Figure 4.

Platinum-mediated drug decaging from a noninternalizing ADC. a. Cysteine-selective and irreversible modification of the noninternalizing antibody F16 (anti tenascin-C) in IgG format with MMAE conjugating linker 14. IgG(F16) contains a single reactive cysteine at the C-terminal extremity of the light chain ideal for cysteine-specific modification. Briefly, a solution of F16 (7.1 μM) in sodium phosphate buffer (NaP_i) pH 7.4 was treated with 14 (40 equiv) in MeCN to a final concentration of 10% v/v. The reaction was heated to 37 °C for 1 h, and reaction progress was monitored by LC–MS. The ADC was purified by dialysis into fresh NaP_i buffer pH 7.4 with a 10 kDa MWCO overnight. b. Deconvoluted ESI–MS mass spectrum of the light-chain of F16. c. Deconvoluted ESI–MS mass spectrum of the light-chain of F16-14 that shows an exact drug-to-light-chain ratio of 1. d. Schematic of the platinum-mediated decaging of MMAE from a noninternalizing ADC. e. Cell viability of HeLa cells after treatment with F16-14 and subsequent decaging efficiency upon treatment with 20 μM K₂PtCl₄, twice daily. Cell viability was measured at day 3 by using AlamarBlue reagent. The statistical significance of the differences between groups was evaluated by using the unpaired t test. A p value <0.05 (**) was considered statistically significant. Error bars represent ± s.d. (n = 3). Experiments were performed three times.