PRELIMINARY VALIDATION OF AN ACTIVATION ASSAY FOR EX-VIVO ACTIVATED T-CELLS UTILIZED IN CANCER-IMMUNOTHERAPY

An in vitro assay that measures the activation level of ex vivo activa ted (EVA) T cells currently being used in the adoptive immunotherapy o f metastatic renal cell carcinoma has been developed. This assay is ba sed on the ability of activated, but not resting, T cells to prolifera te in response to the protein kinase C activator, phorbol myristate ac etate (PMA). To utilize this assay for in-process monitoring and contr ol, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of H-3-thymidine incorporated, was demonstrated to have intra-assay coefficients of variation (cv's) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at leas t 6 weeks with little or no deterioration in their ability to prolifer ate in response to PMA. Using these cells, inter-assay cv's that were typically less than 15% were obtained by individual analysts, and over all cv's of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is ve ry reasonable for a cellular assay. Further validation of this assay w ill address the issues of sensitivity, linearity and selectivity. To d ate, this assay has been used to analyze over 90 patient EVA cell samp les and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cells used therapeutically. ( C) 1994 John Wiley and Sons, Inc.