Linkers - A Crucial Factor in Antibody–Drug Conjugates
Antibo-drug conjugate (ADCs) combines the high specificity of antibodies with the strong cytotoxicity of small-molecule drugs. This combination combines the antibody's unique and very sensitive targeting ability to distinguish between healthy and cancerous tissue. It also has the cell-killing capabilities of cytotoxic drugs, potentially minimizing dose-limiting toxicity while maximizing the desired therapeutic effect.
The primary advantage of ADCs is that they can be used as drugs in the systemic circulation, ultimately releasing free drugs into target tumor cells. In this process, linkers play a key role in the release of effective drugs targeting tumor cells, determining the pharmacokinetic properties, therapeutic markers and selectivity of the ADC, and even the overall success of the ADC. The linkers currently in use can be divided into two categories: clementable linkers and non-clementable linkers. The difference between them lies in whether they will be degraded in the cell.
1. Cleavable linkers for Linkers
The main category of ADC connectors is the cleavable connector. Cleavable linkers are designed to exhibit chemical instability to extracellular and intracellular environmental differences (pH, REDOX potential, etc.) or can be cleaved by specific lysosomal enzymes. In most cases, the linker is designed to release the payload molecule after the bond breaks. This untraceable drug release mechanism enables researchers to estimate the cytotoxicity of the conjugated payload based on known pharmacological parameters of the free payload.
1.1 Tpyes of cleavable linkers
Hydrazone linker
Hydrazone is an acid-unstable group that is used as a cleavable linker to release free drugs by hydrolysis when ADC is transported to endosomes (pH 5.0-6.0) and lysosomes (pH about 4.8).
Cathepsin B-responsive linker
Cathepsin B, a lysosomal protease, is overexpressed in many cancer cells and is involved in many human carcinogenic processes. Cathepsin B has a relatively broad range of substrates, but it preferentially recognizes certain sequences, such as phenylalanine-lysine (Phe-Lys) and Valine-citrulline (Val-Cit). The C-terminal of this sequence cuts the peptide bond.
The Val-Cit and Val-Ala ligand coupled to P-amino-benzyl carbonyl (Val-Cit- pabc and Val-Ala-pABC) are the most successful adc cleavable ligand. The PABC fragment enables free payload molecules to be released in a traceless manner.
Disulfide linker
Glutathione-sensitive linkers are another common lysed linker whose strategy relies on a higher concentration of reducing molecules (such as glutathione) in the cytoplasm (1-10 mmol/L). Disulfide bonds are embedded in the linker and resist reducing cleavage in the cycle. However, after internalization, large amounts of intracellular glutathione reduce disulfide bonds and release free payload molecules. To further improve stability in the cycle, a methyl group is usually installed next to the disulfide bond.
Pyrophosphate diester linker
The anionic linker has higher water solubility and excellent cycling stability than the traditional linker. In addition, after internalization, pyrophosphodiester is rapidly cleaved through the core-lysosome pathway to release unmodified payload molecules.
Figure 1: Types of cleavable linkers. (Kyoji Tsuchikama & Zhiqiang An. 2018)
2. Non-cleavable linkers for Linkers
Non-cleavable linkers consist of stable bonds that resist proteolytic degradation, ensuring higher stability than cleavable linkers. Non-cleavable linkers depend on the complete degradation of ADC antibody components by cytoplasmic and lysosomal proteases and the eventual release of payload molecules linked to amino acid residues derived from the degraded antibody.
The greatest advantage of non-cleavable linkers over cleavable linkers is their enhanced plasma stability, which may provide a larger window of treatment compared to cleavable linkers. In addition, it is expected to reduce off-target toxicity compared to sectionable couplings, as non-sectionable AdCs can provide greater stability and tolerance.
Figure 2. Uncleable linkers. The chemical stability of non-detachable connections can withstand proteolytic degradation. Cytoplasmic/lysosomal degradation of monoclonal antibodies can release payload molecules linked to degraded monoclonal amino acid residues.（Kyoji Tsuchikama & Zhiqiang An. 2018）
3. Conclusion in summary
The ultimate goal of Linker selection is to ensure the specific release of free drugs in tumor cells. This linker is important for ADC stability, toxicity, PK characteristics and pharmacodynamics. Each link has its advantages and disadvantages. Many factors must be considered in the selection of linkers, including existing, reactive, and derived functional groups in monoclonal antibodies and cytotoxic drugs. Finally, case-by-case analysis is needed to determine how to optimize the selection of appropriate connectors, targets and toxic molecules to balance the effectiveness and toxicity of ADC drugs.
Table1: Linker types and comparison of advantages and disadvantage
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Reference
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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.
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