Antibody-drug conjugates (ADCs) represent a significant advancement in targeted cancer therapy, combining the specificity of monoclonal antibodies with the potent cytotoxic effects of chemotherapy. A critical component of these conjugates is the linker that connects the antibody to the cytotoxic payload. Among various linker types, disulfide linkers have garnered attention for their unique properties, which facilitate selective drug release in the tumor microenvironment. This article explores the chemical properties of disulfide linkers, their mechanisms of action within ADCs, and the various payloads that can be utilized to enhance therapeutic outcomes in cancer treatment.

Antibody Drug Conjugate

Antibody-drug conjugates are complex molecules that connect targeted antibodies and cytotoxic toxin small molecules (payloads) through chemical linkers. In recent years, research on payloads and linkers has gradually deepened and made breakthrough progress. ADCs represented by Enhertu (DS-8201) have broken through people's understanding of traditional ADCs and greatly improved clinical treatment effects, becoming blockbuster products. In the past two years, ADC drugs have also ushered in an explosive period of listing, becoming one of the hottest tracks in the field of innovative drugs. The linker of ADC must not only connect antibodies and toxins, but also be able to cleave and release toxins at tumor cells. These two functions determine that the linker must undertake two tasks: first, it must ensure that ADC has good stability in the blood; second, it must ensure that ADC can accurately release the payload at the target location. Therefore, the linker is required to have the following three characteristics:

• The linker has good stability, which can maintain the drug concentration of ADC in the blood circulation and will not be released before the cytotoxic drug reaches the target, resulting in minimal off-target effects and improving the safety of ADC drugs;
• The linker allows the rapid release of the payload at the target site after internalization;
• The appropriate hydrophilicity/lipophilicity of the linker can enhance the properties of payload binding and reduce immunogenicity.

Fig. 1. ADC linker (Acta Pharm Sin B. 2021, 11(12): 3889-3907).

ADC Linker Technology

ADC linkers are divided into two types: non-cleavable linkers and cleavable linkers. Non-cleavable linkers mean that the linker remains intact during intracellular metabolism. ADCs with such linkers require lysosomal degradation of the antibody to release the payload. Cleavable linkers mean that the linker can be split during intracellular metabolism, which will produce metabolites containing the cytotoxic agent, which may have part of the linker.

What is Disulfide Linker?

In chemistry, a disulfide is a compound containing the functional group R−S−S−R′. This bond is also called an SS bond or sometimes a disulfide bridge and is usually derived from two thiol groups. Disulfide linkers are a specialized class of chemically cleavable linkers used in the design of ADCs. These linkers contain disulfide bonds, which are unique due to their ability to remain stable in the bloodstream while selectively releasing their payload within the reducing environment of tumor cells. This feature makes disulfide linkers an attractive option for the design of more effective and safer cancer therapies.

In physiological conditions, disulfide bonds are thermodynamically stable, allowing them to withstand degradation in the bloodstream. This stability is essential as it prevents the premature release of the drug before it reaches the targeted cells. Disulfide linkers remain intact while circulating, minimizing off-target effects and potential toxicity to normal tissues. When the drug is internalized by cancer cells, the level of glutathione in the cytoplasm is higher (1–10 mmol/L) than in plasma (∼5 μmol/L), so the reduced disulfide linker is reduced and cleaved by glutathione in the cell to release the payload.

Disulfide Linker from BOC Sciences

Advantages of Disulfide Linkers

• Redox Sensitivity: The most significant advantage of disulfide linkers is their redox sensitivity. They remain stable in extracellular environments but readily break down in the presence of intracellular reducing agents like glutathione. This property is exploited in targeted drug delivery, ensuring that drugs are released within the target cells.
• Biocompatibility: Disulfide linkers are biocompatible, minimizing immunogenic and cytotoxic responses when used in vivo. Since disulfide bonds naturally occur in proteins, they are well-tolerated by biological systems.
• Versatility: Disulfide linkers can be incorporated into various molecular architectures, including polymers, peptides, and nanoparticles. This versatility allows them to be used in a wide range of applications, from drug delivery to biosensors.
• Functionalization: These linkers can be easily modified to introduce other functional groups, enhancing their applicability. For instance, they can be functionalized with fluorescent tags for imaging or with targeting moieties for precise delivery.

Disulfide Linker for ADC Drugs

In the context of ADCs, disulfide linkers serve as a critical bridge connecting the cytotoxic drug to the antibody that targets specific cancer cells. However, the current disulfide bond structure cannot achieve a perfect combination of high circulatory stability and efficient intracellular release. In 2017, Thomas et al. attempted to solve this problem by directly connecting small molecule drugs to engineered cysteine in the Thiomab antibody. Thiomab technology, also known as the introduction of reactive cysteine engineering technology, was first developed by Genentech. Simply put, it is to insert cysteine residues at specific points of the antibody through genetic engineering, and then couple the hydroxyl group on the cysteine with the toxin to form a site-specific antibody-drug conjugate. Thiomab technology will neither interfere with the folding and assembly of immunoglobulins nor change the binding mode of antibody antigens. The obtained ADC drug not only retains its in vivo anti-tumor activity, but also improves tolerance and reduces systemic toxicity. This technology has the advantages of high uniformity, good reactivity and stability, but the disadvantage is that it requires genetic modification, usually DAR 2, 4, 6, 8 (2N).

Fig. 2. Structure of the glutathione cleavage linker (Acta Pharm Sin B. 2021, 11(12): 3889-3907).

The application of disulfide linkers in ADCs offers several advantages. They exhibit enhanced serum stability compared to pH-sensitive linkers, reducing the risk of off-target drug release and associated toxicity. Additionally, the selective release mechanism ensures that the drug is activated only within the target cells, increasing the therapeutic index of the ADC. Currently, a variety of payloads can be conjugated to antibodies via disulfide linkers, including paclitaxel, calicheamicin, and maytansinoid derivatives. These payloads are chosen for their potent cytotoxic effects and ability to induce cell death once released. For instance, maytansinoid derivatives, such as DM1 and DM4, are commonly used in clinical trials and are linked via disulfide bonds to achieve effective therapeutic outcomes.

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