NH₂-trimethyl lock (NH₂-TML): a stable and effective adapter for exatecan-based antibody–drug conjugates

The stability of the linker plays a critical role in the development of antibody–drug conjugates (ADCs). A stable linkage strategy can mitigate off-target toxicity and enhance therapeutic efficacy. In this study, an NH ₂ -TML adapter, capable of directly connecting peptide chains via an amide bond, was developed based on the trimethyl lock (TML) system. Using this adapter, three peptide chains—Val-Ala, Gly-Gly-Phe-Gly, and Gly-Phe-Leu-Gly—were conjugated to synthesize the linker-payloads W1507 , W2707 , and W2907 , respectively. For comparison, compounds JOEP4 and JOEP8 , which contain the widely used p -aminobenzyl carbamate (PABC) spacer, were also synthesized, for demonstrating that the amide linkage exhibits superior stability over ester bonds. Studies indicated that W1507 possesses better aqueous solubility compared to W2707 , W2907 , and JOEP4 , affording it greater advantages in ADC development. Although the compound W1507 did not release free payload Exatecan was detected in in vitro cathepsin B-mediated release assay, the ADC Her2-W1507 designed and synthesized using the NH ₂ -TML adapter exhibited antitumor activity comparable to that of ADC Her2-JOEP8 with PABC spacers in vivo the NCI-N87 xenograft tumor models. This suggests the involvement of additional enzymatic processes in vivo . These findings underscore the potential of the NH₂-TML system in ADC design and cancer therapy.