US20170298322A1 - Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases - Google Patents

Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases Download PDF

Info

Publication number
US20170298322A1
US20170298322A1 US15/094,467 US201615094467A US2017298322A1 US 20170298322 A1 US20170298322 A1 US 20170298322A1 US 201615094467 A US201615094467 A US 201615094467A US 2017298322 A1 US2017298322 A1 US 2017298322A1
Authority
US
United States
Prior art keywords
activator
cells
tregs
stimulator
subpopulation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US15/094,467
Inventor
Tinghua Cao
Li Li
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mallinckrodt Hospital Products IP Unlimited Co
Original Assignee
Mallinckrodt Hospital Products IP Unlimited Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mallinckrodt Hospital Products IP Unlimited Co filed Critical Mallinckrodt Hospital Products IP Unlimited Co
Priority to US15/094,467 priority Critical patent/US20170298322A1/en
Assigned to DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT reassignment DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT NOTICE OF GRANT OF SECURITY INTEREST IN PATENTS Assignors: Mallinckrodt Hospital Products IP Limited
Publication of US20170298322A1 publication Critical patent/US20170298322A1/en
Priority to US15/981,332 priority patent/US11186823B2/en
Priority to US17/515,985 priority patent/US20220056410A1/en
Assigned to MALLINCKRODT US POOL LLC, CNS THERAPEUTICS, INC., LAFAYETTE PHARMACEUTICALS LLC, MALLINCKRODT PHARMA IP TRADING UNLIMITED COMPANY (F/K/A MALLINCKRODT PHARMA IP TRADING D.A.C.), MALLINCKRODT BRAND PHARMACEUTICALS LLC (F/K/A MALLINCKRODT BRAND PHARMACEUTICALS, INC.), OCERA THERAPEUTICS LLC (F/K/A OCERA THERAPEUTICS, INC.), MALLINCKRODT CARRIBEAN, INC., MALLINCKRODT US HOLDINGS LLC, ST SHARED SERVICES LLC, IMC EXPLORATION COMPANY, SpecGx LLC, MALLINCKRODT VETERINARY, INC., MALLINCKRODT US HOLDINGS LLC (F/K/A MALLINCKRODT US HOLDINGS INC.), MALLINCKRODT CB LLC, MALLINCKRODT ENTERPRISES HOLDINGS LLC (F/K/A MALLINCKRODT ENTERPRISES HOLDINGS, INC.), MALLINCKRODT FINANCE GMBH, MALLINCKRODT PHARMACEUTICALS IRELAND LIMITED, THERAKOS, INC., MALLINCKRODT INTERNATIONAL FINANCE S.A., LUDLOW LLC (F/K/A LUDLOW CORPORATION), IKARIA THERAPEUTICS LLC, MALLINCKRODT ARD IP UNLIMITED COMPANY (F/K/A MALLINCKRODT ARD IP LIMITED), STRATATECH CORPORATION, SUCAMPO PHARMA AMERICAS LLC, MALLINCKRODT LLC, LIEBEL-FLARSHEIM COMPANY LLC, MALLINCKRODT ENTERPRISES LLC, VTESSE LLC (F/K/A VTESSE INC.), INO THERAPEUTICS LLC, MALLINCKRODT HOSPITAL PRODUCTS IP UNLIMITED COMPANY (F/K/A MALLINCKRODT HOSPITAL PRODUCTS IP LIMITED), MEH, INC., MNK 2011 LLC (F/K/A MALLINCKRODT INC.), INFACARE PHARMACEUTICAL CORPORATION reassignment MALLINCKRODT US POOL LLC RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025 Assignors: DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • 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]
    • 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

Definitions

  • the invention relates to methods for enhancing the suppressive function of regulatory T cells.
  • the invention provides a method for ex-vivo expansion of regulatory T cells, which cells demonstrate enhanced suppressive characteristics.
  • Tregs are known to be critical in maintaining a tolerance to self-antigens by suppressing the activation of the immune system.
  • inflammatory/autoimmune diseases such as systemic lupus erythematous, multiple sclerosis, rheumatoid arthritis, asthma, ulcerative colitis, and Crohn's Disease
  • Foxp3 expressing CD4+CD25+Tregs there is a functional defect or frequency decrease in the transcription factor Foxp3 expressing CD4+CD25+Tregs.
  • the person's immune system fails to recognize cells, or parts of cells as the person's own resulting in tissue destruction.
  • Tregs have been shown in the treatment of a variety of animal disease models including, without limitation, rheumatoid arthritis, asthma, and graft-versus-host disease (“GVHD”).
  • GVHD graft-versus-host disease
  • Use of Tregs in treatment applications is problematic because they are present as only a very small percentage, approximately 1 to 2%, of human peripheral blood mononuclear cells.
  • methods of activating and expanding, or proliferating, Tregs ex-vivo have been developed for use in the treatment of certain diseases.
  • FIG. 1 is a graph showing the frequency of CD4+Foxp3+ Tregs from the population of Example 1.
  • FIG. 2 is a graph showing the purity CD4+ Tregs of Example 2.
  • FIGS. 3A-3F are a graphs showing the Treg expansion of Example 3.
  • FIG. 4 is a graph showing the percentage of ex-vivo expanded cells of Example 3 that expressed Foxp3.
  • FIGS. 5A-5L are a graphs showing the inhibitory activity of the expanded Tregs of Example 4.
  • the invention provides methods for the ex-vivo expansion of CD4+CD25+ Tregs, compositions resulting from the method and methods for use of the compositions.
  • the cells resulting from use of the method demonstrate enhanced suppressive activities compared to fresh Treg cells.
  • the invention provides a method for producing ex vivo expanded Tregs that may be used to inhibit unwanted human immune responses against self-antigens or allergens. Additionally, the ex vivo expanded Tregs may provide treatment for inflammatory/automimmune diseases.
  • the invention provides a methods comprising, consisting essentially of, and consisting of: a) obtaining a population of T cells from an individual; b) isolating and purifying from the T cell population a subpopulation of CD4+CD25+ Tregs; and c) expanding the Treg cells of the subpopulation, wherein the expanded Treg cells exhibit enhanced suppressive activity compared to a population of freshly purified, unexpanded Tregs from the individual.
  • Tregs taken from a person with autoimmune/inflammatory diseases including, without limitation Systemic Lupus Erythematous (“SLE”), Multiple Sclerosis (“MS”), asthma, Rheumatoid Arthritis (“RA”), Crohn's Disease (“CD”), and Ulcerative Colitis (“UC)
  • SLE Systemic Lupus Erythematous
  • MS Multiple Sclerosis
  • RA Rheumatoid Arthritis
  • CD Crohn's Disease
  • UC Ulcerative Colitis
  • the Tregs can be expanded 100 to 1,000 fold ex-vivo while having enhanced suppressive function.
  • a population of T cells is obtained.
  • the T cells may be obtained from any suitable source including, without limitation, an individual's peripheral blood, thymus, lymph nodes, spleen or bone marrow.
  • the T cells are obtained from the peripheral blood of a person.
  • the T cells are obtained from an individual with an autoimmune/inflammatory diseases and most preferably from an individual with one or more of SLE, MS, asthma, RA, CD and UC whether the disease is in remission or during a period in which the disease is active.
  • human CD4+CD25+ T regs are purified from whole blood units or leukpaks.
  • a subpopulation of CD4+CD25+ Tregs is then isolated from the T cell population using any convenient separation techniques based on Treg specific cell markers, including, without limitation, flow cytometry by any convenient method.
  • any number of kits for carrying out such isolation and purification are commercially available.
  • the commercially available kits include, without limitation, Miltenyi Treg kit with Auotmacs, ClinMACS, and the like.
  • the isolation and purification is carried out until a population that is greater than 40% positive for Foxp3 and greater than 90% positive for CD4 is obtained.
  • the Treg subpopulation is then expanded ex-vivo using an autoantigen specific regulatory T cell stimulatory composition.
  • the composition antigen-specifically binds and activates the T cell receptor complex.
  • the expansion is carried out in the presence of effective amounts of a first and a second activator and a co-stimulator activator.
  • effective amount is meant an amount effective to stimulate the regulatory T cells to the degree desired.
  • the first activator is a TCR/CD3 activator that may be a multivalent antibody or ligand for TCR/CD3 including, without limitation, antigen non-specific activators such as an anti-CD antibody, and antigen-specific activators, such as an MHC-peptide multimer in which the peptide is an autoimmune/inflammatory disease associated peptide.
  • the TCR/CD3 activator is an anti-CD3 antibody.
  • the composition includes a second activator that is an additional suitable regulatory T cell stimulator.
  • This component may include, without limitation an interleukin.
  • IL-2 is used.
  • the IL-2 typically is used in recombinant form and used in an amount of about 1,000 IU/ml.
  • a multivalent antibody or ligand specific for a TCR co-stimulator may be used as the co-stimulator activator.
  • the co-stimulator activator is CD28.
  • the activator may be enhanced by use of one or more additives including, without limitation, rapamycin, a P13 kinase inhibitor, anit-IL6 and the like.
  • the TCR/CD3 activator and TCR co-stimulator activator preferably are immobilized on a three-dimensional solid surface, more preferably on a bead.
  • Suitable beads are commercially available such as cell Expander Dynalbeads (Invitrogen).
  • the Treg to bead ratio is preferably is 1:3.
  • Optimal bead size will depend on a consideration of the size required to efficiently congregate the antibodies and typically is about 1 to 10 microns in diameter.
  • the anti-CD3 and anti-CD28 antibody ratio on the surface of the bead may vary from about 1 to 20 or about 20 to 1.
  • the expansion is carried out to at least a 100 fold expansion, preferably to a greater than 1,000.
  • the expansion will depend upon the stimulation and length of the culture. However, other variations may be introduced into the culture the longer the length of the culture and, thus, a culture time that permits obtaining the most cells without a substantial introduction of other unfavorable factors is preferred.
  • peripheral blood samples of 50 cc were collected from 9 persons with SLE, 7 persons with RA, 7 persons with asthma, 10 persons with MS, and 7 persons with CD as well as from 11 persons with no autoimmune disease.
  • CD4+ Tregs were isolated and purified from the peripheral blood mononuclear cells (“PBMCs”) of the samples.
  • PBMCs peripheral blood mononuclear cells
  • the PBMCs were isolated from blood samples by density gradient centrifiguation with Ficoll Hypaque (Amersham).
  • the CD4+CD25+ Tregs were purified from PBMCs by autoMACS using the human CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec, Auburn, Calif.) according to the manufacturer's instructions. Briefly, CD4+ T cells were first negatively isolated from PBMCs by depleting non CD4 cells with the mixture of monoclonal antibodies against human CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCRy/8 and CD235a.
  • peripheral blood samples of 50 cc were collected from 9 persons with SLE, 7 persons with RA, 7 persons with asthma, 8 persons with MS, and 7 persons with CD as well as from 25 persons with no autoimmune disease.
  • CD4+ Tregs were purified from the PBMCs of the samples. Purification was carried out using as set forth in Example 1. The purity of purified CD4+ Tregs at day 0 was assessed using intracellular Foxp3 staining and analyzed by flow cytometry and the results are displayed on the graph in FIG. 2 . Approximately 40 to 75% of the purified cells expressed Foxp3.
  • FIG. 3A Approximately 5 to 10 peripheral blood samples of 50 cc each were collected from persons with SLE ( FIG. 3A ), RA ( FIG. 3C ), asthma ( FIG. 3B ), MS ( FIG. 3D ), CD ( FIG. 3E ), and UC ( FIG. 3F ).
  • CD4+ Tregs were purified from the PBMCs of the samples. Purification was carried out using as set forth in Example 1.
  • the CD4+Fox3+ Tregs were cultured in X-VIVO 15TM media (Cambrex Bio-Whitaker, East Rutherford, N.J.) supplemented with 10% pooled human AB serum (Cambrex) in the presence of 1000 IU/Ml of human rIL-2 (Proleukin).
  • anti-CD3/ant-CD28 coated beads (Dynal, Oslo, Norway) were added at a 1:3 cell to bead ratio.
  • the expanded Tregs reached over 100 to 1,000 fold expansion, or 1 billion cells, as shown in the graphs in FIG. 3 .
  • FIG. 4 is a graph depicting that an average of 40 to 50% of the ex-vivo expanded cells expressed Foxp3 by intracellular staining at 2 weeks.
  • the Tregs of Example 3 were evaluated at week 2 with standardized suppressive assays as follows.
  • allogenic CD4+CD25+ human T cells (5 ⁇ 10 4 cells/well) from the same donor were used as responder cells, 1 mg/ml anti-CD3 (OKT3), and allogenic human dendritic cells (from one donor, 5 ⁇ 10 3 cells/well) and serially diluted expanded Tregs were put in 96-well plates in triplicate.
  • CD4+CD25+ human Treg celss (5 ⁇ 10 4 cells/well) from one donor, 5 ⁇ 10 3 human dendritic cells from another donor, and serially diluted expanded Tregs were plated in triplicate in 96-well plates.
  • FIGS. 5A and 5B The results, shown in the graphs of FIGS. 5A and 5B (SLE), FIGS. 5C and 5D (asthma), FIGS. 5E and 5F (RA), FIGS. 5G and 5H (MS), FIGS. 5I and 5J (CD), and FIGS. 5K and 5L (UC), demonstrate that the ex vivo expanded human Tregs had potent inhibitory activity to inhibit T cell proliferation in in vitro functional assays. Additionally, the Tregs' inhibitory activity was enhanced as compared to that of freshly purified Tregs from the same persons. This suppressive activity may be used to inhibit unwanted human immune responses in autoimmune/inflammatory diseases and provide a clinical therapeutic potential for autoimmune/inflammatory diseases.

Abstract

The invention provides methods for the ex-vivo expansion of CD4+CD25+ Tregs. The invention provides a method for producing ex vivo expanded Tregs that may be used to inhibit unwanted human immune responses against self-antigens or allergens. Additionally, the ex vivo expanded Tregs may provide treatment for inflammatory/autoimmune diseases.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application is a continuation of U.S. patent application Ser. No. 12/781,451, filed on May 17, 2010, which claims the benefit of U.S. Provisional Application No. 61/179,165, filed May 18, 2009. The entire disclosure of each application that is set forth in this Cross Reference to Related Applications section is hereby incorporated by reference.
    • Field of the Invention
  • The invention relates to methods for enhancing the suppressive function of regulatory T cells. In particular, the invention provides a method for ex-vivo expansion of regulatory T cells, which cells demonstrate enhanced suppressive characteristics.
    • Background of the Invention
  • Regulatory T cells, or Tregs, are known to be critical in maintaining a tolerance to self-antigens by suppressing the activation of the immune system. In person with inflammatory/autoimmune diseases, such as systemic lupus erythematous, multiple sclerosis, rheumatoid arthritis, asthma, ulcerative colitis, and Crohn's Disease, there is a functional defect or frequency decrease in the transcription factor Foxp3 expressing CD4+CD25+Tregs. In such autoimmune diseases, the person's immune system fails to recognize cells, or parts of cells as the person's own resulting in tissue destruction.
  • The therapeutic effects of administration of CD4+CD25+ Tregs has been shown in the treatment of a variety of animal disease models including, without limitation, rheumatoid arthritis, asthma, and graft-versus-host disease (“GVHD”). Use of Tregs in treatment applications is problematic because they are present as only a very small percentage, approximately 1 to 2%, of human peripheral blood mononuclear cells. Thus, methods of activating and expanding, or proliferating, Tregs ex-vivo have been developed for use in the treatment of certain diseases.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a graph showing the frequency of CD4+Foxp3+ Tregs from the population of Example 1.
  • FIG. 2 is a graph showing the purity CD4+ Tregs of Example 2.
  • FIGS. 3A-3F are a graphs showing the Treg expansion of Example 3.
  • FIG. 4 is a graph showing the percentage of ex-vivo expanded cells of Example 3 that expressed Foxp3.
  • FIGS. 5A-5L are a graphs showing the inhibitory activity of the expanded Tregs of Example 4.
    •  DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
  • The invention provides methods for the ex-vivo expansion of CD4+CD25+ Tregs, compositions resulting from the method and methods for use of the compositions. The cells resulting from use of the method demonstrate enhanced suppressive activities compared to fresh Treg cells. The invention provides a method for producing ex vivo expanded Tregs that may be used to inhibit unwanted human immune responses against self-antigens or allergens. Additionally, the ex vivo expanded Tregs may provide treatment for inflammatory/automimmune diseases.
  • In one embodiment, the invention provides a methods comprising, consisting essentially of, and consisting of: a) obtaining a population of T cells from an individual; b) isolating and purifying from the T cell population a subpopulation of CD4+CD25+ Tregs; and c) expanding the Treg cells of the subpopulation, wherein the expanded Treg cells exhibit enhanced suppressive activity compared to a population of freshly purified, unexpanded Tregs from the individual.
  • It is a discovery of the invention that Tregs taken from a person with autoimmune/inflammatory diseases including, without limitation Systemic Lupus Erythematous (“SLE”), Multiple Sclerosis (“MS”), asthma, Rheumatoid Arthritis (“RA”), Crohn's Disease (“CD”), and Ulcerative Colitis (“UC), display enhanced suppressive activities to inhibit T cell proliferation when compared to freshly purified Tregs from the same person. It is a further discovery of the invention that the Tregs can be expanded 100 to 1,000 fold ex-vivo while having enhanced suppressive function.
  • In a first step of the method of the invention, a population of T cells is obtained. The T cells may be obtained from any suitable source including, without limitation, an individual's peripheral blood, thymus, lymph nodes, spleen or bone marrow. Preferably, the T cells are obtained from the peripheral blood of a person. More preferably, the T cells are obtained from an individual with an autoimmune/inflammatory diseases and most preferably from an individual with one or more of SLE, MS, asthma, RA, CD and UC whether the disease is in remission or during a period in which the disease is active. Most preferably, human CD4+CD25+ T regs are purified from whole blood units or leukpaks.
  • A subpopulation of CD4+CD25+ Tregs is then isolated from the T cell population using any convenient separation techniques based on Treg specific cell markers, including, without limitation, flow cytometry by any convenient method. For example, any number of kits for carrying out such isolation and purification are commercially available. The commercially available kits include, without limitation, Miltenyi Treg kit with Auotmacs, ClinMACS, and the like. Preferably, the isolation and purification is carried out until a population that is greater than 40% positive for Foxp3 and greater than 90% positive for CD4 is obtained.
  • The Treg subpopulation is then expanded ex-vivo using an autoantigen specific regulatory T cell stimulatory composition. Preferably, the composition antigen-specifically binds and activates the T cell receptor complex. More preferably, the expansion is carried out in the presence of effective amounts of a first and a second activator and a co-stimulator activator. By effective amount is meant an amount effective to stimulate the regulatory T cells to the degree desired.
  • The first activator is a TCR/CD3 activator that may be a multivalent antibody or ligand for TCR/CD3 including, without limitation, antigen non-specific activators such as an anti-CD antibody, and antigen-specific activators, such as an MHC-peptide multimer in which the peptide is an autoimmune/inflammatory disease associated peptide. Preferably, the TCR/CD3 activator is an anti-CD3 antibody.
  • The composition includes a second activator that is an additional suitable regulatory T cell stimulator. This component may include, without limitation an interleukin. Preferably IL-2 is used. The IL-2 typically is used in recombinant form and used in an amount of about 1,000 IU/ml.
  • A multivalent antibody or ligand specific for a TCR co-stimulator may be used as the co-stimulator activator. Preferably, the co-stimulator activator is CD28. The activator may be enhanced by use of one or more additives including, without limitation, rapamycin, a P13 kinase inhibitor, anit-IL6 and the like.
  • The TCR/CD3 activator and TCR co-stimulator activator preferably are immobilized on a three-dimensional solid surface, more preferably on a bead. Suitable beads are commercially available such as cell Expander Dynalbeads (Invitrogen). The Treg to bead ratio is preferably is 1:3. Optimal bead size will depend on a consideration of the size required to efficiently congregate the antibodies and typically is about 1 to 10 microns in diameter. The anti-CD3 and anti-CD28 antibody ratio on the surface of the bead may vary from about 1 to 20 or about 20 to 1.
  • The expansion is carried out to at least a 100 fold expansion, preferably to a greater than 1,000. The expansion will depend upon the stimulation and length of the culture. However, other variations may be introduced into the culture the longer the length of the culture and, thus, a culture time that permits obtaining the most cells without a substantial introduction of other unfavorable factors is preferred.
  • The invention will be clarified by consideration of the following, non-limiting examples.
    • EXAMPLES Example 1
  • Approximately 5 to 10 peripheral blood samples of 50 cc each were collected from 9 persons with SLE, 7 persons with RA, 7 persons with asthma, 10 persons with MS, and 7 persons with CD as well as from 11 persons with no autoimmune disease.
  • CD4+ Tregs were isolated and purified from the peripheral blood mononuclear cells (“PBMCs”) of the samples. The PBMCs were isolated from blood samples by density gradient centrifiguation with Ficoll Hypaque (Amersham). The CD4+CD25+ Tregs were purified from PBMCs by autoMACS using the human CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec, Auburn, Calif.) according to the manufacturer's instructions. Briefly, CD4+ T cells were first negatively isolated from PBMCs by depleting non CD4 cells with the mixture of monoclonal antibodies against human CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCRy/8 and CD235a. Human CD4+CD25+ Tregs were then positively isolated with anti-human CD25 antibody-conjugated microbeads from the enriched CD4+ T cell population. The frequency of Foxp3-expressing CD4+ Tregs was assessed using intracellular Foxp3 staining and analyzed by flow cytometry. The results are displayed in FIG. 1 below in which the percentage of CD4+Fox3+ Tregs in the CD4+ pool population from each person is shown.
    • Example 2
  • Approximately 5 to 10 peripheral blood samples of 50 cc each were collected from 9 persons with SLE, 7 persons with RA, 7 persons with asthma, 8 persons with MS, and 7 persons with CD as well as from 25 persons with no autoimmune disease.
  • CD4+ Tregs were purified from the PBMCs of the samples. Purification was carried out using as set forth in Example 1. The purity of purified CD4+ Tregs at day 0 was assessed using intracellular Foxp3 staining and analyzed by flow cytometry and the results are displayed on the graph in FIG. 2. Approximately 40 to 75% of the purified cells expressed Foxp3.
    • Example 3
  • Approximately 5 to 10 peripheral blood samples of 50 cc each were collected from persons with SLE (FIG. 3A), RA (FIG. 3C), asthma (FIG. 3B), MS (FIG. 3D), CD (FIG. 3E), and UC (FIG. 3F). CD4+ Tregs were purified from the PBMCs of the samples. Purification was carried out using as set forth in Example 1.
  • The CD4+Fox3+ Tregs were cultured in X-VIVO 15™ media (Cambrex Bio-Whitaker, East Rutherford, N.J.) supplemented with 10% pooled human AB serum (Cambrex) in the presence of 1000 IU/Ml of human rIL-2 (Proleukin). In addition, anti-CD3/ant-CD28 coated beads (Dynal, Oslo, Norway) were added at a 1:3 cell to bead ratio. After 3 weeks of culture at 37° C. in an incubator, the expanded Tregs reached over 100 to 1,000 fold expansion, or 1 billion cells, as shown in the graphs in FIG. 3. In FIG. 4 is a graph depicting that an average of 40 to 50% of the ex-vivo expanded cells expressed Foxp3 by intracellular staining at 2 weeks.
    • Example 4
  • The Tregs of Example 3 were evaluated at week 2 with standardized suppressive assays as follows. For anti-CD3 stimulated cultures, allogenic CD4+CD25+ human T cells (5×104 cells/well) from the same donor were used as responder cells, 1 mg/ml anti-CD3 (OKT3), and allogenic human dendritic cells (from one donor, 5×103 cells/well) and serially diluted expanded Tregs were put in 96-well plates in triplicate. For alloantigen stimulated cultures, CD4+CD25+ human Treg celss (5×104 cells/well) from one donor, 5×103 human dendritic cells from another donor, and serially diluted expanded Tregs were plated in triplicate in 96-well plates.
  • The results, shown in the graphs of FIGS. 5A and 5B (SLE), FIGS. 5C and 5D (asthma), FIGS. 5E and 5F (RA), FIGS. 5G and 5H (MS), FIGS. 5I and 5J (CD), and FIGS. 5K and 5L (UC), demonstrate that the ex vivo expanded human Tregs had potent inhibitory activity to inhibit T cell proliferation in in vitro functional assays. Additionally, the Tregs' inhibitory activity was enhanced as compared to that of freshly purified Tregs from the same persons. This suppressive activity may be used to inhibit unwanted human immune responses in autoimmune/inflammatory diseases and provide a clinical therapeutic potential for autoimmune/inflammatory diseases.

Claims (15)

1-10. (canceled)
11. A method comprising: a) obtaining a population of T cells from an individual with asthma; b) isolating and purifying from the T cell population a subpopulation of CD4+CD25+ Tregs; and c) expanding the Treg cells of the subpopulation in the presence of effective amounts of a first and a second activator and a co-stimulator activator, wherein the expanded Treg cells exhibit enhanced suppressive activity compared to a population of freshly purified, unexpanded Tregs from the individual.
12. The method of claim 11, wherein the expansion is between about 100 to 1,000 fold.
13. The method of claim 11, wherein the expansion was carried out for a period of about 3 weeks.
14. The method of claim 11, wherein the first activator is an anti-CD3 antibody.
15. The method of claim 11, wherein the second activator is IL-2.
16. The method of claim 11, wherein the co-stimulator activator is CD28.
17. The method of claim 11, wherein the first activator is an anti-CD3 antibody, the second activator is IL-2 and the co-stimulator activator is CD28.
18. The method of claim 11, wherein the isolating and purifying are carried out until the subpopulation of CD4+CD25+, Tregs is greater than 40% positive for Foxp3 and greater than 90% for CD4.
19. A method of treating an individual with immune-mediated disease, the method comprising: a) obtaining a population of T cells from an individual with asthma; b) isolating and purifying from the T cell population a subpopulation of CD4+CD25+ Tregs; c) expanding the Treg cells of the subpopulation in the presence of effective amounts of a first and a second activator and a co-stimulator; and d) administering a portion of the CD4+CD25+ regulatory T cells to a human being treated for the immune-mediated disease.
20. The method of claim 19, wherein the first activator is an anti-CD3 antibody.
21. The method of claim 19, wherein the second activator is IL-2.
22. The method of claim 19, wherein the co-stimulator activator is CD28.
23. The method of claim 19, wherein the first activator is an anti-CD3 antibody, the second activator is IL-2 and the co-stimulator activator is CD28.
24. The method of claim 19, wherein the isolating and purifying are carried out until the subpopulation of CD4+CD25, Tregs is greater than 40% positive for Foxp3 and greater than 90% for CD4.
US15/094,467 2009-05-18 2016-04-08 Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases Abandoned US20170298322A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US15/094,467 US20170298322A1 (en) 2009-05-18 2016-04-08 Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases
US15/981,332 US11186823B2 (en) 2009-05-18 2018-05-16 Method for ex-vivo expansion of regulatory T cells with enhanced suppressive function for clinical application in immune mediated diseases
US17/515,985 US20220056410A1 (en) 2009-05-18 2021-11-01 Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US17916509P 2009-05-18 2009-05-18
US12/781,451 US20100291117A1 (en) 2009-05-18 2010-05-17 Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases
US15/094,467 US20170298322A1 (en) 2009-05-18 2016-04-08 Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US12/781,451 Continuation US20100291117A1 (en) 2009-05-18 2010-05-17 Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/981,332 Continuation US11186823B2 (en) 2009-05-18 2018-05-16 Method for ex-vivo expansion of regulatory T cells with enhanced suppressive function for clinical application in immune mediated diseases

Publications (1)

Publication Number Publication Date
US20170298322A1 true US20170298322A1 (en) 2017-10-19

Family

ID=43068677

Family Applications (4)

Application Number Title Priority Date Filing Date
US12/781,451 Abandoned US20100291117A1 (en) 2009-05-18 2010-05-17 Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases
US15/094,467 Abandoned US20170298322A1 (en) 2009-05-18 2016-04-08 Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases
US15/981,332 Active US11186823B2 (en) 2009-05-18 2018-05-16 Method for ex-vivo expansion of regulatory T cells with enhanced suppressive function for clinical application in immune mediated diseases
US17/515,985 Pending US20220056410A1 (en) 2009-05-18 2021-11-01 Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US12/781,451 Abandoned US20100291117A1 (en) 2009-05-18 2010-05-17 Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases

Family Applications After (2)

Application Number Title Priority Date Filing Date
US15/981,332 Active US11186823B2 (en) 2009-05-18 2018-05-16 Method for ex-vivo expansion of regulatory T cells with enhanced suppressive function for clinical application in immune mediated diseases
US17/515,985 Pending US20220056410A1 (en) 2009-05-18 2021-11-01 Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases

Country Status (5)

Country Link
US (4) US20100291117A1 (en)
EP (1) EP2446022A4 (en)
JP (1) JP5937003B2 (en)
CN (1) CN102459577B (en)
WO (1) WO2010135255A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020157129A1 (en) * 2019-01-30 2020-08-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Optimisation of the scurfy model for in vivo testing of innovative treatments of autoimmunity
US11384336B2 (en) 2016-12-07 2022-07-12 East Carolina University Compositions and methods for in vitro cultivation and/or expansion of regulatory T cells

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014515258A (en) * 2011-05-19 2014-06-30 ティゲニックス エス．エー．ユー Cell population having immunomodulating activity, preparation method thereof, and use thereof
US20130273089A1 (en) * 2011-11-03 2013-10-17 Tolera Therapeutics, Inc. Antibody and methods for selective inhibition of t-cell responses
US20190062706A1 (en) * 2015-10-28 2019-02-28 Life Technologies As Selective expansion of different subpopulations of t cells by the alteration of cell surfacing signals and signal ratio
PL236046B1 (en) 2015-12-17 2020-11-30 Gdanski Univ Medyczny Method of in-vitro multiplication of regulatory T cells (Treg)
CN107519207A (en) * 2017-08-31 2017-12-29 广东颜值科技有限公司 A kind of immunosuppressant cell preparation and its preparation method and application
WO2019166658A1 (en) * 2018-03-01 2019-09-06 Fundación Para La Investigación Biomédica Del Hospital Gregorio Marañón Method for obtaining regulatory t cells derived from thymic tissue and use of said cells as cell immunotherapy in immune system disorders
EP3985106A1 (en) 2020-10-16 2022-04-20 Uniwersytet Gdanski Conditioned regulatory t cell population with enhanced therapeutic potential, method for obtaining of regulatory t cell population and the medical use of regulatory t cell population
EP3985105A1 (en) 2020-10-16 2022-04-20 Uniwersytet Gdanski Conditioned regulatory t cell population tregs with enhanced therapeutic potential, method for obtaining of conditioned tregs and the medical use of the tregs population

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1409650B1 (en) * 2001-05-30 2006-04-12 Fondazione Telethon Ex-vivo isolated cd25+cd4+ t cells with immunosuppressive activity and uses thereof
US20030049696A1 (en) * 2001-06-07 2003-03-13 Norment Anne M. Regulatory T cells and uses thereof
CA2369250A1 (en) * 2001-09-10 2003-03-10 Andrew Hoi-Tao Wong Novel anti-viral and anti-proliferative agents derived from stat1 transcription factor
CN1953767B (en) * 2004-01-08 2011-03-16 加利福尼亚大学董事会 Regulatory T cells suppress autoimmunity
US8563308B2 (en) * 2004-03-10 2013-10-22 The Rockefeller University Culture-expanded T suppressor cells and methods of use thereof
US20070009497A1 (en) * 2004-03-10 2007-01-11 Steinman Ralph M Dendritic cell expanded T suppressor cells and methods of use thereof
DE102007039429A1 (en) * 2007-08-21 2009-02-26 Imtm Gmbh Method for activating regulatory T cells
CN101605839B (en) 2007-11-21 2013-10-09 莫门蒂夫性能材料股份有限公司 Organosilicone compositions and methods for preparing them
JP2011519271A (en) * 2008-04-11 2011-07-07 ユニバーシティ オブ サザン カリフォルニア Methods and compositions for accelerating the generation of regulatory T cells ex vivo
MX2011011290A (en) * 2009-04-28 2012-02-13 Boehringer Ingelheim Int Ex-vivo treatment of immunological disorders with pkc-theta inhibitors.
US20170096636A1 (en) 2014-06-05 2017-04-06 Wake Forest University Health Sciences Methods and compounds for phototherapy with chalcogenorhodamine photosensitizers

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11384336B2 (en) 2016-12-07 2022-07-12 East Carolina University Compositions and methods for in vitro cultivation and/or expansion of regulatory T cells
WO2020157129A1 (en) * 2019-01-30 2020-08-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Optimisation of the scurfy model for in vivo testing of innovative treatments of autoimmunity

Also Published As

Publication number Publication date
EP2446022A1 (en) 2012-05-02
US11186823B2 (en) 2021-11-30
US20220056410A1 (en) 2022-02-24
JP2012527237A (en) 2012-11-08
EP2446022A4 (en) 2013-06-26
CN102459577B (en) 2014-08-20
CN102459577A (en) 2012-05-16
US20100291117A1 (en) 2010-11-18
JP5937003B2 (en) 2016-06-22
US20180258393A1 (en) 2018-09-13
WO2010135255A1 (en) 2010-11-25

Similar Documents

Publication Publication Date Title
US20220056410A1 (en) Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases
US20230280341A1 (en) Cd127 expression inversely correlates with foxp3 and suppressive function of cd4+ tregs
Putnam et al. Clinical grade manufacturing of human alloantigen-reactive regulatory T cells for use in transplantation
US9114100B2 (en) Methods of treatment using ex vivo expansion of cord blood T cells
US20180036345A1 (en) Expansion of alloantigen-reactive regulatory t cells
Alsuliman et al. A robust, good manufacturing practice–compliant, clinical-scale procedure to generate regulatory T cells from patients with amyotrophic lateral sclerosis for adoptive cell therapy
JP2004529631A (en) CD4 + CD25 + regulatory T cells from human blood
JP2006506987A (en) Compositions and methods for immune repertoire recovery in patients with autoimmunity and immunological deficits associated with organ or hematopoietic stem cell transplantation
Lukas Yani et al. CD8+ HLADR+ regulatory T cells change with aging: they increase in number, but lose checkpoint inhibitory molecules and suppressive function
RU2627445C2 (en) METHODS FOR IDENTIFICATION, SELECTION, ENRICHMENT AND DEPLETION OF Tr1 CELLS POPULATIONS, Tr1 CELLS POPULATIONS, PHARMACEUTICAL COMPOSITIONS, METHODS FOR THERAPY EFFECT MONITORING
US20070258959A1 (en) CD4+CD25- T CELLS AND Tr1-LIKE REGULATORY T CELLS
Holzer et al. Influence of a mutation in IFN-γ receptor 2 (IFNGR2) in human cells on the generation of Th17 cells in memory T cells
Wichlan et al. Efficient and reproducible large-scale isolation of human CD4+ CD25+ regulatory T cells with potent suppressor activity
Williams et al. Choice of resident costimulatory molecule can influence cell fate in human naïve CD4+ T cell differentiation
US8323969B2 (en) Preparation of regulatory T cells using ICAM-1 co-stimulation
CA2549394A1 (en) Methods for the identification and preparation of regulator/suppressor t lymphocytes, compositions and uses thereof
WO2018032088A2 (en) Regulation of tumor-associated t cells
US20210087530A1 (en) Compositions and methods for culturing and expanding cells
Yunger et al. Modulating the proliferative and cytotoxic properties of patient-derived TIL by a synthetic immune niche of immobilized CCL21 and ICAM1
Gerstner et al. Specific phenotype and function of CD56-expressing innate immune cell subsets in human thymus

Legal Events

Date Code Title Description
AS Assignment

Owner name: DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT, NEW YORK

Free format text: NOTICE OF GRANT OF SECURITY INTEREST IN PATENTS;ASSIGNOR:MALLINCKRODT HOSPITAL PRODUCTS IP LIMITED;REEL/FRAME:043601/0025

Effective date: 20170817

Owner name: DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AG

Free format text: NOTICE OF GRANT OF SECURITY INTEREST IN PATENTS;ASSIGNOR:MALLINCKRODT HOSPITAL PRODUCTS IP LIMITED;REEL/FRAME:043601/0025

Effective date: 20170817

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: INO THERAPEUTICS LLC, MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: IKARIA THERAPEUTICS LLC, NEW JERSEY

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: THERAKOS, INC., MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: ST SHARED SERVICES LLC, MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: INFACARE PHARMACEUTICAL CORPORATION, MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT PHARMA IP TRADING UNLIMITED COMPANY (F/K/A MALLINCKRODT PHARMA IP TRADING D.A.C.), IRELAND

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT PHARMACEUTICALS IRELAND LIMITED, IRELAND

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: VTESSE LLC (F/K/A VTESSE INC.), MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: SUCAMPO PHARMA AMERICAS LLC, MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: STRATATECH CORPORATION, WISCONSIN

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: SPECGX LLC, MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: OCERA THERAPEUTICS LLC (F/K/A OCERA THERAPEUTICS, INC.), MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT ARD IP UNLIMITED COMPANY (F/K/A MALLINCKRODT ARD IP LIMITED), IRELAND

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT HOSPITAL PRODUCTS IP UNLIMITED COMPANY (F/K/A MALLINCKRODT HOSPITAL PRODUCTS IP LIMITED), IRELAND

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MEH, INC., MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: IMC EXPLORATION COMPANY, MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT US HOLDINGS LLC, MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT VETERINARY, INC., MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT BRAND PHARMACEUTICALS LLC (F/K/A MALLINCKRODT BRAND PHARMACEUTICALS, INC.), MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: LIEBEL-FLARSHEIM COMPANY LLC, MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: LAFAYETTE PHARMACEUTICALS LLC, MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT LLC, MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT ENTERPRISES LLC, MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT ENTERPRISES HOLDINGS LLC (F/K/A MALLINCKRODT ENTERPRISES HOLDINGS, INC.), MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: CNS THERAPEUTICS, INC., MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: LUDLOW LLC (F/K/A LUDLOW CORPORATION), MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MNK 2011 LLC (F/K/A MALLINCKRODT INC.), MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT US POOL LLC, MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT CARRIBEAN, INC., MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT US HOLDINGS LLC (F/K/A MALLINCKRODT US HOLDINGS INC.), MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT FINANCE GMBH, SWITZERLAND

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT CB LLC, MISSOURI

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114

Owner name: MALLINCKRODT INTERNATIONAL FINANCE S.A., LUXEMBOURG

Free format text: RELEASE OF PATENT SECURITY INTERESTS RECORDED AT REEL 043601, FRAME 0025;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH, AS COLLATERAL AGENT;REEL/FRAME:065610/0233

Effective date: 20231114