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.

What is claimed is: 
     
         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 . The method of  claim 1 , wherein said second medium additionally comprises one or more of fetal calf serum (FCS), transferrin, insulin, ethanolamine, oleic acid, linoleic acid, palmitic acid, bovine serum albumin (BSA) and phytohemagglutinin. 
     
     
         14 . The method of  claim 1 , wherein the hematopoietic stem or progenitor cells are CD34 + . 
     
     
         15 . The method of  claim 1 , wherein the hematopoietic stem or progenitor cells comprise hematopoietic stem or progenitor cells from human placental perfusate and hematopoietic stem or progenitor cells from umbilical cord, wherein said placental perfusate and said umbilical cord blood are from the same placenta. 
     
     
         16 . The method of  claim 1 , wherein the feeder cells in step (b) comprise mitomycin C-treated peripheral blood mononuclear cells (PBMC), K562 cells or tissue culture-adherent stem cells. 
     
     
         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 . A method of suppressing the proliferation of tumor cells comprising contacting the tumor cells with a plurality of CD94 + CD117 +  NK cells, wherein said NK cells have been produced by the method of  claim 1 . 
     
     
         25 . The method of  claim 24 , wherein said tumor cells are primary ductal carcinoma cells, glioblastoma cells, leukemia cells, acute T cell leukemia cells, chronic myeloid lymphoma (CML) cells, acute myelogenous leukemia cells, chronic myelogenous leukemia (CML) cells, lung carcinoma cells, colon adenocarcinoma cells, histiocytic lymphoma cells, multiple myeloma cells, colorectal carcinoma cells, colorectal adenocarcinoma cells, prostate cancer cells, or retinoblastoma cells.