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US 12,692,478 B2Granted
Populations of enriched regulatory T cells and methods for producing same
AssigneeCellenkos, Inc.
InventorSimrit Parmar
Life of this patent
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Abstract
Provided herein are populations of enriched ex vivo expanded umbilical cord blood-derived regulatory T cells. Also provided are methods of making and using the same.
Claims28
A method for producing an expanded population of human T regulatory (Treg) cells enriched for CXCR4+ Treg cells from a cryopreserved human umbilical cord blood unit, the method comprising:
- (a) thawing the cryopreserved human umbilical cord blood unit;
- (b) diluting and washing the thawed umbilical cord blood unit;
- (c) isolating CD25+ Treg cells using double selection for CD25+ cell surface expression with a reagent that specifically binds to CD25;
- (d) ex vivo expanding the isolated CD25+ Treg cells in a culture medium, in a gas permeable cultureware, in the presence of:
- (1) an effective amount of interleukin-2 (IL-2);
- (2) a reagent that specifically binds to CD3 and CD28;
- and
- (3) magnetic microbeads coated with anti-CXCR4 antibody,
- for up to 10 days or up to 12 days, wherein the culture medium is replaced about every 48 hours, to produce a CXCR4-enriched culture of CD25+ Treg cells;
- and
- (e) harvesting the CD25+ CXCR4+ cells from the culture medium to produce an expanded population of human Treg cells enriched for CXCR4+ Treg cells.
8 more elements
A method for producing an expanded population of human Treg cells enriched for CXCR4+ Treg cells from a cryopreserved human umbilical cord blood unit, the method comprising:
- (a) thawing the cryopreserved human umbilical cord blood unit;
- (b) diluting and washing the thawed umbilical cord blood unit;
- (c) isolating Treg cells using double selection for CD25+ cell surface expression with a reagent that specifically binds to CD25;
- (d) ex vivo expanding the isolated CD25+ Treg cells in a culture medium, in a gas permeable cultureware, wherein the ex vivo expansion step comprises:
- (1) at day 0, adding beads coated with an anti-CD3antibody and beads coated with an anti-CD28 antibody to the culture medium;
- (2) at day 2, adding about 1000 IU/ml IL-2 to the culture medium;
- (3) at day 3 or 4, removing the beads coated with an anti-CD3 antibody and the beads coated with an anti-CD28 antibody from the culture medium and adding anti CXCR4 magnetic microbeads coated with anti-CXCR4 antibody to the culture medium;
- and
- (4) at day 3 or 4, removing the magnetic microbeads coated with anti-CXCR4 antibody attached to CXCR4+ Treg cells from the culture medium, and adding fresh beads coated with an anti-CD3 antibody and beads coated with an anti-CD28 antibody to the CXCR4+ Treg cells,
- wherein the ex vivo expansion takes place for up to 10 days or up to 12 days, wherein the culture medium is replaced about every 48 hours, to produce a CXCR4-enriched culture of CD25+ Treg cells;
- and
- (e) harvesting the CD25+ CXCR4+ cells from the culture medium to produce an expanded population of human Treg cells enriched for CXCR4+ Treg cells.
9 more elements
Dependencies are inferred from the claim text.