^a CD8+ PBL from patient 3713 were stimulated in vitro with mature autologous DCs pulsed with mutated peptides predicted to bind with high affinities to HLA-A*0201 and were restimulated twice with autologous peptide-pulsed PBMCs. Recognition of relevant target cells was evaluated on the basis of IFNγ secretion after overnight coculture. 2 peptides, one derived from a mutation in the AHNAK protein and one derived from a mutation in the SRPX protein, stimulated T cells that specifically recognized peptide, COS7 cells expressing HLA-A*0201 that had been transfected with the relevant minigene, and the autologous tumor cell line.

^b TIL from patient 3713 were cocultured overnight with autologous DCs electroporated with in vitro transcribed (IVT) RNA encoding a fragment of the mutated SRPX protein. CD3+ CD8+ 41BB+ cells were sorted by FACS and expanded in the presence of allogeneic feeder cells, α-CD3, and IL2. The resulting T cell population specifically recognized peptide, COS7 cells expressing HLA-A*0201 that had been transfected with the relevant minigene, and the autologous tumor cell line.

^c TCR α and β chains in T cell populations were identified by 5' RACE using degenerate constant region primers and were cloned into retroviral vectors. These were then used to transduce PBL from patient 3713, and the function of the resulting genetically modified T cells was evaluated on the basis of IFNγ secretion after overnight coculture.

^d HBV: hepatitis B core virus peptide used as a negative control with high binding affinity to HLA-A*0201.

^e AHNAK: neuroblast differentiation-associated protein, also known as desmoyokin.

^f SRPX: sushi repeat-containing protein.