Methods of generating natural killer cells

Abstract

Provided herein are methods of producing natural killer cells using a two-step expansion and differentiation method. Also provided herein are methods of suppressing tumor cell proliferation, of treating individuals having cancer or a viral infection, comprising administering the NK cells produced by the method to an individual having the cancer or viral infection.

1 . A method of producing a population of activated natural killer (NK) cells, comprising:
 (a) seeding a population of hematopoietic stem or progenitor cells in a first medium comprising interleukin-15 (IL-15) and, optionally, one or more of stem cell factor (SCF) and interleukin-7 (IL-7), wherein said IL-15 and optional SCF and IL-7 are not comprised within an undefined component of said medium, such that the population expands, and a plurality of hematopoietic stem or progenitor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expanding; and   (b) expanding the cells from the step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells.   
     
     
         2 . A two-step method of producing a population of activated natural killer (NK) cells, wherein a first step of said method comprises expanding a population of hematopoietic stem or progenitor cells in a first medium comprising one or more of stem cell factor (SCF), interleukin-7 (IL-7) and interleukin-15 (IL-15), and wherein said SCF, IL-7 and IL-15 are not comprised within an undefined component of said medium, and wherein a plurality of hematopoietic stem or progenitor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expanding; and
 wherein a second step of said method comprises expanding the cells from the first step in a second medium comprising interleukin-2 (IL-2), to produce activated NK cells.   
     
     
         3 . The method of  claim 1 , wherein the first medium further comprises one or more of Fms-like-tyrosine kinase 3 ligand (Flt3-L), thrombopoietin (Tpo), interleukin-2 (IL-2), or heparin. 
     
     
         4 . The method of  claim 3 , wherein the first medium further comprises fetal bovine serum or human serum. 
     
     
         5 . The method of  claim 3 , wherein the SCF is present at a concentration of about 1 to about 150 ng/mL in the first medium. 
     
     
         6 . The method of  claim 3 , wherein the Flt3-L is present at a concentration of about 1 to about 150 ng/mL in the first medium. 
     
     
         7 . The method of  claim 3 , wherein the IL-2 is present at a concentration of about 50 to about 1500 IU/mL in the first medium. 
     
     
         8 . The method of  claim 3 , wherein the IL-7 is present at a concentration of about 1 to about 150 ng/mL in the first medium. 
     
     
         9 . The method of  claim 3 , wherein the IL-15 is present at a concentration 1 to about 150 ng/mL in the first medium. 
     
     
         10 . The method of  claim 3 , wherein the Tpo is present at a concentration of about 1 to about 150 ng/mL in the first medium. 
     
     
         11 . The method of  claim 3 , wherein the heparin is present at a concentration of about 0.1 to about 30 U/mL in the first medium. 
     
     
         12 . The method of  claim 1 , wherein said IL-2 in the second step is present at a concentration 50 to about 1500 IU/mL in the second medium. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the hematopoietic stem or progenitor cells are CD34 + . 
     
     
         15 .- 16 . (canceled) 
     
     
         17 . The method of  claim 1 , wherein the NK cells are CD3 − CD56 + CD16 − . 
     
     
         18 . The method of  claim 17 , wherein the NK cells are additionally CD94 + CD117 + . 
     
     
         19 . The method of  claim 17 , wherein the NK cells are additionally CD161 − . 
     
     
         20 . The method of  claim 17 , wherein the NK cells are additionally NKG2D + . 
     
     
         21 . The method of  claim 17 , wherein the NK cells are additionally NKp46 + . 
     
     
         22 . The method of  claim 17 , wherein the NK cells are additionally CD226 + . 
     
     
         23 . A population of activated NK cells obtained by a method comprising:
 (a) seeding a population of hematopoietic stem or progenitor cells in a first medium comprising interleukin-15 (IL-15) and, optionally, one or more of stem cell factor (SCF) and interleukin-7 (IL-7), wherein said IL-15 and optional SCF and IL-7 are not comprised within an undefined component of said medium, such that the population expands, and a plurality of hematopoietic stem or progenitor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expanding; and   (b) expanding the cells from the step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells.   
     
     
         24 .- 25 . (canceled)