Effector cells (E = PBMCs) obtained from a melanoma patient (for ImmTAC-gp100) or healthy donors (for ImmTAC-nybr1, ImmTAC-mageA3 and ImmTAC-nyeso) were incubated with Ag+ cell lines that present target peptide-HLA or Ag- cell lines that are HLA-relevant but do not present target peptide. Cells were incubated in the presence or absence of ImmTAC molecules. IFNγ release was assessed by ELISpot assay. Ag+ cells: Mel526, CAMA1 A2b2m, EJM and IM9 for ImmTAC-gp100, ImmTAC-nybr1, ImmTAC-mageA3 and ImmTAC-nyeso respectively. Ag- cells: A375, MDA MB 231, Colo205 and Mel526 for ImmTAC-gp100, ImmTAC-nybr1, ImmTAC-mageA3 and ImmTAC-nyeso, respectively. Statistical differences between Ag+ and Ag- cells in the presence of effector cells (E) + ImmTAC was measured using a Two-way ANOVA where *** p<0.0001, **p<0.01. If unmarked, results were not significant.