METHODS AND COMPOSITIONS FOR DETERMINING THE POTENCY OF A THERAPEUTIC CELLULAR COMPOSITION
This disclosure provides a potency assay for evaluating the potency of facilitating cells and/or alpha beta TCR+ T cells.
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.