4.11. can deliver an extremely cytotoxic DNA mono-alkylating payload to CSPG4-expressing tumors at doses tolerated in vivo. < 0.0001; Scale bar 10 m, 40 magnification. To engender selective cytotoxicity for target cells, ADCs need to: a) recognize a tumor antigen expressed at higher levels by cancer cells compared with healthy cells and b) to be internalized by the target cells upon recognizing the antigen in order to expose the cell to the toxic payload. CSPG4-expression on target cells was confirmed by flow cytometry (Figure 2C). To evaluate targeting cancer cells with our ADC, we selected CSPG4 high-expressing melanoma cells (A375, A2058) and CSPG4 low-expressing melanoma (SBCL-2) and breast cancer (SKBR-3) cell lines. To confirm that the antibody was internalized by cancer cells, a reporter assay was employed for which the anti-CSPG4 IgG1 was linked to streptavidin and then conjugated to biotinylated TRi-1 Saporin (anti-CSPG4-SB-Saporin). Saporin is a 30 kDa ribosome-inhibitor unable to cross a cell membrane unaided, however Saporin is only toxic once taken up by cells, a process known to happen when it is conjugated to an internalizing antibody, as previously described [34,35]. Treatment with anti-CSPG4-SB-Saporin for 4 days decreased tumor cell viability in CSPG4-high A375 and A2058 melanoma cell lines, while it had low toxic effects on the CSPG4-low SBCL-2 melanoma and SKBR-3 breast cancer cells. As expected, none of the cell lines studied showed any loss in cell viability when treated with naked antibody or with Saporin alone (Figure 2D). In concordance, we confirmed antibody internalization by A375 melanoma cells in a time-dependent manner by confocal microscopy analysis of fluorescently labelled anti-CSPG4 antibody (Figure 2E). Together the reporter and imaging findings suggest that anti-CSPG4-IgG1 internalization occurred in CSPG4- expressing melanoma cells. These data confirmed the generation of intact anti-CSPG4-IgG1 able to be internalized in CSPG4-high expressing melanoma cells, but TRi-1 less so in CSPG4-low expressing melanoma or breast cancer cell lines. 2.2. Evaluation of Payload Toxicity across Different Cancer Cell Types We next investigated the suitability of the PDD (Figure 3A) as a potent payload for this antibody. This molecule is designed to covalently bind to the C2-amino groups of guanine bases WBP4 in the minor groove of DNA to form mono-adducts. TRi-1 Cell viability assays TRi-1 were performed in different cell types, specifically melanoma (A375, A2058), ovarian (IGROV1, TOV21G) and immune (U937, THP-1) cell lines with the PDD-based agent, a dummy payload (aniline) and mc-peg8-aniline (linker-dummy payload). The aim was to assess toxicity of the payload and of controls across different cancer cell and immune cell types. Results showed cytotoxicity for the PDD-based agent only, with IC50 values in the low nanomolar to picomolar range across multiple cell target types. As expected, there were no effects on cell viability for aniline or mc-peg8-aniline (Figure 3B). Furthermore, confocal microscopy confirmed the intracellular localization of the PDD in the nucleus of cancer cells after 3 hours incubation (Figure 3C). The results therefore show that the PDD alone affects cell viability in various cancer and monocytic-derived cell lines at different levels (Figure 3B) and may suggest that the efficacy of a PDD-bearing ADC may not only depend on the antibody target expression but also on the potency of the PDD itself. Our findings may also support the use of the PDD as a payload to target melanoma cells due to its picomolar IC50 profile in both melanoma.