ADC Drugs: Concepts of Linker Cleavage and Payload Release

Linker Cleavage and Payload Release in ADC Drugs

Antibody-drug conjugates (ADCs) rely on specialized linkers that connect targeting antibodies to small-molecule toxin payloads. These linkers are categorized as cleavable or non-cleavable, depending on how they degrade within the cell.

Cleavable Linkers

Cleavable linkers are designed to be chemically unstable, taking advantage of differences between extracellular and intracellular conditions such as pH levels and redox potential. They can also be enzymatically degraded within lysosomes.

Chemically cleavable linkers primarily include acid-sensitive linkers (such as perylene or carbonate bonds) and reducible linkers (such as disulfide bonds). Acid-sensitive linkers break down in acidic environments, specifically in endosomes (pH 5.5–6.2) and lysosomes (pH 4.5–5.0), triggering payload release.

Another key type of cleavable linker is the enzyme-sensitive linker, which is broken down by intracellular hydrolytic enzymes. Among these, peptide-based linkers facilitate ADC drug internalization via endocytosis and rely on lysosomal cathepsins for cleavage. Common enzyme-sensitive linkers include GGFG (Gly-Gly-Phe-Gly) and VC (Val-Cit) linkers. The GGFG linker is particularly responsive to cathepsin L, which enables the nearly complete release of DXd from its ADC within 72 hours, while cathepsin B exhibits minimal activity in this process. However, both GGFG and VC linkers undergo cleavage by cathepsin B in lysosomes or endosomes of tumor cells, ensuring efficient drug release.

Compared to acid-cleavable and glutathione (GSH)-cleavable linkers, the GGFG linker offers greater stability in the bloodstream, minimizing unintended payload release and enhancing drug safety. Similarly, the VC linker remains stable in plasma and effectively releases the drug in tumor cells, ensuring targeted delivery.

Since cleavable linkers vary in mechanism, selecting the right cleavage system is crucial for effective drug testing. Common in vitro test systems for payload release include acidified liver homogenate (pH 5.0–6.0), acidified liver S9 fraction (pH 5.0–6.0), liver lysosomes, tumor cells, and specific enzymes such as cathepsins B, M, and L, glucuronidase, glutathione (GSH), and legumain. The choice of system depends on the type of linker being teste

Non-Cleavable Linkers

Non-cleavable linkers depend on the complete breakdown of ADC antibodies by cytoplasmic and lysosomal proteases, ultimately releasing the payload attached to an antibody degradation product. However, their effectiveness relies heavily on high antigen expression in tumor cells and the strong internalization capability of the conjugated drug to ensure efficient payload release.

For in vitro studies, test systems such as acidified liver homogenate, acidified liver S9 fraction, and lysosomes can effectively degrade the antibody component, facilitating payload release. These systems are essential for assessing the pharmacokinetic properties of ADCs using non-cleavable linkers.

Keywords: ADC linker, Payload release, liver lysosome, lysosomal stability, lysosome catabolism, Cathepsin B, DS8201a, GGFG-DxD