Notch induced natural killer cell generation and therapeutic uses

Abstract

A method of preparing differentiated NK cells by ex vivo expansion includes the steps of; (1) isolating a plurality of CD34 + hematopoietic cells; (2) culturing the cells in a medium, wherein the medium includes an effective amount of a notch ligand and one or more cytokines selected from the group consisting of IL-7, IL-15, SCF, Flt-3, IL-3 and IL-6; and (3) maintaining the cells in culture for a duration of time sufficient to produce NK cells.

Having described the invention, the following is claimed: 
     
         1 . A method of preparing differentiated NK cells by ex vivo expansion comprising:
 isolating a plurality of CD 34+ hematopoietic cells;   culturing the cells in a medium free of stromal cells, wherein the medium includes an effective amount of a notch ligand and one or more cytokines selected from the group consisting of IL-7, IL-15, SCF, Flt-3 and IL-3, the notch ligand being immobilized on a solid substrate that facilitates interaction of the CD34+ cells and the notch ligand, wherein the solid substrate is suspended in the medium; and   maintaining the cells in culture for a duration of time sufficient to produce NK cells.   
     
     
         2 . The method of  claim 1 , the notch ligand comprising delta4 notch ligand. 
     
     
         3 . The method of  claim 1 , wherein the NK cells lack MHC class I inhibitory signals. 
     
     
         4 . The method of  claim 1 , wherein the NK cells do not express an inhibitory receptor surface protein selected from KIR and NKG2A. 
     
     
         5 . The method of  claim 1 , the solid substrate comprising a plurality of protein A beads. 
     
     
         6 . The method according to  claim 5 , wherein the protein A beads are used at a concentration of at least 0.01 μg/ml to 100 μg/ml. 
     
     
         7 . The method of  claim 1  wherein the hematopoietic cells are derived from umbilical cord blood. 
     
     
         8 . The method of  claim 1 , the medium further comprising IL-6. 
     
     
         9 . The method of  claim 1 , the predetermined time period comprising between about 2 weeks and about 6 weeks. 
     
     
         10 . The method of  claim 1 , wherein the NK cells have at least the following characteristics: CD56 bright , CD16 − , CD117 + , KIR − , CD94 low , CD11b−. 
     
     
         11 . A method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a substantially homogenous population of NK cells, wherein the NK cells are alloreactive and have at least the following characteristics: CD56 bright , CD16 − , CD117 + , KIR − , CD94 low , CD11b−. 
     
     
         12 . The method of  claim 11 , wherein the NK cells lack MHC class I inhibitory signals. 
     
     
         13 . The method of  claim 12 , the substantially homogenous population of NK cells comprising greater than about 60% homogeneous NK cells. 
     
     
         14 . The method of  claim 11 , wherein the NK cells are administered to the subject systemically. 
     
     
         15 . The method of  claim 11 , wherein the NK cells are administered directly to a tumor site in the subject. 
     
     
         16 . The method of  claim 11 , wherein the cancer is selected from the group consisting of a solid tumor, carcinoma, and a hematological malignancy. 
     
     
         17 . The method of  claim 11 , further comprising administering to the subject a therapeutically effective amount of hematopoietic stem cells. 
     
     
         18 . A method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of hematopoietic stem cells and a substantially homogeneous cell population of NK cells, wherein the NK cells have at least the following characteristics: CD56 bright , CD16 − ,CD117 + , KIR − , CD94 low , CD11b−. 
     
     
         19 . The method of  claim 18 , wherein the NK cells are alloreactive and have specific cytotoxic activity towards a host's antigen presenting cells, thereby inhibiting GVHD in the subject following hematopoietic stem cell transplantation. 
     
     
         20 . The method of  claim 19 , wherein the NK cells lack MHC class I inhibitory signals. 
     
     
         21 . The method of  claim 18 , the substantially homogenous population of NK cells comprising greater than about 60% homogeneous NK cells. 
     
     
         22 . The method of  claim 18 , wherein the cancer is selected from the group consisting of a solid tumor, carcinoma, and a hematological malignancy.