Modified rapid expansion methods ("modified-REM") for in vitro propagation of T lymphocytes

Abstract

The present invention provides a modified rapid expansion method (termed “low-PBMC-REM” or “modified-REM”), for quickly generating large numbers of T lymphocytes, including cytolytic and helper T lymphocytes, without using the large excesses of peripheral blood mononuclear cells (PBMC) or EBV-transformed lymphoblastoid cells (LCL) characteristic of high-PBMC-REM. Clonal expansions of greater than 500-fold can be achieved within a single stimulation cycle of about 8-14 days.

1 . A method for rapidly expanding an initial T lymphocyte population in culture medium in vitro, comprising the steps of: 
 adding an initial T lymphocyte population to a culture medium in vitro;    adding to the culture medium a non-dividing mammalian cell line expressing at least one T-cell-stimulatory component, wherein said cell line is not an EBV-transformed lymphoblastoid cell line (LCL); and    incubating the culture.    
     
     
         2 . A rapid expansion method of  claim 1 , wherein said T-cell-stimulatory component is selected from the group consisting of an Fc-γ receptor, a cell adhesion-accessory molecule and a cytokine.  
     
     
         3 . A rapid expansion method of  claim 1 , wherein said T-cell-stimulatory component is selected from the group consisting of an Fc-γ receptor, a cell adhesion-accessory molecule and a cytokine, and wherein said initial T lymphocyte population is expanded at least 200-fold after an incubation period of less than about two weeks.  
     
     
         4 . A rapid expansion method of  claim 1 , wherein said T-cell-stimulatory component is selected from the group consisting of an Fc-γ receptor, a cell adhesion-accessory molecule and a cytokine, and wherein said initial T lymphocyte population is expanded at least 500-fold after an incubation period of less than about two weeks.  
     
     
         5 . A rapid expansion method of  claim 1 , wherein said T-cell-stimulatory component is selected from the group consisting of an Fc-γ receptor, a cell adhesion-accessory molecule and a cytokine, and wherein said initial T lymphocyte population is expanded at least 1000-fold after an incubation period of less than about two weeks.  
     
     
         6 . A rapid expansion method of  claim 1 , further comprising the step of adding anti-CD3 monoclonal antibody to the culture medium wherein the concentration of anti-CD3 monoclonal antibody is at least about 1.0 ng/ml.  
     
     
         7 . A rapid expansion method of  claim 1 , further comprising the step of adding IL-2 to the culture medium, wherein the concentration of IL-2 is at least about 10 units/ml.  
     
     
         8 . A rapid expansion method of  claim 1 , wherein said mammalian cell line comprises at least one cell type that is present at a frequency at least three times that found in human peripheral blood mononuclear cells (human PBMCs).  
     
     
         9 . A rapid expansion method of  claim 1 , wherein said T-cell-stimulatory component is selected from the group consisting of an Fc-γ receptor and a cell adhesion-accessory molecule.  
     
     
         10 . A rapid expansion method of  claim 1 , wherein said T-cell-stimulatory component is selected from the group consisting of a cell adhesion-accessory molecule and a cytokine.  
     
     
         11 . A rapid expansion method of  claim 1 , wherein said T-cell-stimulatory component is selected from the group consisting of an Fc-γ receptor and a cytokine.  
     
     
         12 . A rapid expansion method of  claim 1 , wherein said mammalian cell line expresses a cell adhesion-accessory molecule.  
     
     
         13 . A rapid expansion method of  claim 12 , wherein said cell adhesion-accessory molecule is selected from the group consisting of Class II MHC, Class I MHC, ICAM 1, ICAM 2, ICAM 3, CD58, CD72, fibronectin, ligand to CD27, CD80, CD86 and hyaluronate.  
     
     
         14 . A rapid expansion method of  claim 1 , wherein said mammalian cell line expresses a cytokine.  
     
     
         15 . A rapid expansion method of  claim 1 , wherein said T-cell-stimulatory component is a molecule that binds to CD21.  
     
     
         16 . A rapid expansion method of  claim 14 , wherein said cytokine is selected from the group consisting of IL-1, IL-2, IL-4, IL-6, IL-7, IL-12 and IL-15.  
     
     
         17 . A rapid expansion method of  claim 1 , further comprising the step of adding a soluble T-cell-stimulatory factor to the culture medium.  
     
     
         18 . A rapid expansion method of  claim 17 , wherein said soluble T-cell-stimulatory factor is selected from the group consisting of a cytokine, an antibody specific for a T cell surface component, and an antibody specific for a component capable of binding to a T cell surface component.  
     
     
         19 . A rapid expansion method of  claim 17 , wherein said soluble T-cell-stimulatory factor is a cytokine selected from the group consisting of IL-1, IL-2, IL-4, IL-6, IL-7, IL-12 and IL-15.  
     
     
         20 . A rapid expansion method of  claim 17 , wherein said soluble T-cell-stimulatory factor is an antibody specific for a T cell surface component, and wherein said T cell surface component is selected from the group consisting of CD4, CD8, CD11a, CD2, CD5, CD49d, CD27, CD28 and CD44.  
     
     
         21 . A rapid expansion method of  claim 17 , wherein said soluble T-cell-stimulatory factor is an antibody specific for a component capable of binding to a T cell surface component, and wherein said T cell surface component is selected from the group consisting of CD4, CD8, CD11a, CD2, CD5, CD49d, CD27, CD28 and CD44.  
     
     
         22 . A rapid expansion method of  claim 17 , wherein said soluble T-cell-stimulatory factor is a molecule that binds to CD21.  
     
     
         23 . A rapid expansion method of  claim 22 , wherein said molecule that binds to CD21 is an anti-CD21 antibody.  
     
     
         24 . A rapid expansion method of  claim 1 , further comprising the step of adding to the culture a multiplicity of peripheral blood mononuclear cells (PBMCs).  
     
     
         25 . A rapid expansion method of  claim 24 , wherein the ratio of PBMCs to initial T cells to be expanded is less than about 40:1.  
     
     
         26 . A rapid expansion method of  claim 24 , wherein the ratio of PBMCs to initial T cells to be expanded is less than about 10:1.  
     
     
         27 . A rapid expansion method of  claim 24 , wherein the ratio of PBMCs to initial T cells to be expanded is less than about 3:1.  
     
     
         28 . A rapid expansion method of  claim 1 , further comprising the step of adding to the culture a multiplicity of EBV-transformed lymphoblastoid cells (LCLs).  
     
     
         29 . A rapid expansion method of  claim 28 , wherein the ratio of LCLs to initial T cells to be expanded is less than about 10:1.  
     
     
         30 . A rapid expansion method of  claim 1 , wherein the initial T lymphocyte population comprises at least one human CD8+ antigen-specific cytotoxic T lymphocyte (CTL).  
     
     
         31 . A rapid expansion method of  claim 1 , wherein the initial T lymphocyte population comprises at least one human CD4+ antigen-specific helper T lymphocyte.  
     
     
         32 . A method of genetically transducing a human T cell, comprising the steps of: adding an initial T lymphocyte population to a culture medium in vitro; adding to the culture medium a non-EBV-transformed mammalian cell line expressing a T-cell-stimulatory component; and incubating the culture; and adding a vector to the culture medium.  
     
     
         33 . A genetic transduction method of  claim 32 , wherein the vector is a retroviral vector containing a selectable marker providing resistance to an inhibitory compound that inhibits T lymphocytes, and wherein the method further comprises the steps of: continuing incubation of the culture for at least one day after addition of the retroviral vector; and adding said inhibitory compound to the culture medium after said continued incubation step.  
     
     
         34 . A genetic transduction method of  claim 32 , further comprising adding a multiplicity of human PBMCs.  
     
     
         35 . A genetic transduction method of  claim 34 , wherein the ratio of PBMCs to initial T cells is less than about 40:1.  
     
     
         36 . A genetic transduction method of  claim 32 , further comprising adding non-dividing EBV-transformed lymphoblastoid cells (LCL).  
     
     
         37 . A genetic transduction method of  claim 36 , wherein the ratio of LCL to initial T cells is less than about 10:1.  
     
     
         38 . A method of generating a REM cell line capable of promoting rapid expansion of an initial T lymphocyte population in vitro, comprising the steps of: depleting one or more cell types from a human PBMC population to produce a cell-type-depleted PBMC population, using said cell-type-depleted PBMC population in place of non-depleted PBMCs in an hp-REM protocol to determine the contribution of the depleted cell type to the activity provided by the non-depleted PBMCs, identifying a T cell stimulatory activity provided by said depleted cell type, and transforming a mammalian cell line with a gene allowing expression of said T cell stimulatory activity.  
     
     
         39 . A method of generating a REM cell line according to  claim 38 , wherein said T-cell-stimulatory component is selected from the group consisting of an Fc-γ receptor, a cell adhesion-accessory molecule and a cytokine.  
     
     
         40 . A REM cell line capable of stimulating rapid expansion of an initial T lymphocyte population in vitro, comprising a mammalian cell line generated according to the method of  claim 38 .  
     
     
         41 . A REM cell line according to  claim 40 , wherein said cell line expresses a cell adhesion-accessory molecule.  
     
     
         42 . A REM cell line according to  claim 41 , wherein said cell adhesion-accessory molecule is selected from the group consisting of Class II MHC, Class I MHC, ICAM 1, ICAM 2, ICAM 3, CD58, CD72, fibronectin, ligand to CD27, CD80, CD86 and hyaluronate.  
     
     
         43 . A REM cell line according to  claim 40 , wherein said cell line expresses an Fc-γ receptor.  
     
     
         44 . A REM cell line according to  claim 40 , wherein said cell line expresses at least one T cell stimulatory cytokine.  
     
     
         45 . A REM cell line according to  claim 44 , wherein said T cell stimulatory cytokine is selected from the group consisting of IL-1, IL-2, IL-6, IL-7, IL-12 and IL-15.  
     
     
         46 . A REM cell line according to  claim 40 , wherein said cell line expresses a molecule that binds to CD21.  
     
     
         47 . A culture medium capable of rapidly expanding an initial T lymphocyte population in vitro comprising a REM cell line according to  claim 40 .  
     
     
         48 . A culture medium according to  claim 47 , further comprising an exogenous cytokine.  
     
     
         49 . A culture medium according to  claim 47 , further comprising a multiplicity of exogenous cytokines, wherein said multiplicity comprises at least one interleukin.  
     
     
         50 . A culture medium according to  claim 49 , wherein said interleukin is selected from the group consisting of IL-1, IL-2, IL-6, IL-7, IL-12 and IL-15.  
     
     
         51 . A culture medium according to  claim 47 , further comprising a molecule that binds to CD21.  
     
     
         52 . A culture medium according to  claim 51 , wherein said molecule that binds to CD21 is an anti-CD21 antibody.  
     
     
         53 . A culture medium according to  claim 49 , further comprising an anti-CD3 monoclonal antibody.