Immolation of p-Aminobenzyl Ether Linker and Payload Potency and Stability Determine the Cell-Killing Activity of Antibody–Drug Conjugates with Phenol-Containing Payloads

The valine-citrulline (Val-Cit) dipeptide and p-aminobenzyl (PAB) spacer have been commonly used as a cleavable self-immolating linker in ADC design including in the clinically approved ADC, brentuximab vedotin (Adcetris). When the same linker was used to connect to the phenol of the cyclopropabenzindolone (CBI) (P1), the resulting ADC1 showed loss of potency in CD22 target-expressing cancer cell lines (e.g., BJAB, WSU-DLCL2). In comparison, the conjugate (ADC2) of a cyclopropapyrroloindolone (CPI) (P2) was potent despite the two corresponding free drugs having similar picomolar cell-killing activity. Although the corresponding spirocyclization products of P1 and P2, responsible for DNA alkylation, are a prominent component in buffer, the linker immolation was slow when the PAB was connected as an ether (PABE) to the phenol in P1 compared to that in P2. Additional immolation studies with two other PABE-linked substituted phenol compounds showed that electron-withdrawing groups accelerated the immolation t...