Linkers - A Crucial Factor in Antibody–Drug Conjugates

Linkers - A Crucial Factor in Antibody–Drug Conjugates

Antibody-drug conjugate (ADC) combines the high specificity of antibodies with the strong cytotoxicity of small molecule drugs. This combination combines the unique and very sensitive target capabilities of antibodies, which can distinguish healthy tissues from cancerous tissues. It also has the cell-killing ability of cytotoxic drugs, potentially minimizing dose-limiting toxicity, while maximizing the desired therapeutic effect.

The main advantage of ADCs is that they can be used as drugs during systemic circulation and eventually release free drugs in target tumor cells. In this process, the linker plays a key role in releasing effective drugs to target tumor cells, determining the pharmacokinetic properties, therapeutic indicators and selectivity, and even overall success of ADCs. The currently used linkers can be divided into two categories, namely cleavable linkers, and non-cleavable linkers, and the difference between them is whether they will be degraded in the cell.

Cleavable linkers for Linkers

The main category of ADC linkers is cleavable linkers. The cleavable linker is designed to show chemical instability to the environmental differences between the extracellular and intracellular environments (pH, redox potential, etc.) or it can be cleaved by specific lysosomal enzymes. In most cases, such linkers are designed to release the payload molecule after the bond is broken. This traceless drug release mechanism allows researchers to estimate the cytotoxicity of the conjugate payload based on the known pharmacological parameters of the free payload.

Types of cleavable linkers

  • Hydrazone linker 

Hydrazone, an acid-labile group, is used as a cleavable linker that releases free drug through hydrolysis once an ADC is transported to endosomes (pH 5.0–6.0) and lysosomes (pH about 4.8).

  • Cathepsin B-responsive linker 

Cathepsin B is a lysosomal protease, which is overexpressed in various cancer cells and participates in many carcinogenic processes in humans. Cathepsin B has a relatively wide substrate range, but it preferentially recognizes certain sequences, such as phenylalanine-lysine (Phe-Lys) and valine-citrulline (Val-Cit). The C-terminus of such sequences cleaves the peptide bond.

Val-Cit and Val-Ala linkers coupled with p -aminobenzyloxycarbonyl (Val-Cit-PABC and Val-Ala-PABC) are the most successful cleavable linkers for ADCs. The PABC moiety enables the free payload molecules release in a traceless manner.

  • Disulfide linker

Glutathione-sensitive linkers are another commonly cleaved linker, which strategy relies on a higher concentration of reducing molecules (such as glutathione) in the cytoplasm (1-10 mmol/L). The disulfide bond is embedded in the linker and resists reductive cleavage in circulation. However, after internalization, a large amount of intracellular glutathione reduces disulfide bonds to release free payload molecules. In order to further enhance the stability in the cycle, a methyl group is usually installed next to the disulfide bond.

  • Pyrophosphate diester linker

This anionic linker has higher water solubility than traditional linkers and has excellent cycle stability. In addition, after internalization, the pyrophosphodiester is rapidly cleaved through the endosome-lysosome pathway, releasing unmodified payload molecules.

Figure 1: Types of cleavable linkers. (Kyoji Tsuchikama & Zhiqiang An. 2018)

Non-cleavable linkers for Linkers

The non-cleavable linker is composed of stable bonds that resist proteolytic degradation, ensuring higher stability than the cleavable linker. The non-cleavable linker relies on the complete degradation of ADC antibody components by cytoplasm and lysosomal proteases, and finally releases payload molecules linked to amino acid residues derived from the degraded antibody.

Compared with cleavable linkers, the biggest advantage of non-cleavable linkers is that their plasma stability is enhanced, which can potentially provide a larger therapeutic window compared to cleavable linkers. In addition, compared with cleavable linker conjugates, it is expected to reduce off-target toxicity, since the non-cleavable ADCs can provide greater stability and tolerance.

Figure 2: Non-cleavable linker. The chemical stability of the non-cleavable link can withstand proteolytic degradation. Cytoplasmic/lysosomal degradation of the mAb can release payload molecules linked to the degraded mAb-derived amino acid residues. (Kyoji Tsuchikama & Zhiqiang An. 2018)

Conclusion in summary

Ensuring the specific release of free drugs in tumor cells is the ultimate-goal of choosing Linker. The linker is very important for the stability, toxicity, PK characteristics, and pharmacodynamics of ADC. Each linker has its advantages and disadvantages. When choosing linkers, many factors must be considered, including the existing groups in the monoclonal antibodies and cytotoxic drugs, the reactive groups and derived functional groups. In the end, it is necessary to determine how to optimize the selection of appropriate linkers, targets, and toxic molecules through case by case analysis to balance the effectiveness and toxicity of ADC drugs.

Table 1: Linker types and comparison of advantages and disadvantages.

LinkerStrategyAdvantagesDisadvantages
Cleavable LinkerHydrazone linkerStable at the neutral pH of blood circulationInsufficiently stable under physiological conditions
Cathepsin B-responsive linkerGreater systemic stability with rapid enzymatic release of the drug in the target cell 
DisulfideIntracellular release of payloadPotential premature cleavage during circulation
Pyrophosphate diesterstability during circulation
Hydrophilicity 
Traceless release of payload
Unknown mechanism of lysosomal cleavage
Non-cleavable linkerStable linker without cleavage mechanismStability during circulation  An amino acid residue attached on the released payload

References:

  1. Kyoji Tsuchikama & Zhiqiang An. Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein & Cell. 2018; 9:33-46.
  2. Jun Lu. Feng Jiang. Aiping Lu. and Ge Zhang. Linkers Having a Crucial Role in Antibody–Drug Conjugates. Int J Mol Sci. 2016 Apr; 17(4):561.
  3. Monteiro Ide P, Madureira P, de Vasconscelos A, Pozza DH, de Mello RA. Targeting HER family in HER2-positive metastatic breast cancer: potential biomarkers and novel targeted therapies. Pharmacogenomics. 2015; 16(3):257-71.

* Only for research. Not suitable for any diagnostic or therapeutic use.

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