A methods study to define the performance specifications of MHC tetramer assays for immune monitoring of tumor immunotherapy

9682 Background: Monitoring tumor antigen-specific immunotherapy requires accurate and reproducible assays to identify pts that elicit an immune response. However, the performance specifications of cellular immunology assays, such as MHC-restricted tetramer assays, have not been well defined. Methods: Following informed consent and screening, 10 healthy subjects and 20 pts with melanoma (all HLA-A2.1) not receiving active therapy underwent 4 blood draws within 2 months. Peripheral blood mononuclear cells were cryopreserved and later subjected to iTAg™ Tetramer assays. Tetramers tested using sequential gating FACS analysis: MART-1 26–35, HIVgag77–85, Flu M1 58–66, CMVpp65, EBV BMLF1, AFP 325–334 and a Negative Tetramer (a non-relevant peptide control, all from Beckman Coulter). Optimal signal was obtained when at least 50,000 CD8+ events were accrued. Results: Primary objectives were to define the performance specifications of the MART-1 tetramer assay, and to identify positive and negative controls. Using...