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Antibody-drug conjugates (ADCs) are composed of monoclonal antibodies and cytotoxins covalently linked through a linker. The linker is the bridge between antibodies and cytotoxic drugs and plays a key role in ADC drugs because its properties greatly affect the therapeutic indicators, efficacy, and pharmacokinetics of these drugs. The ideal conjugation must be stable in vitro or in the blood circulation to prevent systemic toxicity caused by premature release of cytotoxic drugs, while at the same time being able to enter and kill cancer cells through the rapid release of effective cytotoxic drugs. BOC Sciences can provide ADC linkers with multiple cleavage mechanisms according to your project needs, including enzymatic cleavage linkers, chemical cleavage linkers and peptide linkers. We also support the integrated design of linkers to modulate payload release and ADC stability for optimal efficacy of ADC drugs.
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The linker is an essential component in ADC design, which connects the antibody to the cytotoxic payload through covalent coupling. The ADC linker should be stable enough in the circulation to maintain the ADC drug concentration in the blood circulation and not be released before the cytotoxic drug reaches the target, thereby causing minimal off-target effects and improving the safety of the ADC drug. At the same time, appropriate hydrophilicity or lipophilicity of the linker can enhance payload coupling and reduce immunogenicity properties and is also a key aspect of the linker. Therefore, in the design of ADC drugs, these important parameters of the linker must be correctly adjusted to achieve a balance between ADC stability and payload release efficiency, in order to achieve the expected effects of ADC drugs.
Fig. 1. ADC structure diagram.
The linker is composed of three parts: isolation rod, cleavage zone and release zone. Among them, the cleavage zone is the core part of the linker, and the cleavage site of the linker is usually in the cleavage zone. The function of the isolation rod and the release zone is mainly to modify the hydrophilic/hydrophobicity of the linker. Commonly used modifiers include MC (maleimidocaproyl), PEG (polyethylene glycol) and PABC (p-aminobenzyl alcohol). The release zone will be released together with the toxin after the linker is broken, so the release zone can also modify the hydrophilicity/hydrophobicity of the toxin after the linker is broken. The ideal ADC linker is mainly reflected in:
Two types of linkers are used in ADC development, cleavable linkers and non-cleavable linkers. These linkers play an important role in determining the pharmacokinetic properties, selectivity, therapeutic index, and overall success of ADCs. Both types of linkers have been shown to be safe in preclinical and clinical trials. In addition, linkers can be classified based on their drug release mechanisms and their stability in circulation. In the past few years, many new linkers have been developed, including cathepsin-cleavable linkers, acid-cleavable linkers, GSH-cleavable linkers, Fe(II)-cleavable linkers, novel enzyme-cleavable linkers, photoresponsive cleavable linkers and bioorthogonal cleavable linkers. Among them, cathepsins, GSH, and acid-cleavable linkers have been well studied and used in approved ADCs.
The linker may be cleavable. Here, a chemical bond (or multiple chemical bonds) between the payload and the antibody attachment site (usually an amino acid) will be cleaved intracellularly. The cleavable linker can be degraded under different pH values or the action of intracellular enzymes to achieve the separation of chemical drugs from antibodies. However, because the chemical may escape from the target cell after the drug is released, the drug can also kill tissue adjacent to the target cell.
Chemically cleaved linkers include acid-cleavable linkers (hydrazone bonds) and reducible linkers (disulfide bonds).
Acid-cleavable linker is highly sensitive to the acidic environment in the body and uses the acidic environment of endosomes and lysosomes to trigger cleavage of the linker and subsequently release the payload. However, in fact, linkers with hydrazone bonds can also undergo slow hydrolysis under physiological conditions (pH=7.4, 37 °C), resulting in slow release of toxic loads.
The other type is the reducible linker-disulfide bond linker. The disulfide bond is dependent on reduced glutathione. Compared with plasma (∼5 μmol/L), the level of glutathione in the cytoplasm is higher (1-10 mmol/L), therefore the reduced disulfide linker is relatively stable in the blood circulation, and intracellular glutathione reductively cleaves the disulfide linker to release the payload.
Unlike chemically unstable linkers, enzymatically cleaved linkers exploit the unique high concentration of hydrolases in cells to cleave and release drugs, achieving clinical success in controlled drug release.
The first category is peptide linkers, which mainly include dipeptide linkers and tetrapeptide linkers. The mechanism of peptide linker cleavage is that after internalization of ADC through endocytosis and transport to lysosomes, cathepsin B selectively cleaves the linker, thereby releasing the payload. The most commonly used dipeptide linkers among marketed ADC drugs include Val-Cit and Val-Ala dipeptides. The stability and cell activity of the two linkers are equivalent. In addition to dipeptide linkers, the tetrapeptide Gly-Gly-Phe-Gly has also been successfully used in ADC drugs. Compared with dipeptides, tetrapeptide linkers are more stable in blood circulation, and the marketed ADC drug Enhertu uses this type of linker.
The second type of enzyme-cleavable linkers is glucosidase linkers, which mainly include β-glucosidase linkers and β-galactosidase linkers. β-glucuronidase-sensitive ADCs are covalently bound to cytotoxic drugs and antibodies by combining a β-glucuronidase linker with a self-eliminating PABC spacer molecule. Similar to β-glucuronidase, β-galactosidase is overexpressed in certain tumors, where it hydrolyzes β-galactosidic bonds, releasing the drug. The difference is that β-galactosidase only exists in lysosomes, while β-glucuronidase is expressed in lysosomes and also in the microenvironment of solid tumors.
A third type of enzymatically cleavable linker is sulfatase cleavable linkers. Sulfatases are overexpressed in multiple cancer types, exhibiting potential selectivity. The study involved Her2 antibodies with MMAE as the payload. Compared with the classic cleavable Val-Ala and Val-Cit linkers, the sulfatase linker showed similar potency on HER2+ cell lines.
The final category is phosphatase-cleaved linkers. Phosphatases belong to another important class of enzymes that cleave connexins and are target enzymes expressed exclusively in the lysosomal compartment. These linkers target pyrophosphatase and acid phosphatase, which hydrolyze the pyrophosphatase and terminal monophosphate into their respective alcohols, thereby releasing the payload.
Non-cleavable linkers fall into two groups, thioether or maleimidocaproyl (MC). They consist of stable bonds that protect against proteolytic cleavage and ensure greater plasma stability than their cleavable counterparts. ADCs containing such linkers rely on complete lysosomal enzymatic degradation of the antibody, releasing the payload upon internalization, resulting in simultaneous detachment of the linker. The advantage of non-cleavable linkers over cleavable linkers is their improved stability. Genentech/Immunogen has successfully explored this linkage strategy, with Trastuzumab emtansine (Kadcyla/T-DM1) gaining clinical approval. This ADC contains a non-cleavable SMCC (N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate) linker to link warhead DM1 cytotoxin to an anti-HER2 mAb Lys residue of Trastuzumab. The drug conjugate exhibits greater activity than traditional trastuzumab-DM1 or trastuzumab conjugated to other maytansinoid alkaloids via reducible disulfide bonds. Likewise, MMAF drug conjugates with non-reducible thioether linkers were found to be more stable than Val-Cit conjugates and also retained their potency.
Notably, non-cleavable linkers allow the chemical properties of small molecules to be altered to adjust transporter affinity or improve potency. The advantage of non-cleavable linkers over cleavable linkers is their increased plasma stability. Overall, non-cleavable linkers provide a larger therapeutic window than cleavable linkers because payload derivatives from non-cleavable ADCs can kill target cells.
Given that linkers often affect the stability, toxicity, pharmacokinetic properties, and pharmacodynamics of ADCs, great care must be taken when selecting linkers in ADC design. In addition, linkers must consider reactive groups on the cytotoxic drug, including mAbs and derivatized functional groups. Currently, most ADCs in the clinical stage use common Lys and Cys residues for conjugation. In this case, researchers are working on developing new linkers. For example, photosensitive ADC linkers and bioorthogonal cleavable linkers, etc. Although these linkers offer several advantages, such as specificity, potency, and low toxicity, ADCs containing them are still in their infancy.
In view of this, in addition to providing a full range of ADC linkers, BOC Sciences also provides customized development services for special linkers. We provide one-stop ADC linker development services, including linker design, synthesis, characterization and optimization. Our team of experienced chemists and scientists works closely with customers to develop custom connector solutions that meet their specific requirements, providing expert guidance and support throughout the development process.
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