Combining targeting of the cancer-metabolome with cancer-associated stress antigens impacts transcriptomic heterogeneity, dynamics, and efficacy of engineered T-cells

The majority of cancers cannot be efficiently targeted with engineered T cell strategies. In this line, we explored how and whether γδTCR-mediated cancer metabolome targeting can be combined with the attack of cancer-associated stress antigens, like NKG2D-ligands, in order to overcome limited targeting of many tumors, and at the same time skew heterogeneous transcriptomic signatures of engineered immune cells towards more active, and less exhausted phenotypes.Single cell transcriptomic analysis revealed that only NKG2D-4-1BBCD28TM chimera reprogrammed TEGs significantly, by skewing CD4+ TEG heterogeneity towards more adhesive, proliferative, cytotoxic, and less exhausted transcriptional signatures. TEGs (T cells engineered with defined gamma delta TCRs), 'CD28 chimeras' (TEGs expressing the NKG2D-CD28wt chimera), '41BB chimeras' (TEGs expressing the NKG2D-4-1BBCD28TM chimera), '103_41BB chimeras' (TEGs expressing the 103-4-1BB chimera) were co-cultured together with RPMI 8226 cells in the presence of 10 µM PAM during 48h. The '_t' label marks samples that originated from a shared donor. After incubation, cells were sorted by expression of γδTCR+CD4+ or γδTCR+CD8+ (pan-γδTCR-PE, clone IMMU510; CD4-PB, clone RPA-T4; CD8-PerCP Cy5.5, RPA-T8) into 384-well PCR plates using ARIA II. Cells were sequenced according to the SORT-seq method based on CEL-Seq2, and libraries were sequenced with paired end sequencing on Nextseq 500.