The RIPK2 PROTAC 1 was attached to the VC-PAB S6 via a carbonate linkage, which was then linked to the diBrPD by an amide bond (Scheme S3). Conjugation to the anti-HER2 mAb, trastuzumab, was achieved by reduction of the interchain disulfides by TCEP, followed by addition of the diBrPD rebridging reagent S10 to form conjugate ADC-2, which identified a drug-to-Ab ratio (DAR) of 4.0 (Figure ). These exist as an interchain bridged species, and an intrachain "half body" (HB) species, where the cysteines have bridged within a single heavy chain. The control anti-IL4 ADC-3 was synthesised in a similar manner, which resulted in a DAR of 3.7. G0F and G1F correspond to glycan modifications on the mAb. RIPK2 degradation using ADC-2 and ADC-3 were assessed in a SKOV3 HER2+ breast cancer cell line. The anti-HER2 ADC-2 showed similar levels of RIPK2 degradation compared to the parent PROTAC whereas the anti-IL4 ADC-3 showed no degradation at 10 nM. An unexpected observation was RIPK2 degradation using ADC-3 at concentrations above 100 nM (Figure ). As the SKOV3 cells do not have membrane-bound IL-4, we rationalised that the observed degradation might be due to non-specific uptake mechanisms, such as macropinocytosis. No degradation was observed for all compounds, confirming that degradation is via a proteasome dependent pathway (Figure ). No cytoxicity was observed up to 1 μM for both ADC-1 and ADC-2 (Figure ).