Disulfide re-bridging reagents for single-payload antibody-drug conjugates

Abstract

Numerous antibody-drug conjugate (ADC) linker technologies exist for the synthesis of ADCs with drug-to-antibody ratios (DARs) being an even integer (typically 2, 4 or 8). However, ADCs with odd-integer DARs are significantly harder to synthesise. Here, we report the synthesis of ADCs loaded with a single warhead, using TetraDVP linkers which simultaneously re-bridge all four interchain disulfides of an IgG1 antibody.

Fig. 1. TetraDVP linkers reported by Dannheim et al. enabled the simultaneous re-bridging of all four interchain disulfides of an IgG1 antibody. However, subsequent CuAAC reaction failed to produce the desired ADCs in high conversion, postulated to be due to steric hindrance of the pendant alkyne.

Fig. 2. TetraDVP linkers synthesised in this work. PEG spacers were included in the linker backbones (a–d) and azide-containing side chains of different lengths were employed (series 1 and 2, respectively).

Scheme 1. SDS-PAGE analysis, under reducing conditions, of trastuzumab (Ab) re-bridged with the two series of linkers. The major species is the fully re-bridged antibody for all linkers tested. Proteins were stained with Coomassie Brilliant Blue before imaging.

Fig. 3. DBCO reagent 8 used for the addition of a pyrrolobenzodiazepine (PBD) dimer payload to ALCs 4a–d.