High efficiency transduction of T lymphocytes using rapid expansion methods ("REM")

• US 6,040,177 A
• Filed: 03/13/1996
• Issued: 03/21/2000
• Est. Priority Date: 08/31/1994
• Status: Expired due to Term

• Current Assignee: Fred Hutchinson Cancer Research Center (Fred Hutchinson Cancer Research Center|Argus Genetics|Mars)
• Original Assignee: Fred Hutchinson Cancer Research Center (Fred Hutchinson Cancer Research Center|Argus Genetics|Mars)
• Inventors: Riddell, Stanley R., Greenberg, Philip D.
• Primary Examiner(s): Saunders, David
• Assistant Examiner(s): VANDERVEGT, FRANCOIS P
• Application Number:
  US08/614,585
• Time in Patent Office: 1,469 Days
• Field of Search: 424/93.71, 435/2, 435/240.21, 435/320.1, 435/455, 435/372.3, 435/374, 435/375, 435/377, 435/383, 435/384, 435/373, 530/351, 530/338.75, 530/388.85, 530/389.6, 536/23.72, 536/23.1, 935/23
• US Class Current: 435/372.3
• 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 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 39/46434 Antigens related to induction of tolerance to non-self
• CPC Class Codes: A61K 39/464838 Viral antigens
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/515 CD3, T-cell receptor complex
• CPC Class Codes: C12N 2502/11 blood or immune system cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2799/027 where the vector is derived from a retrovirus
• CPC Class Codes: C12N 5/0636 T lymphocytes

Abstract

The present invention provides a rapid expansion method (termed "REM"), for quickly generating large numbers of T lymphocytes, including cytolytic and helper T lymphocytes. REM involves culturing the T cells in association with a disproportionately large concentration of nondividing feeder cells, preferably γ-irradiated peripheral blood mononuclear cells ("PBMC") present at an excess of at least 40-fold (relative to the number of target T cells), more preferably at an excess of at least about 200-fold. Cultures grown under REM exhibit dramatically enhanced expansion rates that can be even further elevated by the use of appropriate concentrations of an additional feeder cell, an anti-CD3 monoclonal antibody and IL-2, as described herein. Clonal expansions in the range of 500-fold to 3000-fold can be achieved within a single stimulation cycle of about 10-13 days, which is more than 100-fold more efficient than currently employed methods of culturing human T cell clones. Genetic transduction efficiencies were also enhanced using REM-expanded T lymphocytes. Several examples involving human bone marrow transplant recipients illustrate the effective use of REM-expanded antigen-specific cytotoxic T lymphocytes for adoptive immunotherapy in humans.

Claims

1. A method of genetically transducing a T cell, comprising:

(a) adding initial isolated T lymphocytes to a culture medium in vitro, said T lymphocytes comprising a T cell receptor complex, wherein said T lymphocytes are isolated by use of an affinity reagent that selects antigen-specific T cells or are isolated by limiting dilution culture of antigen-stimulated T lymphocytes;

(b) adding to the culture medium a disproportionately large number of non-dividing peripheral blood mononuclear cells (PBMC) as feeder cells such that the resulting population of cells contains a ratio of at least about 40 PBMC feeder cells for each T lymphocyte in the initial population to be expanded;

(c) activating said T cell receptor complex;

(d) adding a transduction vector to said culture medium;

(e) incubating the culture, thereby producing a population of transduced T lymphocytes which become reversibly quiescent upon withdrawal of exogenous cytokine and are antigen-specific and MHC restricted.

2. The method of claim 1 wherein said non-dividing PBMC are gamma-irradiated PBMC feeder cells.

3. The method of claim 1 wherein the ratio of PBMC feeder cells to initial T lymphocytes is at least about 200:

1.

4. The method of claim 3, wherein the ratio of PBMC feeder cells to initial T lymphocytes is between about 400:

1 and 800;

1.

5. The method of claim 4 further comprising adding IL-2 to said culture medium.

6. The method of claim 5 wherein said adding IL-2 to said culture medium comprises adding at least about 10 units/ml IL-2 to said culture medium.

7. The method of claim 5, wherein the step of activating the T cell receptor complex comprises adding anti-CD3 antibody to the culture medium.

8. The method of claim 7, wherein the concentration of anti-CD3 antibody is at least about 0.5 ng/ml.

9. The method of claim 7 further comprising adding non-dividing Epstein Barr virus-transformed lymphoblastoid cells (LCL) as additional feeder cells.

10. The method of claim 9 wherein the LCL are added at a ratio LCL to initial T lymphocytes is at least about 10:

1.

11. The method of claim 1 further comprising adding IL-2 to said culture medium.

12. The method of claim 11 wherein the concentration of IL-2 is at least about 10 units/ml.

13. The method of claim 12, wherein the incubation is continued for at least about 9 days and wherein the step of adding IL-2 to thee culture medium is repeated after each 3-5 day interval.

14. The method of claim 1, wherein the step of activating the T cell receptor complex comprises adding anti-CD3 antibody to the culture medium.

15. The method of claim 14, wherein the concentration of anti-CD3 antibody is at least about 0.5 ng/ml.

16. The method of claim 15, wherein the concentration of anti-CD3 antibody is at least about 1.0 ng/ml.

17. The method of claim 1 further comprising adding non-dividing Epstein Barr virus-transformed lymphoblastoid cells (LCL) as additional feeder cells.

18. The method of claim 17 wherein the LCL are added at a ratio LCL, to initial T lymphocytes is at least about 10:

1.

19. The method of claim 18 wherein the ratio of LCL to initial T lymphocytes is between about 50:

1 and 200;

1.

20. The method of claim 1, wherein said transduction vector is a retroviral vector.

21. The method of claim 20 wherein said retroviral vector comprises a selectable marker which provides resistance to a compound that inhibits T lymphocytes.

22. The method of claim 21, further comprising the step of adding said inhibitory compound to the culture medium at least one day after addition of said transduction vector.

23. The method of claim 21 wherein said transduction vector comprises both a selectable marker which provides resistance to a compound that inhibits T lymphocytes and a selectable marker that confers sensitivity to a negative selection compound.

24. The method of claim 23 wherein said transduction vector comprises a fusion gene encoding hygromycin phosphotransferase and Herpes simplex virus thymidine kinase.