Glo substrate

To explore the functional response of MAGE-A4-targeted TCR T cell therapies to common HLA-A 02 subtypes, MAGE-A4+ HLA-A 02-negative tumor cell lines were transduced using lentiviral vectors expressing HLA-A 02 alleles, green fluorescent protein, and puromycin-N-acetyltransferase. These lines were selected through culture in puromycin, and comparable levels of transgene expression were confirmed by flow cytometry (data not shown). The ability of these lines to induce an afami-cel response was subsequently assessed by IFN-γ cell enzyme-linked immunoassay (ELISA). Generation of afami-cel TCR T cells was described previously. 17 For the cell-based ELISA, 384-well plates were coated with IFN-γ capture antibodies overnight followed by plating of target cells (10 4 /well), effector T cells (10 4 /well) and/or peptide, or IFN-γ standards. After 48 h, the plates were washed and the assay was carried out following the manufacturer's protocol (Human IFN-gamma DuoSet ELISA, R&D Systems, Minneapolis, MN), with the use of a luminescent HRP substrate (Glo Substrate, R&D Systems). Luminescence was measured using a FLUOstar Omega plate reader (BMGLabtech, Cary, NC). Peptide response curve fitting was performed using the drc R package using a three-parameter log-logistic function. 25