Attachment Site Cysteine Thiol p<i>K</i>_a Is a Key Driver for Site-Dependent Stability of THIOMAB Antibody–Drug Conjugates

The incorporation of cysteines into antibodies by mutagenesis allows for the direct conjugation of small molecules to specific sites on the antibody via disulfide bonds. The stability of the disulfide bond linkage between the small molecule and the antibody is highly dependent on the location of the engineered cysteine in either the heavy chain (HC) or the light chain (LC) of the antibody. Here, we explore the basis for this site-dependent stability. We evaluated the in vivo efficacy and pharmacokinetics of five different cysteine mutants of trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide bonds. A significant correlation was observed between disulfide stability and efficacy for the conjugates. We hypothesized that the observed site-dependent stability of the disulfide-linked conjugates could be due to differences in the attachment site cysteine thiol p<i>K</i>_a. We measured the cysteine thiol p<i>K</i>_a using isothermal titration calorimetry (ITC) and found that the variants with the highest thiol p<i>K</i>_a (LC K149C and HC A140C) were found to yield the conjugates with the greatest in vivo stability. Guided by homology modeling, we identified several mutations adjacent to LC K149C that reduced the cysteine thiol p<i>K</i>_a and, thus, decreased the in vivo stability of the disulfide-linked PBD conjugated to LC K149C. We also present results suggesting that the high thiol p<i>K</i>_a of LC K149C is responsible for the sustained circulation stability of LC K149C TDCs utilizing a maleimide-based linker. Taken together, our results provide evidence that the site-dependent stability of cys-engineered antibody-drug conjugates may be explained by interactions between the engineered cysteine and the local protein environment that serves to modulate the side-chain thiol p<i>K</i>_a. The influence of cysteine thiol p<i>K</i>_a on stability and efficacy offers a new parameter for the optimization of ADCs that utilize cysteine engineering

Immolation of <i>p</i>‑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 <i>p</i>-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) (<b>P1</b>), the resulting <b>ADC1</b> showed loss of potency in CD22 target-expressing cancer cell lines (e.g., BJAB, WSU-DLCL2). In comparison, the conjugate (<b>ADC2</b>) of a cyclopropapyrroloindolone (CPI) (<b>P2</b>) was potent despite the two corresponding free drugs having similar picomolar cell-killing activity. Although the corresponding spirocyclization products of <b>P1</b> and <b>P2</b>, 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 <b>P1</b> compared to that in <b>P2</b>. Additional immolation studies with two other PABE-linked substituted phenol compounds showed that electron-withdrawing groups accelerated the immolation to release an acidic phenol-containing payload (to delocalize the negative charge on the anticipated anionic phenol oxygen during immolation). In contrast, efficient immolation of <b>LD4</b> did not result in an active <b>ADC4</b> because the payload (<b>P4</b>) had a low potency to kill cells. In addition, nonimmolation of <b>LD5</b> did not affect the cell-killing potency of its <b>ADC5</b> since immolation is not required for DNA alkylation by the center-linked pyrrolobenzodiazepine. Therefore, careful evaluation needs to be conducted when the Val-Cit-PAB linker is used to connect antibodies to a phenol-containing drug as the linker immolation, as well as payload potency and stability, affects the cell-killing activity of an ADC