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 NH₂-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 NH₂-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.