Comparative Enzymatic and Stability Assays Reveal GPLG as an Effective Cathepsin B Cleavable Linker for Tumor-Targeting Drug Conjugates

In the past decade, targeted drug delivery systems have significantly advanced cancer therapy. A key component of these constructs is the chemical linker that covalently connects a targeting unit to a potent cytotoxic payload. Among approved and investigational antibody-drug conjugates (ADCs) and small molecule-drug conjugates (SMDCs), lysosomal-cleavable peptide sequences such as Val-Cit (VCit), Val-Ala (VA), and Gly-Phe-Leu-Gly (GFLG) are widely used for tumor-specific drug release. However, premature drug release and instability often cause off-target toxicity and poor selectivity. Since lysosomal proteases are still considered optimal for the drug release within the tumor site, the quest for new and more stable lysosomal-sensitive peptide sequences is currently an ongoing challenge. This work investigates the enzymatic susceptibility, cleavage kinetics, and metabolic stability of the peptide sequence Gly-Pro-Leu-Gly (GPLG) as a novel Cathepsin B-cleavable linker for tumor-targeting drug conjugates. Compared to GFLG, VCit, and VA, all conjugated to paclitaxel via a PABC-<i>N</i>,<i>N</i>'-dimethylethylenediamine spacer, GPLG exhibited the fastest Cathepsin B cleavage within the first 30 min of the assay, and higher stability at pH 5.4 and in both human and rat plasma samples. These results highlight GPLG as a promising lysosomal-sensitive linker for next-generation SMDCs and ADCs.