Ex vivo nk cell differentiation from cd34+ hematopoietic cells

Abstract

The present invention relates to the ex vivo differentiation of NK cells from CD34+ hematopoietic stem cells. Such NK cells and their progenitor cells can be used in therapies of a broad range of malignancies. In the present invention it is shown that IL-12 modulates ex vivo NK cell differentiation. Specific, we achieved significantly higher expression of KIR, CD16 and CD62L in the presence of IL-12 in the cell culture system. The induction of receptor expression by IL-12 occurred predominantly on an augmented population of CD33+NKG2A+ NK cells early during NK cell differentiation. These cells further show enhanced cytolytic activity against MHC class I positive AML targets. In line with the enhanced CD16 expression, IL-12 modulated ex vivo generated NK cells exhibit an improved antibody-dependent-cytotoxicity, using anti CD20 antibody on various B cell targets. Additional to the enhanced expression of CD62L, we show that this cell population consists of a specific chemokine receptor profile. By showing an increased capacity for adhesion to lymphendothelial cells and a specific chemokine receptor profile, we show that IL-12 provided the ex vivo generated NK cells with specific tissue-homing abilities.

1 . A method for producing NK cells said method comprising
 i—providing a sample of human CD34 positive cells,   ii—expanding said CD34 positive cells ex vivo,   iii—culturing CD34 positive cells obtained in step ii ex vivo in an NK-cell differentiation medium,   said method characterized in that said NK-differentiation medium comprises IL-12.   
     
     
         2 . A method according to  claim 1 , wherein said NK-differentiation medium comprises between 20 pgram/ml and 20 ngram/ml IL-12. 
     
     
         3 . A method according to  claim 2 , wherein said NK-differentiation medium comprises between 0.2 ngram/ml and 2 ngram/ml IL-12. 
     
     
         4 . A method according to  claim 1 , wherein step ii) is performed with a culture medium comprising three or more of stem cell factor (SCF), flt-3Ligand (FLT-3L), thrombopoietin (TPO) and interleukin-7 (IL-7) and three or more of granulocyte-macrophage-colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), interleukin-6 (IL-6), leukaemia-inhibitory factor (LIF) and Macrophage-inflammatory protein-1 alpha (MIP-I alpha). 
     
     
         5 . A method according to  claim 1 , wherein step iii) is performed with an NK-cell differentiation medium comprising one or more of IL-2 and IL-15; and one or more of IL-7 and SCF; and three or more GM-CSF, G-CSF, IL-6, LIF and MIP-I alpha. 
     
     
         6 . A method according to  claim 1 , wherein step ii) is performed in culture medium comprising low molecular weight heparin. 
     
     
         7 . A collection of NK-cells obtainable by a method according to  claim 1 . 
     
     
         8 . A collection of NK-cells according to  claim 7 , wherein at least 20% of the CD56 positive cells express CD62L; at least 10% of the CD56 positive cells express KIR; or at least 20% of the CD56 positive cells express CD16. 
     
     
         9 . A collection of NK-cells according to  claim 8 , that comprises NKG2A and CD33 positive cells and wherein at least 50% of the CD56 positive, NKG2A positive and CD33 positive cells are positive for both KIR and CD62L. 
     
     
         10 . A cell bank comprising a collection of NK-cells according to  claim 7 . 
     
     
         11 . A method for killing cancer cells with NK-cells, said method characterized in that said NK-cells comprise a collection according to  claim 7 . 
     
     
         12 . A collection of NK-cells according to  claim 7 , for the preparation of a cell transplant. 
     
     
         13 . A collection of NK-cells according to  claim 7 , for use in the treatment of cancer, wherein preferably said cancer treatment further comprises treatment with an antibody specific for an antigen present on cells of said cancer. 
     
     
         14 . A collection of NK-cells according to  claim 7 , for use in the treatment of cancer, wherein said cancer treatment further comprises treatment with an antibody specific for an antigen present on cells of said cancer, and wherein said cancer is a cancer of hematopoietic origin. 
     
     
         15 . A collection of NK-cells according to  claim 7 , for use, in the treatment of cancer, wherein preferably said cancer treatment further comprises treatment with an antibody specific for an antigen present on cells of said cancer, and wherein said antibody is specific for CD20.