An Introduction to Linkers in Antibody-Drug Conjugates (ADCs)

July 2, 2024
Posted by axispharm

Antibody-drug conjugates (ADCs) are “biological bullets” that combine cytotoxic drugs (payloads) with antibodies through “linkers.” ADCs consist of five key elements: target, antibody, linker, payload, and conjugation method. Each part is crucial to the function of ADCs, but today we will focus on the linkers that connect the antibodies and payloads to see how far research on linkers has progressed.

Characteristics of Linkers

For ADC drugs, linkers need to fulfill two tasks: first, they must ensure good stability of the ADC in the bloodstream; second, they must ensure that the ADC can precisely release the payload at the target site.

Therefore, linkers are required to have the following three characteristics:

Good stability to maintain the drug concentration of the ADC in the bloodstream and prevent premature release of the cytotoxic drug before reaching the target, thereby minimizing off-target effects and improving the safety of ADC drugs.

Classification of Linkers

ADCs have two types of linkers: non-cleavable linkers and cleavable linkers (Table 1). Non-cleavable linkers remain intact during intracellular metabolism. ADCs with these linkers require lysosomal degradation of the antibody to release the payload. Cleavable linkers, on the other hand, can be split during intracellular metabolism, producing metabolites containing the cytotoxic agent, which may include part of the linker.

Characteristics of Non-Cleavable Linkers

Non-cleavable linkers are divided into two types: thioether or maleimidocaproyl (MC), composed of stable bonds that prevent proteolytic cleavage (Figure 1). ADCs with this type of linker rely on lysosomal enzyme degradation to release the internalized payload, resulting in the simultaneous separation of the linker.

Early exploration of this type of linker was successfully conducted by Genentech/ImmunoGen, such as in the ADC drug trastuzumab emtansine (T-DM1 or Kadcyla®) for treating HER2-positive metastatic breast cancer. This ADC contains a non-cleavable SMCC (N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate) linker that connects the DM1 cytotoxin to trastuzumab.

Characteristics of Cleavable Linkers

Chemically Cleavable Linkers

Chemically cleavable linkers include acid-cleavable linkers (hydrazone bonds) and reducible linkers (disulfide bonds). The former are highly sensitive to acidic environments in the body, utilizing the acidic environment of endosomes and lysosomes to trigger linker cleavage and release the payload. However, hydrazone linkers can also undergo slow hydrolysis under physiological conditions (pH 7.4, 37°C), leading to the slow release of the toxic payload. A representative ADC drug is Mylotarg (Figure 2), the first approved ADC. However, due to the instability of the linker and the heterogeneity of the payload complex, the drug was prematurely released before reaching the target site, leading to its voluntary withdrawal by the FDA in 2010.

Another type is reducible linkers—disulfide bonds, which are dependent on reduced glutathione. Compared to plasma (~5 μmol/L), the cytoplasm has higher levels of glutathione (1–10 mmol/L), making reducible disulfide linkers relatively stable in the bloodstream and cleavable by intracellular glutathione to release the payload.

If you want to buy cleavable linker related products, here are some provided by AxisPharm:

Cleavable Linkers

Dde Cleavable Linkers

Cleavable Peptide

S-S- Cleavable

Disulfide Linkers

β-Glucuronide Linkers

Enzyme-Cleavable Linkers

Unlike chemically unstable linkers, enzyme-cleavable linkers utilize the unique high concentration of hydrolases in cells to cleave and release the drug, achieving clinical success in controlled drug release.

The first type is peptide linkers, mainly including dipeptide and tetrapeptide linkers. The cleavage mechanism of peptide linkers involves selective cleavage by cathepsin B after ADC internalization and transport to lysosomes, releasing the payload.

The most commonly used dipeptide linkers in approved ADC drugs include Val-Cit and Val-Ala dipeptides. Both linkers have comparable stability and cellular activity. However, due to precipitation and aggregation, Val-Cit is challenging to achieve high DAR (drug-to-antibody ratio), whereas Val-Ala linkers can achieve DAR as high as 7.4 with limited aggregation (<10%). Compared to Val-Cit, Val-Ala has higher hydrophilicity, making it advantageous in the context of lipophilic payloads, such as PBD dimers. Approved ADC drugs using these dipeptide linkers include Adcetris (Figure 3), Polivy, Padcev, and Disitamab Vedotin (RC48).

In addition to dipeptide linkers, the tetrapeptide Gly-Gly-Phe-Gly has also been successfully applied in ADC drugs. Compared to dipeptides, tetrapeptide linkers are more stable in the bloodstream. The approved ADC drug Enhertu (Figure 4) uses this type of linker, and Hengrui’s soon-to-be-launched HER2-targeting ADC drug SHR-A1811 also employs this tetrapeptide linker.

The second type of enzyme-cleavable linkers is glucuronide linkers, mainly including β-glucuronidase linkers and β-galactosidase linkers. β-glucuronidase-sensitive ADCs covalently bind cytotoxic drugs and antibodies through β-glucuronidase linkers and self-immolative PABC spacers. Similar to β-glucuronidase, β-galactosidase is overexpressed in certain tumors, hydrolyzing β-galactosidic bonds to release the drug. However, β-galactosidase is only present in lysosomes, whereas β-glucuronidase is expressed in lysosomes and the microenvironment of solid tumors.

Figure 5. Structure and Release Mechanism of β-Glucuronidase Linkers

Linkers affect the stability, toxicity, pharmacokinetics, and pharmacodynamics of ADCs, so careful selection of appropriate linkers is crucial in ADC design. The development of new linkers is steadily progressing, further enriching ADC design strategies to enhance their clinical value in the future.