Activation and expansion of T-cells using an engineered multivalent signaling platform as a research tool

• US 7,745,140 B2
• Filed: 06/13/2003
• Issued: 06/29/2010
• Est. Priority Date: 01/03/2002
• Status: Active Grant

• Current Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Thomas, Anna, Riley, James, Vonderheide, Robert, Maus, Marcela, June, Carl
• Primary Examiner(s): Haddad; Maher M.
• Assistant Examiner(s): DAHLE, CHUN WU
• Application Number:
  US10/461,283
• Publication Number:
  US 20040101519A1
• Time in Patent Office: 2,573 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: A61K 2039/5158 Antigen-pulsed cells, e.g. T-cells
• CPC Class Codes: A61K 2239/31 characterized by the route of administration
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 2239/49 Breast
• CPC Class Codes: A61K 2239/58 Prostate
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• CPC Class Codes: A61K 39/4615 Dendritic cells
• CPC Class Codes: A61K 39/4622 Antigen presenting cells
• CPC Class Codes: A61K 39/464457 Telomerase or [telomerase reverse transcriptase [TERT]
• CPC Class Codes: A61K 39/464838 Viral antigens
• CPC Class Codes: C07K 14/70535 Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
• CPC Class Codes: C12N 2501/51 B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
• CPC Class Codes: C12N 2501/515 CD3, T-cell receptor complex
• CPC Class Codes: C12N 2501/599 with CD designations not provided for elsewhere
• CPC Class Codes: C12N 2502/99 genetically modified cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 5/0634 Cells from the blood or the immune system
• CPC Class Codes: C12N 5/0636 T lymphocytes

Abstract

Provided are a system and methods for selectively inducing expansion of a population of T cells in the absence of exogenous growth factors, such as lymphokines, and accessory cells for research purposes. The cell based expansion system and methods permit the long-term growth of CTLs, preferably human CTLs. In addition, T cell proliferation can be induced without the need for antigen, thus providing an expanded T cell population that is polyclonal with respect to antigen reactivity. Further provided are methods for using the system and methods to screen and identify antigens related to specific diseases or conditions, tumors, autoimmune disorders, or an infectious disease or pathogen, and to identify target molecule for research purposes, or for developing a vaccine based thereon.

Claims

1. An in vitro method for activating or stimulating a population of T cells, the method comprising:

contacting said population of cells with an engineered K562 cell followed by restimulation of said T cells with said engineered K562 cell, wherein said engineered K562 cell comprises;

     1) an exogenous antibody that binds to CD3 or CD2, wherein the antibody is loaded onto a human Fcγ

receptor wherein the Fcγ

receptor is expressed from an expression vector in said engineered K562 cell,

     2) a molecule that binds CD28 selected from the group consisting of anti-CD28 antibody, CD80, and CD86, and

     3) 4-1BBL;

thereby stimulating or activating said T cells.

2. The method of claim 1, further comprising in vitro expanding said T cells by incubating in the presence of said engineered K562 cell.

3. The method of claim 1, further comprising separating said T cell from said engineered K562 cell and subsequently incubating said T cells in vitro with an agent that facilitates T cell expansion.

4. The method of claim 1, wherein said T cells are a T cell line.

5. The method of claim 1, wherein said T cells are a T cell clone.

6. An in vitro method for maintaining T cell repertoire, the method comprising providing a population of T cells with an engineered K562 cell for a time sufficient to induce activation and subsequently expanding said T cells followed by restimulation of said T cell with said engineered K562 cell, thereby maintaining said T cell repertoire, wherein said engineered K562 cell comprises:

1) an exogenous antibody that binds to CD3 or wherein the antibody is loaded onto a human Fcγ

receptor wherein the Fcγ

receptor is expressed from an expression vector in said engineered K562 cell,

     2) a molecule that binds CD28 selected from the group consisting of anti-CD28 antibody, CD80, and CD86, and

     3) 4-1BBL, thereby maintaining said T cell repertoire.

7. The method of claim 6, further comprising in vitro expanding said T cells by incubating said T cells in the presence of said engineered K562 cell.

8. A method for maintaining the viability of T cells during culture, the method comprising contacting said T cells with an engineered K562 cell followed by restimulation of said T cells with said engineered K562 cell, wherein said engineered K562 cell comprises:

1) an exogenous antibody that binds to CD3 or wherein the antibody is loaded onto a human Fcγ

receptor wherein the Fcγ

receptor is expressed from an expression vector in said engineered K562 cell,

     2) a molecule that binds CD28 selected from the group consisting of anti-CD28 antibody, CD80, and CD86, and

     3) 4-1BBL, thereby maintaining the viability of said T cells during culture.

9. The method of claim 8, wherein said T cells are CD4⁺ T cells.

10. The method of claim 8, wherein said T cells are CD8⁺ T cells.

11. The method of claim 8, wherein said contacting occurs by sequential stimulation of T cells by said engineered K562 cell.

12. An in vitro method for expanding a population of T cells, the method comprising:

contacting said population of T cells with an engineered K562 cell followed by restimulation of said T cells with said engineered K562 cell, wherein said engineered K562 cell comprises;

     1) an exogenous antibody that binds to CD3 or wherein the antibody is loaded onto a human Fcγ

receptor wherein the Fcγ

receptor is expressed from an expression vector in said engineered K562 cell,

     2) a molecule that binds CD28 selected from the group consisting of anti-CD28 antibody; and

     3) 4-1BBL; and

culturing said T cells under conditions and time sufficient to induce cell division, wherein said contacting occurs in the absence of exogenously added cytokines, thereby expanding said population of T cells.

13. The method of claim 12, further comprising sequentially stimulating said T cells in vitro with said engineered K562 cell either by previously purifying T cells from originally added engineered K562 cell and subsequently adding additional engineered K562 cell or by adding additional engineered K562 cell to previously stimulated cells without separation of originally added engineered K562 cell from said previously stimulated cells.

14. The method of claim 12, further comprising separating said T cells from said engineered K562 cell and subsequently incubating said T cells with an agent that facilitates T cell expansion, followed by restimulation of said T cells with said engineered K562 cell in vitro.

15. An in vitro method for expanding a population of T cells, said method comprising:

contacting said population of T cells with an engineered K562 cell followed by restimulation of said T cells with said engineered K562 cell, wherein said engineered K562 cell comprises;

     1) an exogenous antibody that binds to CD3 or wherein the antibody is loaded onto a human Fcγ

receptor wherein the Fcγ

receptor is expressed from an expression vector in said engineered K562 cell,

     2) a molecule that binds CD28 selected from the group consisting of anti-CD28 antibody, CD80, CD86; and

     3) 4-1BBL; and

culturing said T cells under conditions and time sufficient to induce cell division thereby expanding said population of T cells.

16. The method of claim 15, further comprising sequentially stimulating said T cells in vitro with said engineered K562 cell either by previously purifying T cells from originally added engineered K562 cell and subsequently adding additional engineered K562 cell or by adding additional engineered K562 cell to previously stimulated cells without separation of originally added engineered K562 cell.

17. The method of claim 15, further comprising separating said T cells from said engineered K562 cell and subsequently incubating said T cells with an agent that facilitates T cell expansion, followed by restimulation with said engineered K562 cell in vitro.

18. The method of claim 15, wherein following initial stimulation or activation said T cells are separated from said engineered K562 cell and then expanded in vitro in the presence of exogenously added cytokines.

19. The method of claim 18, wherein said T cells are substantially free of said engineered K562 cell prior to in vitro expansion.

20. An ex vivo method for expanding antigen specific T cells, the method comprising:

a. contacting a population of T cells with an antigen for a time sufficient to induce activation of T cells specific to said antigen; and

b. contacting said population of antigen-specific T cells ex vivo with an engineered K562 cell under conditions and for time sufficient to induce proliferation of said antigen-specific T cells followed by restimulation of said T cells with said engineered K562 cell, wherein said engineered K562 cell comprises;

     1) an exogenous antibody that binds to CD3 or CD2, wherein the antibody is loaded onto a human Fcγ

receptor wherein the Fcγ

receptor is expressed from an expression vector in said engineered K562 cell,

     2) a molecule that binds CD28 selected from the group consisting of anti-CD28 antibody, CD80, and CD86; and

     3) 4-1BBL, thereby expanding said antigen specific T cells.

21. An in vitro method of activating antigen specific T cells, the method comprising contacting T cells with an antigen and an engineered K562 cell under conditions and for time sufficient to induce activation of T cells specific to said antigen followed by restimulation of said T cells with said engineered K562 cell, wherein said engineered K562 cell comprises:

1) an exogenous antibody that binds to CD3 or CD2,

     2) a molecule that binds CD28 selected from the group consisting of anti-CD28, CD80, and CD86; and

     3) 4-1BBL, thereby activating said antigen specific T cells.

22. The method of claim 21, wherein said antigen is a tumor antigen.

23. The method of claim 22, wherein said antigen is human telomerase reverse transcriptase (hTERT).

24. The method of claim 21, wherein said antigen is pulsed on or expressed by an antigen-presenting cell.

25. The method of claim 21, further comprising at least one in vitro round of peptide-MHC tetramer sorting of said antigen-specific T cells.

26. The method of claim 21, further comprising at least one in vitro round of peptide-MHC tetramer magnetic selection of said antigen-specific T cells.