Methods and compositions for determining the potency of a therapeutic cellular composition

Abstract

This disclosure provides a potency assay for evaluating the potency of facilitating cells and/or alpha beta TCR+ T cells.

What is claimed is: 
     
         1 . A method comprising:
 contacting a sample comprising FCs and/or alpha beta TCR+ T cells with a mitogenic stimulus; and   determining the number of FCs and/or alpha beta TCR+ T cells in the sample in which ribosomal S6 protein is phosphorylated (pS6).   
     
     
         2 . The method of  claim 1 , wherein the sample comprises about 100,000 to about 2,000,000 FCs and/or alpha beta TCR+ T cells. 
     
     
         3 . The method of  claim 1 , wherein the mitogenic stimulus is selected from the group consisting of phorbol 12-myristate 13-acetate (PMA) with ionomycin, phytohaemagglutinin (PHA), concanavalin A (conA), and pokeweed mitogen (PWM). 
     
     
         4 . The method of  claim 1 , wherein the sample is contacted with the mitogenic stimulus for a period of time from about 5 mins to about 60 mins. 
     
     
         5 . The method of  claim 1 , wherein the sample is contacted with the mitogenic stimulus for a period of time from about 20 mins to about 30 mins. 
     
     
         6 . The method of  claim 1 , where the number of FCs and/or alpha beta TCR+ T cells in the sample in which pS6 is phosphorylated is determined using a fluorescently-labeled antibody against pS6. 
     
     
         7 . The method of  claim 6 , wherein binding of the fluorescently-labeled antibody is detected using FACS. 
     
     
         8 . The method of  claim 1 , wherein at least 30% of the FCs in the sample are pS6+. 
     
     
         9 . The method of  claim 1 , wherein at least 30% of the alpha beta TCR+ T cells in the sample are pS6+. 
     
     
         10 . The method of  claim 1 , wherein the method is performed prior to freezing the FCs and/or alpha beta TCR+ T cells. 
     
     
         11 . The method of  claim 1 , wherein the method is performed on FCs and/or alpha beta TCR+ T cells that have been frozen. 
     
     
         12 . A method of determining the potency of a biological sample comprising facilitating cells (FCs) and/or alpha beta TCR+ T cells, the method comprising:
 contacting a portion of the biological sample comprising FCs and/or alpha beta TCR+ T cells with a mitogenic stimulus;   determining the number of FCs and/or alpha beta TCR+ T cells in which ribosomal S6 protein is phosphorylated (pS6) in the portion of the sample, wherein the number of FCs and/or alpha beta TCR+ T cells in which pS6 is phosphorylated in the portion of the sample is indicative of the potency of the FCs and/or alpha beta TCR+ T cells, respectively, in the biological sample; and   determining a therapeutic dose of FCs and/or alpha beta TCR+ T cells in the biological sample based on the number of FCs and/or alpha beta TCR+ T cells in which ribosomal S6 protein is phosphorylated in the portion of the sample, wherein a therapeutic dose is a dose of FCs and/or alpha beta TCR+ T cells in the biological sample that allows engraftment in the absence of graft-vs-host-disease.   
     
     
         13 . The method of  claim 12 , wherein the sample comprises about 100,000 to about 2,000,000 FCs and/or alpha beta TCR+ T cells. 
     
     
         14 . The method of  claim 12 , wherein the mitogenic stimulus is selected from the group consisting of phorbol 12-myristate 13-acetate (PMA) with ionomycin, phytohaemagglutinin (PHA), concanavalin A (conA), and pokeweed mitogen (PWM). 
     
     
         15 . The method of  claim 12 , wherein the sample is contacted with the mitogenic stimulus for a period of time from about 5 mins to about 60 mins. 
     
     
         16 . The method of  claim 12 , wherein the sample is contacted with the mitogenic stimulus for a period of time from about 20 mins to about 30 mins. 
     
     
         17 . The method of  claim 12 , where the number of FCs and/or alpha beta TCR+ T cells in the sample in which pS6 is phosphorylated is determined using a fluorescently-labeled antibody against pS6. 
     
     
         18 . The method of  claim 17 , wherein binding of the fluorescently-labeled antibody is detected using FACS. 
     
     
         19 . The method of  claim 12 , wherein at least 30% of the FCs in the sample are pS6+. 
     
     
         20 . The method of  claim 12 , wherein at least 30% of the alpha beta TCR+ T cells in the sample are pS6+. 
     
     
         21 . The method of  claim 12 , wherein the method is performed prior to freezing the FCs and/or alpha beta TCR+ T cells. 
     
     
         22 . The method of  claim 12 , wherein the method is performed on FCs and/or alpha beta TCR+ T cells that have been frozen.