Trimethyl Lock-2Cl (TML-2Cl): A self-cyclizing adapter for molecular release in antibody-drug conjugates

The amide bond provides superior stability in chemical structures, enhances selectivity for drug loading, and reduces off-target toxicity during drug delivery. Conventional enzyme-cleavable antibody-drug conjugates (ADCs) typically rely on cathepsin B-sensitive linkers conjugated to self-immolative spacers, such as p-aminobenzyl carbamate (PABC), p-aminobenzyl ether (PABE) spacers or ethylenediamine-derived motifs, to facilitate payload release. In this work, we introduced the trimethyl lock (TML) system as a versatile adapter to expand the scope of applicable payloads. To meet the pKa requirements of 1,6-elimination and subsequent rapid self-cyclization reactions, dichloro-modified trimethyl lock (TML-2Cl) was further developed and applied for the first time in drug delivery systems. Using Exatecan as the payload, hydrophilic and cathepsin B-cleavable TML linker-payloads were designed and synthesized. Among these, L10081 and L10082, incorporating the hydrophilic units PSAR-10 or PEG-8, exhibited enhanced capacity for Exatecan release. Furthermore, the ADCs Her2-L10081 and Her2-L10082 significantly inhibited tumor growth in an NCI-N87 xenograft mouse model. However, although this adapter TML-2Cl exhibits excellent stability, its intrinsic hydrophobicity may limit its suitability for the development of ADCs with high drug-to-antibody ratios (DAR), particularly those incorporating poorly water-soluble payloads.