Method for culturing natural killer cells using t cells

Abstract

The present invention relates to a method for producing natural killer cells using T cells, and more particularly, to a method for producing natural killer cells, which comprises culturing seed cells using CD4(+) T cells as feeder cells. The method for producing natural killer cells using T cells according to the present invention is a method capable of producing natural killer cells by selectively proliferating only natural killer cells from a small amount of seed cells while maintaining the high killing activity of the natural killer cells. The method of the present invention can produce a large amount of natural killer cells that can be frozen, and thus is useful for commercialization of cell therapeutic agents.

1 . A method of preparing natural killer cell comprising culturing seed cells by using CD4(+) T cells as feeder cells. 
     
     
         2 . The method of  claim 1 , wherein the CD4(+) T cells are CD4(+) T cells isolated ex vivo, CD4(+) T cells expansion-cultured ex vivo, or a CD4(+) T cell line. 
     
     
         3 . The method of  claim 2 , wherein the CD4(+) T cell line is H9 or HuT78. 
     
     
         4 . The method of  claim 1 , wherein the CD4(+) T cells are inactivated whose division and proliferation are inhibited, or non-inactivated cells. 
     
     
         5 . The method of  claim 1 , wherein the seed cells are one or more types selected from the group consisting of peripheral blood cells, peripheral blood leukocytes, PBMCs (peripheral blood mononuclear cells), enriched natural killer cells, and isolated natural killer cells. 
     
     
         6 . The method of  claim 5 , wherein the seed cells are CD3(+) cell-depleted cells. 
     
     
         7 . The method of  claim 1 , wherein the culturing is carried out by mixing feeder cells and the seed cells with a ratio of at least 1:1. 
     
     
         8 . The method of  claim 7 , wherein the culturing is carried out by mixing feeder cells and the seed cells with a ratio of 2:1-20:1. 
     
     
         9 . The method of  claim 1 , wherein the culturing is carried out for 5-60 days. 
     
     
         10 . The method of  claim 1 , wherein the culturing is carried out in a medium containing a T cell-stimulating antibody, which has a low affinity for T cells, and an interleukin protein. 
     
     
         11 . The method of  claim 10 , wherein the antibody is at least one selected from the group consisting of OKT3, UCHT1 and HIT3a antibodies. 
     
     
         12 . The method of  claim 10 , wherein the interleukin protein is at least one selected from the group consisting of IL-2, IL-12, IL-15, IL-18, and IL-21. 
     
     
         13 . The method of  claim 1 , further comprising re-stimulating CD3(+) cell-depleted seed cells by using 2˜20 times CD4(+) T cells as feeder cells, compared to the seed cell. 
     
     
         14 . The method of  claim 13 , wherein the re-stimulation is performed at intervals of 5-12 days. 
     
     
         15 . The method of  claim 13 , wherein the re-stimulation is repeated at least once. 
     
     
         16 . The method of  claim 13 , wherein the CD4(+) T cells used in the re-stimulation step are H9 or HuT78. 
     
     
         17 . The method of  claim 13 , wherein the CD4(+) T cells used in the re-stimulation step are inactivated whose division and proliferation are inhibited, or non-inactivated cells. 
     
     
         18 . The method of  claim 13 , the method comprising culturing seed cells in a medium containing a T cell-stimulating antibody, which has a low affinity for T cells, and an interleukin protein. 
     
     
         19 . The method of  claim 18 , wherein the antibody is at least one selected from the group consisting of OKT3, UCHT1 and HIT3a antibodies. 
     
     
         20 . The method of  claim 18 , wherein the interleukin protein is at least one selected from the group consisting of IL-2, IL-12, IL-15, IL-18, and IL-21. 
     
     
         21 . Natural killer cells produced by the method of  claim 1 . 
     
     
         22 . A composition for preventing or treating cancer or infectious disease, which comprises the natural killer cells of  claim 21  as an active ingredient. 
     
     
         23 . A method for preventing or treating cancer or infectious disease, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising the natural killer cells of  claim 21  as an active ingredient.