System and methods for promoting expansion of polyclonal and antigen-specific cytotoxic T lymphocytes in response to artificial antigen presenting cells

• US 20030147869A1
• Filed: 01/03/2003
• Published: 08/07/2003
• 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: June, Carl, Riley, James, Maus, Marcela
• Granted Patent: US 7,670,781 B2
• Time in Patent Office: Days
• US Class Current: 424/93.21
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/4621 immunosuppressive or immunotolerising
• CPC Class Codes: A61K 39/46433 Antigens related to auto-immune diseases; Preparations to induce self-tolerance
• CPC Class Codes: A61K 39/464499 Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
• 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 5/0634 Cells from the blood or the immune system
• CPC Class Codes: C12N 5/0636 T lymphocytes

Abstract

Provided is 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 artificial antigen presenting cell (aAPC) for inducing rapid, long term proliferation of a population of T cells for research purposes, comprising a selected population of K562 cells engineered to express Fcγ

receptor CD32 (thereby creating K32 cells), transfected by a co-stimulatory ligand, then coating the K-32-ligand cells with anti-CD3 antibodies or anti-CD28 antibodies, or a combination thereof.

2. The aAPC of claim 1, wherein the ligand is 4-1BB (4-1BBL).

3. The aAPC of claim 1, wherein the the K32 cells, the antibody(ies) and the ligand are of human origin.

4. A method of preparing the aAPC of claim 1, comprising:

selecting a population of K562 cells and engineering the cells to express Fcγ

receptor CD32 (thereby creating K32 cells);

transfecting the K32 cells with the co-stimulatory ligand;

cloning the K32 cells to permit exogenous loading of the anti-CD3 or anti-CD28 antibodies, or a combination thereof; and

then

loading of the anti-CD3 or anti-CD28 antibodies, or a combination thereof.

5. The method of claim 4, wherein the ligand is 4-1BB (4-1BBL).

6. A system for inducing a population of T cells to rapidly proliferate long term to sufficient numbers for use in research, comprising exposing the population of T cells to the aAPC of claim 2.

7. A system for inducing a population of CD8⁺ T cells to rapidly proliferate long term to sufficient numbers for use in research, comprising exposing the population of T cells to the aAPC of claim 3.

8. A method for inducing a population of T cells to rapidly proliferate exponentially for a long term to sufficient numbers for research purposes, comprising:

a) activating the population of T cells by contacting the T cells ex vivo with at least one exogenous first agent that provides a primary activation signal to the T cells; and

b) stimulating the activated T cells with at least one second agent that provides a co-stimulatory signal, such that T cells that have received a primary activation signal are stimulated to rapidly proliferate.

9. The method for inducing a population of T cells of claim 8, wherein the activating step further comprises activating the population of T cells by contacting the T cells ex vivo with exogenous anti-CD3 antibody or exogenous anti-CD28 antibody, or a combination thereof.

10. The method for inducing a population of T cells of claim 9, wherein the stimulating step further comprises stimulating the activated T cells with a co-stimulatory ligand.

11. The method of claim 10, wherein the ligand is 4-1BB (4-1BBL).

12. The method of claim 9, wherein the anti-CD3 antibody and the anti-CD28 antibody are human.

13. The method of claim 10, wherein the co-stimulatory ligand is human.

14. The method of claim 10, further comprising monitoring exponential proliferation of the T cells in response to continuing exposure to the ligand;

and re-activating and re-stimulating the T cells when the rate of T cell proliferation has decreased, thereby inducing further proliferation of the T cells.

15. The method of claim 10, further comprising repeating the steps to produce a population of T cells increased in number of from about 100- to about 100,000-fold the original T cell population.

16. The method of claim 10, wherein the T cells comprise CD4⁺ T cells or CD8⁺ T cells, or a combination thereof.

17. A method for stimulating a population of CD8⁺ T cells to rapidly proliferate exponentially for a long term, comprising:

a) primarily activating the population of CD8⁺ T cells by contacting the CD8⁺ T cells ex vivo with at least one exogenous first agent which stimulates a T cell receptor/CD3 complex-associated signal in the T cells; and

b) stimulating the activated T cells with at least one second agent which is a ligand providing a co-stimulatory signal, such that T cells that have received the primary activation signal are stimulated to rapidly proliferate.

18. The method for inducing a population of T cells of claim 17, wherein the activating step further comprises activating the population of T cells by contacting the T cells ex vivo with exogenous anti-CD3 antibody or exogenous anti-CD28 antibody, or a combination thereof.

19. The method for inducing a population of T cells of claim 17, wherein the stimulating step further comprises stimulating the activated T cells with a co-stimulatory ligand.

20. The method of claim 19, wherein the ligand is 4-1BB.

21. The method of claim 20, wherein the antibody(ies) and the ligand are of human origin.

22. A method of inducing a population of T cells from a subject to rapidly proliferate exponentially for a long term to sufficient numbers for research purposes, comprising:

a) isolating a population of T cells from a subject;

b) activating the population of T cells by contacting the T cells ex vivo with at least one exogenous first agent that provides a primary activation signal to the T cells; and

c) stimulating the activated T cells with at least one second agent that provides a co-stimulatory signal, such that T cells that have received a primary activation signal are stimulated to rapidly proliferate.

23. The method for inducing a population of T cells of claim 22, wherein the activating step further comprises activating the population of T cells by contacting the T cells ex vivo with exogenous anti-CD3 antibody or exogenous anti-CD28 antibody, or a combination thereof.

24. The method for inducing a population of T cells of claim 22, wherein the stimulating step further comprises stimulating the activated T cells with a co-stimulatory ligand.

25. The method of claim 24, wherein the ligand is 4-1BB (4-1BBL).

26. The method of claim 22, wherein the subject is human, and wherein the method further comprises using the activated T cells to identify antigens in the subject.

27. The method of claim 26, wherein the subject is infected with a disease or condition, having at least one antigen related thereto, and wherein the method further comprises using the activated T cells to identify the at least one antigen.

28. The method of claim 27, wherein the antigen comprises a tumor antigen.

29. The method of claim 28, wherein the method further comprises screening the at least one antigen as a target molecule for responding to the subject'"'"'s disease or condition for research purposes.

30. The method of claim 29, wherein the treating step further comprises developing a vaccine for the subject based upon the at least one antigen.

31. The method of claim 27, wherein the antigen comprises an antigen associated with an autoimmune disorder or condition.

32. The method of claim 31, wherein the method further comprises screening the at least one antigen as a target molecule for responding to the subject'"'"'s autoimmune disorder or condition for research purposes.

33. The method of claim 32, wherein the treating step further comprises developing a vaccine for the subject based upon the at least one antigen.

34. The method of claim 27, wherein the antigen comprises an antigen associated with an infectious disease or pathogen.

35. The method of claim 34, wherein the method further comprises screening the at least one antigen as a target molecule for responding to the subject'"'"'s an infectious disease or pathogen for research purposes.

36. The method of claim 35, wherein the treating step further comprises developing a vaccine for the subject based upon the at least one antigen.