Acid-cleavable thiomaleamic acid linker for homogeneous antibody-drug conjugation

Abstract

In this communication we describe a novel acid-cleavable linker strategy for antibody-drug conjugation. Functional disulfide bridging of the single interchain disulfide bond of a trastuzumab Fab fragment yields a homogeneous antibody-drug conjugate bearing a thiomaleamic acid linker. This linker is stable at physiological pH and temperature, but quantitatively cleaves at lysosomal pH to release the drug payload.

Scheme 1. Small molecule partial hydrolysis/cleavage study: (a) PhNH₂, AcOH, rt to 130 °C, 57%; (b) EtSH, NEt₃, CH₂Cl₂, 93%; (c) LiOH·H₂O, CD₃OD : D₂O (1 : 1); (d) 2 M HCl to pH 4, >99% (over two steps).

Scheme 2. Synthesis and cleavage study of thiomaleamate linker 7: (a) MeOCOCl, NMM, THF, 97%; (b) PABA, CH₂Cl₂, 99%; (c) EtSH, NEt₃, CH₂Cl₂, 86%; (d) PhNCO, NEt₃, CH₂Cl₂, 62%; (e) LiOH·H₂O, CD₃OD : D₂O (1 : 1), then 2 M HCl to pH 4, >99%.

Scheme 3. Synthesis of thiomaleamate–PABC–DOX construct 10: (a) PhSH, NEt₃, CH₂Cl₂, 98%; (b) PNPC, py, CH₂Cl₂, 72%; (c) DOX·HCl, NEt₃, NMP, 99%.

Scheme 4. Assembly/cleavage study of Fab ADC 13: (a) TCEP, pH 8.0, 37 °C, 1.5 h, then 10, 37 °C, 1 h; (b) pH 7.4, 20 h; (c) pH 4.5, 72 h.

Fig. 1. ELISA analysis of Fab 11, processed Fab and Fab ADC 13 binding to the HER2 antigen.