Activation and Expansion of T-Cells Using an Engineered Multivalent Signaling Platform as a Research Tool

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.

1 . An engineered K562 cell, comprising at least one membrane bound antibody or fragment thereof capable of activating and/or stimulating a T cell. 
     
     
         2 . The engineered cell of  claim 1 , wherein the at least one membrane bound antibody or fragment thereof comprises a membrane bound anti-CD3 scFv and/or a membrane bound anti-CD28 scFv. 
     
     
         3 . The engineered cell of  claim 2 , wherein the membrane bound anti-CD3 scFv and/or the membrane bound anti-CD28 scFv are each derived from human. 
     
     
         4 . The engineered cell of  claim 1 , further modified to express a co-stimulatory molecule selected from the group consisting of CD80, CD86, 4-1BBL, OX40L, ICOS-L, ICAM, PD-L1, and PD-L2. 
     
     
         5 . The engineered cell of  claim 1 , further modified to express a cytokine selected from the group consisting of IL-2, IL-15, IL-4, GM-CSF, TNF-α, and IFN-γ. 
     
     
         6 . The engineered cell of  claim 1 , wherein said T cell is a CD4+ T cell, a CD8+ T cell, or a regulatory T cell. 
     
     
         7 . A method for activating and/or stimulating a population of T cells, comprising contacting said population of T cells with the engineered K562 cell of  claim 1 . 
     
     
         8 . A method for activating and/or stimulating a population of T cells, comprising:
 contacting said population of T cells with an engineered K562 cell comprising at least one membrane bound antibody or fragment thereof capable of activating and/or stimulating a T cell.   
     
     
         9 . The method of  claim 8 , wherein the at least one membrane bound antibody or fragment thereof comprises a membrane bound anti-CD3 scFv and/or a membrane bound anti-CD28 scFv. 
     
     
         10 . The method of  claim 8 , wherein said engineered K562 cell is further modified to express a co-stimulatory molecule selected from the group consisting of CD80, CD86, 4-1BBL, OX40L, ICOS-L, ICAM, PD-L1, and PD-L2. 
     
     
         11 . The method of  claim 8 , wherein said engineered K562 cell is further modified to express a cytokine selected from the group consisting of IL-2, IL-15, IL-4, GM-CSF, TNF-α, and IFN-γ. 
     
     
         12 . The method of  claim 8 , wherein said T cells comprise a CD4+ T cell, a CD8+ T cell, or a regulatory T cell. 
     
     
         13 . The method of  claim 8 , further comprising culturing the population of activated and/or stimulated T cells under conditions and time sufficient to induce cell division. 
     
     
         14 . A method for expanding a population of T cells, comprising
 contacting said population of T cells with an engineered K562 cell, thereby producing a population of activated and/or stimulated T cells, wherein said engineered K562 cell comprises at least one membrane bound antibody or fragment thereof capable of activating and/or stimulating a T cell; and culturing said population of activated and/or stimulated T cells under conditions and time sufficient to induce cell division.   
     
     
         15 . The method of  claim 14 , wherein the at least one membrane bound antibody or fragment thereof comprises a membrane bound anti-CD3 scFv and/or a membrane bound anti-CD28 scFv. 
     
     
         16 . The method of  claim 14 , wherein said engineered K562 cell is further modified to express a co-stimulatory molecule selected from the group consisting of CD80, CD86, 4-1BBL, OX40L, ICOS-L, ICAM, PD-L1, and PD-L2. 
     
     
         17 . The method of  claim 14 , wherein said engineered K562 cell is further modified to express a cytokine selected from the group consisting of IL-2, IL-15, IL-4, GM-CSF, TNF-α, and IFN-γ. 
     
     
         18 . The method of  claim 14 , wherein said T cells comprise a CD4+ T cell, a CD8+ T cell, or a regulatory T cell. 
     
     
         19 . A method of treating cancer in a subject in need thereof, comprising:
 isolating a population of T cells from said subject; contacting said population of T cells with an engineered K562 cell comprising a membrane bound anti-CD3 scFv and/or a membrane bound anti-CD28 scFv, thereby producing a population of activated T cells;   culturing said population of activated T cells under conditions and time sufficient to induce cell division, thereby producing a population of expanded T cells; and   infusing a therapeutically effective amount of said population of expanded T cells into said subject, thereby treating cancer in said subject.   
     
     
         20 . The method of  claim 19 , wherein said engineered K562 cell is further modified to express a cytokine selected from the group consisting of IL-2, IL-15, IL-4, GM-CSF, TNF-α, and IFN-γ. 
     
     
         21 . A method of treating cancer in a subject in need thereof, comprising:
 isolating a population of antigen-specific T cells from said subject;   contacting said population of antigen-specific T cells with an engineered K562 cell comprising a membrane bound anti-CD3 scFv and/or a membrane bound anti-CD28 scFv, thereby producing a population of activated antigen-specific T cells;   culturing said population of activated antigen-specific T cells under conditions and time sufficient to induce cell division, thereby producing a population of expanded antigen-specific T cells; and   infusing a therapeutically effective amount of said population of expanded antigen-specific T cells into said subject, thereby treating cancer in said subject.   
     
     
         22 . The method of  claim 21 , wherein said engineered K562 cell is further modified to express a cytokine selected from the group consisting of IL-2, IL-15, IL-4, GM-CSF, TNF-α, and IFN-γ.