Rapid expansion method ("REM") for in vitro propagation of T lymphocytes

• US 5,827,642 A
• Filed: 10/03/1994
• Issued: 10/27/1998
• 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): Schwadron, Ronald B.
• Application Number:
  US08/317,100
• Time in Patent Office: 1,485 Days
• Field of Search: 424/93.71, 435/2, 435/240.1, 435/240.2, 435/240.21, 435/240.25, 435/372.3, 435/373, 435/375, 435/383, 435/386, 435/384, 530/788.22, 530/388.75, 530/389.6
• US Class Current: 435/2
• 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 for expanding an initial T lymphocyte population in culture medium in vitro, comprising the steps of:

adding an initial T lymphocyte population comprising at least 10⁴ T lymphocytes to a culture medium in vitro;

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 at least about 40 PBMC feeder cells for each T lymphocyte in the initial population to be expanded;

adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells;

adding anti-CD3 monoclonal antibody and IL-2 to the culture medium; and

incubating the culture, thereby producing an expanded population of antigen specific, MHC-restricted T lymphocytes.

2. The expansion method of claim 1, wherein the initial T lymphocyte population comprises at least one human CD8+ antigen-specific cytotoxic T lymphocyte.

3. The expansion method of claim 1, wherein the non-dividing PBMC feeder cells comprise gamma-irradiated PBMC feeder cells.

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

1.

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

1 and 800;

1.

6. The expansion method of claim 1, wherein the ratio of LCL feeder cells to initial T lymphocytes is at least about 10:

1.

7. The expansion method of claim 6, wherein the ratio of LCL feeder cells to initial T lymphocytes is between about 50:

1 and 200;

1.

8. The expansion method of claim 1, wherein the concentration of anti-CD3 monoclonal antibody is at least about 0.5 ng/ml.

9. The expansion method of claim 1, wherein the concentration of anti-CD3 monoclonal antibody is at least about 1.0 ng/ml.

10. The expansion method of claim 1, wherein the concentration of Il-2 is at least about 10 units/ml.

11. The expansion method of claim 10, wherein the incubation is continued for at least about 9 days and wherein the step of adding IL-2 to the culture medium is repeated after each 3-5 day interval.