Methods for the isolation and expansion of cord blood derived T regulatory cells

Patent metadata

Field	Value
Publication number	US-9273282-B2
Application number	US-22616805-A
Country	US
Kind code	B2
Filing date	Sep 14, 2005
Priority date	Sep 15, 2004
Publication date	Mar 1, 2016
Grant date	Mar 1, 2016

Abstract

The present invention encompasses methods, and kits for the isolation and expansion of T regulatory cells having the CD45RA + phenotype, including such cells from human umbilical cord blood.

First claim

What is claimed:

1. A method of isolating a regulatory T cell (T reg cell) from a population of phenotypically CD45RA + blood cells and culture activating said T reg cell to generate a suppressor cell line, wherein said suppressor cell line suppresses T cell proliferation in a mixed lymphocyte reaction by a factor greater than about 95% at a ratio of 1:32 (Treg: T cell), said method comprising:
a) isolating a population of mononuclear cells from a human umbilical cord blood sample;
b) contacting said population of mononuclear cells with an antibody that specifically binds CD25 under conditions suitable for formation of a mononuclear cell-antibody complex;
c) substantially separating said mononuclear cell-antibody complex from said population of mononuclear cells, thereby isolating a population of CD25 + cells, wherein said CD25 + cells are CD45RA + ;
d) expanding said isolated population of CD25 + cells in the presence of anti-CD3/CD28 antibody coated beads at a 3:1 bead to cell ratio;
e) activating said expanded population of CD25+ cells in the presence of IL-2;
f) substantially separating said activated population of CD25 + cells; thereby generating a suppressor cell line that uniformly express CD25, intracellular CTLA4, CD27 and CD62L.

2. The method of claim 1 , wherein said population comprises an enhanced population of phenotypically CD45RA + blood cells, wherein said blood cells are isolated from umbilical cord blood.

3. The method of claim 1 , wherein said T reg cell expresses the FoxP3 antigen.

4. The method of claim 1 , wherein said antibody in step b is selected from the group consisting of an isolated antibody, a biological sample comprising an antibody, an antibody bound to a physical support and a cell-bound antibody.

5. The method of claim 4 , wherein said antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a synthetic antibody, a biologically active fragment of an antibody, and combinations thereof.

6. The method of claim 5 , wherein said biologically active fragment is selected from the group consisting of an Fab fragment, a F(ab′) 2 fragment, a Fv fragment, and an scFv fragment.

7. The method of claim 4 , wherein said physical support is selected from the group consisting of a microbead, a magnetic bead, an absorption column and an adsorption membrane.

8. The method of claim 1 , wherein said mononuclear cell-antibody complex is substantially separated from said population of mononuclear cells by a method selected from the group consisting of fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS).

9. The method of claim 1 , wherein steps b) and c) are repeated.

10. A method of isolating a regulatory T (T reg ) precursor cell and culture activating said precursor cell to generate a suppressor cell line, wherein said suppressor cell line suppresses T cell proliferation in a mixed lymphocyte reaction by a factor greater than about 95% at a ratio of 1:32 (Treg: T cell), said method comprising:
a) isolating a population of mononuclear cells from a human umbilical cord blood sample;
b) contacting said population of mononuclear cells with an antibody that specifically binds CD25 under conditions suitable for formation of a mononuclear cell-antibody complex; and
c) substantially separating said mononuclear cell-antibody complex from said population of mononuclear cells, thereby isolating a population of CD25 + cells, wherein said CD25 + cells are CD45RA + ;
d) expanding said isolated population of CD25 + cells in the presence of anti-CD3/CD28 antibody coated beads at a 3:1 bead to cell ratio;
e) activating said expanded population of CD25+ cells in the presence of IL-2;
f) substantially separating said activated population of CD25 + cells; thereby generate a suppressor cell line that uniformly express CD25, intracellular CTLA4, CD27 and CD62L.

11. The method of claim 10 , wherein said method produces a population of suppressor T cells increased in number by about 100-fold from the original isolated T cell population.

12. The method of claim 11 , wherein said population comprises an enhanced population of phenotypically CD3 + blood cells, wherein said blood cells are isolated from umbilical cord blood.

13. The method of claim 11 , wherein said T reg cell expresses the FoxP3 antigen.

14. The method of claim 11 , wherein said antibody in step b is selected from the group consisting of an isolated antibody, a biological sample comprising an antibody, an antibody bound to a physical support and a cell-bound antibody.

15. The method of claim 14 , wherein said antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a synthetic antibody, a biologically active fragment of an antibody, and combinations thereof.

16. The method of claim 15 , wherein said biologically active fragment is selected from the group consisting of an Fab fragment, a F(ab′) 2 fragment, a Fv fragment, and an scFv fragment.

17. The method of claim 14 , wherein said physical support is selected from the group consisting of a microbead, a magnetic bead, an absorption column and an adsorption membrane.

18. The method of claim 11 , wherein said mononuclear cell-antibody complex is substantially separated from said population of mononuclear cells by a method selected from the group consisting of fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS).

19. The method of claim 11 , wherein steps b) and c) are repeated.

20. The method of claim 1 , wherein said method produces a population of suppressor T cells increased in number by about 100-fold from the original isolated cell population.

Assignees

• Godfrey Wayne R
• June Carl
• Univ Pennsylvania

Inventors

• Godfrey Wayne R
• June Carl

Classifications

• C12N2501/505

  CD4; CD8 · CPC title

• C12N2501/599

  with CD designations not provided for elsewhere · CPC title

• C12N5/0087 Primary

  Purging against subsets of blood cells, e.g. purging alloreactive T cells · CPC title

• C12N2501/515

  CD3, T-cell receptor complex · CPC title

• C12N2501/23

  Interleukins [IL] · CPC title