Development Of Natural Killer Cells And Functional Natural Killer Cell Lines

Abstract

The invention relates to natural killer cells and methods for the development of immortalized natural killer cells and use of the natural killer cells. A growth and culture system is described that supports increased natural killer cell development, and provides for the establishment of continuous natural killer cell lines. Additionally, the disclosed method for generating natural killer cells may be used to produce large numbers of natural killer cells for therapeutic applications and for natural killer cell research.

1 . A method for producing natural killer (NK) cells in vitro or ex vivo, the method comprising bringing a population of hematopoietic progenitor (HSC) or light-density mononuclear cells (MNC) or stem cells into contact with IL-7, Flt3L and Jagged2, and culturing said population of hematopoietic progenitor (HSC) or stem cells to produce a NK precursor cell. 
     
     
         2 . The method according to  claim 1 , further comprising passaging said culture of hematopoietic progenitor (HSC) or stem cells in media containing Flt3L, IL-7, Jagged2 and stem cell factor (SCF) for a period of two to three months. 
     
     
         3 . The method according to  claim 2 , further comprising producing an immortalized NK precursor. 
     
     
         4 . The method of  claim 2 , wherein a synergistic interaction between IL-7 and SCF allows for the continuous culturing of an immortalized NK precursor. 
     
     
         5 . The method according to  claim 1 , wherein said Jagged2 is provided by a population of cells expressing Jagged2. 
     
     
         6 . The method according to  claims 5 , wherein said population of cells are OP-9 stromal cells. 
     
     
         7 . The method according: to  claim 4  wherein the OP-9 stromal cells are slow growing OP-9 stromal cells. 
     
     
         8 . The method according to  claim 1 , wherein the hematopoietic progenitor (HSC) or stem cell is obtained from bone marrow. 
     
     
         9 . The method according to  claim 8 , further comprising selecting Lin −  Sca-1 +  c-Kit +  hematopoietic progenitor (HSC) or stem cells. 
     
     
         10 . The method according to  claim 1 , wherein the NK precursor cells are NK1.1 + , CD3 + , TCRαβ − , TCRδγ − , CD4 − , CD8 − , CD19 − , CD25 + , CD43 + , CD45 + , CD49b − , CD51 + , CD94 + , NKG2D + , Mac-1 −/low , B220 − , c-kit + , perforin I + , granzyme B + , Notch-1 + , and cytotoxic. 
     
     
         11 . The method according to  claim 1 , further comprising contacting said co-culture with IL-2. 
     
     
         12 . The method according to  claim 1 , wherein Jagged2 is human Jagged2. 
     
     
         13 . The method according to  claim 1 , further comprising obtaining said hematopoietic progenitor (HSC) or stem cell from a human subject. 
     
     
         14 . The method according to  claim 5 , wherein said population of stromal cells expressing Jagged2 comprises a population of stromal cells having a retroviral vector sequence encoding said Jagged2. 
     
     
         15 . The method according to  claim 1 , further comprising modifying said NK precursor cell. 
     
     
         16 . The method according to  claim 15 , wherein modifying said NK precursor cell comprises decreasing or preventing infection by human immunodeficiency virus. 
     
     
         17 . A natural killer (NK) cell produced by the method according to  claim 1 . 
     
     
         18 . A natural killer (NK) cell comprising an immortalized NK precursor cell as deposited with ATCC, deposit number PTA-6651. 
     
     
         19 . A method for preparing a NK precursor cell or NK cell useful for the treatment of cancer or viral infection, comprising:
 bringing a population of Hematopoietic progenitor or stem cells into contact with IL-7,   Flt3L and a population of cells expressing Jagged2 in vitro or ex vivo;   co-culturing said Hematopoietic progenitor or stem cells and said cells expressing Jagged2 to produce a population of cells enriched for NK precursor cell or NK cell;   isolating said NK precursor or NK cell; and   admixing said NK precursor cell or NK cell and a pharmaceutically acceptable vehicle.   
     
     
         20 . The method according to  claim 19 , further comprising obtaining said population of hematopoietic progenitor (HSC) or stem cells from bone marrow of a subject to be treated with the cell preparation. 
     
     
         21 . The method according to  claim 20 , wherein light-density mononuclear cells (MNC) or HSC are obtained from the bone marrow and selecting a Lin −  Sca-1 +  c-Kit +  hematopoietic progenitor (HSC) or stem cell. 
     
     
         22 . The method according to  claim 21 , further comprising passaging said co-culture in media containing Flt3L, IL-7, and SCF for a period of about 3 months. 
     
     
         23 . The method according to  claim 19 , wherein said cell expressing Jagged2 is a stromal cell. 
     
     
         24 . The method according to  claim 23 , wherein said cell expressing Jagged2 comprises an OP-9 stromal cell. 
     
     
         25 . The method according to  claim 24 , wherein the OP-9 stromal cell is a slow growing OP-9 stromal cell. 
     
     
         26 . The method according to  claim 19 , further comprising contacting said co-culture with IL-2. 
     
     
         27 . The method according to  claim 19 , wherein Jagged2 is human Jagged2. 
     
     
         28 . The method according to  claim 19 , wherein said cell expressing Jagged2 comprises a retroviral vector sequence encoding said Jagged2. 
     
     
         29 . The method according to  claim 19 , wherein the NK precursor cell or NK cell are are NK1.1 + , CD3 + , TCRαβ − , TCRδγ − , CD4 − , CD8 − , CD19 − , CD25 + , CD43 + , CD45 + , CD49b − , CD51 + , CD94 + , NKG2D + , Mac-1 −/low , B220 − , c-kit + , perforin I + , granzyme B + , Notch-1 + , and cytotoxic. 
     
     
         30 . The method according to  claim 19 , further comprising modifying said NK precursor cell or NK cell. 
     
     
         31 . The method according to  claim 30 , wherein modifying said NK precursor cell or NK cell comprises decreasing or preventing infection by human irnrnunodeficiency virus. 
     
     
         32 . The method according to  claim 19 , further comprising producing an immortalized NK cell line. 
     
     
         33 . The method according to  claim 29 , further comprising producing an immortalized NK cell line. 
     
     
         34 . The method according to  claim 1 , further comprising administering the NK precursor cell or NK cell to a subject. 
     
     
         35 . The method according to  claim 1 , wherein about 100 to about 160 times the amount of NK precursor cells or NK cells are produced, relative to the amount of NK precursor cells or NK cells produced in the absence of Jagged2. 
     
     
         36 . A method of treating a subject thought to have a viral infection or a cancer, the method comprising:
 producing an NK cell or KIL cell according, to the method of  claim 1 ,   administering an antibody capable of recognizing an antigen produced by a cell infected by a virus or produced by a cancer cell to the subject; and   coadministering the NK cell or KIL cell to the subject.   
     
     
         37 . The method according to  claim 36 , wherein the antibody is selected from the group consisting of rituximab, alemtuzumab, tositumomab, cetuximab, gemtuzumab, trastuzumab, Oncolym, LL2, Natalizumab, 3F8, bevacizumab, ranibizumab, omalizumab, pertuzumab, visilizumab, daclizumab, volociximab, fontolizumab, and combinations thereof. 
     
     
         38 . The method according to  claim 36 , wherein the cancer is a leukaemia. 
     
     
         39 . The method according to  claim 36 , wherein the cancer is a lymphoma.