Once the mutations have been identified (through DNA and RNA sequencing of tumour and normal tissue), they clone them into plamids and express these in E. coli (+/- listeriolysin O to enable presentation via both MHC class I or class II). Then for each patient, these get co-cultured with their APCs (monocyte derived dendritic cells) and then T-cells are added and incubated overnight. After this, neoantigen-specific responses are detected by comparing cytokine production in response to each of these against a non-immunogenic control protein.
GNCA have shown that using inhibitory neoantigens will lead to PD [1], while others have shown they reduce the efficacy of the vaccine [2].