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 the Negative Tetramer, the low limit of detection (LLD) was 0.034% CD8+ cells. Positivity (> 0,034% MART-1-specific CD8+ cells) in at least 2 of 4 samples was found in 26 of 30 subjects. 21 were positive in all samples analyzed. Higher reactivity was noted in pts with melanoma compared to healthy volunteers (p < 0.05). HIVgag77–85 was not a universal negative control, while AFP tetramer was usually negative. The widely used Flu M1 was not a reliable positive control, while the CMV and EBV were positive in seropositive subjects. Using the MART-1 and EBV tetramers, a functional sensitivity of 0.025–0.050% and a reportable range up to 7% CD8+ cells were defined. Conclusions: MART-1-tetramer assay is precise enough at the LLD and within the reportable range to allow the definition of a positive response, making it a robust endpoint for immunotherapy studies. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Beckman Coulter, Inc.; Pfizer Pfizer Pfizer Inc.