Antibody-drug conjugates (ADCs), as emerging anticancer drugs, can deliver highly cytotoxic molecules directly to cancer cells to kill them. ADC is a monoclonal antibody covalently bound to cytotoxic chemical substances (payload) through linker. ADC linker plays a key role in the therapeutic effect of ADC, and its characteristics greatly affect the therapeutic index, pharmacodynamics and pharmacokinetics of ADC. For example, the linkage between linker-mAb determines the drug-antibody ratio (DAR), which determines the homogeneity and stability of ADC. In order to ensure the selectivity and efficacy of ADC, linker should strive to achieve three key features :

(1) High cycle stability: Payload will not be released before it reaches the target, thereby minimizing the off-target effects.

(2) High water solubility: it is helpful for coupling and avoids the formation of inactive ADC aggregates.

(3) Efficient release: allowing efficient release of highly cytotoxic linker-payload metabolites.

Cleavable and Non-cleavable Linker

ADC linker can be divided into cleavable linker and non-cleavable linker. Mechanistically, when the non-cleavable linker reaches the lysosome, the mAb is metabolized through the proteolytic mechanism, and the payload, linker and amino acid appendages are released. Substantial modifications to the payload can also yield potent ADCs, such as Kadcyla®, if the key pharmacophore of the payload is not affected. However, non-cleavable linkers are often unable to exert bystander effects due to the lack of cell permeability of charged amino acid appendages. Therefore, the application range of ADCs containing non-cleavable linkers is limited, and they are mainly used for the treatment of hematological cancers or tumors with high antigen expression.

Compared with non-cleavable linkers, cleavable linkers use specific conditions to release drugs at target cells. Cleavable linkers can be further subdivided into chemically cleavable linkers or enzymatically cleavable linkers. Although having a wider range of applications than non-cleavable linkers, cleavable linkers are more unstable in blood circulation. Cleavable linkers' performance thus hinges on their capacity to effectively distinguish between target cell circumstances and blood circulation conditions.

Chemically Cleavable Linker

There are three main types of chemically induced cleavable linkers: acid cleavable, cleavable under reducing conditions (disulfide, etc.), and linkers that can be cleaved by exogenous stimuli.

Acid-cleavable Linker

Acid-cleavable linkers are designed to utilize the acidity of endosomes (pH 5.5-6.2) and lysosomes (pH 4.5-5.0), while maintaining circulation stability under physiological conditions at pH 7.4. This strategy achieved the earliest clinical success with Pfizer's Mylotarg® (AcBut Linker). Although reducible disulfides are also employed, linker contains an acid-sensitive N-acylhydrazone linkage. Therefore, under acid catalysis, Mylotarg® is hydrolyzed into ketone and hydrazide-payload. In addition, during the development of Mylotarg®, the researchers also tested the stability of a series of hydrazone-containing linkers at pH 4.5 and pH 7.4, as well as their in vitro and in vivo stability in mice as part of ADC. Studies have shown that linker, which is stable at pH 7.4 and unstable at pH 4.5, provides the most effective ADC. This kind of linker-payload is also applied to Besponsa®.

In addition to the hydrazone bond mentioned above, the carbonate linker used by Trodelvy® is also a kind of acid cleavage linker. Although ester bonds are theoretically more stable than carbonates in blood circulation, experimental results show that ADCs constructed from the former are less stable in human serum. The serum stability of the ADC was significantly improved (t1/2=36 hrs) by introducing a p-aminobenzyl (PABC) spacer, which showed some selectivity for the acidic lysosomal compartment, with t1/2 at pH 5 2 for 10 hours.

Reductive Cleavage Linker

Despite the clinical success of Mylotarg®, Besponsa®, and Trodelvy®, acid-cleavable linkers are no longer an option for most ADC ligation techniques. Linker's requirement to strictly distinguish between pH 5 and pH 7.4 environments is very difficult. Although in some cases, slow release of payload can produce beneficial results, this method is usually only able to adopt payload of moderate cytotoxicity, and the highly toxic payload preferred by ADC now requires a more stable linker.

The release of payload in Mylotarg ® and Besponsa ® requires not only the acid-sensitive hydrazone bond to play a role, but also the disulfide bond in the linker. The disulfide bond linker is stable at physiological pH, but is susceptible to nucleophilic attack by thiols. In plasma, the main thiol species is the reduced form of human serum albumin (HSA, ~422 μM ), but its reactivity with macromolecules is hindered due to limited exposure of solvents containing free thiol residues. Contrary to the limited reduction capacity of plasma, the cytoplasm contains high levels of glutathione (GSH, 1-10 mM); the reduction conditions of plasma and cytoplasm provide an opportunity for ADC to selectively release the effective load. In addition, compared to normal tissues, tumor-associated oxidative stress typically leads to elevated levels of glutathione, which increases the selectivity of cancer cells. At present, the payload of linkers involving disulfide bond hydrolysis mainly includes the effective load of maytansinoids (DM1, DM3, DM4) and disulfide bond carbamate.

Exogenous Stimulus Cleavable Linker

Although the use of endogenous cleavage ADC linker is the simplest drug release method, the release of payload through external stimuli can have the following advantages: (1) avoid the difference in linker cleavage rate due to biological differences between patients, and (2) ) ADCs can also function when endogenous cleavage is insufficient to effectively release the payload. Akalux®, which has been launched in Japan, uses a near-infrared light-sensitive linker as a cleavage method. After irradiation with 690 nm red light, it releases toxic IRDye700 to play a therapeutic role, which belongs to near-infrared photoimmunotherapy. In addition, there are currently more studies on metal ion (Pt, Pd, Fe, Lu) catalyzed cleavage, UV/Vis and NIR light-sensitive linkers.

Enzyme-mediated Cleavable Linker

According to the traditional mechanism of action of ADC, the enzyme-cleavable linker can be selectively cleaved inside the cell by transporting ADC to the lysosome, which contains a high concentration of certain hydrolytic enzymes. The most successful method at the moment is the use of cathepsin B cleavable linkers. The peptide linker broken by cathepsin B is used by the commercially available medications Adcetris®, Polivy®, Padcev®, Tivdak®, Aidixi®, Lumoxiti®, Zynlonta®, and Enhertu®. Among them, p-aminobenzyl carbamate (PABC) was used as a self-degrading spacer, which spontaneously undergoes 1,6-elimination after proteolysis, releasing payload, CO₂ and azaquinone methide. PABC maintains the enzyme activity independent of payload, which increases the scope of application of the peptide linker. All of these linker combinations are stable in isolated human plasma. In addition to cathepsin B cleavable linker, phosphatase cleavable linker, sulfatase cleavable linker, β-galactosidase cleavable linker, β-glucosidase cleavable linker and nitroreductase cleavable linker are also widely used in the construction of ADCs.

Other Cleavable Linker

Based on the characteristics of bioorthogonal chemistry that does not interfere with normal biological processes, high selectivity, fast and simple processing, and non-toxic by-products, bioorthogonal cleavage pairs can also be used as linker cleavage triggers. In addition, IEDDA reactions can also be used for payload release. Introduced by Tagworks Pharmaceuticals, this method uses trans-cyclooctene (TCO) as a cleavable linker to undergo a click reaction with a tetrazine activator to generate a 4,5-dihydropyridazine intermediate, which is then converted to 2,5- and 1 ,4-Tautomers, of which only the latter can undergo subsequent electron cascade reactions, thereby releasing different payloads. The IEDDA and tautomerization steps are affected by substituents on the tetrazine moiety, respectively, and show better drug release properties through the combination of two functional groups with opposite electron-withdrawing and electron-repelling properties.

In Conclusion

A good linker is a crucial assurance of the security and efficiency of ADC. Despite the prevalence of non-cleavable linkers, most ADC medicines prefer cleavable linkers because to the cytotoxicity of free payloads and the significance of bystander effects. Acid-cleavable linkers were initially promising, but the stringent stability requirements for highly toxic payloads reduced their usefulness. In contrast, the vast majority of ADCs currently use peptide technology because they can effectively distinguish blood circulation and target cell conditions, but this aspect still needs further development to better address key issues such as solubility and plasma stability. Furthermore, the applicability of extracellular cleavage to non-internalizing ADCs, eliminating the requirement for antigen internalization, greatly increases the number of possible antigen targets, which may open the door to many new ADC therapies.

References

1. Albericio, F. et al. Linkers: An Assurance for Controlled Delivery of Antibody-Drug Conjugate. Pharmaceutics. 2022, 14: 396.

2. Joubert, N. et al. Antibody–Drug Conjugates: The Last Decade. Pharmaceuticals. 2020, 13: 245.

3. Pillow, T.H. et al. Decoupling Stability and Release in Disulfide Bonds with Antibody-Small Molecule Conjugates. J. Name. 2013, 00: 1-3.

4. Spring, D.R. et al. Cleavable linkers in antibody–drug conjugates. Chem. Soc. Rev. 2019, 48(16): 4361-4374.

5. Spring, D.R. et al. Sulfatase-cleavable linkers for antibody-drug conjugates. Chem Sci. 2020, 11(9): 2375-2380.