3d adcc nk facs assay

Abstract

Herein is reported a cell analytical technology based on a three-dimensional spheroid/aggregate co-culture assay, wherein the spheroid or aggregate is formed of tumor and natural killer cells. This method is useful for the in vitro functional analysis of antibodies in single and high-throughput format.

1 . A method for the in vitro detection of effector function of an antibody comprising incubating a three-dimensional spheroid comprising tumor cells and natural killer cells or a three-dimensional aggregate comprising tumor cells and natural killer cells with the antibody. 
     
     
         2 . The method according to  claim 1  comprising the following steps:
 a) mixing natural killer cells and tumor cells, 
 b) adding about 10 4  cells per 200 μl to the wells of a multi-well plate, 
 c) centrifuging the multi-well plate to induce the formation of a three-dimensional spheroid or a three-dimensional aggregate, 
 d) adding the antibody to the wells of the multi-well plate, 
 e) incubating the multi-well plate for about 20 hours to about 72 hours, 
 f) analyzing the cells in the wells of the multi-well plate by fluorescence activated cell sorting, and 
 g) detecting the effector function of the antibody. 
 
     
     
         3 . The method according to  claim 2 , wherein the natural killer cells are human natural killer cells. 
     
     
         4 . The method according to  claim 2 , wherein the natural killer cells and tumor cells are mixed at a ratio of from 10:1 to 1:10. 
     
     
         5 . The method according to  claim 4 , wherein the ratio is from 1:2 to 1:4. 
     
     
         6 . The method according to  claim 1  or  claim 2 , wherein the incubating is for about 20 hours to about 28 hours. 
     
     
         7 . The method according to  claim 2 , wherein the centrifuging is at 1,000 rpm for 10 min. 
     
     
         8 . The method according to  claim 1 , wherein the tumor cell is a lymphoma cell. 
     
     
         9 . The method according to  claim 8 , wherein the lymphoma cell is selected from a Raji-cell, a SU-DHL4 cell, and a Z138 cell. 
     
     
         10 . The method according to  claim 2 , wherein the antibody is added at a concentration of from 15 μg/ml to 0.1 μg/ml. 
     
     
         11 . The method according to  claim 10 , wherein the antibody is added at a concentration of from 8 μg/ml to 12 μg/ml. 
     
     
         12 . A method comprising use of a three-dimensional spheroid comprising a multitude of tumor cells and natural killer cells or a three-dimensional aggregate comprising a multitude of tumor cells and natural killer cells for the determination of effector function of a combination of a multitude of antibodies with the multitude of tumor cells. 
     
     
         13 . A method for determining in vitro an antibody with effector function from a multitude of provided antibodies comprising
 a) mixing natural killer cells and tumor cells,   b) adding about 10 4  cells per 200 μl to the wells of a multi-well plate,   c) centrifuging the multi-well plate to induce the formation of a three-dimensional spheroid or a three-dimensional aggregate,   d) adding each of the provided antibodies to an individual well of the multi-well plate,   e) incubating the multi-well plate for about 20 hours to about 72 hours, and   f) determining the antibody with a ratio of more than 1 of dead cells to viable cells as the antibody with effector function.   
     
     
         14 . A kit comprising:
 a) a tumor cell labeled with a fluorescent dye,   b) isolated natural killer cells,   c) a 96-well multi-well plate, and   d) propidium iodide.