Introduction
Antibody-drug conjugates (ADCs) combine the targeting precision of antibodies with the high potency of cytotoxic payloads, making linker design a critical determinant of therapeutic performance. An ideal ADC linker must remain stable during systemic circulation while enabling efficient payload release at the tumor site. Premature cleavage can lead to off-target toxicity, whereas insufficient release may limit tumor antitumor efficacy.
Scheme 1. Structural role of linkers in antibody-drug-conjugates.
Enzyme-responsive linkers have been widely explored to address this balance by leveraging biological difference between tumor and normal tissues. While peptide-based linkers cleaved by lysosomal proteases such as cathepsin B remain the most established approach, their performance can be affected by protease expression variability and extracellular instability. In this context, glycosidase-cleavable linkers have emerged as an attractive alternative.
Glycosidase-cleavable linkers are designed to undergo enzyme hydrolysis of glycosidic bonds by specific glycosidase, which are predominantly localized in lysosomal and, in certain tumors, enriched or released into the tumor microenvironment. This biological feature enables controlled payload release. In addition, the hydrophilic modular nature of sugar-based linkers allows flexible integration of self-immolative spacers and diverse payloads, supporting a broad range of ADC designs. Based on the sugar motif and the corresponding glycosidase involved, these linkers can be broadly grouped into three categories: β-glucuronidase, β-galactosidase, and other less established glycosidase-sensitive designs.
Scheme 2. Classification of glycosidase-cleavable linkers.
β-Glucuronidase-Cleavable Linkers
β-Glucuronidase-cleavable linkers represent the most established class of glycosidase-responsive linkers in ADC design. β-Glucuronidase is a hydrolytic lysosomal enzyme responsible for cleaving β-glucuronic acid residues from glycosylated substrates. Under physiological conditions, this enzyme is predominantly localized in the lysosomal compartment, where it operates in a hydrophilic environment to catalyze glycosidic bond hydrolysis.
Importantly, beyond intracellular lysosomes, β-glucuronidase has been reported to be enriched or released in the necrotic regions of certain solid tumors. Unlike many lysosomal proteases, β-glucuronidase can retain enzymatic activity in the extracellular tumor microenvironment. This unique feature enables β-glucuronide-based linkers to support not only intracellular payload release following ADC internalization, but also extracellular drug liberation, thereby contributing to a potential bystander effect.
Scheme 3. Mechanism of β-glucuronidase-cleavable linkers in ADCs
A representative and widely cited implementation of this strategy was reported by Jeffrey and coworkers, who established β-glucuronide-based linkers as a robust platform for ADC payload delivery. In their initial studies, β-glucuronic acid was incorporated as an enzymatic trigger and connected to cytotoxic payloads through a self-immolative p-aminobenzyl carbamate (PABC) spacer, enabling efficient release of auristatin derivatives (MMAE and MMAF) as well as doxorubicin following enzymatic activation.
In this linker architecture, drug release follows a well-defined two-step process. Enzymatic cleavage of the β-glucuronide moiety by β-glucuronidase generates an unstable phenolic or aniline intermediate, which subsequently undergoes rapid self-immolation of the PABC spacer to liberate the free drug. This design decouples enzymatic recognition from payload chemistry, providing both clean release kinetics and high stability during systemic circulation.
β-Glucuronide-based ADCs derived from this framework demonstrated favorable developability profiles, including high achievable drug-to-antibody ratios, reduced aggregation driven by the hydrophilic carbohydrate motif, and improved plasma stability relative to conventional protease-cleavable dipeptide linkers. Subsequent extensions of this strategy further showed that, through appropriate spacer engineering, β-glucuronide linkers can support not only amine-containing payloads but also phenolic cytotoxins, substantially broadening the accessible payload space. Collectively, these studies established β-glucuronidase-cleavable linkers as a versatile and chemically adaptable class within the broader family of enzyme-responsive ADC linkers.
β-Galactosidase-Cleavable Linkers
β-Galactosidase-cleavable linkers constitute another important class of glycosidase-responsive linkers explored in ADC design. β-Galactosidase is a hydrolytic lysosomal enzyme that catalyzes the cleavage of β-glycosidic bonds between galactose residues and their associated aglycones. Similar to other lysosomal glycosidases, β-galactosidase primarily operates within the lysosomal compartment, where it contributes to the degradation of glycosylated biomolecules under hydrophilic conditions.
In addition to its intracellular role, β-galactosidase has been reported to be overexpressed in certain tumor types. This differential expression provides a biological basis for exploiting β-galactosidase activity as a trigger for selective payload release. Compared with β-glucuronidase-based systems, β-galactosidase-cleavable linkers are generally considered to favor intracellular activation, offering a release profile that may enable more controlled drug liberation with reduced reliance on extracellular bystander effects.
Scheme 4. Mechanism of β-galactosidase-cleavable linkers in ADCs
A representative implementation of β-galactosidase-responsive linkers was reported by Kolodych and coworkers, who developed trastuzumab–MMAE ADCs incorporating a galactoside trigger and a self-immolative release module. Payload conjugation was achieved through reduced interchain cysteines using either conventional maleimide chemistry or a more plasma-stable arylpropiolonitrile (APN) handle, resulting in well-defined ADCs with moderate drug loading.
These galactoside-linked ADCs demonstrated antigen-dependent cytotoxicity in vitro and selective activation upon exposure to β-galactosidase, while remaining stable toward non-relevant enzymatic triggers. In vivo evaluation in HER2-positive xenograft models further confirmed effective tumor growth inhibition following single-dose administration, supporting the functional relevance of intracellular β-galactosidase-mediated activation.
Collectively, these studies position β-galactosidase-cleavable linkers as a practical intracellularly activated option within the broader family of glycosidase-sensitive ADC designs, particularly in applications where controlled, cell-restricted payload release is preferred.
Other Glycosidase-Sensitive Linkers
In addition to β-glucuronidase- and β-galactosidase-cleavable systems, other glycosidase-sensitive linker designs, such as those responsive to mannosidases or fucosidases, have been proposed. However, these approaches remain largely exploratory, with limited biological validation and few reported ADC examples. As a result, current glycosidase-cleavable linker development continues to center on the two established platforms discussed above.
Application Considerations and Our Linker Portfolio
From an application perspective, the key distinction between β-glucuronidase- and β-galactosidase-cleavable linkers lies in their payload release profiles. β-Glucuronidase-based systems can support both intracellular and extracellular drug release, enabling bystander effects that are advantageous in heterogeneous solid tumors. In contrast, β-galactosidase-cleavable linkers primarily rely on intracellular activation following ADC internalization, offering a more controlled release profile with reduced dependence on extracellular enzymatic activity.
At Precise PEG, we focus on the development and production of glycosidase-cleavable linkers for ADC applications, with a portfolio encompassing both β-glucuronidase- and β-galactosidase-cleavable designs. (https://precisepeg.com/collections/novel-enzymes-cleavable-linkers) This coverage enables rational linker selection based on payload properties and desired release behavior, supporting ADC programs from early discovery through lead development.
Reference
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