The development of antibody-drug conjugates (ADCs) has progressed significantly over the past decade due to improvements in payloads, linkers, and conjugation methods. In particular, linker design plays a key role in regulating ADC stability in systemic circulation and payload release efficiency in tumors, thereby affecting ADC pharmacokinetics (PK), efficacy, and toxicity profiles.
Some key parameters, such as coupling chemistry, linker length, and linker steric hindrance, all have an impact on the PK and efficacy of ADC drugs. An ideal linker should remain stable in the circulation and release cytotoxic payloads in the tumor. However, existing linkers often unspecifically release payloads and inevitably lead to off-target toxicity. Therefore, in the design of ADC drugs, it is necessary to correctly adjust these important parameters of the linker to achieve the balance between ADC stability and payload release efficiency, so as to achieve the desired effect of ADC drugs.
New Type of ADC Linkers
In the past few years, many new linkers have been developed, including cathepsin cleavable linkers, acid cleavable linkers, GSH cleavable linkers, Fe (II) cleavable linkers, novel enzyme cleavable linkers, light-responsive cleavable linkers, and bioorthogonal cleavable linkers. Among them, cathepsin, GSH, and acid-cleavable linkers have been well studied and used in approved ADCs. Many important advances in linker design have been made, and these researches will help to indicate the future direction of linker development.
1. Cathepsin Cleavable Linkers
In 2017, Caculitan et al. found that the valine-citrulline (Val-Cit) linker exhibits broad sensitivity to a variety of cathepsins, including cathepsin B, cathepsin K, cathepsin L, and more. To improve selectivity, Wei et al. designed a linker using the cyclobutane-1,1-dicarboxamide (cBu) structure, which is largely dependent on cathepsin B.
In cells, cathepsin B inhibitors effectively inhibited (over 75%) drug release, which contains cBu-Cit linkers, while cathepsin K inhibitors had no significant effect. In contrast, conventional Val-Cit-containing linkers appear to be resistant to all single protease inhibitors (cathepsin B, L, and K inhibitors are all less than 15%). Meanwhile, compared with ADC containing Val-Cit linker, ADC containing cBu-Cit linker showed greater tumor inhibition in vitro.
Furthermore, the optimization of peptide linkers is not limited to the development of new structures. Peptide linkers can be optimized with minimal structural changes, such as the type of amino acids and structural chemistry. Several studies have compared Val-Cit and Val-Ala dipeptide structures with MMAE payload connections. In the case of non-internalized antibodies, both Val-Cit and Val-Ala linkers bound to engineered cysteine exhibit similar characteristics and exhibit better performance than Val-Lys and Val-Arg analogs. In the case of a random cysteine-conjugated anti-HER2 ADC, Val-Ala shows less aggregation in high DAR structures than Val-Cit. On the other hand, both linkers showed similar buffer stability, cathepsin B release efficiency, cellular activity, and histopathological characteristics.
The tetrapeptide Gly-Gly-Phe-Gly exhibits all the properties of a stable and efficient cleavable linker used in the marketed ADC drug Enhertu. Daiichi Sankyo’s Enhertu is a plasma-stable ADC with a DAR of 7.7 that undergoes protease degradation in the lysosome to release DX-8951f, a potent topoisomerase I inhibitor derived from exatecan. Since the linker does not contain a solubilizer, achieving such a high DAR is very impressive, as it contradicts the widely established principle that high DAR conjugates may have poorer pharmacokinetic profiles. The self-degrading spacer used here is a simple and compact hemi-amination rather than the PABC used by the Val-Cit linker.
2. Acid Cleavable Linkers
Acid-cleavable linkers selectively deliver payloads to tumor tissue by taking advantage of the pH difference between tumor tissue (4.0-5.0) and plasma (~ 7.4). This strategy had its earliest clinical success with Mylotarg and was later used in Besponsa. However, the stability of the acid-cleavable linker severely limits its application in ADCs. Phenylketone-derived hydrazone ligand hydrolyzed with a 2-day half-life in human and mouse plasma, and the serum stability of sacituzumab- govitecan (Trodelvy) was also not satisfied with a 36-hour half-life.
Therefore, acid-cleavable ADCs require more stable linkers, or can only use payloads of moderate cytotoxicity. In 2019, a novel silyl ether-based acid-cleaving ADC was developed, carrying the highly cytotoxic monomethyl auristatin E (MMAE). This design greatly improves the stability of the acid-cleavable linker, in addition, this ADC using the novel silyl ether linker has a half-life of more than 7 days in human plasma and has shown promising therapeutic efficacy in mouse xenograft models.
3. GSH Cleavable Linkers
GSH-cleaved linkers depend on higher levels of glutathione (1–10 mmol/L) in the cytoplasm compared to plasma (~5 μmol/L). Disulfide bonds are most commonly used among these linkers, however, the current disulfide bond structures cannot achieve a perfect combination of high cycling stability and efficient intracellular release. In 2017, Thomas et al. attempted to solve this problem by linking small molecule drugs directly to engineered cysteine in thiomumab. By directly linking the antibody, the spatial protection of the antibody will improve circulatory stability.
4. Fe (II) Cleavable Linkers
Abnormal iron metabolism can increase the level of free iron, and based on this strategy, increasing the concentration of unconjugated iron has been used in prodrug design. In 2018, Spangler et al. reported a Fe (II) reactive 1,2, 4-trioxane (TRX) ligand and used this linker for ADCs.
5. Novel Enzyme Cleavable Linkers
Like cathepsins, pyrophosphatases and phosphatases are hydrolases that are selectively expressed in lysosomes. In 2016, researchers at Merck designed phosphate- and pyrophosphate-containing linkers paired with cathepsin B-sensitive Val-Cit-PABA to deliver glucocorticoids. The advantage of this hydrophilic and permanently charged group is solubility, which not only enables bioconjugation with lipophilic glucocorticoid derivatives but also facilitates ADC purification with less than 0.10% residual linkers in ADC. ADCs containing both phosphate and pyrophosphate were active in vitro.
In addition to the classic β-glucuronidase-cleavable linker developed for ADCs in 2006, β-galactosidase was also found to be overexpressed and hydrolytically active in tumor cells. An ADC using a β-galactosidase-cleavable linker was recently reported, which contained a PEG10 spacer. The spacer was replaced with a nitro group to increase the rate of autodegradation. Analogous to the β-glucuronidase linker, the dissociation mechanism involves hydrolysis of the β-galactosidase moiety, which confers hydrophilicity to the chemical precursor. Another advantage is that β-galactosidase is only present in lysosomes, whereas β-glucuronidase is expressed in lysosomes and also in the microenvironment of solid tumors. The study demonstrated that ADCs containing a β-galactosidase linker were more potent than T-DM1 in vitro and in vivo in the context of MMAE released from anti-HER2-ADCs.
6. Light-responsive Cleavable Linkers
In recent years, payload release strategies based on light-responsive cleavage have gradually emerged. Photoresponsive cleavable has the following advantages, including low toxicity, rapid response, high sensitivity, and specificity.
7. Bioorthogonal Cleavable Linkers
Bioorthogonal chemistry has the characteristics of high selectivity, fast and easy processing, and non-toxic by-products. In 2019, Wang et al. developed a bioorthogonal cleavable linker using the classical bioorthogonal cleavage pair Cu(I)-BTTAA and dsProc. However, bioorthogonal cleavable linkers are currently mainly focused on in vitro exploration. There are still problems in reaction efficiency, reaction rate, substrate stability, biocompatibility, and ease of operation, which are far from clinical application.
