
  • CAR Tregs targeted to human CD6 (“CD6 CAR Tregs”).
  • CAR Tregs can be targeted to a variety of other antigens such as CD19 or an IL-13 receptor (IL-13R; e.g., IL-13Ra2).
  • IL-13R IL-13 receptor
  • a wide variety of CARs targeted to CD6, CD19 and IL-13 are described in WO 2021/138454, hereby incorporated by reference.
  • a method for generating a composition comprising regulatory T cells comprising: (a) providing a population of peripheral blood mononuclear cells (PBMC); (b) treating the population of PBMC to select for cells expressing CD25 (CD25+ cells); (c) treating the population of cells selected for cells expressing CD25 to select for cells expressing CD4 (CD4+ cells), cells highly expressing CD25 (CD25hi cells) and have low or no expression of CD127 (CD1271ow/- cells) thereby producing a cell population selected for cells that are CD25+, CD4+ and CD1271ow/- (CD25+, CD4+,CD1271ow/- cells); (d) activating the CD25+, CD4+, CD1271ow/- cells by exposing them to antibodies targeted to CD3, antibodies targeted to CD28 and antibodies targeted to CD2 to create an activated cell population; (e) introducing a viral vector into the activated cell population to create a population of transduced Tre
  • the method does not include a step of enriching for cells that are CD45RO and does not include low or negative and does not include a step of enriching for cells that are CD45RA high or positive; the cells are not cultured in the presence of exogenously added IL-2 until after step (f); there are no cell selection or enrichment steps other than selection for cells expressing CD25 and a selection for cells that are CD25+, CD4+ and CD1271ow/-;
  • the CAR comprises or consists of an amino acid sequence of any of SEQ ID Nos: 145. 147, 149. 151, 154, 155, 156, 157 and 159 with no more than 5 single amino acid substitutions;
  • the spacer region is selected from SEQ ID NOs: 116-126 and the transmembrane domain is selected from SEQ ID NOs: 128- 136;
  • the scFv is selected from SEQ ID NOs: 101, 104 and 107;
  • the scFv has a VH comprising SEQ ID NO: 102 and an VL comprising SEQ ID NO: 103;
  • the scFv has a VH comprising SEQ ID NO: 105 and an VL comprising SEQ ID NO: 106;
  • scFv has a VH comprising SEQ ID NO: 108 and an VL comprising SEQ ID NO: 109; wherein a linker comprising S
  • a method for generating a composition comprising regulatory T cells comprising: (a) providing a population of peripheral blood mononuclear cells (PBMC); (b) treating the population of PBMC to select for cells expressing CD25 (CD25+ cells); (c) treating the population of cells selected for cells expressing CD25 to select for cells that express CD4 (CD4+ cells), express CD25 and have low or no expression of CD127 (CD1271ow/- cells) thereby producing a cell population selected for cells that are CD25hi, CD4+ and CD1271ow/- (CD25hi, CD4+, CD1271ow/- cells); (d) activating the CD25hi, CD4+, CD1271ow/- cells by exposing them to antibodies targeted to CD3, antibodies targeted to CD28 and antibodies targeted to CD2 to create an activated cell population; (e) introducing a vector into the activated cell population to create a population of transduced Treg; (I) culturing the transduced Treg
  • PBMC peripheral blood mono
  • the cells are not cultured in the presence of exogenously added IL-2 prior to step (e); the cells are not cultured in the presence of exogenously added IL-2 until after step (I); there are no cell selection or enrichment steps other than selection for cells expressing CD25 and a selection for cells that are CD25+, CD4+ and CD1271ow/-; the cells are cultured without exogenously added IL-2 between steps (d) and (e) and after step (e); the cells are cultured in the presence of exogenously added IL-2 for the first time at least 6 hours after step (e) and for the remaining of the expansion; exogenously added IL-2 is present at 250-500 U/ml of culture medium (e.g., during expansion); the population of transduced Treg expressing is cultured for at least 10 days to create an expanded population of transduced Treg; the expanded population of transduced Treg is cryopreserved; the vector encodes a chimeric antigen receptor; the vector is a viral
  • the scFv has a VH comprising SEQ ID NO: 102 and an VL comprising SEQ ID NO: 103; the scFv has a VH comprising SEQ ID NO: 105 and an VL comprising SEQ ID NO: 106; the scFv has a VH comprising SEQ ID NO: 108 and an VL comprising SEQ ID NO: 10; a linker comprising SEQ
  • step (g) takes place 7-10 days after step (e); after step (e), the cells are cultured at 750,000 - 1,250,000 cells/ml; steps (b) and (c) comprise fluorescent activated cell sorting using anti-CD127 antibodies, anti-CD25 antibodies, anti-CD4 antibodies; the transduced Treg population have at least 60% or 70% viability, at least 70%, 80% or 90% express CD4.
  • transduced Treg population cells are transduced with a viral vector encoding a selected polypeptide; and the transduced Treg population have at least 70% viability', at least 90% express CD4, at least 60% express (preferably unmethylated) FOXP3, and less than 5% express CD8.
  • graft versus host disease or diabetes (Type 1 or Type 2) comprising administering to a patient in need thereof, a therapeutically effective amount of Treg produced by the method described herein.
  • a population of Treg prepared by a method described herein.
  • CD6 CAR Tregs target the CD6 molecule overexpressed in pro- inflammatory T-cells, e.g., in cGVHD or Type 1 Diabetes (T1D) patients.
  • the current appoach employs a CAR or polypeptide that includes an scFv derived from Itolizumab, an immunomodulatory anti-CD6 monoclonal antibody (US 6,572,857).
  • a number of useful CAR include a CD 152 (CTLA-4) cytoplasmic domain (in addition to a CD3 zeta signalling domain) that can drive inhibitory signaling in transduced host cells and reinforce the immunomodulatory activity of CAR-Tregs.
  • the binding between a CAR or described herein and the antigen is of intermediate affinity.
  • the KD of the interaction is between about 10 nM and 140 nM.
  • the binding between a CD6 CAR expressed by a Treg and CD6 can be relatively low affinity. Higher expressing CD6+ cells will be more susceptible to be immune modulated by CAR-Tregs and CD6 low/ " subset of cells will be spared.
  • the CAR is expressed in an CD6 low/ " subset of Tregs.
  • Tregs can produce anti-inflammatory molecules such as, IL- 10, TGF-beta, and IDO.
  • the Tregs expressing a CAR described herein unexpectedly display potent tumor cell cytotoxicity, maintaining the distinct features of human regulatory cells such as persistent FOXP3 expression.
  • a targeting domain e.g., an scFv that binds a target antigen, e.g., a CD6 scFV
  • the transmembrane domain is selected from: a CD4 transmembrane domain or variant thereof having 1-5 single amino acid substitutions, a CD8 transmembrane domain or variant thereof having 1-5 single amino acid substitutions, a CD28 transmembrane domain or a variant thereof having 1-5 single amino acid substitutions;
  • the spacer comprises 20-150 amino acids and is located between the tumor-targeting domains and the transmembrane domain;
  • the transmembrane domain is a CD4 transmembrane domain or variant thereof having 1-5 single amino acid substitutions;
  • the transmembrane domain is a CD4 transmembrane domain;
  • the chimeric antigen receptor comprises a transmembrane domain selected from: a CD4 transmembrane domain or variant thereof having 1-2 single amino acid substitutions, a CD8 transmembrane domain or variant thereof having 1-2 single amino acid substitutions, a CD28 transmembrane domain or a variant thereof having 1-2 single amino acid substitutions;
  • a CD6-targeted CAR (CD6 CAR, also called anti-CD6 CAR herein) described herein includes a CD6 targeting scFv (e.g., an scFv comprising the amino acid sequence:
  • the scFv comprises the sequence EVQLVESGGGLVKPGGSLKLSCAASGFKFSRYAMSWVRQAPGKRLEWVATISSGGSYI YYPDSVKGRFTISRDNVKNTLYLQMSSLRSEDTAMYYCARRDYDLDYFDSWGQGTLV TV (SEQ ID NO: 102) or variant thereof with 1, 2.
  • a CD6-targeted CAR (CD6 CAR, also called anti-CD6 CAR herein) described herein include a CD6 targeting scFv (e.g., an scFv comprising the amino acid sequence:
  • the scFv comprises the sequence EVQLVESGGGLVKPGGSLKLSCAASGFKFSRYAMSWVRQAPGKGLEWVATISSGGSYI YYPDSVKGRFTISRDNVKNTLYLQMSSLRSEDTAMYYCARRDYDLDYFDSWGQGTLV TVSS (SEQ ID NO: 105) or variant thereof with 1, 2, 3, 4. or 5 single amino acid substitutions and the sequence
  • DIQMTQSPSSLSASVGDRVTITCKASRDIRSYLTWYQQKPGKAPKTLIYYATSLADGVPS RFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPFTFGSGTKMEIKRA (SEQ ID NO: 106) or variant thereof with 1, 2, 3, 4, or 5 single amino acid substitutions joined by a flexible linker, e.g., GGGSGGGSGGGGSS (SEQ ID NO: 115).
  • a CD6-targeted CAR (CD6 CAR, also called anti-CD6 CAR herein) described herein include a CD6 targeting scFv (e.g., an scFv comprising the amino acid sequence:
  • the scFv comprises the sequence DIQMTQSPSSLSASVGDRVTITCKASRDIRSYLTWYQQKPGKAPKTLIYYATSLADGVPS RFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPFTFGSGTKMEIKRA (SEQ ID NO: 108) or variant thereof with 1, 2, 3, 4, or 5 single amino acid substitutions and the sequence EVQLVESGGGLVKPGGSLKLSCAASGFKFSRYAMSWVRQAPGKGLEWVATISSGGSYI YYPDSVKGRFT1SRDNVKNTLYLQMSSLRSEDTAMYYCARRDYDLDYFDSWGQGTLV TVSS (SEQ ID NO:109) or variant thereof with 1, 2, 3, 4, or 5 single amino acid substitutions joined by a flexible linker, e.g., GGGSGGGSGGGGSS (SEQ ID NO: 115).
  • the CD6 scFv comprises a VH that is at least 90%
  • VH CDR1 comprising the sequence RY AMS (SEQ ID NO: 158); a VH CDR2 comprising the sequence TISSGGSYIYYPDSVKG (SEQ ID NO: 159); a VH CDR3 sequence comprising the sequence RDYDLDYFDS (SEQ ID NO: 160) or has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 single amino acid substitutions compared to SEQ ID NO: 102, excluding changes in VH CDR1, VH CDR2 and VH CDR3; and has a VL that is at least 90%, 95%, 98% or 99% identical to
  • VL CDR1 comprising the sequence KASRDIRSYLT (SEQ ID NO: 161); a VL CDR2 comprising the sequence YATSLAD (SEQ ID NO: 162); a VL CDR3 comprising the sequence LQHGESPFT (SEQ ID NO: 163) or has no more than 1, 2, 3. 4, 5, 6, 7.
  • VH can precede the VL and can be joined to the VL by a flexible linker, e g., GGGSGGGSGGGGSS (SEQ ID NO: 115).
  • T cells transduced by a vector comprising a nucleic acid described herein. Also described herein is a population of T cells expressing a CAR or polypeptide encoded by a nucleic acid described herein. In some embodiments, the T cells are regulator ⁇ ' T cells or effector T cells.
  • the population of Treg cells are such that least 70%, 80%, or 90% of the cells are: 1) CD4 + CD25 high CD127 low / neg ; 2) CD4 + CD25 high CD127 1OW CD6 low/neg ;4) CD4 + CD25 high CD127 neg CD6 low/neg ; 5) CD4 + CD25 high CD127 low CD6 neg ; or 6) CD4 + CD25 hlgh CD127 neg CD6 neg .
  • the population of T cells are human T cells. In some embodiments, the population of T cells are autologous human T cells or allogenic human T cells.
  • At least 70% of the cells in the population of Treg cells are CD4 + CD25 hlgh CD127 low/neg . In some embodiments, at least 70%, 80%, or 90% of cells are CD6 low or CD6 neg . . In some embodiments, the population of Treg are autologous to the patient being treated. In some embodiments they are allogenic to the person being treated.
  • compositions comprising any of the population of Tregs described herein. Also described herein are compositions comprising a population of Tregs harboring a nucleic acid described herein. Also described herein are compositions comprising a population of Tregs comprising a CAR or polypeptide described herein. Also described herein are compositions comprising a population of Tregs expressing a CAR or polypeptide described herein. In some embodiments, the population of T cells are human
  • Also described herein is a method of treating a patient, the method comprising administering to a subject in need thereof a population of Tregs harboring a nucleic acid described herein, a population of Tregs described herein, or a composition comprising a population of Tregs described herein.
  • the population of Tregs are human Tregs.
  • Also described herein is a method of treating an autoimmune disease, the method comprising administering to a subject in need thereof a population of Tregs harboring a nucleic acid described herein, a population of Tregs described herein.
  • the population of Tregs are human Tregs.
  • the population of Tregs are autologous human Tregs or allogenic human Tregs.
  • the autoimmune disease is Type I Diabetes or Graft-versus-Host Disease.
  • the population of Tregs or composition are administered in single or repeat dosing.
  • effective amount is administered to a subject.
  • at least one symptom is reduced, ameliorated, or relieved.
  • Also described herein is a method of killing, eliminating, or reducing cells expressing CD6, the method comprising administering to a subject in need thereof a population of Tregs harboring a nucleic acid described herein, a population of T cells described herein, or a composition T cells described herein.
  • the population of Tregs are human Tregs.
  • the population of Tregs are autologous human Tregs or allogenic human Tregs.
  • the cells expressing CD6 are cancerous.
  • the population of Tregs or composition are administered in single or repeat dosing.
  • effective amount is administered to a subject.
  • at least one symptom is reduced, ameliorated, or relieved.
  • Also descnbed herein is a method of killing, eliminating, or reducing cells expressing CD 19, the method comprising administering to a subject in need thereof a population of Tregs harboring a nucleic acid described herein, a population of T cells described herein, or a composition T cells described herein.
  • the population of Tregs are human Tregs.
  • the population of Tregs are autologous human Tregs or allogenic human Tregs.
  • the cells expressing CD19 are cancerous.
  • the population of Tregs or composition are administered in single or repeat dosing.
  • effective amount is administered to a subject.
  • at least one symptom is reduced, ameliorated, or relieved.
  • Also described herein is a method of killing, eliminating, or reducing cells expressing an IL-13R (e.g. IL-13Ra2), the method comprising administering to a subject in need thereof a population of Tregs harboring a nucleic acid described herein, a population of T cells described herein, or a composition T cells described herein.
  • the population of Tregs are human Tregs.
  • the population of Tregs are autologous human Tregs or allogenic human Tregs.
  • the cells expressing IL-13Ra2 are cancerous.
  • the population of Tregs or composition are administered in single or repeat dosing.
  • effective amount is administered to a subject.
  • at least one symptom is reduced, ameliorated, or relieved.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton. Proteins (1984)).
  • FIG 1 Phenotype of test run products. Cells w ere stained with fluorochrome- conjugated antibodies to detect intracellular FOXP3, or surface CD4, CD8, or the CD19t transgene marker. Percentages of viable (Zombie Aqua negative) CD3+-gated cells with immunoreactivity above control staining are indicated in each histogram
  • FIG 2 Immunomodulatory Activity of CD6-CAR Treg Cells against autologous PBMC.
  • A Viable cell numbers of CD6-CAR Treg cells per w ell at the time of seeding (Day 0) or after four days of culture at the indicated E:T ratios are depicted as mean + S.D. of duplicate wells.
  • B Measuring proliferation by the dilution of CFSE signal in either the CD3/CD4-gated or CD3/CD8-gated population, the percent inhibition of target cell proliferation after 4 days of coculture with CD6-CAR Treg cells at the indicated E:T ratios, and as calculated based on cultures of target cells alone, is depicted as mean + S.D. of duplicate w ells.
  • C Levels of IFNy (red) or IL- 10 (green) in the supernatants harvested after 4 days of culture are depicted at each E:T ratio.
  • FIG 4 Control of GvHD upon administration of CD6-CAR Treg cells.
  • A Quantification of whole-body weight (mean ⁇ SEM depicted at each timepoint) shows that CD6-CAR Treg cell treated mice maintained their body weight longer than those that received vehicle only, or control Treg cells.
  • Immunohistochemistry analysis of the lungs shows less infiltration of CD4+ (purple) and CD8+ (green) Teff cells in CD6-CAR Treg cell-treated mice compared to mice treated with non-transduced Treg cells or vehicle alone.
  • FIG 5 Map of CD6scFvIgG4(L235E,N297Q)-CTLA4-Zeta-T2A-CD19t-epHIV
  • A Diagram of the cDNA open reading frame of the 3066 nt construct indicating: the GMCSF signal sequence preceding the CD6 CAR, the various domains of the CD6 CAR, the T2A skip sequence, the CD 19 signal sequence preceding the truncated CD 19 marker, and the truncated CD 19 marker.
  • B Map of the plasmid.
  • C Diagram of the sequences flanked by long terminal repeats (indicated by ‘R’) that will integrate into the host genome.
  • FIG 6 Amino acid sequence of the immature CD6 CAR expressed by pF03496.
  • CD6scFv(VH_VL)-IgG4(L235E,N297Q)-CTLA4-Zeta is shown with the various domains marked.
  • FIG 7 Amino acid sequence of the immature CD6 CAR expressed by pF03497.
  • CD6scFv(VL_VH)-IgG4(L235E,N297Q)-CTLA4-Zeta is shown with the various domains marked.
  • FIG 8 Amino acid sequence of the immature CD6 CAR expressed by pF03488.
  • CD6scFv(VH-VL)-IgG4(L235E, N297Q)-41BB-Zeta is shown with the various domains marked.
  • FIG 9 Amino acid sequence of the immature CD6 CAR expressed by pF03491.
  • CD6scFv(VL-VH)-IgG4(L235E, N297Q)-41BB-Zeta is shown with the various domains marked.
  • FIG 10 Amino acid sequence of a mature IL13 CAR.
  • IL13-IgG4(L235E,N297Q)- CD4tm-CTLA4-Zeta is shown with the various domains marked.
  • FIG 11 Amino acid sequence of a mature CD6 CAR.
  • CD6scFv(VH- VL)IgG4(L235E,N297Q)-CD4tm-41BB-Zeta is shown with the various domains marked.
  • FIG 12 Amino acid sequence of a mature CD6 CAR.
  • VL)IgG4(L235E.N297Q)-CD4tm-CLTA4-Zeta is shown with the various domains marked.
  • FIG 13 Amino acid sequence of a mature CD6 CAR.
  • CD6scFvmhi(VH- VL)IgG4(L235E,N297Q)-CD4tm-41BB-Zeta is shown with the various domains marked. The three mutations in the scFv are bolded and double underlined. SEQ ID NO: 155.
  • FIG 14 Amino acid sequence of a mature CD6 CAR.
  • CD6scFvmhi(VH- VL)IgG4(L235E.N297Q)-CD4tm-CTLA4-Zeta is shown with the various domains marked. The three mutations in the scFv are bolded and double underlined. SEQ ID NO: 156.
  • FIG 15 Amino acid sequence of a mature CD19 CAR.
  • CD19scFvop(VH-VL) IgG4(L235E,N297Q)-CD4tm-CLTA4-Zeta is shown with the various domains marked.
  • FIG 16 Amino acid sequence of a CD6 CAR with a signal sequence. The signal sequence is underlined SEQ ID NO: 158 with the signal sequence and SEQ ID NO: 159 without the signal sequence.
  • FIG 17 Nucleotide sequence of a lentiviral vector encoding a CD6 CAR. This lentiviral vector encodes the CAR depicted in FIG 16. (SEQ ID NO: 160)
  • FIG 18 Schematic depiction of a lentiviral vector. This is a map of the lentiviral vector of FIG 17.
  • FIG 19 Preparation and Assessment of Tregs.
  • A Schematic depiction of the gating strategy for sorting Tregs from CD25-enriched cells.
  • B Purity assessment of isolated Tregs: Lymphocyte population gating (Top-left), Doublet-exclusion gating (Top-right), CD4 Gating (Bottom-left), and CD4 + > CD25 hi CD127 10 (Bottom-right).
  • CD4 + CD25 + CD127 10 Treg Purity 95.33%.
  • C Culture morphology images taken at 40X magnification of CD6-CAR Treg QR2 cultures on Days 1 (post-initial activation), 9 (prior to reactivation), 10 (post-reactivation), and 14 of expansion.
  • D Culture morphology images taken at 40X magnification of CD6-CAR Treg QR2 cultures on Days 1 (post-initial activation), 9 (prior to reactivation), 10 (post-reactivation), and 14 of expansion.
  • D Culture morphology images
  • E. Cell product stabi 1 i ty was assessed at the end of the expansion penod.
  • Tregs were incubated at RT at concentrations of 10-50xl0 6 cells/ml in 2% purified human saline. Cell viability, identity, potency, and purity samples were analyzed at time-points, 0, 4, and 6 hours after preparation.
  • Percent suppression is normalized to maximum target cell proliferation and target cell reduction is normalized to incubated target cells without Tregs and depicted as mean ⁇ S.D; Two-way ANOVA compared CD4 vs CD8 PBMC suppression and target cell reduction (i and ii). Luminex analysis of IFNy (yellow) or IL-10 (green) in the supernatants collected after 4 days of co-incubation at E:T ratios indicated; Two-way ANOVA compared IFNy and IL-10 production at the L I vs 2: 1 E:T ratios (hi).
  • CAR-Treg survival is measured by viable CD3+ cell numbers of Treg cells at E:T ratios; One-way ANOVA was used to show similarities in the survival of Tregs in each condition (iv).
  • CD6-CAR-Tregs were co-incubated with autologous and allogenic target cells for a 4-day period by which suppression and target cell reduction were compared at E:T ratios indicated (v and vi).
  • C A representative dot plot depiction of CAR detection with ddPCR consists of primers that separately detect the CD6 and CD19t sequence portions in both Fresh and Frozen CAR-Treg cell products.
  • tSNE clustering and FLOWS OM analysis was done on the indicated markers after a 4-day incubation period of activated Tregs or PBMC targets alone, or at a 1: 1 effector to autologous (auto) or allogenic (allo) target ratio.
  • Pie charts depict the presence of each population in both activated targets (ii) and Tregs (iii) alone or at a 1: 1 effector to autologous or allogenic target ratio.
  • the Treg is a Treg that includes CD4positive-CD25high-CD1271ow or negative expression. In some cases, the Treg is a Treg that includes CD4positive-CD25high- CD1271ow or negative, CD61ow or negative expression. In some cases, the Treg is a Treg that includes CD4positive-CD25high-CDl 271ow or negative-CD61ow or negative expression. In some cases, the Treg is a Treg that includes CD45RA+ expression. In some cases, the Treg is a Treg that does not include CD45RA+ expression. In some cases, the Treg is a Treg that includes FOXP3 demethylation.
  • the Treg is a Treg that does not include FOXP3 demethylation. In some cases, the Treg is a Treg that can be expanded either with IL-2 and/or rapamycin and/or retinoic acid. In some cases, the Treg is a Treg that can be expanded with IL-2 and/or rapamycin and/or retinoic acid. In some cases, the Treg is a Treg that can be expanded with IL-2. In some cases, the Treg is a Treg that can be expanded with rapamycin. In some cases, the Treg is a Treg that can be expanded with retinoic acid.
  • a CAR described herein can include a spacer located between the targeting domain (i.e. , a CD6 targeted scFv or variant thereof, a CD 19 targeted scFv or variant thereof, an IL- 13 or a variant thereof that binds an IL-13R) and the transmembrane domain.
  • the targeting domain i.e. , a CD6 targeted scFv or variant thereof, a CD 19 targeted scFv or variant thereof, an IL- 13 or a variant thereof that binds an IL-13R
  • a variety of different spacers can be used. Some of them include at least portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain or variants thereof. Table 2 below provides various spacers that can be used in the CARs and polypeptides described herein.
  • Some spacer regions include all or part of an immunoglobulin (e.g., IgGl, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CHI and CH2 domains of an immunoglobulin, e g., an IgG4 Fc hinge or a CD8 hinge.
  • Some spacer regions include an immunoglobulin CH3 domain (called CH3 or ACH2) or both a CH3 domain and a CH2 domain.
  • the immunoglobulin derived sequences can include one or more amino acid modifications, for example, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off-target binding.
  • Any flexible linker described herein can be a repeat of GGGS (SEQ ID NO: 127).
  • Any flexible linker can comprise one. two, three, four, five, or more repeats of SEQ ID NO: 127.
  • Any flexible linker can also have 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to the repeats of SEQ ID NO: 127.
  • transmembrane domains can be used in any CAR or polypeptide described herein.
  • Table 3 includes examples of suitable transmembrane domains. Where a spacer region is present, the transmembrane domain (TM) is located carboxy tenninal to the spacer region.
  • CAR expressed by a Treg includes a CTLA4 cytoplasmic domain (AVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN; SEQ ID NO: 137) located between the transmembrane domain and the CD3c signaling domain.
  • CTLA4 cytoplasmic domain AVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN; SEQ ID NO: 137
  • the CD3 ⁇ signaling domain includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to SEQ ID NO: 138. In some cases, the CD3 ⁇ signaling has 1 , 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO: 138.
  • compositions provided herein, including embodiments thereof, are used as effective treatment of autoimmune diseases.
  • a method of treating an autoimmune disease e.g., Type I diabetes, Graft-versus-Host Disease, Lupus
  • the method including administering to a subj ect in need thereof an effective amount of a population of Treg cells provided herein including.
  • the autoimmune disease is associated with reduced islet cell (e.g. , beta cell) function, viability, or survival.
  • the autoimmune disease is Type I Diabetes.
  • the autoimmune disease is Graft-versus-Host Disease.
  • the autoimmune disease includes a subject’s immune system attacking the subject’s islet cells (e.g., beta cells).
  • a chimeric antigen receptor described herein is administered locally or systemically; in some embodiments, a method of treatment includes reducing or eliminating cells expressing a CD6 receptor T cell expressing a CAR or polypeptide (e.g., CAR Teff or CAR Treg) described herein is administered by single or repeat dosing.
  • a CAR or polypeptide e.g., CAR Teff or CAR Treg
  • PBMC Collection and Enrichment for CD25+ Cells (Day -2 to Day -1) Peripheral blood mononuclear cells (PBMC) are collected by leukapheresis, for example, from a patient to later be treated with Tregs expressing a chimeric antigen receptor. The collected cells are then treated to enrich for cells expressing CD25 (CD25+ cells). Briefly, appropriate volume of wash buffer is pumped into the starting leukapheresis product bag, mixed well, and centrifuged at 200xg for 15 minutes at room temperature. The supernatant is removed by gravity 7 flow, and the remaining cell pellet resuspended in the re-sealed starting bag. The cells are then CD25 microbead-labeled.
  • the volume of the labeling and CD25 enrichment beads is determined using the white blood cell counts (WBC) recorded at the time of the leukapheresis procedure, and the appropriate volume of cold CliniMACs Buffer + 0.5% HAS (labeling buffer) added to the resuspended cells.
  • WBC white blood cell counts
  • HAS labeling buffer
  • Up to 40 - 120 xlO 6 labeled cells are enriched using Miltenyi’s Program Enrichment 3.2 on the CliniMACs system, where the final suspension of CD25+ cells will be collected in a 250 mL Jensen tube and counted.
  • the enriched cells suspension is centrifuged at 1200 rpm for 10 minutes. The supernatant is removed, and the cell pellet resuspended in T cell culture media without added IE-2 and cultured overnight at 1 x 10 6 cells/ml at 37°C with 5 % CO2.
  • the enriched cells are be prepared for Treg cell sorting. Briefly, , the cells are mixed and transferred to a 50 rnE or 250 mL tube (depending on volume) and counted. The cells are centrifuged again and resuspended in the buffer to a concentration of 20x10 6 cells/mL in the appropriate tubes.
  • Treg Sort Panel 1 CD4-FITC (BD Biosciences, Catalog 340133), CD25- APC (BD Biosciences, Catalog 340939), and CD127-PE (BD Biosciences, Catalog 561028) or Treg Sort Panel 2: MACS GMP CD4-PerCP (Miltenyi Biotec, Catalog 170-076-506 ), MACS GMP CD25-PE (Miltenyi Biotec, Catalog 170-076-503), MACS GMP CD127-FITC (Miltenyi Biotec, Catalog 170-076-512) 20 minutes in the dark at room temperature.
  • CD4-FITC BD Biosciences, Catalog 340133
  • CD25- APC BD Biosciences, Catalog 340939
  • CD127-PE BD Biosciences, Catalog 561028
  • Treg Sort Panel 2 MACS GMP CD4-PerCP (Miltenyi Biotec, Catalog 170-076-506 ), MACS GMP CD25-PE (Miltenyi Biotec, Catalog 170-076-503), MACS GMP
  • the cells are washed (1200 rpm for 10 minutes) with buffer, the cell pellet is resuspended at a concentration of 10xl0 6 cells/mL in the appropriate tubes and filtered through a 70 pm cell strainer. The resulting volume of cells is divided into FACs tubes, each containing no more than 4 mL/tube, and is then ready for sorting.
  • the Sony FX-500 Sorter can used for Treg cell sorting.
  • the sorted cells are collected in prepared tubes containing complete culture media (X-VIVO15 + 10% HABS). All the tubes are be consolidated into one 50 mL or 250 mL tube and mixed. This process results in the preparation of a population of cells that are CD4+, CD127 low/ ", CD25 hlgh . A sample can be taken for FACs analysis to confirm successful isolation of desired cell population and viability.
  • the post-sort Treg cells are held on wet ice in a 50 mL conical tube before proceeding. If the cell suspension volume is below 25 mL, then the suspension is supplemented wi th complete culture media. The cell suspension is then centrifuged at 1500 RPM for 5 minutes at 25°C ⁇ 2°C. the supernatant is decanted, the pelleted cells are vortexed and resuspended to adjust the concentration to approximately 3xl0 6 /mL, and a sample is harvested for total cell viability assessment. Based on the total viable cells, the cell suspension is adjusted to a final concentration of lx!0 6 /mL for activation.
  • ImmunoCult CD3/CD28/CD2 (bead-free antibodies targeted to CD3, CD28 and CD2 for cell activation , Stemcell, RUO 10970, cGMP-grade 100-0785) is added to the suspension for cell activation at 25yAnL and swirled to mix the reagent into the suspension.
  • the activated cell suspension is either plated or placed in a culture flask (e.g., T-25 culture flasks at a volume not to exceed 15 mL/flask). Then, after transferring the cell suspension to culture container, the container(s) is placed in a 37°C ⁇ 2°C incubator
  • the plateZflask(s) containing activated Treg cells are supplemented with protamine sulfate at a final concentration of 0. 1 mg/mL.
  • Virus encoding a protein of interest e.g. GMP-grade lentivirus (CD6(EQ)CTLA4Z-T2A-CD19t) is thawed, thoroughly vortexed, and then using a pipettor and sterile pipet tips, the required volume of virus is added to each Treg plate/flask to achieve an MOI of 1.0.
  • Each culture vessel is then gently swirled to ensure that the protamine sulfate- vims additions are well mixed into the cell suspension before being returned to the 37°C ⁇ 2°C incubator and incubated for 5-6hours.
  • a master mix is be prepared containing complete culture media supplemented with IL-2 for the final working concentration of 500 U/mL.
  • the cultures are supplemented at a ratio of 1:2 master mix; on days 5 and 7, the cultures are supplemented at a ratio of 1:3 master mix; cultures are split into anew culture container if the volume exceeds the capacity of the current container and returned to the 37°C ⁇ 2°C incubator.
  • the transduced Treg cultures are harvested from their container(s) and placed in the appropriately sized conical tubes.
  • the conical tubes are centrifuged using the appropriate parameters for the tube size. Post-centrifugation, the supernatants are removed, and each cell pellet is vortexed and resuspended in complete culture media.
  • a well-mixed sample is harvested for cell counts and viability using the appropriate counting device. Samples containing cells is harvested for in-process testing. Based on cell counts, the volume for the final concentration for activation is calculated.
  • the calculated volume of viable cells for activation is harvested into the appropriately sized tubes (if the concentration is too low, the cell suspension is centrifuged), and the cell suspension is supplemented with complete culture media to adjust the suspension to the calculated final volume for activation.
  • the cell suspension is swirled or mixed with a serological pipet and then aliquoted into appropriately sized culture vessel(s). At the end of the reactivation, the culture is returned to the 37°C ⁇ 2°C incubator.
  • a sample is harvested form one representative flask and counted using the appropriate counting device. Based on the count, the transduced Treg culture is harvested from their containers for Mycoplasma testing (from the pooled cell suspension), and two samples containing cells are harvested for in-process testing (from the pooled suspension). Next, the suspension is transferred to appropriately sized tubes. The conical tubes are centrifuged using the appropriate parameters for the tube size that is used. Post-centrifugation, the supernatant is removed. Once the supernatant is removed, each cell pellet is vortexed and resuspended in wash buffer, and the cell suspension well mixed. A well- mixed sample is harvested for counting using the appropriate counting device.
  • the conical tube(s) is centrifuged using the appropriate parameters for the tube size that is used. During centrifugation, calculations are made for the final total cell concentration of the cell product, where the concentration is 10xl0 6 /mL. and the maximum concentration is 50xl0 6 /mL in a volume of 10-20mL in the bag(s). These products are cryopreserved and stored in CryoMACS50 bags (Miltenyi Biotec #200-074-400:) for infusion, with cryopreserved vial (Cryovials, Nunc #375418) aliquots for QC. Post-centrifugation, the supernatant is removed.
  • each cell pellet is vortexed and resuspended in cry opreservation media, with 5% DMSO (CiyoStorCS5, Stemcell Technologies, 07933). Sterility samples are harvested from this cell suspension.
  • the cell suspension is transferred to the appropriate size cryopreservation container, such as CryoMACS50 bags, as well as cryovials.
  • cryopreservation containers Upon filling, cryopreservation containers are placed in a chilled cassete, and the cryovials are placed in Mr. Frostys.
  • the bags/cryovials are be transferred to the cooled (approximately 4°C) controlled rate freezer (CRF).
  • CRF controlled rate freezer
  • the CRF probe is placed into the cassette of a filled appropriately sized cryopreservation container or into a mock ciyovial containing cCryoStorCS5.
  • the frozen bags/cryovials are immediately be transferred into a GMP LN2 freezer for storage. If CRF is not used, bags/ciyovials can be transferred directly into -80°C freeze; after three days in the -80°C freezer, bags/cryovials are transferred into a GMP LN2 freezer for storage.
  • CD6 CAR Treg cells were prepared from PBMC from four healthy donors. The cells were reactivated as described in Example 1 and maintained with the addition of complete X-VIVO 15 media and cytokine every other day until the last day of culture. Two of the test runs (Test Runs 3 and 4) were cryopreserved as described in Example 1. Subsequently, cells from each of the four test runs were expanded for 14-19 days. These results of this study (Table 5) demonstrate that the cells, including cryopreserved cells, can expand up to 1464-fold in 14 days, demonstrating that the cells can be expanded to numbers sufficient for clinical application.
  • Table 5 Expansion of Test Run Products Cells from Test Run 3 and Test Run 4 were stained with fluorochrome-conjugated antibodies to detect intracellular FOXP3, or surface CD4, CD8, or the CD19t transgene marker. The results of this analysis are presented in FIG 1 in which percentages of viable (Zombie Aqua negative) CD3+-gated cells with immunoreactivity above control staining are indicated in each histogram.
  • Tregs from Test Run 3 and Test Run 4 underwent general safety testing that included analysis for vector copy number, as neoplastic transformation would be anticipated to increase with higher numbers of vector copies/insertions.
  • qPCR quantitative polymerase chain reaction
  • Quantitative PCR for the VSV-G DNA copy number was also carried out in an effort to detect overt replication competent lentivirus (RCL).
  • the average VSV-G copy numbers in Test Run Products 3 and 4 were ⁇ 0.7 copy per 50 ng gDNA.
  • Transduced Treg compositions for therapeutic use are those that are: > 70% cell viability; > 60% FOXP3 expression and > 90% CD4 expression by flow cytometric analysis; ⁇ 5% CD8 expression by flow cytometric analysis; > 20% CAR expression (as assessed, for example by surface marker expression) by flow cytometric analysis.
  • Example 3 Activity Assessment of Treg Cells Transduced with CD6(EQ)CTLA4Z-T2A- CD19t epHIV7
  • the objective of this study was to evaluate the immunomodulatory activity of regulatory T (Treg) cell products that were transduced with CD6(EQ)CTLA4Z-T2A-CD19t_epHIV7 GMP- grade lentivirus and expanded in vitro as described in Example 1.
  • the immunomodulatory activity 7 of CD6-CAR Tregs against autologous peripheral blood mononuclear cells (PBMC) or CD6-CAR transduced autologous effector T (Tefl), as well as allogenic PBMC cells was evaluated using proliferation assays and cytokine production assays.
  • PBMC peripheral blood mononuclear cells
  • Tefl CD6-CAR transduced autologous effector T
  • CD6-CAR Treg cell qualification run products were generated from healthy donors using methodologies suited for clinical use. Briefly, CD4+, CD25+, CD127 low/neg Treg cells that had been enriched and sorted from PBMC were transduced with GMP-grade CD6(EQ)CTLA4Z- T2A-CD19t_epHIV7 lentivirus, expanded for 14 days and then cryopreserved. Using either autologous PBMC or an autologous CD25/CD14-depleted, CD6-CAR transduced Teff population as ‘target' cells, the CD6-CAR Treg products were then evaluated for effector function using flow cytometry-based assays for target cell proliferation and cytokine expression.
  • Treg cells were isolated, transduced with CD6(EQ)CTLA4Z-T2A-CD19t_epHIV7 lentivirus, and expanded with IL-2 and cryopreserved on day 14 as described above. For future use as target cells, a portion of the source PBMC were cryopreserved prior to the isolation/production of CD6-CAR Treg cells.
  • PBMC or CD6-CAR Teff ‘target’ cells were stained with CellTrace CFSE, and CD6-CAR Treg ‘effector’ cells were stained with CellTrace Violet.
  • the CD6-CAR Treg cells were seeded at either 2: 1 or 1 : 1 effector-to-target (E:T) cell ratios, in a 96-well round bottom plate.
  • PBMC were activated with 0.02 pgAvell of plate-bound anti-human CD3 antibody
  • CD6-CAR Teff cells were activated with 2.5 pL/well of CD3/CD28 T cell activator.
  • the final volumes of co-cultures were 200 pL X-VIVO 15 media supplemented with 10% FBS per well.
  • Treg cells transduced with CD6(EQ)CTLA4Z-T2A-CD19t_epHIV7 suppress of Teff cell proliferation and modulate cytokine secretion upon co-incubation with activated Teff cells.
  • PBMC peripheral blood mononuclear cell proliferation
  • CD6-CAR Treg numbers are higher than that seen with cultures of Tregs alone (1:0), indicating that activation of CD6-CAR Treg cells promotes their survival and/or prevents their depletion.
  • Example 4 In vivo anti-GVHD and immunomodulatory efficacy of IV administered CAR Tref cells
  • 5x10 6 CD6-CAR Treg cells 50% CAR+; prepared essentially as described above
  • PBS vehicle
  • mice were monitored for graft vs. host disease (GVHD) by measuring body weight over time up to 51 days, and serum cytokine levels were measured on Day 13. Tissue samples for immunohistochemistry studies were also collected from designated euthanized mice on Day 13. Euthanasia was carried out in accordance with the recommendations of the AVMA Guidelines for the Euthanasia of Animals: 2020 Edition, where criteria for mouse survival included maintaining > 80% of initial body weight, and survival was monitored by Kaplan Meier curve analysis.
  • GVHD graft vs. host disease
  • mice treated with CD6-CAR Treg cells better maintained their body weight (FIG 4A) and had a survival advantage (FIG 4B).
  • Treg treated mice and CD6-CAR Treg treated mice had a reduction in serum proinflammatory cytokines (IL-2, IFN-gamma, TNF, GzmB, IL-6, CG-CSF and IL-10) (FIG 4C), as well as a reduction of tissue-infiltrating CD4+ and CD8+ Teff cells in the lungs (FIG 4D)
  • mice While these were not formal toxicity studies, the mice were monitored daily for any obvious signs of distress or general toxicity. Mice treated intravenously with CD6-CAR Treg cells were bright, alert and reactive throughout the experiment, and did not exhibit weight loss until they began to succumb to GVHD.
  • Example 5 Characterization of CD6 CAR Tregs Generated by GMP Compliant Flow Cytometry CD6 CTLA4 CAR-Treg cells are isolated, transduced, and expanded by GMP-compliant flow cytometry-based procedures for clinical application and characterized. The results of this analysis are presented in FIG. 19A-L and FIG. 20A-G.
  • CD6 CTLA4 CAR-Treg is a combination of the cell therapy field of Treg and established CAR T cell therapy procedures used in prior and ongoing clinical trials at City of Hope. Qualification studies were conducted to support the manufacturing process, evaluate critical parameters, and assess investigational cellular product using methodologies proposed for clinical use.
  • CD6 CTLA4 Tregs were generated from healthy donors through leukapheresis blood products from StemCell (Vancouver, Canada). Concentrated leukocytes were CD25 microbead- labeled for CliniMACS (Miltenyi Biotec, San Jose, USA) positive selection to generate CD25 + enriched cells that were cultured overnight without cytokines then sorted for CD4 + . CD25 + , and CD127 lo/ ’ using the FX500 Cell Sorter (Sony, San Jose, USA) and aseptic technique in a cGMP- level clean room facility (FIG 19A). Purity assessment of all qualification runs resulted in >95% pure Tregs depicted by a representative diagram in FIG 19B.
  • Tregs sorted by the FX500 Cell Sorter were within range of Treg products analyzed by the BD FACSAria II used by other cGMP Treg manufacturers (Putnam, A.L., et al.. Clinical grade manufacturing of human alloantigen-reactive regulatory T cells for use in transplantation. Am J Transplant, 2013. 13(11): p. 3010-20).
  • Sorted Tregs were cultured at IxlO 6 viable cells/ml, activated overnight with CD3/CD28/CD2 Immunocult (StemCell, Vancouver, Canada) prior to transduction with vCD6(EQ)CTLA4Z- T2A-CD19t_epHIV7 GMP-grade lentivirus using an MOI of 1.0. Cytokines were added to the transduced CD6 CTLA4 CAR-Treg culture at 5-6 hours post-transduction. Cultures were expanded with the addition of HABS and IL-2 supplemented Xvivol5 media every 2 days. Reactivation was completed on Day 9 and expanded for another 5 days for a total of 14 days of culture expansion.
  • FIG 19C Culture morphology images of Days 1, 9 (prior to reactivation), 10, and 14 of expansion are shown in FIG 19C.
  • Cell clusters are observed following the activation of cells on Days 1 and 10, which are days that follow the initial activation at post-isolation (Day 0) and reactivation (Day 9).
  • Treg culture morphologies are different from those of T effector cells, in which Treg cell clustering occurs throughout the expansion, which is also observed at Days 9 and 14 (FIG 19C). Further data is currently being collected to assess if culture morphologies correlate with modulatory function of Tregs. Using this method of GMP-compliant standard operation procedures, the expansion of CD6 CTLA4 CAR-Treg cells to numbers sufficient for clinical application is attainable.
  • FIG 19D depicts the expansion of qualification productions that exceeded IxlO 9 total Tregs and up to 8.8xl0 9 .
  • CD6 CTLA4 CAR-Treg cells were designated for cryopreserved or phenotyping and functional testing.
  • Treg cell culture expansions are highly dependent on high doses of IL-2 and dense cell concentrations. Cytokine-dependence assessments of CD6 CTLA4 CAR-Treg cells were done upon reactivation (Day 9) of Treg cell cultures and upon thawing of cryopreserved cells. When cells were reactivated on Day 9, 10xl0 6 Treg cells were designated to be cultured without IL -2 supplementation while following the maintenance schedule of Days 10-14 of Treg culture expansion, and in parallel lOxlO 6 Treg cells progressed with the expansion following the standard operation procedure placed for the clinical production of CD6 clLA4 CAR-Treg cells.
  • Treg cells that were designated to be cultured without IL -2 supplementation after reactivation have shown cell viability decreases by about 80% at the e 30% after 2 days of culture without IL- 2 after thaw and by more than 85% after 7 days, compared to CAR-Tregs that were thawed and maintained with IL-2, decreases in viability were 12% after 2 days and 64% after 7 days (FIG. 20A), cytokine-dependent).
  • CD6-CAR-specific detection by PCR is used as a release criterion for CD6-CAR Treg cells.
  • Treg cells transduced with the lentiviral vector vCD6(EQ)CTLA4Z-T2A-CD19t_epHIV7 was distinguished from cells transduced with other lentiviral vectors containing CD19t by a sequence of two PCR assays (FIG.19G).
  • the unique binding domain sequence within the CD6(EQ)CTLA4 ⁇ CAR is targeted by specific primers.
  • the second PCR assay targets the region between CD3 ⁇ and CD19t, the CAR signaling domain and transduction efficiency surface marker respectively.
  • CD6-C AR Treg samples must be PCR positive for both the CD6scFv-EQ portion of the CD6(EQ)CTLA4 ⁇ CAR sequence (PCR1) and CD19t (PCR2) to meet accepted parameters.
  • primers detecting the CD6-CAR transgene (954bp, Lanes 1-3) and CD19t (578bp, Lanes 4-6) were used to analyze genomic DNA isolated from CD6-CAR Treg samples from QR2 (Lanes 1 and 4), alongside FACS-confirmed CD6-CAR+ cells (positive control, Lanes 2 and 5) and non-transduced cells (negative control, Lanes 3 and 6).
  • PCR products are compared to lOObp ladder (Lane L) with indicated lOOObp and 600bp.
  • Tregs demonstrate effectiveness in preventing and treating Graft Versus Host Disease (GVHD) (Di lanni, M., et al., Tregs prevent GVHD and promote immune reconstitution in HLA- haploidentical transplantation. Blood, 2011. 117(14): p. 3921-8). Additionally, multiple studies have shown the potential of this type of treatment in autoimmune diseases such as Type 1 diabetes (Marek-Trzonkowska, N.. et al., Administration of CD4+CD25highCD127- regulatory T cells preserves beta-cell function in type 1 diabetes in children. Diabetes Care, 2012. 35(9): p. 1817-20).
  • GVHD Graft Versus Host Disease
  • CAR-Treg cell products intended for clinical use undergo rigorous microbial safety and criteria testing that may take several hours after cell products have been dosed and prepared for infusion. Therefore, stability tests were done on two qualification runs at the end of the 14- day culture period to assess the resilience of expanded CD6-CAR Tregs over time after being dose-prepared following a similar methodology proposed for clinical use. Phenotypic assessments via How cytometry of qualification run CAR-Tregs at the end of expansion included the evaluation of percent CD4-positive cells, 97.6% (93.4-100), and FOXP3-positve cells, 98.4% (95.7-99.9), data is reported as average and (min-max).
  • Viability was evaluated at 98.43% (97.7- 99.1) with 67.2% (44.4-80.5) of Tregs expressing CAR.
  • Expanded CAR-Tregs were placed into an infusion bag or syringe at a concentration of 10-50xl0 6 viable cells/ml in 2% purified human saline.
  • Each vessel contained 10ml (bag) or 1ml (syringe) of cell suspension and was placed on a rocking apparatus at room temperature for a duration of specific time-points: 0, 4, and 6 hours. Vessels were rotated every 30 minutes to prevent possible settling of cells. Assessment samples at each time-point were acquired from the vessels via adapters. Samples were tested for viability', identity, and purity' markers summarized in FIG 19E.
  • CD6-CAR Treg viability’, identity, and purity markers remained stable for a duration of up to 6 hours at room temperature.
  • CD6-CAR Treg cells exhibit suppressive function tow ards autologous and allogenic targets. Functional assessment of CD6-CAR Treg cells was done at the end of the 14-day culture expansion using a mix lymphocyte incubation and flow cytometry-based analysis. Briefly, CAR-Treg cells were incubated with autologous or allogenic PBMC, labeled as targets, in a series dilution of 1:64 to 2: 1 Treg to target cell ratio with activation by plate-bound anti-human CD3 antibody for 4 days at 37°C.
  • CD6-CAR Treg was calculated for each ratio condition and normalized to maximum proliferation (anti -human CD3 activated target cells alone) to calculate percent suppression of Tregs depicted on FIG 19F.
  • the figure illustrates the ability of CD6-CAR Treg to suppress both activated autologous and allogenic target cells at indicated effector to target cell ratios (E:T).
  • CD6-CAR Tregs were able to suppress autologous target cell proliferation at the 2:1 E:T up to 56% (CD4) and 31% (CD8), and up to 65% (CD4) and 47% (CD8) in allogenic target cells.
  • CD6-CAR Treg prepared for clinical application meets release criteria.
  • Cryopreservation provides multiple benefits to the clinical use of CAR T cell therapy, including long-term storage until needed, flexibility in administration of therapy doses and timing of infusion, as well as giving ample time for quality release testing. Limited studies have shown the effects of cry opreservation on the modulatory effects of Tregs (Golab, K._ et al., Challenges in cryopreservation of regulatory T cells (Tregs) for clinical therapeutic applications. Int Immunopharmacol, 2013. 16(3): p. 371 -5; Hippen, K.L., et al.. Massive ex vivo expansion of human natural regulatory' T cells (T(regs)) with minimal loss of in vivo functional activity 7 . Sci Transl Med, 2011. 3(83): p.
  • CAR-Treg Accepted release criteria of cryopreserved CAR-Treg were established using a combination of current and past clinical trials and our experiences with pre-clinical CD6-CAR Treg productions (Bluestone, J.A., et al., Type 1 diabetes immunotherapy using polyclonal regulatory T cells. Sci Transl Med, 2015. 7(315): p. 315ral89).
  • CAR-Treg must be within the accepted parameters of >70% viability 7 , >90% CD4, >60% FOXP3, and >20% CAR expression, as well as maintain CD8 contamination ⁇ 5%. These parameters were analyzed in cryopreserved CD6-CAR Tregs from these qualification runs.
  • cryopreserved CD6-CAR Treg cells using a lab method or a clinical protocol, are within the accepted release criteria of viability, CD4 purity, FOXP3, and CAR expression, and below the parameter set for CD8 contamination.
  • CD6-CAR Treg cells maintain vector copy number below accepted safety parameters.
  • VSV-G Vesicular stomatitis virus glycoprotein
  • RCL Replication Competent Lentivirus
  • Data obtained from the qualification run products indicate generating gene-modified CAR Treg cell products with an average vector copy 7 number less than 5 per CAR+ cell, after transduction with CD6(EQ)CTLA4Z-T2A- CD19t_epHIV7 using an MOI of 1.0, is attainable and can be generated without evident RCL.
  • CD6-CAR Treg modulatory markers are retained pre- and post-crv opreservation.
  • FOXP3 Transcription factor, FOXP3, is a widely known marker that designates Tregs from other T cell populations. Commonly studied by flow cytometry-based assays, FOXP3 expression is linked to suppressive capabilities of Tregs. In addition to FOXP3 expression, epigenetic modifications to FOXP3, assessed by measurement of unmethylated FOXP3, may also serve as a surrogate measurement of Treg stability and function. Qualification run samples were collected after 14- day expansion, fresh CD6-CAR-Treg (IxlO 6 cells) stored as cell pellets at -80°C until assessment.
  • CAR-Tregs were thawed in Xvivol5 + 10% HABS, washed to remove DMSO, then allocated for assessment.
  • Genomic DNA was isolated from cell pellets (Fresh) and thawed cells (Frozen) and 200-500 ng of DNA was underwent bisulfite treatment in preparation for analysis using droplet digital methylation-specific PCR (ddPCR) previously described (Husseiny, M L, et al., Development of Quantitative Methylation-Specific Droplet Digital PCR (ddMSP) for Assessment of Natural Tregs. Front Genet, 2020. 11: p. 300).
  • ddPCR droplet digital methylation-specific PCR
  • Relative expression of unmethylated FOXP3 was calculated by dividing the total copies of positive unmethylated FOXP3 by the total copies of TSDR region and summarized in FIG. 19H.
  • Fresh and frozen CD6-CAR Treg displayed high percentages of unmethylated FOXP3, fresh 98.1% (94.4-100) and frozen 94.7% (90.3-100). which can be correlated with the expression of FOXP3 analyzed previously by FACS.
  • the permanence of unmethylated FOXP3 TSDR pre- and post-cryopreservation indicates that modulatory markers are not altered by the preservation process.
  • CD6-CAR specific stimulation activates CD3 signaling pathway.
  • CD6-CAR positive cells were purified from total cultured Tregs and rested overnight in Xvivol5 media supplemented with 10% HABS and IL-2. Fresh samples w ere immediately acquired from cultures at the end of expansion and frozen samples were cells that were released from cGMP and cryopreserved in the lab using 10% DMSO.
  • CD6-CAR Tregs was plated in wells coated with CD6 Fc for Bit at 37°C prior to cell lysis. Fifteen micrograms of protein were loaded onto pre-cast 4-12% Bis-Tris gels (NuPAGE, Invitrogen) and transferred onto a nitrocellulose membrane. Antibodies for CD3 pY142 (Abeam), SLP76p(Ser376), pERKl/2(Thr202/Tyr204), pAKT (Ser 473), and pPI3k (Tyr467/199) were used for immunoblotting and nonnalized to P Tubulin. Bands were detected using a chemiluminescence substrate.
  • Stimulation increased the expression of pCD3 ⁇ Y142 in fresh CD6-CAR Treg as well as in the frozen group of cells.
  • Constitutive phosphorylation of the CAR CD3 ⁇ domain or tonic signaling has been shown to occur with some CARs.
  • Stimulation of the CAR is known for Lck-activation of ZAP70 which phosphory lates number of proteins including SLP76 required for signal propagation.
  • SLP76 phosphory lates number of proteins including SLP76 required for signal propagation.
  • the SLP76 complex further activate critical signaling pathways downstream of the TCR, including MAPK/Erkl/2.
  • Cell mediated immunomodulatory activity 7 is observed in fresh and frozen CD6-C AR Treg.
  • CAR-Tregs were co-cultured with autologous PBMC cells, labeled as targets, at a ratio of 2: 1 and 1 : 1 Treg:PBMC ratio (E:T) in wells with plate-bound anti-human CD3. It was at these two E:T ratios in which maximum function was observed, either by cellular analysis by FACS or detection of cytokine production by multi-plex Luminex.
  • Modulatory' activity' defined by the suppression of target cell proliferation and cellular reduction, is summarized in FIG 19L.
  • Immune-modulation was observed by in fresh and frozen CAR-Treg when co-incubated with target cells. Highest suppression is observed in CD4 target cells with the fresh sample, 56.5% at the 2:1, and 46.9% at the 1 : 1 E:T ratio. The ability for CAR-Tregs to suppress CD4 target cells was decreased in the frozen cell product, 19.5% at the 2: 1 and 12.0% at the 1: 1 E:T. However CD8 target cell suppression was maintained between the fresh (30.6% at the 2: 1 and 22.2% at the 1 : 1 E:T) and frozen (38.9% at the 2: 1 and 20.2% at the 1 : 1 E:T).
  • Target cell depletion of CD4 and CD8 target cells was detected in the fresh and frozen CAR-Tregs, in which an increased reduction of CD8 targets, 71.2% and 68.5% reduction at 2: 1 and 1 : 1 E:T with fresh and 72.4% and 53.7% with the frozen CAR-Treg, was observed compared to 66.8% and 65.1% CD4 target reduction at the 2: 1 and 1 : 1 E:T with fresh and 50.4% and 45.5% with the frozen.
  • Cytokine analysis of supernatants collected at the end of the functional assay indicates the reduction of pro-inflammatory cytokine, IFNy, from activated target cells alone compared to when co-cultured with both fresh and frozen CAR-Treg. Although slightly lower in the frozen cells, IL-10 production was detected when CAR-Tregs were activated (1 :0 E:T) and increased when CAR-Tregs were exposed to target cells (2: 1 and 1 : 1).
  • CD6-CAR cell products may contain a small contaminating CD6-CAR Teff cell population. Non-Treg contamination is rarely detected during expansion, therefore it would be difficult to assess how CAR-Tregs maintain phenotypic stability and modulatory function in the presence of non-Treg culture contaminants.
  • Autologous T effector cells were isolated, transduced, expanded, and cryopreserved in parallel to Treg cultures. Fresh and frozen CD6-CAR Treg cells were assessed for their modulatory' activity' on non-Treg contaminants that were emulated by these CD6-CAR Teff. In a 2: 1 and 1 : 1 ratio of CD6-CAR Treg to CAR Teff (E:T).
  • CAR Treg cells were able to immunomodulate contaminating CAR Teff cells by means of suppression of proliferation and reduction of proinflammatory cytokine, IFNy (FIG 20C). Cytokine analysis also showed the production of IL- 10 in the modulatory' activity' of fresh and frozen cells.
  • CD6-CAR Treg cells were done at the end of the 4-day functional analysis. After four days, the number of fresh and frozen CD6-CAR Treg cells were reduced compared to what was seeded at Day 0, as expected. CAR-Treg numbers were > 3-fold higher upon co-incubation with PBMC (E:T of 2: 1 or 1 : 1) than when cultured alone (E:T of 1:0), further indicating the dependence of Treg on a source of IL-2 for survival. Frozen CAR-Treg exhibited slightly lower survival compared to the fresh. This decrease in survival may be attributed to the effects of cryopreservation. Few studies have assessed how cryopreservation affects CAR-Treg phenotype and function.
  • Cryopreservation was done in two of these qualification runs, using methods proposed for clinical use, which includes the use of CS5, a cryopreservation solution that composed of 5% DMSO, and monitoring of the freezing process using a control rate freezer, one of which functional data is summarized in FIG 19 J.
  • CD6-CAR Treg are depleted by targeting CD19t
  • Antibody -dependent cellular cytotoxicity is commonly used to eliminate target cells in a micro environment by means of immune cytotoxicity. This method can be instrumental to deplete CD6-CAR Treg cells in the case of a post-infusion adverse event.
  • ADCC Antibody -dependent cellular cytotoxicity
  • a CD19-directed cytolytic antibody that harnesses cellular phagocytosis and cytotoxicity of macrophages and NK cells, it was observ ed that CD6-CAR Treg cells that are marked by CD19t can be depleted (FIG ZOE).
  • Flow cytometry assays confirmed that Tafasitamab binds to the same epitope as the CD 19 antibody used to routinely detect CD19t in CAR proteins. Due to its enhanced affinity' for CD19, the detection of bound Tafasitamab was visualized by FACS even after 4 days of incubation.
  • Treg cells were co-incubated with immediately thawed PBMC in wells containing plate-bound anti-human CD3 at a 1 : 1 and 4: 1 E:T ratio, where Tregs were the target cells and PBMC served as effectors, with or without Tafasitamab. After a 4-day incubation, Treg counts were analyzed by CellTrace technology and FACS. Cell counts were used to calculated percent of depleted Tregs at specified E:T ratios when incubated with Tafasitamab compared to incubations without.
  • Treg count reduction with Tafasitamab at the 1 : 1 E:T ratio and a further reduction at the 4: 1 compared to co-incubations without. This analysis indicates the use of Tafasitamab as an “off-s itch” in case CD6-CAR Treg therapy must be terminated.

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Abstract

Provided herein are, inter alia, CAR-T cell compositions targeting CD6, CD19, and/or an IL-13R, and methods useful for treating autoimmune diseases and cancer.

Description

METHODS OF MAKING AND USING REGULATORY T CELLS FOR TREATMENT OF AUTOIMMUNE DISORDERS AND CANCERS
BACKGROUND
[001] Regulatory T cells (Tregs) play an important role in controlling autoimmunity and maintaining immune homeostasis. Because antigen-specific Tregs are localized to sites of antigen presentation, they are attractive candidates for inducing or re-establishing immune tolerance in diseases such as graft versus host disease (GVHD). Adoptive transfer of antigen specific Tregs have been shown to be more potent than polyTregs in preclinical models of GVHD and Type 1 diabetes. There are a variety of approaches used for generating antigenspecific Tregs. However, it can be challenging to produce a useful number of active Tregs. It can also be difficult to preserve Tregs. These challenges can make it difficult to develop Tregs for therapeutic use.
SUMMARY
[002] Described herein are methods for preparing and preserving Tregs. including Tregs that express a chimeric antign receptor (‘'CAR”) targeted to an antigen expressed on a human cell (“CAR Tregs”). The methods can be used to produce both Tregs and CAR Tregs that have a number of desirable characterisics and produce them in therapeutically useful quantities. Importantly, the Tregs and CAR Tregs can be cryopreserved and retain many desirable characteristics once thawed. This has imporant advantages. For example, Tregs or CAR Tregs can be produced from cells isolated from a patient and the Tregs or CAR Tregs can be preserved in a manner that allows aliquots of the stored cells to be expanded so that they can be used to treat the patient on multiple occassions over a period of many months without the need for isolating additional cells from the patient for the preparation of additional Tregs or CAR Tregs.
[003] Of particular interest are CAR Tregs targeted to human CD6 ("CD6 CAR Tregs”). However, CAR Tregs can be targeted to a variety of other antigens such as CD19 or an IL-13 receptor (IL-13R; e.g., IL-13Ra2). A wide variety of CARs targeted to CD6, CD19 and IL-13 are described in WO 2021/138454, hereby incorporated by reference.
[004] Described herein is a method for generating a composition comprising regulatory T cells (Tregs), comprising: (a) providing a population of peripheral blood mononuclear cells (PBMC); (b) treating the population of PBMC to select for cells expressing CD25 (CD25+ cells); (c) treating the population of cells selected for cells expressing CD25 to select for cells expressing CD4 (CD4+ cells), cells highly expressing CD25 (CD25hi cells) and have low or no expression of CD127 (CD1271ow/- cells) thereby producing a cell population selected for cells that are CD25+, CD4+ and CD1271ow/- (CD25+, CD4+,CD1271ow/- cells); (d) activating the CD25+, CD4+, CD1271ow/- cells by exposing them to antibodies targeted to CD3, antibodies targeted to CD28 and antibodies targeted to CD2 to create an activated cell population; (e) introducing a viral vector into the activated cell population to create a population of transduced Treg; (f) culturing the transduced Treg in a culture medium that comprises exogenously added IL-2 for at least one day; and (g) activating the cultured, transduced Treg by exposing them to antibodies targeted to CD3, antibodies targeted to CD28 and antibodies targeted to CD2 to create an activated cell population.
[005] In various embodiments: the method does not include a step of enriching for cells that are CD45RO and does not include low or negative and does not include a step of enriching for cells that are CD45RA high or positive; the cells are not cultured in the presence of exogenously added IL-2 until after step (f); there are no cell selection or enrichment steps other than selection for cells expressing CD25 and a selection for cells that are CD25+, CD4+ and CD1271ow/-;
[006] the cells are cultured without exogenously added IL-2 between steps (d) and (e) and after step (e); cells are cultured in the presence of exogenously added IL-2 for the first time at least 6 hour after step (e) and for the remaining of the expansion; exogenously added IL-2 is present at 250-500 U/ml of culture medium; the population of transduced Treg expressing is cultured for at least 10 days to create an expanded population of transduced Treg; the expanded population of transduced Treg is cryopreserved; the viral vector encodes a chimeric antigen receptor; the viral vector is introduced at an MOI between 0.8 and 1.3, preferably 1.0; the cell population enriched for cells that are CD25+, CD4+ and CD1271ow/- are cultured at 1 x 106 cells/ml; all culturing steps prior to transduction take place in the absence of exogenously added IL-2; the step of treating the population of cells enriched for CD25+ cells to enrich for cells expressing CD4 (CD4+ cells) and have low or no expression of CD 127 (CD1271ow/- cells) comprises fluorescence activated cell sorting (FACS); the step of treating the population of cells enriched for CD25+ cells to enrich for cells expressing CD4 (CD4+ cells), having low or no expression of CD127 (CD1271ow/- cells), and expressing CD25 comprises FACS and the cells are not exposed to FACS for more than 5 hours; the step of treating the population of cells enriched for CD25+ cells to enrich for cells expressing CD4 (CD4+ cells), having low or no expression of CD127 (CD1271ow/- cells) and expressing CD25 produces a population of cells comprising cells expressing a high level of CD25 expression (CD25high); the cryopreservation medium contains 5% DMSO; step (b) treating the population of PBMC to select for cells expressing CD25 (CD25+ cells) comprises selecting cells expressing CD25 using an anti-CD25 antibody attached to a solid support; step (c) treating the population of cells selected for cells expressing CD25 to select for cells expressing CD4 (CD4+ cells) and have low or no expression of CD127 (CD1271ow/- cells) thereby producing a cell population selected for cells that are CD25+, CD4+ and CD1271ow7- (CD25+, CD4+,CD1271ow/- cells) comprises cell sorting using an anti-CD25 antibody, and anti-CD127 antibody and an anti-CD4 antibody; the anti-CD25 antibody of step (a) binds to a different epitope than the anti-CD25 antibody of step (b); the viral vector encodes a chimeric antigen receptor (CAR) comprising a single chain variable fragment (scFv) targeted to CD6, a spacer region, a transmembrane domain, a CTLA4 signaling domain, and a CD3 zeta signaling domain; the transmembrane domain comprises a CD4 transmembrane domain or a variant thereof, a CD8 transmembrane domain or a variant thereof, a CD28 transmembrane domain or a variant thereof, or a CD3^ transmembrane domain or a variant thereof; CAR comprises or consists of an amino acid sequence at least 90, 95, 96, 97, 98, or 99% identical to any of SEQ ID Nos: 145. 147, 149, 151, 154, 155, 156, 157 and 159; the CAR comprises or consists of an amino acid sequence of any of SEQ ID Nos: 145. 147, 149. 151, 154, 155, 156, 157 and 159 with no more than 5 single amino acid substitutions; the spacer region is selected from SEQ ID NOs: 116-126 and the transmembrane domain is selected from SEQ ID NOs: 128- 136; the scFv is selected from SEQ ID NOs: 101, 104 and 107; the scFv has a VH comprising SEQ ID NO: 102 and an VL comprising SEQ ID NO: 103; the scFv has a VH comprising SEQ ID NO: 105 and an VL comprising SEQ ID NO: 106; scFv has a VH comprising SEQ ID NO: 108 and an VL comprising SEQ ID NO: 109; wherein a linker comprising SEQ ID NO: 115 is located between the V L and VH.
Also described is a method for generating a composition comprising regulatory T cells (Tregs), comprising: (a) providing a population of peripheral blood mononuclear cells (PBMC); (b) treating the population of PBMC to select for cells expressing CD25 (CD25+ cells); (c) treating the population of cells selected for cells expressing CD25 to select for cells that express CD4 (CD4+ cells), express CD25 and have low or no expression of CD127 (CD1271ow/- cells) thereby producing a cell population selected for cells that are CD25hi, CD4+ and CD1271ow/- (CD25hi, CD4+, CD1271ow/- cells); (d) activating the CD25hi, CD4+, CD1271ow/- cells by exposing them to antibodies targeted to CD3, antibodies targeted to CD28 and antibodies targeted to CD2 to create an activated cell population; (e) introducing a vector into the activated cell population to create a population of transduced Treg; (I) culturing the transduced Treg in a culture medium that comprises exogenously added IL-2 for at least one day, wherein the cells are not treated to select for or against cells expressing CD45RO and the cells are not treated to select for or against cells expressing CD45RA. In various embodiments of this method: the cells are not cultured in the presence of exogenously added IL-2 prior to step (e); the cells are not cultured in the presence of exogenously added IL-2 until after step (I); there are no cell selection or enrichment steps other than selection for cells expressing CD25 and a selection for cells that are CD25+, CD4+ and CD1271ow/-; the cells are cultured without exogenously added IL-2 between steps (d) and (e) and after step (e); the cells are cultured in the presence of exogenously added IL-2 for the first time at least 6 hours after step (e) and for the remaining of the expansion; exogenously added IL-2 is present at 250-500 U/ml of culture medium (e.g., during expansion); the population of transduced Treg expressing is cultured for at least 10 days to create an expanded population of transduced Treg; the expanded population of transduced Treg is cryopreserved; the vector encodes a chimeric antigen receptor; the vector is a viral vector introduced at an MOI between 0.8 and 1.3, preferably 1.0; the cell population enriched for cells that are CD25+, CD4+ and CD1271ow/- are cultured at 1 x 106 cells/ml; all culturing steps prior to transduction take place in the absence of exogenously added IL-2; wherein the step of treating the population of cells enriched for CD25+ cells to enrich for cells expressing CD4 (CD4+ cells) and have low or no expression of CD127 (CD1271ow/- cells) comprises fluorescence activated cell sorting (FACS); wherein the step of treating the population of cells enriched for CD25+ cells to enrich for cells expressing CD4 (CD4+ cells), having low or no expression of CD 127 (CD1271ow/- cells), and expressing CD25 comprises FACS and the cells are not exposed to FACS for more than 5 hours: the step of treating the population of cells enriched for CD25+ cells to enrich for cells expressing CD4 (CD4+ cells), having low or no expression of CD 127 (CD1271ow/- cells) and expressing CD25 produces a population of cells comprising cells expressing a high level of CD25 expression (CD25high); the cry opreservation medium contains 5% DMSO; treating the population of PBMC to select for cells expressing CD25 (CD25+ cells) comprises selecting cells expressing CD25 using an anti-CD25 antibody attached to a solid support; treating the population of cells selected for cells expressing CD25 to select for cells expressing CD4 (CD4+ cells) and have low or no expression of CD127 (CD1271ow/- cells) thereby producing a cell population selected for cells that are CD25+, CD4+ and CD1271ow/- (CD25+, CD4+,CD1271ow/- cells) comprises cell sorting using an anti-CD25 antibody, and antiCD 127 antibody and an anti-CD4 antibody; wherein the anti-CD25 antibody of step (a) binds to a different epitope than the anti-CD25 antibody of step (b);the viral vector encodes a chimeric antigen receptor (CAR) comprising a single chain variable fragment (scFv) targeted to CD6, a spacer region, a transmembrane domain, a CTLA4 signaling domain, and a CD3 zeta signaling domain; transmembrane domain comprises a CD4 transmembrane domain or a variant thereof, a CD8 transmembrane domain or a variant thereof, a CD28 transmembrane domain or a variant thereof, or a CD3^ transmembrane domain or a variant thereof; the CAR comprises or consists of an amino acid sequence at least 90, 95, 96, 97, 98, or 99% identical to any of the CAR described herein (optionally with no more than 5 single amino acid substitutions); the spacer region is selected from SEQ ID NOs: 116-126 and the transmembrane domain is selected from SEQ ID NOs: 128-136; scFv is selected from SEQ ID NOs: 101. 104 and 107; the scFv has a VH comprising SEQ ID NO: 102 and an VL comprising SEQ ID NO: 103; the scFv has a VH comprising SEQ ID NO: 105 and an VL comprising SEQ ID NO: 106; the scFv has a VH comprising SEQ ID NO: 108 and an VL comprising SEQ ID NO: 10; a linker comprising SEQ
ID NO: 115 is located between the V L and VH; step (g) takes place 7-10 days after step (e); after step (e), the cells are cultured at 750,000 - 1,250,000 cells/ml; steps (b) and (c) comprise fluorescent activated cell sorting using anti-CD127 antibodies, anti-CD25 antibodies, anti-CD4 antibodies; the transduced Treg population have at least 60% or 70% viability, at least 70%, 80% or 90% express CD4. at least 50% or 60% express (preferably unmethylated) FOXP3, and less than 10% or 5% express CD8; transduced Treg population cells are transduced with a viral vector encoding a selected polypeptide; and the transduced Treg population have at least 70% viability', at least 90% express CD4, at least 60% express (preferably unmethylated) FOXP3, and less than 5% express CD8.
[007] Also described is a method of treating graft versus host disease or diabetes (Type 1 or Type 2) comprising administering to a patient in need thereof, a therapeutically effective amount of Treg produced by the method described herein. [008] Also described is a population of Treg prepared by a method described herein.
[009] In some embodiments, CD6 CAR Tregs target the CD6 molecule overexpressed in pro- inflammatory T-cells, e.g., in cGVHD or Type 1 Diabetes (T1D) patients. In some embodiments, the current appoach employs a CAR or polypeptide that includes an scFv derived from Itolizumab, an immunomodulatory anti-CD6 monoclonal antibody (US 6,572,857). A number of useful CAR include a CD 152 (CTLA-4) cytoplasmic domain (in addition to a CD3 zeta signalling domain) that can drive inhibitory signaling in transduced host cells and reinforce the immunomodulatory activity of CAR-Tregs. In some cases, the binding between a CAR or described herein and the antigen is of intermediate affinity. In some embodiments, the KD of the interaction is between about 10 nM and 140 nM. For example, the binding between a CD6 CAR expressed by a Treg and CD6 can be relatively low affinity. Higher expressing CD6+ cells will be more susceptible to be immune modulated by CAR-Tregs and CD6low/" subset of cells will be spared. In some embodiments, the CAR is expressed in an CD6low/" subset of Tregs. In some embodiments, Tregs can produce anti-inflammatory molecules such as, IL- 10, TGF-beta, and IDO. In some embodiments, the Tregs expressing a CAR described herein unexpectedly display potent tumor cell cytotoxicity, maintaining the distinct features of human regulatory cells such as persistent FOXP3 expression.
[010] Described herein are Tregs harboring a nucleic acid molecule comprising a nucleotide sequence encoding a CAR comprising: a targeting domain (e.g., an scFv that binds a target antigen, e.g., a CD6 scFV), a spacer, a transmembrane domain, a CTLA4 cytoplasmic domain, and a CD3 signaling domain.
[OH] In some embodiments: the transmembrane domain is selected from: a CD4 transmembrane domain or variant thereof having 1-5 single amino acid substitutions, a CD8 transmembrane domain or variant thereof having 1-5 single amino acid substitutions, a CD28 transmembrane domain or a variant thereof having 1-5 single amino acid substitutions; the spacer comprises 20-150 amino acids and is located between the tumor-targeting domains and the transmembrane domain; the transmembrane domain is a CD4 transmembrane domain or variant thereof having 1-5 single amino acid substitutions; the transmembrane domain is a CD4 transmembrane domain; the chimeric antigen receptor comprises a transmembrane domain selected from: a CD4 transmembrane domain or variant thereof having 1-2 single amino acid substitutions, a CD8 transmembrane domain or variant thereof having 1-2 single amino acid substitutions, a CD28 transmembrane domain or a variant thereof having 1-2 single amino acid substitutions; the spacer comprises an IgG hinge region; the spacer comprises 10-50 amino acids; the CTLA-4 cy toplasmic domain is a variant thereof having 1-5 single amino acid substitutions; the CD3C, signaling domain or a variant thereof having 1-5 single amino acid substitutions; a linker of 3 to 15 amino acids can be located between the CTLA-4 cytoplasmic domain and the CD3(^ signaling domain, or variant thereof; the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 145, 147, 149, 151, 154, 155, 156, 157 and 159 or a variant thereof having 1-5 or 1-2 single amino acid substitutions; the CD6 scFv comprises the amino acid sequence of any one of SEQ ID NOS: 101, 104 and 106, or a variant thereof having 1-5 or 1-2 single amino acid substitutions. In some embodiments, the nucleic acid sequences and/or amino acid sequences described herein have been codon optimized.
[012] In some embodiments, a CD6-targeted CAR (CD6 CAR, also called anti-CD6 CAR herein) described herein includes a CD6 targeting scFv (e.g., an scFv comprising the amino acid sequence:
EVQLVESGGGLVKPGGSLKLSCAASGFKFSRYAMSWVRQAPGKRLEWVATISSGGSYI YYPDSVKGRFTISRDNVKNTLYLQMSSLRSEDTAMYYCARRDYDLDYFDSWGQGTLV TVSSGSTSGGGSGGGSGGGGSSDIQMTQSPSSLSASVGDRVTITCKASRDIRSYLTWYQQ KPGKAPKTLIYYATSLADGVPSRFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPFTFG SGTKLEIKRA (SEQ ID NO: 101) or variant thereof with 1, 2, 3, 4. 5, 6, 7. 8, 9, or 10 single amino acid substitutions. In some embodiments the scFv comprises the sequence EVQLVESGGGLVKPGGSLKLSCAASGFKFSRYAMSWVRQAPGKRLEWVATISSGGSYI YYPDSVKGRFTISRDNVKNTLYLQMSSLRSEDTAMYYCARRDYDLDYFDSWGQGTLV TV (SEQ ID NO: 102) or variant thereof with 1, 2. 3, 4, or 5 single amino acid substitutions and the sequence DIQMTQSPSSLSASVGDRVTITCKASRDIRSYLTWYQQKPGKAPKTLIYYATSLADGVPS RFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPFTFGSGTKLEIKRA (SEQ ID NO: 103) or variant thereof with 1, 2, 3, 4, or 5 single amino acid substitutions joined by a flexible linker, e g., GGGSGGGSGGGGSS (SEQ ID NO: 115).
[013] In some embodiments, a CD6-targeted CAR (CD6 CAR, also called anti-CD6 CAR herein) described herein include a CD6 targeting scFv (e.g., an scFv comprising the amino acid sequence:
EVQLVESGGGLVKPGGSLKLSCAASGFKFSRYAMSWVRQAPGKGLEWVATISSGGSYI YYPDSVKGRFTISRDNVKNTLYLQMSSLRSEDTAMYYCARRDYDLDYFDSWGQGTLV TVSSGSTSGGGSGGGSGGGGSGDIQMTQSPSSLSASVGDRVTITCKASRDIRSYLTWYQQ KPGKAPKTLIYYATSLADGVPSRFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPFTFG SGTKMEIKRA (SEQ ID NO: 104) or variant thereof with 1, 2, 3. 4, 5, 6. 7, 8, 9, or 10 single amino acid substitutions. In some embodiments the scFv comprises the sequence EVQLVESGGGLVKPGGSLKLSCAASGFKFSRYAMSWVRQAPGKGLEWVATISSGGSYI YYPDSVKGRFTISRDNVKNTLYLQMSSLRSEDTAMYYCARRDYDLDYFDSWGQGTLV TVSS (SEQ ID NO: 105) or variant thereof with 1, 2, 3, 4. or 5 single amino acid substitutions and the sequence
DIQMTQSPSSLSASVGDRVTITCKASRDIRSYLTWYQQKPGKAPKTLIYYATSLADGVPS RFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPFTFGSGTKMEIKRA (SEQ ID NO: 106) or variant thereof with 1, 2, 3, 4, or 5 single amino acid substitutions joined by a flexible linker, e.g., GGGSGGGSGGGGSS (SEQ ID NO: 115).
[014] In some embodiments, a CD6-targeted CAR (CD6 CAR, also called anti-CD6 CAR herein) described herein include a CD6 targeting scFv (e.g., an scFv comprising the amino acid sequence:
DIQMTQSPSSLSASVGDRVTITCKASRDIRSYLTWYQQKPGKAPKTLIYYATSLADGVPS RFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPFTFGSGTKLEIKRAGSTSGGGSGGGS GGGGSSEVQLVESGGGLVKPGGSLKLSCAASGFKFSRYAMSWVRQAPGKRLEWVATIS SGGSYIYYPDSVKGRFTISRDNVKNTLYLQMSSLRSEDTAMYYCARRDYDLDYFDSWG QGTLVTVSS (SEQ ID NO: 107) or variant thereof with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 single amino acid substitutions. In some embodiments the scFv comprises the sequence DIQMTQSPSSLSASVGDRVTITCKASRDIRSYLTWYQQKPGKAPKTLIYYATSLADGVPS RFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPFTFGSGTKMEIKRA (SEQ ID NO: 108) or variant thereof with 1, 2, 3, 4, or 5 single amino acid substitutions and the sequence EVQLVESGGGLVKPGGSLKLSCAASGFKFSRYAMSWVRQAPGKGLEWVATISSGGSYI YYPDSVKGRFT1SRDNVKNTLYLQMSSLRSEDTAMYYCARRDYDLDYFDSWGQGTLV TVSS (SEQ ID NO:109) or variant thereof with 1, 2, 3, 4, or 5 single amino acid substitutions joined by a flexible linker, e.g., GGGSGGGSGGGGSS (SEQ ID NO: 115). [015] In some cases, the CD6 scFv comprises a VH that is at least 90%, 95%, 98% or 99% identical to:
EVQLVESGGGLVKPGGSLKLSCAASGFKFSRYAMSWVRQAPGKRLEWVATISSGGSYI YYPDSVKGRFTISRDNVKNTLYLQMSSLRSEDTAMYYCARRDYDLDYFDSWGOGTLV TVSSGSTS (SEQ ID NO: 102); a VH CDR1 comprising the sequence RY AMS (SEQ ID NO: 158); a VH CDR2 comprising the sequence TISSGGSYIYYPDSVKG (SEQ ID NO: 159); a VH CDR3 sequence comprising the sequence RDYDLDYFDS (SEQ ID NO: 160) or has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 single amino acid substitutions compared to SEQ ID NO: 102, excluding changes in VH CDR1, VH CDR2 and VH CDR3; and has a VL that is at least 90%, 95%, 98% or 99% identical to
DIOMTOSPSSLSASVGDRVTITCKASRDIRSYLTWYOQKPGKAPKTLIYYATSLADGVPS RFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPFTFGSGTKLEIKRA (SEQ ID NO: 103); VL CDR1 comprising the sequence KASRDIRSYLT (SEQ ID NO: 161); a VL CDR2 comprising the sequence YATSLAD (SEQ ID NO: 162); a VL CDR3 comprising the sequence LQHGESPFT (SEQ ID NO: 163) or has no more than 1, 2, 3. 4, 5, 6, 7. 8, 9 or 10 single amino acid substitutions compared to SEQ ID NO: 103, excluding changes in VL CDR1, VL CDR2 and VL CDR3. The VH can precede the VL and can be joined to the VL by a flexible linker, e g., GGGSGGGSGGGGSS (SEQ ID NO: 115).
Table 1: CD6 ScFv in pF03496
Figure imgf000010_0001
[016] Also described herein is a population of T cells transduced by a vector comprising a nucleic acid described herein. Also described herein is a population of T cells expressing a CAR or polypeptide encoded by a nucleic acid described herein. In some embodiments, the T cells are regulator}' T cells or effector T cells. In some embodiments, the population of Treg cells are such that least 70%, 80%, or 90% of the cells are: 1) CD4+ CD25high CD127low/neg; 2) CD4+ CD25high CD1271OW CD6low/neg ;4) CD4+ CD25high CD127neg CD6low/neg; 5) CD4+ CD25high CD127low CD6neg; or 6) CD4+ CD25hlgh CD127neg CD6neg. In some embodiments, the population of T cells are human T cells. In some embodiments, the population of T cells are autologous human T cells or allogenic human T cells.
[017] In some embodiments, at least 70% of the cells in the population of Treg cells are CD4+ CD25hlgh CD127low/neg. In some embodiments, at least 70%, 80%, or 90% of cells are CD6low or CD6neg. . In some embodiments, the population of Treg are autologous to the patient being treated. In some embodiments they are allogenic to the person being treated.
[018] Also described herein are compositions comprising any of the population of Tregs described herein. Also described herein are compositions comprising a population of Tregs harboring a nucleic acid described herein. Also described herein are compositions comprising a population of Tregs comprising a CAR or polypeptide described herein. Also described herein are compositions comprising a population of Tregs expressing a CAR or polypeptide described herein. In some embodiments, the population of T cells are human
[019] Also described herein is a method of treating a patient, the method comprising administering to a subject in need thereof a population of Tregs harboring a nucleic acid described herein, a population of Tregs described herein, or a composition comprising a population of Tregs described herein. In some embodiments, the population of Tregs are human Tregs.
[020] Also described herein is a method of treating an autoimmune disease, the method comprising administering to a subject in need thereof a population of Tregs harboring a nucleic acid described herein, a population of Tregs described herein. In some embodiments, the population of Tregs are human Tregs. In some embodiments, the population of Tregs are autologous human Tregs or allogenic human Tregs. In some embodiments, the autoimmune disease is Type I Diabetes or Graft-versus-Host Disease. In some embodiments, the population of Tregs or composition are administered in single or repeat dosing. In some embodiments, effective amount is administered to a subject. In some embodiments, at least one symptom is reduced, ameliorated, or relieved.
[021] Also described herein is a method of killing, eliminating, or reducing cells expressing CD6, the method comprising administering to a subject in need thereof a population of Tregs harboring a nucleic acid described herein, a population of T cells described herein, or a composition T cells described herein. In some embodiments, the population of Tregs are human Tregs. In some embodiments, the population of Tregs are autologous human Tregs or allogenic human Tregs. In some embodiments, the cells expressing CD6 are cancerous. In some embodiments, the population of Tregs or composition are administered in single or repeat dosing. In some embodiments, effective amount is administered to a subject. In some embodiments, at least one symptom is reduced, ameliorated, or relieved.
[022] Also descnbed herein is a method of killing, eliminating, or reducing cells expressing CD 19, the method comprising administering to a subject in need thereof a population of Tregs harboring a nucleic acid described herein, a population of T cells described herein, or a composition T cells described herein. In some embodiments, the population of Tregs are human Tregs. In some embodiments, the population of Tregs are autologous human Tregs or allogenic human Tregs. In some embodiments, the cells expressing CD19 are cancerous. In some embodiments, the population of Tregs or composition are administered in single or repeat dosing. In some embodiments, effective amount is administered to a subject. In some embodiments, at least one symptom is reduced, ameliorated, or relieved.
[023] Also described herein is a method of killing, eliminating, or reducing cells expressing an IL-13R (e.g. IL-13Ra2), the method comprising administering to a subject in need thereof a population of Tregs harboring a nucleic acid described herein, a population of T cells described herein, or a composition T cells described herein. In some embodiments, the population of Tregs are human Tregs. In some embodiments, the population of Tregs are autologous human Tregs or allogenic human Tregs. In some embodiments, the cells expressing IL-13Ra2are cancerous. In some embodiments, the population of Tregs or composition are administered in single or repeat dosing. In some embodiments, effective amount is administered to a subject. In some embodiments, at least one symptom is reduced, ameliorated, or relieved.
[024] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton. Proteins (1984)). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. All headings, sections, subheadings, and subsections are solely present for organization purposes and not meant to limit the scope therein. The content of each section can be applied to any and all aspects of the disclosure presented under any other heading, section, subheading, and subsection. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
DESCRIPTION OF THE DRAWINGS
[025] FIG 1: Phenotype of test run products. Cells w ere stained with fluorochrome- conjugated antibodies to detect intracellular FOXP3, or surface CD4, CD8, or the CD19t transgene marker. Percentages of viable (Zombie Aqua negative) CD3+-gated cells with immunoreactivity above control staining are indicated in each histogram
[026] FIG 2: Immunomodulatory Activity of CD6-CAR Treg Cells against autologous PBMC. A, Viable cell numbers of CD6-CAR Treg cells per w ell at the time of seeding (Day 0) or after four days of culture at the indicated E:T ratios are depicted as mean + S.D. of duplicate wells. B, Measuring proliferation by the dilution of CFSE signal in either the CD3/CD4-gated or CD3/CD8-gated population, the percent inhibition of target cell proliferation after 4 days of coculture with CD6-CAR Treg cells at the indicated E:T ratios, and as calculated based on cultures of target cells alone, is depicted as mean + S.D. of duplicate w ells. C, Levels of IFNy (red) or IL- 10 (green) in the supernatants harvested after 4 days of culture are depicted at each E:T ratio.
[027] FIG 3: Immunomodulatory Activity of CD6-CAR Treg Cells against CD6-CAR Teff Cells. A, Measuring proliferation by the dilution of CGSE signal in either the CD3/CD4- gated or CD3/CD8-gated population, the percent inhibition of CD6-CAR Teff cell proliferation after 4 days of co-culture with CD6-CAR Treg cells at the indicated E:T ratios, and as calculated based on cultures of Teff cells alone, is depicted as mean ± S.D. of duplicate wells. B, Levels if IFNy (red) or IL-10 (green) in the supernatants harvested after 4 days of culture are depicted at each E:T ratio.
[028] FIG 4: Control of GvHD upon administration of CD6-CAR Treg cells. NSG mice first received 5xl06 PBMCs i.v., and were immediately treated i.v. with either 5xl06 CD6-CAR Treg cells (50% CAR transduced), non-transduced Treg cells, or vehicle alone, n=4 per group. A, Quantification of whole-body weight (mean ± SEM depicted at each timepoint) shows that CD6-CAR Treg cell treated mice maintained their body weight longer than those that received vehicle only, or control Treg cells. Weight loss in the vehicle and Treg control groups was significantly greater than CD6-CAR Treg treated group by days 12 and 13, Kruskal-Wallis test p=0.0012 and p=0.0047, respectively. B. Kaplan Meier analysis showed a median survival of 12 days for the control group treated with vehicle-only. Non-transduced Treg control group extended the median survival to 23 days. The median survival further improved to 47.5 days in the CD6-CAR Treg cell-treated mice group, Log-rank (Mantel-Cox) test p=0.0082. C, Lower levels of serum proinfl ammatory cytokines as measured by Luminex assay were observed in both Treg cell and CD6-CAR Treg cell-treated mice. D. Immunohistochemistry’ analysis of the lungs shows less infiltration of CD4+ (purple) and CD8+ (green) Teff cells in CD6-CAR Treg cell-treated mice compared to mice treated with non-transduced Treg cells or vehicle alone.
[029] FIG 5: Map of CD6scFvIgG4(L235E,N297Q)-CTLA4-Zeta-T2A-CD19t-epHIV A, Diagram of the cDNA open reading frame of the 3066 nt construct indicating: the GMCSF signal sequence preceding the CD6 CAR, the various domains of the CD6 CAR, the T2A skip sequence, the CD 19 signal sequence preceding the truncated CD 19 marker, and the truncated CD 19 marker. B, Map of the plasmid. C, Diagram of the sequences flanked by long terminal repeats (indicated by ‘R’) that will integrate into the host genome.
[030] FIG 6: Amino acid sequence of the immature CD6 CAR expressed by pF03496. CD6scFv(VH_VL)-IgG4(L235E,N297Q)-CTLA4-Zeta is shown with the various domains marked. SEQ ID NO: 144 with the signal sequence and SEQ ID NO: 145 without the signal sequence.
[031] FIG 7: Amino acid sequence of the immature CD6 CAR expressed by pF03497.
CD6scFv(VL_VH)-IgG4(L235E,N297Q)-CTLA4-Zeta is shown with the various domains marked. SEQ ID NO: 146 with the signal sequence and SEQ ID NO: 147 without the signal sequence.
[032] FIG 8: Amino acid sequence of the immature CD6 CAR expressed by pF03488.
CD6scFv(VH-VL)-IgG4(L235E, N297Q)-41BB-Zeta is shown with the various domains marked. SEQ ID NO: 148 with the signal sequence and SEQ ID NO: 149 without the signal sequence.
[033] FIG 9: Amino acid sequence of the immature CD6 CAR expressed by pF03491.
CD6scFv(VL-VH)-IgG4(L235E, N297Q)-41BB-Zeta is shown with the various domains marked. SEQ ID NO: 150 with the signal sequence and SEQ ID NO: 151 without the signal sequence.
[034] FIG 10: Amino acid sequence of a mature IL13 CAR. IL13-IgG4(L235E,N297Q)- CD4tm-CTLA4-Zeta is shown with the various domains marked. SEQ ID NO: 152.
[035] FIG 11: Amino acid sequence of a mature CD6 CAR. CD6scFv(VH- VL)IgG4(L235E,N297Q)-CD4tm-41BB-Zeta is shown with the various domains marked. SEQ ID NO: 153.
[036] FIG 12: Amino acid sequence of a mature CD6 CAR. CD6scFv(VH-
VL)IgG4(L235E.N297Q)-CD4tm-CLTA4-Zeta is shown with the various domains marked. SEQ ID NO: 154.
[037] FIG 13: Amino acid sequence of a mature CD6 CAR. CD6scFvmhi(VH- VL)IgG4(L235E,N297Q)-CD4tm-41BB-Zeta is shown with the various domains marked. The three mutations in the scFv are bolded and double underlined. SEQ ID NO: 155.
[038] FIG 14: Amino acid sequence of a mature CD6 CAR. CD6scFvmhi(VH- VL)IgG4(L235E.N297Q)-CD4tm-CTLA4-Zeta is shown with the various domains marked. The three mutations in the scFv are bolded and double underlined. SEQ ID NO: 156.
[039] FIG 15: Amino acid sequence of a mature CD19 CAR. CD19scFvop(VH-VL) IgG4(L235E,N297Q)-CD4tm-CLTA4-Zeta is shown with the various domains marked. SEQ ID NO: 157. [040] FIG 16: Amino acid sequence of a CD6 CAR with a signal sequence. The signal sequence is underlined SEQ ID NO: 158 with the signal sequence and SEQ ID NO: 159 without the signal sequence.
[041] FIG 17: Nucleotide sequence of a lentiviral vector encoding a CD6 CAR. This lentiviral vector encodes the CAR depicted in FIG 16. (SEQ ID NO: 160)
[042] FIG 18: Schematic depiction of a lentiviral vector. This is a map of the lentiviral vector of FIG 17.
[043] FIG 19: Preparation and Assessment of Tregs. A. Schematic depiction of the gating strategy for sorting Tregs from CD25-enriched cells. B. Purity assessment of isolated Tregs: Lymphocyte population gating (Top-left), Doublet-exclusion gating (Top-right), CD4 Gating (Bottom-left), and CD4+ > CD25hiCD12710 (Bottom-right). CD4+CD25+CD12710 Treg Purity = 95.33%. C. Culture morphology images taken at 40X magnification of CD6-CAR Treg QR2 cultures on Days 1 (post-initial activation), 9 (prior to reactivation), 10 (post-reactivation), and 14 of expansion. D. Expansion of Tregs were calculated as Fold Expansion and monitored at Days 0, 9, and 14 (end of expansion). (N=7). The Y-axis, depicted in LoglO, depicts the exponential growth of the cells from the initiation of expansion (Day 0). E. Cell product stabi 1 i ty was assessed at the end of the expansion penod. Tregs were incubated at RT at concentrations of 10-50xl06 cells/ml in 2% purified human saline. Cell viability, identity, potency, and purity samples were analyzed at time-points, 0, 4, and 6 hours after preparation. Functional assessments, suppression of CD3-activated target cells (PBMC) (ii) and cytokine analysis of IFNy and IL- 10 (iii), were done at each time-point. Each symbol represents a Treg production. F. Functional assessment of CD6-CAR Treg cells analyzed after culture expansion by their ability to suppress CD3-activated autologous and allogenic at Treg to target cell. Suppression of autologous CD4 (•) and CD8 (•), and allogenic CD4 (O) and CD8 (O) target cells were calculated for each ratio condition and depicted as mean ± S.D. G. Primers detecting the CD6-CAR transgene (954bp, Lanes 1-3) and CD19t (578bp, Lanes 4-6) analyzed genomic DNA isolated from CD6-CAR Treg samples from QR2 (Lanes 1 and 4), alongside FACS- confirmed CD6-CAR+ cells (positive control, Lanes 2 and 5) and non-transduced cells (negative control, Lanes 3 and 6). PCR products are compared to lOObp ladder (Lane L) with lOOObp and 600bp indicated with arrows. H. Cryopreserved cells from qualification runs (N=6). were assessed for release criteria parameters. Y-axis depicts percent expression or measurement of each criterion with dashed lines indicating accepted release parameter. *WPRE measured as copies per CAR+ cell. ** VS V-G presented as copies/50ng gDNA. I. Unmethylated FOXP3 was measured in genomic DNA of qualification run fresh and frozen samples (N=6). Paired t- test was used to compare Fresh vs Frozen CD6-CAR-Treg cell products. J. CAR detection with ddPCR consists of primers that separately detect the CD6 (i) and CD 19t(ii) portions in both Fresh and Frozen CAR-Treg cell products (N=4). Paired t-test was used to compare Fresh vs Frozen CD6-CAR-Treg cell products. K. Western Blot analysis of pCD3zTyrl42, pSLP76 Ser376, pERKl/2 (Thr204/Tyr202), pAKT Ser473, pPIk3 (Tyr467/199) in protein lysates of CD6-CAR Tregs were stimulated with CD6Fc for 1 hr. Blots and Quantification of fold change of normalized band intensity densitometric values are representative of 2 independent experiments from 2 different donors. L. Treg suppression measured by inhibition of proliferation and reduction of CD4 and CD8-gated PBMC target cells after 4-day co-incubation at indicated E:T ratios. Percent suppression is normalized to maximum target cell proliferation and target cell reduction is normalized to incubated target cells without Tregs and depicted as mean ± S.D; Two-way ANOVA compared CD4 vs CD8 PBMC suppression and target cell reduction (i and ii). Luminex analysis of IFNy (yellow) or IL-10 (green) in the supernatants collected after 4 days of co-incubation at E:T ratios indicated; Two-way ANOVA compared IFNy and IL-10 production at the L I vs 2: 1 E:T ratios (hi). CAR-Treg survival is measured by viable CD3+ cell numbers of Treg cells at E:T ratios; One-way ANOVA was used to show similarities in the survival of Tregs in each condition (iv). CD6-CAR-Tregs were co-incubated with autologous and allogenic target cells for a 4-day period by which suppression and target cell reduction were compared at E:T ratios indicated (v and vi).
[044] FIG. 20 A. Viability (i) and cell count (ii) assessment of cytokine-dependent CD6- CAR Treg cells with (0) and without (0) IL-2 supplementation after reactivation (Day 9) until Day 24 of culture maintenance (N=l). Cytokine-dependence assessment of cryopreserved CD6- CAR Treg cultured with (black symbols) or without (gray symbols) IL-2 supplementation for up to 21 days after thaw (N=5). Viability (iii) and viable cell counts (iv) were measured at the time of culture maintenance with or without IL-2 supplementation. B. Treg suppression and target cell reduction (i) of CD4 and CD8 PBMC by Fresh and Frozen CD6-CAR Tregs from the same expansion were analyzed at the 1: 1 and 2: 1 E:T ratio (N=4). Additionally, cytokine analysis of IFNgamma and IL-10 were assessed between Fresh and Frozen CD6-CAR Treg function at the 1: 1 and 2: 1 E:T ratio. Two-way ANOVA analysis compared Fresh vs Frozen function. C. A representative dot plot depiction of CAR detection with ddPCR consists of primers that separately detect the CD6 and CD19t sequence portions in both Fresh and Frozen CAR-Treg cell products. D. FOXP3 TSDR percentages of CD6-CAR Treg cells after 14 and 36 days of culture (i). tSNE analysis of CD6-CAR Treg cells at days 14 and 36 (ii). E. Immunofluorescence staining of CD6-CAR Tregs with tafasitamab (i) and nuclear counter stain (ii). tSNE analysis illustrating the presence of CD6-CAR Treg cells through the detection of CD19t at 1:4 E:T ratio (iii), and the depletion of CD19t positive CD6-CAR-Treg cells after incubation with tafasitamab at 1:4 E:T ratio (iv). F. Suppression of CD3/CD28 activated CD6-CAR Teff cells after 4 days of incubation with CD6-CAR Treg cells at a 1 : 1 and 2: 1 E:T ratios (i). Levels of IFND and IL-10 in the supernatants analyzed by Luminex at each E:T ratio (ii). tSNE analysis of activated CD6- CAR Teff targets alone or at 1 : 1 E:T ratio with CD6-C AR Treg illustrating the reduction of Teff target cells indicated by rectangles. G. tSNE clustering and FLOWS OM analysis was done on the indicated markers after a 4-day incubation period of activated Tregs or PBMC targets alone, or at a 1: 1 effector to autologous (auto) or allogenic (allo) target ratio. Cells were clustered into populations (pop) 1-8 with a heatmap indicating the expression intensity7 or absence of markers (blue = low to no presence and red = high expression) (i). Pie charts depict the presence of each population in both activated targets (ii) and Tregs (iii) alone or at a 1: 1 effector to autologous or allogenic target ratio.
DETAILED DESCRIPTION
[045] In some cases, the Treg is a Treg that includes CD4positive-CD25high-CD1271ow or negative expression. In some cases, the Treg is a Treg that includes CD4positive-CD25high- CD1271ow or negative, CD61ow or negative expression. In some cases, the Treg is a Treg that includes CD4positive-CD25high-CDl 271ow or negative-CD61ow or negative expression. In some cases, the Treg is a Treg that includes CD45RA+ expression. In some cases, the Treg is a Treg that does not include CD45RA+ expression. In some cases, the Treg is a Treg that includes FOXP3 demethylation. In some cases, the Treg is a Treg that does not include FOXP3 demethylation. In some cases, the Treg is a Treg that can be expanded either with IL-2 and/or rapamycin and/or retinoic acid. In some cases, the Treg is a Treg that can be expanded with IL-2 and/or rapamycin and/or retinoic acid. In some cases, the Treg is a Treg that can be expanded with IL-2. In some cases, the Treg is a Treg that can be expanded with rapamycin. In some cases, the Treg is a Treg that can be expanded with retinoic acid.
[046] Determining “high” and “low ” expression is well know n in the art. A description of high and low expression of the markers referred to supra, and how high and low expression are determined and quantified, may be found, for example, in Putnam et al. Expansion of human regulatory T-cells from patients with type 1 diabetes. Diabetes 58(3): 652-662, 2009; Bluestone et al. . Clinical Grade Regulatory' CD4(+) T Cells (Tregs): Moving Toward Cellular-Based Immunomodulatory' Therapies. Front Immunol 9: 252, 2018; the entire contents of each of which are incorporated herein by reference in their entireties and for all purposes.
Chimeric Antigen Receptors
[047] A CAR described herein can include a spacer located between the targeting domain (i.e. , a CD6 targeted scFv or variant thereof, a CD 19 targeted scFv or variant thereof, an IL- 13 or a variant thereof that binds an IL-13R) and the transmembrane domain. A variety of different spacers can be used. Some of them include at least portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain or variants thereof. Table 2 below provides various spacers that can be used in the CARs and polypeptides described herein.
[048] Some spacer regions include all or part of an immunoglobulin (e.g., IgGl, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CHI and CH2 domains of an immunoglobulin, e g., an IgG4 Fc hinge or a CD8 hinge. Some spacer regions include an immunoglobulin CH3 domain (called CH3 or ACH2) or both a CH3 domain and a CH2 domain. The immunoglobulin derived sequences can include one or more amino acid modifications, for example, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off-target binding.
Table 2: Examples of Spacers
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0002
[049] The hinge/linker region can also comprise an IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO: 118) or ESKYGPPCPPCP (SEQ ID NO: 117). The hinge/linger region can also comprise the sequence ESKYGPPCPPCP (SEQ ID NO: 117) followed by the linker sequence GGGSSGGGSG (SEQ ID NO: 116) followed by IgG4 CH3 sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 126). Thus, the entire linker/spacer region can comprise the sequence:
ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK (SEQ ID NO: 125). In some cases, the spacer has 1, 2, 3, 4. or 5 single amino acid changes (e.g., conservative changes) compared to SEQ ID NO: 125. In some cases, the IgG4 Fc hinge/linker region that is mutated at two positions (L235E; N297Q) in a manner that reduces binding by Fc receptors (FcRs).
[050] Any flexible linker described herein can be a repeat of GGGS (SEQ ID NO: 127). Any flexible linker can comprise one. two, three, four, five, or more repeats of SEQ ID NO: 127. Any flexible linker can also have 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to the repeats of SEQ ID NO: 127.
[051] A variety of transmembrane domains can be used in any CAR or polypeptide described herein. Table 3 includes examples of suitable transmembrane domains. Where a spacer region is present, the transmembrane domain (TM) is located carboxy tenninal to the spacer region.
Table 3: Examples of Transmembrane Domains
Figure imgf000021_0001
Figure imgf000022_0001
[052] CAR expressed by a Treg includes a CTLA4 cytoplasmic domain (AVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN; SEQ ID NO: 137) located between the transmembrane domain and the CD3c signaling domain.
Table 4: CI)3» Signaling Domain and CTLA4 Cytoplasmic Domain
Figure imgf000022_0002
[053] In some cases, the CD3^ signaling domain includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to SEQ ID NO: 138. In some cases, the CD3^ signaling has 1 , 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO: 138.
METHODS OF TREATMENT
[054] In some embodiments, the compositions provided herein, including embodiments thereof, are used as effective treatment of autoimmune diseases. Thus, in an aspect is provided a method of treating an autoimmune disease (e.g., Type I diabetes, Graft-versus-Host Disease, Lupus), the method including administering to a subj ect in need thereof an effective amount of a population of Treg cells provided herein including.
[055] In some cases, the autoimmune disease is associated with reduced islet cell (e.g. , beta cell) function, viability, or survival. In some cases, the autoimmune disease is Type I Diabetes. In some cases, the autoimmune disease is Graft-versus-Host Disease. In some cases, the autoimmune disease includes a subject’s immune system attacking the subject’s islet cells (e.g., beta cells).
[056] comprising administering a population of autologous or allogeneic human T cells transduced by a vector compnsing a nucleic acid molecule descnbed herein. In various embodiments: a chimeric antigen receptor described herein is administered locally or systemically; in some embodiments, a method of treatment includes reducing or eliminating cells expressing a CD6 receptor T cell expressing a CAR or polypeptide (e.g., CAR Teff or CAR Treg) described herein is administered by single or repeat dosing.
EXAMPLES
[057] The follow ing examples are intended to further illustrate certain embodiments of the disclosure. The examples are put forth so as to provide one of ordinary skill in the art and are not intended to limit its scope.
[058] Regulatory T cells (Treg) and regulatory T cells expressing a CAR (CAR Treg) have tremendous therapeutic potential. Fully realizing this potential requires methods for obtaining, expanding and preserving such cells in manner that does not meaningfully diminish their activity, for example, their immunomodulatory activity. It also required the ability to generate such cells in quantities that can be used therapeutically in an effective manner. Thus, tuning of the manufacturing process for such cells is highly useful.
[059] We undertook to improve the Treg and CAR Treg manufacturing and preservation methods described in WO 2021/138454. Among other aspects of the manufacturing and preservation method, we examined approaches to cell sorting, culturing conditions, including cell density and cytokine supplementation, conditions for lentiviral transduction, conditions for cell activation and cryopreservation conditions. Based on this multi-dimensional analysis, an improved method for the production of Treg and CAR Treg is described.
Example 1: Preparation of Tregs
Described below in an example of a method for preparing Tregs.
PBMC Collection and Enrichment for CD25+ Cells (Day -2 to Day -1) Peripheral blood mononuclear cells (PBMC) are collected by leukapheresis, for example, from a patient to later be treated with Tregs expressing a chimeric antigen receptor. The collected cells are then treated to enrich for cells expressing CD25 (CD25+ cells). Briefly, appropriate volume of wash buffer is pumped into the starting leukapheresis product bag, mixed well, and centrifuged at 200xg for 15 minutes at room temperature. The supernatant is removed by gravity7 flow, and the remaining cell pellet resuspended in the re-sealed starting bag. The cells are then CD25 microbead-labeled. The volume of the labeling and CD25 enrichment beads is determined using the white blood cell counts (WBC) recorded at the time of the leukapheresis procedure, and the appropriate volume of cold CliniMACs Buffer + 0.5% HAS (labeling buffer) added to the resuspended cells. Using the appropriate tubing setup, the calculated volume of CD25 microbeads is added to the cell bag and incubated at 4°C for 15 minutes. After incubation, the cells are washed with labeling buffer by centrifugation at 200xg for 15 minutes at 15-20°C, and the supernatant is removed. The remaining cell pellet is resuspended in cold labeling buffer and counted. Up to 40 - 120 xlO6 labeled cells are enriched using Miltenyi’s Program Enrichment 3.2 on the CliniMACs system, where the final suspension of CD25+ cells will be collected in a 250 mL Jensen tube and counted. The enriched cells suspension is centrifuged at 1200 rpm for 10 minutes. The supernatant is removed, and the cell pellet resuspended in T cell culture media without added IE-2 and cultured overnight at 1 x 106 cells/ml at 37°C with 5 % CO2.
Isolation of Tregs (Dav -1 to DO)
After overnight culture, the enriched cells are be prepared for Treg cell sorting. Briefly, , the cells are mixed and transferred to a 50 rnE or 250 mL tube (depending on volume) and counted. The cells are centrifuged again and resuspended in the buffer to a concentration of 20x106 cells/mL in the appropriate tubes. The cells are then stained with an appropriate amounts of antibodies designed for Treg Sort Panel 1: CD4-FITC (BD Biosciences, Catalog 340133), CD25- APC (BD Biosciences, Catalog 340939), and CD127-PE (BD Biosciences, Catalog 561028) or Treg Sort Panel 2: MACS GMP CD4-PerCP (Miltenyi Biotec, Catalog 170-076-506 ), MACS GMP CD25-PE (Miltenyi Biotec, Catalog 170-076-503), MACS GMP CD127-FITC (Miltenyi Biotec, Catalog 170-076-512) 20 minutes in the dark at room temperature. Once complete, the cells are washed (1200 rpm for 10 minutes) with buffer, the cell pellet is resuspended at a concentration of 10xl06 cells/mL in the appropriate tubes and filtered through a 70 pm cell strainer. The resulting volume of cells is divided into FACs tubes, each containing no more than 4 mL/tube, and is then ready for sorting. The Sony FX-500 Sorter can used for Treg cell sorting. Once the procedure is complete, the sorted cells are collected in prepared tubes containing complete culture media (X-VIVO15 + 10% HABS). All the tubes are be consolidated into one 50 mL or 250 mL tube and mixed. This process results in the preparation of a population of cells that are CD4+, CD127low/", CD25hlgh. A sample can be taken for FACs analysis to confirm successful isolation of desired cell population and viability.
Seeding Culture Post CD25 Isolation (Dav 0)
On day 0, the post-sort Treg cells are held on wet ice in a 50 mL conical tube before proceeding. If the cell suspension volume is below 25 mL, then the suspension is supplemented wi th complete culture media. The cell suspension is then centrifuged at 1500 RPM for 5 minutes at 25°C ± 2°C. the supernatant is decanted, the pelleted cells are vortexed and resuspended to adjust the concentration to approximately 3xl06/mL, and a sample is harvested for total cell viability assessment. Based on the total viable cells, the cell suspension is adjusted to a final concentration of lx!06/mL for activation. Once cell concentration has been adjusted appropriately, ImmunoCult CD3/CD28/CD2 (bead-free antibodies targeted to CD3, CD28 and CD2 for cell activation , Stemcell, RUO 10970, cGMP-grade 100-0785) is added to the suspension for cell activation at 25yAnL and swirled to mix the reagent into the suspension. Depending on the final cell suspension volume, the activated cell suspension is either plated or placed in a culture flask (e.g., T-25 culture flasks at a volume not to exceed 15 mL/flask). Then, after transferring the cell suspension to culture container, the container(s) is placed in a 37°C ± 2°C incubator
Transduction (Day 1) and Culture Maintenance (Davs 3 to 7)
[060] On Day 1 of cell culture, the plateZflask(s) containing activated Treg cells are supplemented with protamine sulfate at a final concentration of 0. 1 mg/mL. Virus encoding a protein of interest (e.g. GMP-grade lentivirus (CD6(EQ)CTLA4Z-T2A-CD19t) is thawed, thoroughly vortexed, and then using a pipettor and sterile pipet tips, the required volume of virus is added to each Treg plate/flask to achieve an MOI of 1.0. Each culture vessel is then gently swirled to ensure that the protamine sulfate- vims additions are well mixed into the cell suspension before being returned to the 37°C ± 2°C incubator and incubated for 5-6hours. During the incubation period, a master mix is be prepared containing complete culture media supplemented with IL-2 for the final working concentration of 500 U/mL.
[061] Following the incubation period, the cell suspension containers are retrieved from the incubators and supplemented with the prepared master mix at a ratio of 1:2 (culture: master mix) before being returned to the 37°C ± 2°C incubator. If the new volume exceeds the capacity of the container, then the cell suspension is transferred to a larger container or split into two equal parts doubling the number of containers. Subsequent culture maintenance occurs on days 3, 5, and 7 using a master mix containing 500 U/mL IL-2. On day3, the cultures are supplemented at a ratio of 1:2 master mix; on days 5 and 7, the cultures are supplemented at a ratio of 1:3 master mix; cultures are split into anew culture container if the volume exceeds the capacity of the current container and returned to the 37°C ± 2°C incubator.
Reactivation (Dav 9) and Culture Maintenance (Davs 10 to 13)
[062] On day 9 of cell culture, the transduced Treg cultures are harvested from their container(s) and placed in the appropriately sized conical tubes. The conical tubes are centrifuged using the appropriate parameters for the tube size. Post-centrifugation, the supernatants are removed, and each cell pellet is vortexed and resuspended in complete culture media. A well-mixed sample is harvested for cell counts and viability using the appropriate counting device. Samples containing cells is harvested for in-process testing. Based on cell counts, the volume for the final concentration for activation is calculated. Next, the calculated volume of viable cells for activation is harvested into the appropriately sized tubes (if the concentration is too low, the cell suspension is centrifuged), and the cell suspension is supplemented with complete culture media to adjust the suspension to the calculated final volume for activation. The following reagents is added to this cell suspension: (1) IL-2 ([final] = 500 U/mL), (2) ImmunoCult CD3/CD28/CD2 (bead-free antibodies targeted to CD3, CD28 and CD2 for cell activation Stemcell, RUO 10970, cGMP-grade 100-0785) ([final] = 25 p/mL). Following the reagent additions, to ensure the reagents are mixed well, the cell suspension is swirled or mixed with a serological pipet and then aliquoted into appropriately sized culture vessel(s). At the end of the reactivation, the culture is returned to the 37°C ± 2°C incubator.
[063] Subsequent culture maintenance occurs on days 10 and 12, and the cultures are supplemented with complete culture media at a ratio of 1:3 (media: cytokine). First, the cells are split into the appropriate number of culture vessels to accommodate the final total volume. Next, the cultures are then fed with IL-2 ([final] = 333U/mL), and the media is then returned to the 37°C ± 2°C incubator.
Cry opreservation of Final Product (Day 14)
[064] On day 14 of the cell (day of cry opreservation), a sample is harvested form one representative flask and counted using the appropriate counting device. Based on the count, the transduced Treg culture is harvested from their containers for Mycoplasma testing (from the pooled cell suspension), and two samples containing cells are harvested for in-process testing (from the pooled suspension). Next, the suspension is transferred to appropriately sized tubes. The conical tubes are centrifuged using the appropriate parameters for the tube size that is used. Post-centrifugation, the supernatant is removed. Once the supernatant is removed, each cell pellet is vortexed and resuspended in wash buffer, and the cell suspension well mixed. A well- mixed sample is harvested for counting using the appropriate counting device. The conical tube(s) is centrifuged using the appropriate parameters for the tube size that is used. During centrifugation, calculations are made for the final total cell concentration of the cell product, where the concentration is 10xl06/mL. and the maximum concentration is 50xl06/mL in a volume of 10-20mL in the bag(s). These products are cryopreserved and stored in CryoMACS50 bags (Miltenyi Biotec #200-074-400:) for infusion, with cryopreserved vial (Cryovials, Nunc #375418) aliquots for QC. Post-centrifugation, the supernatant is removed. Once the supernatant is removed, each cell pellet is vortexed and resuspended in cry opreservation media, with 5% DMSO (CiyoStorCS5, Stemcell Technologies, 07933). Sterility samples are harvested from this cell suspension.
[065] The cell suspension is transferred to the appropriate size cryopreservation container, such as CryoMACS50 bags, as well as cryovials. Upon filling, cryopreservation containers are placed in a chilled cassete, and the cryovials are placed in Mr. Frostys. The bags/cryovials are be transferred to the cooled (approximately 4°C) controlled rate freezer (CRF). The CRF probe is placed into the cassette of a filled appropriately sized cryopreservation container or into a mock ciyovial containing cCryoStorCS5. At the completion of the CRF freeze procedure, the frozen bags/cryovials are immediately be transferred into a GMP LN2 freezer for storage. If CRF is not used, bags/ciyovials can be transferred directly into -80°C freeze; after three days in the -80°C freezer, bags/cryovials are transferred into a GMP LN2 freezer for storage.
Example 2: Expansion of Tregs and Transduction with a CD6 CAR
Using essentially the methods described in Example 1, CD6 CAR Treg cells were prepared from PBMC from four healthy donors. The cells were reactivated as described in Example 1 and maintained with the addition of complete X-VIVO 15 media and cytokine every other day until the last day of culture. Two of the test runs (Test Runs 3 and 4) were cryopreserved as described in Example 1. Subsequently, cells from each of the four test runs were expanded for 14-19 days. These results of this study (Table 5) demonstrate that the cells, including cryopreserved cells, can expand up to 1464-fold in 14 days, demonstrating that the cells can be expanded to numbers sufficient for clinical application.
Table 5: Expansion of Test Run Products
Figure imgf000028_0001
Cells from Test Run 3 and Test Run 4 were stained with fluorochrome-conjugated antibodies to detect intracellular FOXP3, or surface CD4, CD8, or the CD19t transgene marker. The results of this analysis are presented in FIG 1 in which percentages of viable (Zombie Aqua negative) CD3+-gated cells with immunoreactivity above control staining are indicated in each histogram.
Tregs from Test Run 3 and Test Run 4 underwent general safety testing that included analysis for vector copy number, as neoplastic transformation would be anticipated to increase with higher numbers of vector copies/insertions. Using a quantitative polymerase chain reaction (qPCR) assay it was found that transduction with vCD6(EQ)CTLA4Z-T2A-CD19t_epHIV7 at an MOI of 1.0 resulted in < 3.3 WPRE copies per CAR+ cell (Table 6)
Table 6: Vector Copy Number of Test Run Products using qPCR for WPRE
Figure imgf000029_0001
Quantitative PCR for the VSV-G DNA copy number was also carried out in an effort to detect overt replication competent lentivirus (RCL). The average VSV-G copy numbers in Test Run Products 3 and 4 were < 0.7 copy per 50 ng gDNA.
Transduced Treg compositions for therapeutic use are those that are: > 70% cell viability; > 60% FOXP3 expression and > 90% CD4 expression by flow cytometric analysis; < 5% CD8 expression by flow cytometric analysis; > 20% CAR expression (as assessed, for example by surface marker expression) by flow cytometric analysis.
Example 3: Activity Assessment of Treg Cells Transduced with CD6(EQ)CTLA4Z-T2A- CD19t epHIV7 The objective of this study was to evaluate the immunomodulatory activity of regulatory T (Treg) cell products that were transduced with CD6(EQ)CTLA4Z-T2A-CD19t_epHIV7 GMP- grade lentivirus and expanded in vitro as described in Example 1. The immunomodulatory activity7 of CD6-CAR Tregs against autologous peripheral blood mononuclear cells (PBMC) or CD6-CAR transduced autologous effector T (Tefl), as well as allogenic PBMC cells was evaluated using proliferation assays and cytokine production assays.
CD6-CAR Treg cell qualification run products were generated from healthy donors using methodologies suited for clinical use. Briefly, CD4+, CD25+, CD127low/neg Treg cells that had been enriched and sorted from PBMC were transduced with GMP-grade CD6(EQ)CTLA4Z- T2A-CD19t_epHIV7 lentivirus, expanded for 14 days and then cryopreserved. Using either autologous PBMC or an autologous CD25/CD14-depleted, CD6-CAR transduced Teff population as ‘target' cells, the CD6-CAR Treg products were then evaluated for effector function using flow cytometry-based assays for target cell proliferation and cytokine expression.
Production of CD6-CAR Treg and Target Populations
Treg cells were isolated, transduced with CD6(EQ)CTLA4Z-T2A-CD19t_epHIV7 lentivirus, and expanded with IL-2 and cryopreserved on day 14 as described above. For future use as target cells, a portion of the source PBMC were cryopreserved prior to the isolation/production of CD6-CAR Treg cells. To generate CD6-CAR Teff cell targets, the negative fraction obtained from the CD25 selection process of CD6-CAR Treg cells (i.e., the CD25-depleted population) was further depleted of CD14, activated with CD3/CD28 Dynabeads ™ beads, transduced with CD6(EQ)CTLA4Z-T2A-CD19t_epHIV7 lentivirus and expanded for 14 days in complete X- VIVO 15 media with cytokine supplementation every Monday, Wednesday and Friday (50 U/mL IL-2 + 0.5 ng/mL IL-15).
Overview of the Inhibition of Proliferation Assay
PBMC or CD6-CAR Teff ‘target’ cells were stained with CellTrace CFSE, and CD6-CAR Treg ‘effector’ cells were stained with CellTrace Violet. The CD6-CAR Treg cells were seeded at either 2: 1 or 1 : 1 effector-to-target (E:T) cell ratios, in a 96-well round bottom plate. PBMC were activated with 0.02 pgAvell of plate-bound anti-human CD3 antibody, and CD6-CAR Teff cells were activated with 2.5 pL/well of CD3/CD28 T cell activator. The final volumes of co-cultures were 200 pL X-VIVO 15 media supplemented with 10% FBS per well. After incubation for 4 days at 37°C, cells were harvested and stained with Zombie Aqua™ Fixable Viability dye and fluorochrome-conjugated antibodies specific for the T cell markers CD3, CD4 and CD8. The cells were then fixed with the BD Stabilizing Fixative and stored overnight at 4°C prior to analysis on an Attune NxT Acoustic Focusing Cytometer. The numbers of viable Treg cells and proliferation of target cells, measured by the dilution of the CFSE signal, was determined using FloJo software (FlowJo, LLC). Percent inhibition of target cell proliferation was calculated based on cultures of target cells alone.
Brief Overview of Cytokine Secretion Analysis
Following incubation of the 4-day co-cultures as described above, 100pL of supernatant from each experimental condition was collected and immediately frozen at -80°C. All frozen samples were later analyzed by cytokine bead array in accordance with APCF-SD-019 (Calibration and Operation of the FlexMap 3D System), and the Human XL Cytokine Luminex Performance Panel kit instructions.
Determination of CD6-CAR Treg Cell-mediated Immunomodulatory Activity against Teff Cells
To evaluate the immunomodulatory potential of a qualification run CD6-CAR Treg cell product, it was co-cultured with autologous PBMC at a ratio of 2: 1 and L 1 in wells with plate-bound anti- CD3. After four days, the number of CD6-CAR Treg cells was diminished compared to what was seeded at Day 0, as expected, but their numbers were > 3-fold higher upon co-incubation with PBMC (E:T of 2: 1 or 1 : 1) than when cultured alone (E:T of 1 :0) (FIG 2A). It was also observed that the CD6-CAR Treg cells suppressed the proliferation of the PBMC, especially the CD8+ population (FIG 2B). Furthermore, the co-cultures at either E:T ratio exhibited decreased levels of the pro-inflammatory cytokine IFNy compared to that of PBMC targets cultured alone (E:T of 0:1), and upregulation of the suppressive cytokine IL-10 compared to cultures of either Treg cells alone or PBMC alone (FIG 2C).
Determination of CD6-CAR Treg Cell-mediated Immunomodulatory Activity against CD6-CAR Teff Cells Because CD6-CAR Treg cell products might contain a small contaminating CD6-CAR Teff cell population, the activity of a qualification run product when co-cultured with CD6-CAR Teff cells was evaluated. Using autologous CD6-CAR Teff cells that were 95% CAR+ as target cells, the CD6-CAR Treg cells strongly suppressed the proliferation of both the CD4+ and CD8+ cell populations (FIG 3A). Furthermore, the co-cultures at either E:T ratio exhibited decreased levels of the pro-inflammatory cytokine IFNy and upregulation of the suppressive cytokine IL- 10 compared to that of Teff targets cultured alone (E:T of 0: 1) (FIG 3B).
These data demonstrate that Treg cells transduced with CD6(EQ)CTLA4Z-T2A-CD19t_epHIV7 suppress of Teff cell proliferation and modulate cytokine secretion upon co-incubation with activated Teff cells. Furthermore, upon co-incubation with PBMC (2: 1 and 1:1), the CD6-CAR Treg numbers are higher than that seen with cultures of Tregs alone (1:0), indicating that activation of CD6-CAR Treg cells promotes their survival and/or prevents their depletion.
Importantly, the inhibition of proliferation and altered cytokine profiles seen with CD6-CAR Teff cells were similar to those observed with PBMC. This immune-inhibitory activity is thus suggestive of a low potential for any risks that might be associated with contaminating Teff cells in a CD6-C AR Treg product.
Example 4: In vivo anti-GVHD and immunomodulatory efficacy of IV administered CAR Tref cells
Immunodeficient NSG mice (8-12 weeks old males. n=4 mice per group) were irradiated at 250 cGy on Day -1. The following day (Day 0), 5xl06 healthy donor PBMCs (autologous to the Treg cells) were inoculated i.v. into each mouse. Immediately following PBMC transplantation, mice were injected with a single dose of 5x106 CD6-CAR Treg cells (50% CAR+; prepared essentially as described above), non-transduced Treg cells or vehicle (PBS) only.
Mice were monitored for graft vs. host disease (GVHD) by measuring body weight over time up to 51 days, and serum cytokine levels were measured on Day 13. Tissue samples for immunohistochemistry studies were also collected from designated euthanized mice on Day 13. Euthanasia was carried out in accordance with the recommendations of the AVMA Guidelines for the Euthanasia of Animals: 2020 Edition, where criteria for mouse survival included maintaining > 80% of initial body weight, and survival was monitored by Kaplan Meier curve analysis.
A single intravenous injection of CD6-CAR-Treg cells (5xl06 cells; 50% CAR+) exhibited robust immunomodulatory activity. As compared to NSG mice treated with vehicle only or nontransduced Treg cells, mice treated with CD6-CAR Treg cells better maintained their body weight (FIG 4A) and had a survival advantage (FIG 4B). Compared to vehicle treated mice, both Treg treated mice and CD6-CAR Treg treated mice had a reduction in serum proinflammatory cytokines (IL-2, IFN-gamma, TNF, GzmB, IL-6, CG-CSF and IL-10) (FIG 4C), as well as a reduction of tissue-infiltrating CD4+ and CD8+ Teff cells in the lungs (FIG 4D)
While these were not formal toxicity studies, the mice were monitored daily for any obvious signs of distress or general toxicity. Mice treated intravenously with CD6-CAR Treg cells were bright, alert and reactive throughout the experiment, and did not exhibit weight loss until they began to succumb to GVHD.
These studies demonstrate that in vivo anti-GVHD and immunomodulatory efficacy can be observed with Treg cells that had been transduced with the CD6(EQ)CTLA4Z-T2A- CD19t_epHIV7 vector. Indeed, intravenous administration of the CD6-CAR Treg cell product resulted in significant survival benefits in the GVHD model without inducing any apparent signs of distress or general toxicity in the mice. Improved outcomes were associated with reduced circulating proinflammatory cytokines and decreased lung tissue-infiltrating Teff cells.
These data suggest that intravenous administered CD6-CAR Treg cells exhibit in vivo immunomodulatory in a mouse model of GVHD. Together this work provides the rationale for intravenous delivery of autologous CD6-CAR Treg cell products, for the treatment of patients with GVHD
Example 5: Characterization of CD6 CAR Tregs Generated by GMP Compliant Flow Cytometry CD6CTLA4 CAR-Treg cells are isolated, transduced, and expanded by GMP-compliant flow cytometry-based procedures for clinical application and characterized. The results of this analysis are presented in FIG. 19A-L and FIG. 20A-G.
The GMP manufacturing of CD6CTLA4 CAR-Treg is a combination of the cell therapy field of Treg and established CAR T cell therapy procedures used in prior and ongoing clinical trials at City of Hope. Qualification studies were conducted to support the manufacturing process, evaluate critical parameters, and assess investigational cellular product using methodologies proposed for clinical use.
Briefly, CD6CTLA4 Tregs were generated from healthy donors through leukapheresis blood products from StemCell (Vancouver, Canada). Concentrated leukocytes were CD25 microbead- labeled for CliniMACS (Miltenyi Biotec, San Jose, USA) positive selection to generate CD25+ enriched cells that were cultured overnight without cytokines then sorted for CD4+. CD25+, and CD127lo/’ using the FX500 Cell Sorter (Sony, San Jose, USA) and aseptic technique in a cGMP- level clean room facility (FIG 19A). Purity assessment of all qualification runs resulted in >95% pure Tregs depicted by a representative diagram in FIG 19B. These post-sorting purity assessments on Tregs sorted by the FX500 Cell Sorter were within range of Treg products analyzed by the BD FACSAria II used by other cGMP Treg manufacturers (Putnam, A.L., et al.. Clinical grade manufacturing of human alloantigen-reactive regulatory T cells for use in transplantation. Am J Transplant, 2013. 13(11): p. 3010-20).
Sorted Tregs were cultured at IxlO6 viable cells/ml, activated overnight with CD3/CD28/CD2 Immunocult (StemCell, Vancouver, Canada) prior to transduction with vCD6(EQ)CTLA4Z- T2A-CD19t_epHIV7 GMP-grade lentivirus using an MOI of 1.0. Cytokines were added to the transduced CD6CTLA4 CAR-Treg culture at 5-6 hours post-transduction. Cultures were expanded with the addition of HABS and IL-2 supplemented Xvivol5 media every 2 days. Reactivation was completed on Day 9 and expanded for another 5 days for a total of 14 days of culture expansion. Culture morphology images of Days 1, 9 (prior to reactivation), 10, and 14 of expansion are shown in FIG 19C. Cell clusters are observed following the activation of cells on Days 1 and 10, which are days that follow the initial activation at post-isolation (Day 0) and reactivation (Day 9). Treg culture morphologies are different from those of T effector cells, in which Treg cell clustering occurs throughout the expansion, which is also observed at Days 9 and 14 (FIG 19C). Further data is currently being collected to assess if culture morphologies correlate with modulatory function of Tregs. Using this method of GMP-compliant standard operation procedures, the expansion of CD6CTLA4 CAR-Treg cells to numbers sufficient for clinical application is attainable. FIG 19D depicts the expansion of qualification productions that exceeded IxlO9 total Tregs and up to 8.8xl09. At the end of the 14-day expansion, CD6CTLA4 CAR-Treg cells were designated for cryopreserved or phenotyping and functional testing.
Treg cell culture expansions are highly dependent on high doses of IL-2 and dense cell concentrations. Cytokine-dependence assessments of CD6CTLA4 CAR-Treg cells were done upon reactivation (Day 9) of Treg cell cultures and upon thawing of cryopreserved cells. When cells were reactivated on Day 9, 10xl06 Treg cells were designated to be cultured without IL -2 supplementation while following the maintenance schedule of Days 10-14 of Treg culture expansion, and in parallel lOxlO6 Treg cells progressed with the expansion following the standard operation procedure placed for the clinical production of CD6clLA4 CAR-Treg cells. Treg cells that were designated to be cultured without IL -2 supplementation after reactivation have shown cell viability decreases by about 80% at the e 30% after 2 days of culture without IL- 2 after thaw and by more than 85% after 7 days, compared to CAR-Tregs that were thawed and maintained with IL-2, decreases in viability were 12% after 2 days and 64% after 7 days (FIG. 20A), cytokine-dependent). Additionally, maintaining cell cultures at the activation time-points (Days 0 and 9) at 1x106 viable cells/ml and adapting to the exponential growth maintenance of CAR-Treg cultures by the addition of specific volumes of HABS and IL-2 supplemented Xvivol5 media to sustain cell concentrations as close to IxlO6 was of utmost importance for expansion. In contrast to culture expansions of T effector cells that can be cultured at a lower density of 0.5xl06 viable cells/ml. Decrease in cell concentrations accompanies the decrease in cell viability in both culture conditions with or without IL-2. Although CAR-Tregs cultured with IL-2 were able to maintain cell concentrations at 0.6x106 until Day 7, which is higher than the culture without IL-2 at 0. IxlO6, decreases in viability was observe which further shows the importance of maintaining a dense culture concentration of IxlO6 cells/ml to achieve highly viable CAR-Tregs. CD6-CAR-specific detection by PCR is used as a release criterion for CD6-CAR Treg cells. Treg cells transduced with the lentiviral vector vCD6(EQ)CTLA4Z-T2A-CD19t_epHIV7 was distinguished from cells transduced with other lentiviral vectors containing CD19t by a sequence of two PCR assays (FIG.19G). First, the unique binding domain sequence within the CD6(EQ)CTLA4^ CAR is targeted by specific primers. The second PCR assay targets the region between CD3^ and CD19t, the CAR signaling domain and transduction efficiency surface marker respectively. CD6-C AR Treg samples must be PCR positive for both the CD6scFv-EQ portion of the CD6(EQ)CTLA4^ CAR sequence (PCR1) and CD19t (PCR2) to meet accepted parameters. In FIG 19G, primers detecting the CD6-CAR transgene (954bp, Lanes 1-3) and CD19t (578bp, Lanes 4-6) were used to analyze genomic DNA isolated from CD6-CAR Treg samples from QR2 (Lanes 1 and 4), alongside FACS-confirmed CD6-CAR+ cells (positive control, Lanes 2 and 5) and non-transduced cells (negative control, Lanes 3 and 6). PCR products are compared to lOObp ladder (Lane L) with indicated lOOObp and 600bp. Single PCR products appearing in the lanes assigned for QR2 CAR-Treg cells and at the predicted size marker, signifies that CD6(EQ)CTLA4^ and CD19t are indeed present in the qualification run CD6-CAR Treg cells and meet this criterion for release.
To test the specificity of the CD6(EQ)CTLA4ytargeting primers, gDNA samples from CD6- CAR and various CAR T cell products were analyzed by PCR using primers specific for the CD6-CAR transgene, CD6-CAR forward and CD4tm-CTLA4 reverse primer. Primers were show n to be specific for the CD6scFv-EQ portion of the CD6-CAR transgene with only a single band appearing in Lane 11 with the expected size of 954bp. Additionally, isolated DNA from CD6-CAR Treg cells were PCR-tested using primers specific for CAR-T cell products mentioned on the Supplementary Table. It is shown that CD6-CAR Treg cells can only be detected using specifically the CD6(EQ)CTLA4^-targeting primers.
Stability of CD6-CAR Treg cell vi abi li tv and phenotype is maintained after preparation for clinical application using fresh culture product.
Tregs demonstrate effectiveness in preventing and treating Graft Versus Host Disease (GVHD) (Di lanni, M., et al., Tregs prevent GVHD and promote immune reconstitution in HLA- haploidentical transplantation. Blood, 2011. 117(14): p. 3921-8). Additionally, multiple studies have shown the potential of this type of treatment in autoimmune diseases such as Type 1 diabetes (Marek-Trzonkowska, N.. et al., Administration of CD4+CD25highCD127- regulatory T cells preserves beta-cell function in type 1 diabetes in children. Diabetes Care, 2012. 35(9): p. 1817-20). CAR-Treg cell products intended for clinical use undergo rigorous microbial safety and criteria testing that may take several hours after cell products have been dosed and prepared for infusion. Therefore, stability tests were done on two qualification runs at the end of the 14- day culture period to assess the resilience of expanded CD6-CAR Tregs over time after being dose-prepared following a similar methodology proposed for clinical use. Phenotypic assessments via How cytometry of qualification run CAR-Tregs at the end of expansion included the evaluation of percent CD4-positive cells, 97.6% (93.4-100), and FOXP3-positve cells, 98.4% (95.7-99.9), data is reported as average and (min-max). Viability was evaluated at 98.43% (97.7- 99.1) with 67.2% (44.4-80.5) of Tregs expressing CAR. Expanded CAR-Tregs were placed into an infusion bag or syringe at a concentration of 10-50xl06 viable cells/ml in 2% purified human saline. Each vessel contained 10ml (bag) or 1ml (syringe) of cell suspension and was placed on a rocking apparatus at room temperature for a duration of specific time-points: 0, 4, and 6 hours. Vessels were rotated every 30 minutes to prevent possible settling of cells. Assessment samples at each time-point were acquired from the vessels via adapters. Samples were tested for viability', identity, and purity' markers summarized in FIG 19E. CD6-CAR Treg viability’, identity, and purity markers remained stable for a duration of up to 6 hours at room temperature.
CD6-CAR Treg cells exhibit suppressive function tow ards autologous and allogenic targets. Functional assessment of CD6-CAR Treg cells was done at the end of the 14-day culture expansion using a mix lymphocyte incubation and flow cytometry-based analysis. Briefly, CAR-Treg cells were incubated with autologous or allogenic PBMC, labeled as targets, in a series dilution of 1:64 to 2: 1 Treg to target cell ratio with activation by plate-bound anti-human CD3 antibody for 4 days at 37°C. Using CellTrace technology and FACS analysis, target cell proliferation was calculated for each ratio condition and normalized to maximum proliferation (anti -human CD3 activated target cells alone) to calculate percent suppression of Tregs depicted on FIG 19F. The figure illustrates the ability of CD6-CAR Treg to suppress both activated autologous and allogenic target cells at indicated effector to target cell ratios (E:T). CD6-CAR Tregs were able to suppress autologous target cell proliferation at the 2:1 E:T up to 56% (CD4) and 31% (CD8), and up to 65% (CD4) and 47% (CD8) in allogenic target cells. CD6-CAR Treg prepared for clinical application meets release criteria.
Cryopreservation provides multiple benefits to the clinical use of CAR T cell therapy, including long-term storage until needed, flexibility in administration of therapy doses and timing of infusion, as well as giving ample time for quality release testing. Limited studies have shown the effects of cry opreservation on the modulatory effects of Tregs (Golab, K._ et al., Challenges in cryopreservation of regulatory T cells (Tregs) for clinical therapeutic applications. Int Immunopharmacol, 2013. 16(3): p. 371 -5; Hippen, K.L., et al.. Massive ex vivo expansion of human natural regulatory' T cells (T(regs)) with minimal loss of in vivo functional activity7. Sci Transl Med, 2011. 3(83): p. 83ra41). To address this practical perspective on long-term storage of expanded CAR-Tregs intended for clinical use, initial assessments were accomplished using Qualification Run 1 cell products that were released from the GMP production facility and cryopreserved at the research laboratory with 10% DMSO at a cell concentration of 50xl06 viable cells/ml, and a control-rate freezing method. In the proceeding qualification productions, cryopreservation was done using methods proposed for clinical application, using 5% DMSO, a cell concentration of 10xl06 viable cells/ml. and a control-rate freezer. Accepted release criteria of cryopreserved CAR-Treg were established using a combination of current and past clinical trials and our experiences with pre-clinical CD6-CAR Treg productions (Bluestone, J.A., et al., Type 1 diabetes immunotherapy using polyclonal regulatory T cells. Sci Transl Med, 2015. 7(315): p. 315ral89). To meet release criteria, CAR-Treg must be within the accepted parameters of >70% viability7, >90% CD4, >60% FOXP3, and >20% CAR expression, as well as maintain CD8 contamination <5%. These parameters were analyzed in cryopreserved CD6-CAR Tregs from these qualification runs. The results indicate that the cryopreserved CD6-CAR Treg cells, using a lab method or a clinical protocol, are within the accepted release criteria of viability, CD4 purity, FOXP3, and CAR expression, and below the parameter set for CD8 contamination.
CD6-CAR Treg cells maintain vector copy number below accepted safety parameters.
High numbers of vector copies/insertions increase the risk for neoplastic transformation due to insertional mutagenesis and/or lentiviral vector integration that alters expression of cell regulatory genes, therefore it is a safety concern when using gene-modified cell products. Samples from each qualification run were collected at the end of expansion and tested for Woodchuck hepatitis virus-derived regulatory element (WPRE), an enhancer for transgene expression, within the CD6(EQ)CTLA4Z-T2A-CD19t_epHIV7 vector (nucleotides 5189-5789) by qPCR which will indicate vector copy number per cell and be used in calculating copy number per CAR+ cell. Based on previous experiences with CAR-T cell products, the accepted criterion for vector copies is <5.0 copies of WPRE per CAR positive cell (Wang, X., et al., Phenotypic and functional attributes of lentivirus-modified CD19-specific human CD8+ central memory T cells manufactured at clinical scale. J Immunother. 2012. 35(9): p. 689-701). [7], It is indicated in that CAR-Treg cell products from these qualification runs are within the accepted parameter of <5.0 copies/CAR positive cell.
Vesicular stomatitis virus glycoprotein (VSV-G) is encoded within the pCMV-G plasmid of lentiviral vector expression system and is a surrogate marker for Replication Competent Lentivirus (RCL). VSV-G is essential for mediating viral entry into cells. Samples from each qualification run were collected at the end of expansion and tested for VSV-G DNA with qPCR. Accepted parameters for VSV-G plasmid detection is < 2.5 copies/50 ng gDNA in which qualification run CAR-Treg cell products are within. Data obtained from the qualification run products indicate generating gene-modified CAR Treg cell products with an average vector copy7 number less than 5 per CAR+ cell, after transduction with CD6(EQ)CTLA4Z-T2A- CD19t_epHIV7 using an MOI of 1.0, is attainable and can be generated without evident RCL.
CD6-CAR Treg modulatory markers are retained pre- and post-crv opreservation.
Transcription factor, FOXP3, is a widely known marker that designates Tregs from other T cell populations. Commonly studied by flow cytometry-based assays, FOXP3 expression is linked to suppressive capabilities of Tregs. In addition to FOXP3 expression, epigenetic modifications to FOXP3, assessed by measurement of unmethylated FOXP3, may also serve as a surrogate measurement of Treg stability and function. Qualification run samples were collected after 14- day expansion, fresh CD6-CAR-Treg (IxlO6 cells) stored as cell pellets at -80°C until assessment. Corresponding cryopreserved CAR-Tregs were thawed in Xvivol5 + 10% HABS, washed to remove DMSO, then allocated for assessment. Genomic DNA was isolated from cell pellets (Fresh) and thawed cells (Frozen) and 200-500 ng of DNA was underwent bisulfite treatment in preparation for analysis using droplet digital methylation-specific PCR (ddPCR) previously described (Husseiny, M L, et al., Development of Quantitative Methylation-Specific Droplet Digital PCR (ddMSP) for Assessment of Natural Tregs. Front Genet, 2020. 11: p. 300). Relative expression of unmethylated FOXP3 was calculated by dividing the total copies of positive unmethylated FOXP3 by the total copies of TSDR region and summarized in FIG. 19H. Fresh and frozen CD6-CAR Treg displayed high percentages of unmethylated FOXP3, fresh 98.1% (94.4-100) and frozen 94.7% (90.3-100). which can be correlated with the expression of FOXP3 analyzed previously by FACS. The permanence of unmethylated FOXP3 TSDR pre- and post-cryopreservation indicates that modulatory markers are not altered by the preservation process. Furthermore, the stability of this parameter in combination with Treg identity markers previously analyzed by FACS in both fresh and frozen samples suggests the presence of the CD6-CAR does not deviate CD6-CAR Treg cells from having a modulatory' phenotype. Helios, a transcription factor associated with in vivo function and survival of Tregs, was measured by intra-nuclear staining and FACS (Lam, A. J., et al., Helios is a marker, not a driver, of human Treg stability’. Eur J Immunol, 2022. 52(1): p. 75-84). Fresh and frozen samples were assessed. An increase in Helios expression was observed in the CD6-CAR Treg samples after cryopreservation, an average of 40.4% expression in fresh samples to 74.6% in frozen samples. Previous studies associate Helios with unmethylated FOXP3 TSDR and regulatory function of Tregs (Thornton, A.M., et al., Helios(+) and Helios(-) Treg subpopulations are phenotypically and functionally distinct and express dissimilar TCR repertoires. Eur J Immunol, 2019. 49(3): p. 398-412) [10], Due to the stability of unmethylated FOXP3 TSDR between fresh and frozen samples collected in this analysis, correlations between the two parameters were not observed. However, it can be speculated that CD6-CAR Tregs that maintained stability after cry opreservation retained a high percentage of unmethylated FOXP3 and Helios expression.
CD6-CAR specific stimulation activates CD3 signaling pathway.
Understanding the molecular mechanisms of intracellular signaling pathways that lead to CAR T cell activation could lead to new strategies for designing CAR T cells with higher clinical efficacy and lower toxicity. We analyzed phosphorylation events and calculated the fold change of the known phosphorylated proteins within each group by comparing the stimulated CD6-CAR Tregs with the non- stimulated cells in both fresh and frozen samples. CD6-CAR positive cells were purified from total cultured Tregs and rested overnight in Xvivol5 media supplemented with 10% HABS and IL-2. Fresh samples w ere immediately acquired from cultures at the end of expansion and frozen samples were cells that were released from cGMP and cryopreserved in the lab using 10% DMSO. Purified CD6-CAR Tregs was plated in wells coated with CD6 Fc for Ihr at 37°C prior to cell lysis. Fifteen micrograms of protein were loaded onto pre-cast 4-12% Bis-Tris gels (NuPAGE, Invitrogen) and transferred onto a nitrocellulose membrane. Antibodies for CD3 pY142 (Abeam), SLP76p(Ser376), pERKl/2(Thr202/Tyr204), pAKT (Ser 473), and pPI3k (Tyr467/199) were used for immunoblotting and nonnalized to P Tubulin. Bands were detected using a chemiluminescence substrate. Stimulation increased the expression of pCD3^Y142 in fresh CD6-CAR Treg as well as in the frozen group of cells. We also detected a low level of basal expression of pCD3^Y142 in non-stimulated cells. Constitutive phosphorylation of the CAR CD3^ domain or tonic signaling has been shown to occur with some CARs. Stimulation of the CAR is known for Lck-activation of ZAP70 which phosphory lates number of proteins including SLP76 required for signal propagation. We observed increased phosphorylation of pSLP76(S376) in the stimulated CAR T cells. The SLP76 complex further activate critical signaling pathways downstream of the TCR, including MAPK/Erkl/2. As expected, we observed activation of the ERK/MAPK pathway w hich is known to play a role in CAR T cell signaling. Our results supported phosphorylation at the sites of ERKl/2p (Thr202/Tyr204) in response to CAR activation. Results also provided evidence that PI3k/AKT signaling pathway was activated upon CAR activation. pAKT (Ser473) and PI3Kp (Tyr467/199) was elevated in the fresh and frozen cells after stimulation with CD6Fc. With a better understanding of the signal transduction in CAR Tregs we can tailor CAR design to increase efficacy and reduce off target effects and toxicity.
Cell mediated immunomodulatory activity7 is observed in fresh and frozen CD6-C AR Treg. To evaluate the immunomodulatory potential of a qualification run CD6-CAR Treg fresh and frozen cell product, CAR-Tregs were co-cultured with autologous PBMC cells, labeled as targets, at a ratio of 2: 1 and 1 : 1 Treg:PBMC ratio (E:T) in wells with plate-bound anti-human CD3. It was at these two E:T ratios in which maximum function was observed, either by cellular analysis by FACS or detection of cytokine production by multi-plex Luminex. Modulatory' activity', defined by the suppression of target cell proliferation and cellular reduction, is summarized in FIG 19L. Immune-modulation was observed by in fresh and frozen CAR-Treg when co-incubated with target cells. Highest suppression is observed in CD4 target cells with the fresh sample, 56.5% at the 2:1, and 46.9% at the 1 : 1 E:T ratio. The ability for CAR-Tregs to suppress CD4 target cells was decreased in the frozen cell product, 19.5% at the 2: 1 and 12.0% at the 1: 1 E:T. However CD8 target cell suppression was maintained between the fresh (30.6% at the 2: 1 and 22.2% at the 1 : 1 E:T) and frozen (38.9% at the 2: 1 and 20.2% at the 1 : 1 E:T). Target cell depletion of CD4 and CD8 target cells was detected in the fresh and frozen CAR-Tregs, in which an increased reduction of CD8 targets, 71.2% and 68.5% reduction at 2: 1 and 1 : 1 E:T with fresh and 72.4% and 53.7% with the frozen CAR-Treg, was observed compared to 66.8% and 65.1% CD4 target reduction at the 2: 1 and 1 : 1 E:T with fresh and 50.4% and 45.5% with the frozen. Cytokine analysis of supernatants collected at the end of the functional assay indicates the reduction of pro-inflammatory cytokine, IFNy, from activated target cells alone compared to when co-cultured with both fresh and frozen CAR-Treg. Although slightly lower in the frozen cells, IL-10 production was detected when CAR-Tregs were activated (1 :0 E:T) and increased when CAR-Tregs were exposed to target cells (2: 1 and 1 : 1).
CD6-CAR cell products may contain a small contaminating CD6-CAR Teff cell population. Non-Treg contamination is rarely detected during expansion, therefore it would be difficult to assess how CAR-Tregs maintain phenotypic stability and modulatory function in the presence of non-Treg culture contaminants. Autologous T effector cells were isolated, transduced, expanded, and cryopreserved in parallel to Treg cultures. Fresh and frozen CD6-CAR Treg cells were assessed for their modulatory' activity' on non-Treg contaminants that were emulated by these CD6-CAR Teff. In a 2: 1 and 1 : 1 ratio of CD6-CAR Treg to CAR Teff (E:T). representing 30% and 50% non-Treg contamination, CAR Treg cells were able to immunomodulate contaminating CAR Teff cells by means of suppression of proliferation and reduction of proinflammatory cytokine, IFNy (FIG 20C). Cytokine analysis also showed the production of IL- 10 in the modulatory' activity' of fresh and frozen cells.
Survival assessment of CD6-CAR Treg cells were done at the end of the 4-day functional analysis. After four days, the number of fresh and frozen CD6-CAR Treg cells were reduced compared to what was seeded at Day 0, as expected. CAR-Treg numbers were > 3-fold higher upon co-incubation with PBMC (E:T of 2: 1 or 1 : 1) than when cultured alone (E:T of 1:0), further indicating the dependence of Treg on a source of IL-2 for survival. Frozen CAR-Treg exhibited slightly lower survival compared to the fresh. This decrease in survival may be attributed to the effects of cryopreservation. Few studies have assessed how cryopreservation affects CAR-Treg phenotype and function. Data collected in this analysis have shown the stability of CD6-CAR Treg viability, phenotype and safety after cryopreservation. however decreases in CAR-Treg function was observed. DMSO concentrations impact Treg characteristics, and past studies demonstrate lower concentration of 5% DMSO can significantly improve Treg function after cryopreservation as opposed to commonly used protocols with 10-20% (Kaiser, D., et al.. Freezing Medium Containing 5% DMSO Enhances the Cell Viability and Recovery Rate After Cryopreservation of Regulatory T Cell Products ex vivo and in vivo. Front Cell Dev Biol, 2021. 9: p. 750286). Cryopreservation was done in two of these qualification runs, using methods proposed for clinical use, which includes the use of CS5, a cryopreservation solution that composed of 5% DMSO, and monitoring of the freezing process using a control rate freezer, one of which functional data is summarized in FIG 19 J.
CD6-CAR Treg are depleted by targeting CD19t,
Antibody -dependent cellular cytotoxicity (ADCC) is commonly used to eliminate target cells in a micro environment by means of immune cytotoxicity. This method can be instrumental to deplete CD6-CAR Treg cells in the case of a post-infusion adverse event. By targeting CD19t with Tafasitamab, a CD19-directed cytolytic antibody that harnesses cellular phagocytosis and cytotoxicity of macrophages and NK cells, it was observ ed that CD6-CAR Treg cells that are marked by CD19t can be depleted (FIG ZOE). Flow cytometry assays confirmed that Tafasitamab binds to the same epitope as the CD 19 antibody used to routinely detect CD19t in CAR proteins. Due to its enhanced affinity' for CD19, the detection of bound Tafasitamab was visualized by FACS even after 4 days of incubation.
Cryopreserved CD6-CAR Treg cells were co-incubated with immediately thawed PBMC in wells containing plate-bound anti-human CD3 at a 1 : 1 and 4: 1 E:T ratio, where Tregs were the target cells and PBMC served as effectors, with or without Tafasitamab. After a 4-day incubation, Treg counts were analyzed by CellTrace technology and FACS. Cell counts were used to calculated percent of depleted Tregs at specified E:T ratios when incubated with Tafasitamab compared to incubations without. There was no reduction in Treg with the CD 19- targeting antibody alone, however when Tregs were co-incubated with PBMC, there was a Treg count reduction with Tafasitamab at the 1 : 1 E:T ratio and a further reduction at the 4: 1 compared to co-incubations without. This analysis indicates the use of Tafasitamab as an “off-s itch” in case CD6-CAR Treg therapy must be terminated.
OTHER EMBODIMENTS
[066] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims

Claims

WHAT IS CLAIMED IS:
1. A method for generating a composition comprising regulatory T cells (Tregs), comprising:
(a) providing a population of peripheral blood mononuclear cells (PBMC);
(b) treating the population of PBMC to select for cells expressing CD25 (CD25+ cells);
(c) treating the population of cells selected for cells expressing CD25 to select for cells that express CD4 (CD4+ cells), express CD25 and have low or no expression of CD 127 (CD1271ow/- cells) thereby producing a cell population selected for cells that are CD25hi, CD4+ and CD1271ow/- (CD25hi, CD4+, CD1271ow/- cells);
(d) activating the CD25hi, CD4+, CD1271ow/- cells by exposing them to antibodies targeted to CD3, antibodies targeted to CD28 and antibodies targeted to CD2 to create an activated cell population;
(e) introducing a vector into the activated cell population to create a population of transduced Treg;
(f) culturing the transduced Treg in a culture medium that comprises exogenously added IL-2 for at least one day, wherein the cells are not treated to select for or against cells expressing CD45RO and the cells are not treated to select for or against cells expressing CD45RA.
2. The method of claim 1, wherein the cells are not cultured in the presence of exogenously added IL-2 prior to step (e).
3. The method of claim 1, wherein the cells are not cultured in the presence of exogenously added IL-2 until after step (f).
4. The method of claim 1, wherein there are no cell selection or enrichment steps other than selection for cells expressing CD25 and a selection for cells that are CD25+, CD4+ and CD1271ow/-.
5. The method of claim 4, wherein the cells are cultured without exogenously added IL-2 between steps (d) and (e) and after step (e).
6. The method of claim 5. wherein the cells are cultured in the presence of exogenously added IL-2 for the first time at least 6 hours after step (e) and for the remaining of the expansion.
7 The method of claims 4-6 wherein exogenously added IL-2 is present at 250-500 U/ml of culture medium.
8. The method of claim 1 , wherein the population of transduced Treg expressing is cultured for at least 10 days to create an expanded population of transduced Treg.
9. The method of claim 8. wherein the expanded population of transduced Treg is cryopreserved.
10. The method of claim 1, wherein the vector encodes a chimeric antigen receptor.
11. The method of claim 1. wherein the vector is a viral vector introduced at an MOI between 0.8 and 1.3, preferably 1.0
12. The method of claim 1, wherein the cell population enriched for cells that are CD25+, CD4+ and CD1271ow/- are cultured at 1 x 106 cells/ml.
13. The method of claim 1, wherein all culturing steps prior to transduction take place in the absence of exogenously added IL -2.
14. The method of claim 1 wherein the step of treating the population of cells enriched for CD25+ cells to enrich for cells expressing CD4 (CD4+ cells) and have low or no expression of CD127 (CD1271ow/- cells) comprises fluorescence activated cell sorting (FACS).
15. The method of claim 9. wherein the step of treating the population of cells enriched for CD25+ cells to enrich for cells expressing CD4 (CD4+ cells), having low or no expression of CD127 (CD1271ow/- cells), and expressing CD25 comprises FACS and the cells are not exposed to FACS for more than 5 hours.
16. The method of claim 9. wherein the step of treating the population of cells enriched for CD25+ cells to enrich for cells expressing CD4 (CD4+ cells), having low or no expression of CD127 (CD1271ow/- cells) and expressing CD25 produces a population of cells comprising cells expressing a high level of CD25 expression (CD25high).
17. The method of claim 13, wherein the cry opreservation medium contains 5% DMSO.
18. The method of any of the forgoing claims, wherein (b) treating the population of PBMC to select for cells expressing CD25 (CD25+ cells) comprises selecting cells expressing CD25 using an anti-CD25 antibody attached to a solid support.
19. The method of any of the forgoing claims, wherein (c) treating the population of cells selected for cells expressing CD25 to select for cells expressing CD4 (CD4+ cells) and have low or no expression of CD127 (CD1271ow/- cells) thereby producing a cell population selected for cells that are CD25+. CD4+ and CD1271ow/- (CD25+, CD4+,CD1271ow/- cells) comprises cell sorting using an anti-CD25 antibody, and anti-CD127 antibody and an anti-CD4 antibody.
20. The method of claim 19, wherein the anti-CD25 antibody of step (a) binds to a different epitope than the anti-CD25 antibody of step (b).
21. The method of claim 1, wherein the viral vector encodes a chimeric antigen receptor (CAR) comprising a single chain variable fragment (scFv) targeted to CD6, a spacer region, a transmembrane domain, a CTLA4 signaling domain, and a CD3 zeta signaling domain.
22. The method of claim 21, wherein the transmembrane domain comprises a CD4 transmembrane domain or a variant thereof, a CD8 transmembrane domain or a variant thereof, a CD28 transmembrane domain or a variant thereof, or a CD3^ transmembrane domain or a variant thereof.
23. The method of claim 21, wherein the CAR comprises or consists of an amino acid sequence at least 90, 95, 96, 97, 98, or 99% identical to any of SEQ ID Nos: 145, 147, 149, 151, 154, 155, 156, 157 and 159.
24. The method of claim 21, wherein the CAR comprises or consists of an amino acid sequence of any of SEQ ID Nos: 145, 147, 149, 151, 154, 155, 156, 157 and 159 with no more than 5 single amino acid substitutions.
25. The method of claim 21, wherein the spacer region is selected from SEQ ID NOs: 116- 126 and the transmembrane domain is selected from SEQ ID NOs: 128-136.
26. The method of claim 21, wherein the scFv is selected from SEQ ID NOs: 101, 104 and 107.
27. The method of claim 21, wherein the scFv has a VH comprising SEQ ID NO: 102 and an VL comprising SEQ ID NO: 103.
28. The method of claim 21, wherein the scFv has a VH comprising SEQ ID NO: 105 and an VL comprising SEQ ID NO: 106.
29. The method of claim 21, wherein the scFv has a VH comprising SEQ ID NO: 108 and an VL comprising SEQ ID NO: 109.
30. The method of any of claims 27-29, wherein a linker comprising SEQ ID NO: 115 is located between the V L and VH.
31. The method of claim any of the forgoing claims, wherein step (g) takes place 7-10 days after step (e).
32. The method of claim 1. wherein after step (e), the cells are cultured at 750,000 - 1.250,000 cells/ml.
33. The method of claim 1, wherein steps (b) and (c) comprise fluorescent activated cell sorting using anti-CD127 antibodies. anti-CD25 antibodies, anti-CD4 antibodies.
34. A method of treating graft versus host disease comprising administering to a patient in need thereof, a therapeutically effective amount of Treg produced by the method of any of the forgoing claims.
35. A method of treating diabetes comprising administering to a patient in need thereof, a therapeutically effective amount of Treg produced by the method of any of the forgoing claims.
36. The method of claim 35, wherein the diabetes is Type 1 diabetes.
37. The method of claim 35, wherein the diabetes is Type 2 diabetes.
38. The method of claim 1, wherein the transduced Treg population have at least 60% or 70% viability, at least 70%, 80% or 90% express CD4, at least 50% or 60% express (preferably unmethylated) FOXP3. and less than 10% or 5% express CD8.
39. The method of claim 39, wherein the transduced Treg population cells are transduced with a viral vector encoding a selected polypeptide.
40. The method of claim 39 or 40, wherein the transduced Treg population have at least 70% viability, at least 90% express CD4, at least 60% express (preferably unmethylated) FOXP3, and less than 5% express CD8.
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