US20100291117A1 - 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

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US20100291117A1
US20100291117A1 US12/781,451 US78145110A US2010291117A1 US 20100291117 A1 US20100291117 A1 US 20100291117A1 US 78145110 A US78145110 A US 78145110A US 2010291117 A1 US2010291117 A1 US 2010291117A1
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tregs
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Tinghua Cao
Li Ll
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Mallinckrodt Pharma IP Trading DAC
Mallinckrodt Critical Care Finance Inc
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Priority to US15/094,467 priority patent/US20170298322A1/en
Priority to US15/981,332 priority patent/US11186823B2/en
Priority to US17/515,985 priority patent/US20220056410A1/en
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Priority to US18/584,070 priority patent/US20240191193A1/en
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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 arthtritis, 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.
  • FIG. 3 is a graph 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.
  • FIG. 5 is a graph 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, TCR ⁇ / ⁇ 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.
  • the CD4+Foxp3+ 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/anti-CD28 coated beads 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 cells (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.
  • results 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.

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US12/781,451 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 Abandoned US20100291117A1 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
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
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
US18/584,070 US20240191193A1 (en) 2009-05-18 2024-02-22 Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases

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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
US18/584,070 Pending US20240191193A1 (en) 2009-05-18 2024-02-22 Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases

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AU2016344745A1 (en) * 2015-10-28 2018-05-17 Life Technologies As Selective expansion of different subpopulations of T cells by the alteration of cell surfacing signals and signal ratio
PL236046B1 (pl) 2015-12-17 2020-11-30 Gdanski Univ Medyczny Sposób namnażania in vitro limfocytów T regulatorowych CD4+ FoxP3+
EP3551749B1 (fr) 2016-12-07 2023-09-27 East Carolina University Compositions et méthodes de culture et/ou de croissance in vitro de lymphocytes t régulateurs
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JP2021516052A (ja) * 2018-03-01 2021-07-01 フンダシオン パラ ラ インベスティガシオン ビオメディカ デル オスピタル グレゴリオ マラニョン 胸腺組織に由来する制御性ｔ細胞を入手する方法および免疫系障害における細胞免疫療法としての前記細胞の使用
WO2020157129A1 (fr) * 2019-01-30 2020-08-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Optimisation du modèle scurfy pour l'analyse in vivo de traitements innovants de l'auto-immunité
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US20180258393A1 (en) 2018-09-13
JP5937003B2 (ja) 2016-06-22
US11186823B2 (en) 2021-11-30
JP2012527237A (ja) 2012-11-08
US20170298322A1 (en) 2017-10-19
EP2446022A4 (fr) 2013-06-26
CN102459577A (zh) 2012-05-16
WO2010135255A1 (fr) 2010-11-25
US20220056410A1 (en) 2022-02-24
EP2446022A1 (fr) 2012-05-02
US20240191193A1 (en) 2024-06-13

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