Fig 2: An affinity-enhanced bispecific CD3/TCR ImmTAC® molecule mediates T cell activation and redirected killing of melanoma, lung, and ovarian tumor cells expressing PRAME. HLA-relevant PRAME+ melanoma (A), NSCLC (B), and ovarian (C) tumor cells were incubated with PBMC effector cells in the presence of increasing concentrations of the PRAME ImmTAC® IMC-F106C. Ovarian TYK-nu (PRAME–/HLA-A*02:01+) and lung NCI-H1693 (PRAME+/HLA-irrelevant) cell lines were included as controls. Effector cells were also cultured with 2 nM IMC-F106C in the absence of target cells, as control. ImmTAC®-mediated T cell activation was determined by IFNγ ELISpot (left panels). $ represents spots too numerous to count by the software and attributed the highest counted value. IMC-F106C-mediated T cell killing was determined as % tumor cell cytolysis in xCELLigence impedance-based assays (right panels). Data were obtained using three healthy PBMC donors and representative data are shown.

Fig 3: PRAME-targeting ImmTAC® redirects PD-1+ TILs to kill tumor cells, an effect that is reduced against PD-L1-expressing tumor cells, but alleviated by anti-PD-1 Ab. (A) CD8+ TILs from a melanoma tumor sample were FACS sorted into programmed cell death protein 1 (PD-1)+ and PD-1– populations. (B) ImmTAC®-mediated redirection of PD-1–TILs (left panels) and PD-1+ TILs (right panels) resulted in the killing of PD-L1- tumor cells (MEL624, top panels) and PD-L1+ tumor cells (PD-L1 transduced MEL624 cells, bottom panel). The experiment was performed over 4 days in the absence (white circles) or presence (black circles) of blocking anti-PD-1 antibody. (C) % cytolysis area under the curve (AUC) was calculated for the conditions presented in (B), using increasing ImmTAC® concentrations in the presence (black) or absence (white) of anti-PD-1 antibody. Data are shown as mean ± SEM.

Fig 4: PRAME protein is homogeneously expressed in patient tumors of multiple types and retained in metastasis. (A) Representative IHC staining images of primary tumor types. PRAME prevalence in (B) primary tumor tissues, (C) primary and metastatic melanoma and lung tumor, and (D) acral and mucosal cutaneous melanoma subsets. The number of samples analyzed is indicated for each tumor type.

Fig 5: SLLQHLIGL PRAME-derived peptide is presented in the context of HLA class I across different cancer indications. Mirror plots of LC-MS/MS spectra obtained for native peptide (SLLQHLIGL, top) and stable isotope-labeled peptide (SLLQHL*IGL, bottom) in representative examples of cutaneous melanoma, ovarian carcinoma, and NSCLC samples.