Fig. 5: Combination of the phosphoramidate-based linker systems with structurally diverse hydroxy-containing cytotoxins exhibiting diverse MOAs.

a DAR8 ADCs from trastuzumab (tras/anti-HER2, red), datopotamab (Dato/anti-Trop2, green) or brentuximab (bren/anti-CD30, blue) in combination with linker 9 for aromatic (highlighted with pink background) and linker 15 for aliphatic payloads (highlighted with purple background). b Ganetespib and ON01300 have been conjugated via linker 9; BAY-2402234, DHODH-IN-16, SNX2112, paclitaxel and LSN3154567 have been conjugated via linker 15. Pink shading refers to aromatic payloads, purple shading refers to primary aliphatic payloads, blue shading refers to secondary aliphatic payloads and green shading refers to tertriary aliphatic payloads. c HIC chromatograms to analyze ADC homogeneity from every payload. A DAR of 8 has been confirmed by MS. d Potency and selectivity of the different ADCs for the targeted cell line. Tras-ADCs were tested on N87 (red solid circle), SKBR-3 (red solid downward-pointing triangle) and HCC1569 (red solid upward-pointing triangle) (HER2 + ), bren-ADCs were tested on L-540 (blue half-filled circles) and Karpas-299 cells (blue half-filled diamonds) (CD30 + ), dato-ADCs were tested on H441 (green open circles), MDA-MB-468 (green open diamonds) and HCC-78-cells (green open squares) (Trop2 + ), while the bren conjugates served as isotypes for the HER2+ and Trop2 + -cell lines and vice versa. The ADC selectivity is plotted as the ratio of IC₅₀ isotype-/IC₅₀ targeted ADC. The absolute potencies of the ADCs are plotted for their target-positive cell lines. Pink columns refer to aromatic payloads, purple columns refer to primary aliphatic payloads, blue columns refer to secondary aliphatic payloads and green columns refer to tertiary aliphatic payloads. The black bars show the mean values for the eight cell lines (targeted via three distinct receptor-antibody-pairs) and serve as orientation to compare the different payloads. e Correlation between the proliferation inhibition IC₅₀ of unconjugated payloads (x-axis) over the ADCs on the same cell line (y-axis) (N87, red solid circle; SKBR-3 red solid downward-pointing triangle; HCC1569, red solid upward-pointing triangle; L-540, blue half-filled circles; Karpas-299, blue half-filled diamonds; (CD30 + ); H441, green open circles; MDA-MB-468, green open diamonds; HCC-78-cells, green open squares). ADCs above the dotted line were inactive at the highest tested concentration of 80 nM and excluded from the correlation. Dose-response curves for ADCs and payloads are shown in Fig. S8.