Lysosomal-Cleavable Peptide Linkers in Antibody-Drug Conjugates

Abstract

Antibody-drug Conjugates (ADCs) are a powerful therapeutic modality for cancer treatment. ADCs are multi-functional biologics in which a disease-targeting antibody is conjugated to an effector payload molecule via a linker. The success of currently used ADCs has been largely attributed to the development of linker systems, which allow for the targeted release of cytocidal payload drugs inside cancer cells. Many lysosomal proteases are over expressed in human cancers. They can effectively cleave a variety of peptide sequences, which can be exploited for the design of ADC linker systems. As a well-established linker, valine-citrulline-p-aminobenzyl carbamate (ValCitPABC) is used in many ADCs that are already approved or under preclinical and clinical development. Although ValCitPABC and related linkers are readily cleaved by cathepsins in the lysosome while remaining reasonably stable in human plasma, many studies have shown that they are susceptible to carboxylesterase 1C (Ces1C) in mouse and rat plasma, which hinders the preclinical evaluation of ADCs. Furthermore, neutropenia and thrombocytopenia, two of the most commonly observed dose-limiting adverse effects of ADCs, are believed to result from the premature hydrolysis of ValCitPABC by human neutrophil elastase. In addition to ValCitPABC, the GGFG tetrapeptidyl-aminomethoxy linker is also cathepsin-cleavable and is used in the highly successful ADC drug, DS8201a. In addition to cathepsin-cleavable linkers, there is also growing interest in legumain-sensitive linkers for ADC development. Increasing plasma stability while maintaining lysosomal cleavability of ADC linkers is an objective of intensive current research. This review reports recent advances in the design and structure-activity relationship studies of various peptide/peptidomimetic linkers in this field.

Keywords: antibody–drug conjugate; cathepsin; legumain; linkers; lysosome; payload release; peptidomimetic; self-immolation.

Figure 1

(A) Fate of an ADC before and after internalization. Premature cleavage of the linker in extracellular matrix is often associated with off-target toxicity. Antigen-mediated endocytosis delivers ADC in the endosomal–lysosomal system and lysosomal linker cleavage releases the drug, which acts to exert its cytotoxicity. (B) Effect of amino acid composition in the linker peptide and substitution on the benzene ring of PABC on linker stability in mouse plasma.

Figure 2

Various Linker payload designs for faster lysosomal cleavage and improved plasma stability. (A) Peptidomimetic linkers specifically cleaved by cathepsin B; (B) modifications to central PABC ring.

Figure 3

Various Linker payload designs for faster lysosomal cleavage and improved plasma stability. (A) Tandem cleavable linkers’ glucuronide group masks the linker system to maintain stability in extracellular environment; (B) Aryalsulfatase A (ARSA) and β-galactosidase dual cleavable 3-O-sulfo-β-galactose linker.

Figure 4

Structure and linker cleavage mechanism of trastuzumab deruxtecan (Enhertu).

Figure 5

Asn-containing peptide linkers with improved selectivity towards legumain.