Selection of antigen loss variants during melanoma T-cell therapy

In this study we investigated the dynamics of CRISPR-Cas9 engineered antigen loss variant in a moouse model of T cell therapy of melanoma (HCmel12 transplantation model). Mixtures of wild type and antigen loss variant melanoma cells were inoculated in syngeneic mice and treated with adoptive transfer of Pmel (also known as gp100-specific T cell receptor (TCR) transgenic CD8+ T cells (Pmel-1 T cells). Pmel-1 T cells regonize the endogenous antigen Pmel/gp100 in mouse melanoma cells presented on MHC class I (H2-Db). The selection of Pmel/gp100 antigen loss variants was monitored by amplicon sequencing using the MiSeq platform. In order to normalize for tumor cell content in DNA isolated from whole tumor tissue we determined the frequency of a tumor cell private mutation in p53. In combination with mathematical modeling, we determined the impact of subclone fitness variability on the selection of antigen loss variants during T cell therapy.