Cleavable Linkers Play a Pivotal Role in the Success of Antibody-Drug Conjugates (ADCs)

Cleavable linkers used in ADC development can release drugs under specific conditions, such as changes in pH, the presence of specific enzymes, or reducing environments within target cells. Although cleavable linkers are designed to be stable in circulation, they are diverse and can bind to a wider variety of payloads. They can be further subdivided into chemically labile linkers and enzyme-cleavable linkers. The success of cleavable linkers hinges on their ability to efficiently distinguish between blood circulation conditions and target cell conditions. By optimizing the structure and performance of these cleavable linkers, scientists can develop more efficient and safer ADC drugs, bringing new hope to cancer patients. Overall, the use of cleavable linkers is of great significance in ADC development, as they enhance drug targeting and effectiveness while minimizing toxicity to normal cells, leading to new breakthroughs in cancer treatment.

The structure and function diagram of ADC
The structure and function diagram of ADC

What are cleavable linkers?

Cleavable linkers are a crucial component in ADC development, playing a significant role in the clinical pipeline. These specialized bioconjugation linkers connect therapeutic drugs to antibodies and are designed to release the drug payload under specific conditions within target cells. Cleavable linkers are particularly popular due to their ability to enhance the therapeutic efficacy of ADCs while minimizing toxic effects on normal cells. They can be divided into two main categories based on their cleavage mechanisms: chemically labile linkers and enzyme-cleavable linkers. Chemically labile linkers, such as acid-sensitive hydrazones and silyl ethers, respond to environmental changes like pH or redox conditions. Enzyme-cleavable linkers, on the other hand, are broken down by specific enzymes present in the target cells.

Cleavable linkers are powerful tools in chemical biology, pharmaceutical, and biotech research and development. They can be cleaved once exposed to enzymes, photo-irradiation, or chemical reagents, making them versatile for various applications. The primary characteristic of cleavable linkers is their capacity to be broken down by environmental variations, such as redox potential and pH, as well as specific lysosomal enzymes in response to both extracellular and intracellular conditions. This precise control over drug release makes cleavable linkers the major class of ADC linkers, offering significant potential for targeted cancer therapies and other medical applications.

Cleavable Linker Mechanism

Cleavable linkers are specialized chemical bonds used in antibody-drug conjugates (ADCs) that are designed to release the drug payload under specific conditions. These cleavable linkers are engineered to break down in response to certain stimuli, such as changes in pH, the presence of specific enzymes, or exposure to reducing environments.

For example, acid-cleavable linkers are designed to break down in the acidic environment of lysosomes within cells, ensuring that the drug is released precisely where it is needed. This targeted release mechanism helps to minimize off-target effects and enhances the therapeutic efficacy of the drug.

Cleavable Linker Examples

Several types of cleavable linkers are commonly used in the design of ADCs. Acid-cleavable linkers, such as hydrazone and carbonate linkers, break down in acidic environments and are often used to target the acidic lysosomal compartments within cells.

Enzyme-cleavable linkers, like valine-citrulline (Val-Cit) and glycine-phenylalanine-leucine-glycine (GFLG), are designed to be cleaved by specific enzymes that are overexpressed in certain types of cancer cells. Another example is disulfide linkers, which are cleaved in the reducing environment of the cytoplasm. Each type of cleavable linker offers unique advantages depending on the specific therapeutic application and the biological environment in which the drug is intended to act.

What is the difference between cleavable and non-cleavable links?

Cleavable and non-cleavable linkers are essential components in the design of antibody-drug conjugates (ADCs), each offering unique advantages for targeted cancer therapy. Cleavable linkers are designed to release the cytotoxic drug in response to specific environmental conditions within tumor cells, such as changes in pH, redox potential, or the presence of lysosomal enzymes. This targeted release mechanism ensures that the drug is delivered precisely where it is needed, minimizing damage to healthy cells. Various types of cleavable linkers, including acid-sensitive linkers, lysosomal protease-sensitive linkers, β-glucuronide linkers, and glutathione-sensitive disulfide linkers, exploit the unique microenvironment of cancer cells to achieve this precise drug delivery. These cleavable linkers are particularly effective in ensuring that the cytotoxic payload is only unleashed within the tumor, thereby enhancing the therapeutic efficacy and reducing systemic toxicity.

Common cleavable linkers used in ADCs
Common cleavable linkers used in ADCs

In contrast, non-cleavable linkers provide higher stability by resisting proteolytic degradation and maintaining the drug in an inactive state while in circulation. These linkers do not release the drug until the ADC is internalized and the antibody component is degraded within the lysosome, ensuring that the cytotoxic agent is not unleashed at off-target sites. This mechanism minimizes off-target toxicity and maintains the drug’s efficacy. Non-cleavable linkers, such as the SMCC linker, are particularly useful for modifying the chemical properties of small molecules to enhance their therapeutic efficacy and reduce toxicity. For example, trastuzumab-SMCC-DM1 has demonstrated superior activity and reduced toxicity compared to other conjugates. While cleavable linkers offer the advantage of targeted drug release, non-cleavable linkers provide stability and reduce the risk of harming healthy cells, making both types of linkers indispensable in the development of effective and safe ADCs.

Common non-cleavable linkers used in ADCs
Common non-cleavable linkers used in ADCs

What is a pH-sensitive cleavable linker?

A pH-sensitive cleavable linker is designed to exploit the acidic microenvironment of tumor tissues and the pH variations within cellular compartments to achieve selective drug release. Tumor tissues generally exhibit a lower pH (6–7) compared to normal tissues (pH 7.4) due to elevated glycolysis and poor lymphatic drainage. Additionally, during endocytic or exocytic processes, the pH inside endosomes and lysosomes drops to about 5.5–6.0 and 4.5–5.0, respectively. These pH differences are leveraged by pH-sensitive cleavable linkers to trigger the release of prodrugs or therapeutic payloads specifically within tumor tissues or through endocytosis/exocytosis processes. The linker must be stable enough to avoid premature release and sensitive enough to ensure efficient release upon cellular uptake. The choice of suitable linker chemistry is often based on intuition, but the significant pH difference between endosomes and the extracellular space (ΔpH ≈ 2) makes it easier to achieve selective release.

Imine/Hydrazone/Oxime Linkers

These cleavable linkers utilize acid-catalyzed hydrolysis for drug release. The hydrolysis rate is influenced by the electron-withdrawing properties of the substituents, with acyl hydrazone cleavable linkers showing a unique balance of stability at neutral pH and lability at acidic pH. For example, acyl hydrazone linkers have been used in the first generation of ADCs, such as cBR96-Dox, which demonstrated minimal premature drug release and reduced side effects in clinical trials.

Mechanism of hydrolytic cleavage of hydrazone/oxime linkers
Mechanism of hydrolytic cleavage of hydrazone/oxime linkers

Phosphoramidate Linkers

These cleavable linkers employ general-acid catalysis facilitated by a protonated pyridinyl group, allowing for tunable release rates. The hydrolysis rate is affected by the pKa values of the amine and pyridinyl groups, with the position of the protonated pyridinium nitrogen also playing a role.

Phosphoramidate linker chemistry
Phosphoramidate linker chemistry

Acetal-Based Linkers

Acetal linkers undergo acid-catalyzed hydrolysis and have been assessed for their hydrolysis profiles. Ethoxybenzyl acetal derivatives have shown high selectivity and efficiency, making them suitable for pH-sensitive drug delivery.

Ethoxybenzylimidazole (NEBI) Linkers

These cleavable linkers use the hydrolysis of “aminol” ether for drug release. The release rate is tunable by varying the substitution on the phenyl ring, with electron-donating groups accelerating the release at acidic pH.

Maleic Acid-Derived Linkers

These cleavable linkers rely on the intramolecular cyclization of maleoyl amide at acidic pH for drug release. They offer a highly desirable pH-sensitive release profile, with complete release at pH 4.0 while remaining stable at pH 7.0. This chemistry has been used to develop pH-sensitive phospholipids for drug and gene delivery.

Maleic acid-derived linkers
Maleic acid-derived linkers

In summary, pH-sensitive cleavable linkers are crucial for targeted cancer therapy, allowing for selective drug release in response to the acidic microenvironment of tumors or within cellular compartments. Various chemistries, including imine/hydrazone/oxime, phosphoramidate, acetal, NEBI, and maleic acid-derived linkers, offer different advantages and can be tailored to specific therapeutic needs. Continued development of new pH-sensitive cleavable linkers with tunable release rates is essential for advancing prodrug and gene-delivery systems.

How does a cleavable linker work?

A cleavable linker in an antibody-drug conjugate (ADC) works by covalently tethering the antibody to the cytotoxic payload, ensuring that the drug remains inactive while circulating in the bloodstream. This stability is crucial to prevent premature release of the toxic payload, which could lead to systemic toxicity and damage to healthy cells.

The cleavable linker is designed to exploit specific environmental conditions at the target site, such as the acidic microenvironment of tumor tissues or the presence of certain enzymes within the tumor cells. Upon reaching the tumor site, the linker undergoes controlled cleavage, triggered by these specific conditions. This targeted release mechanism enhances the therapeutic efficacy of the ADC while minimizing off-target effects. Various chemistries, such as hydrazone, disulfide, and peptide-based linkers, are employed to achieve this controlled cleavage, each tailored to respond to different environmental triggers.

The development and optimization of these cleavable linkers are critical for the success of ADCs, as they must balance stability in circulation with the ability to release the drug rapidly and efficiently within the tumor cells.

Different Types of Cleavable Linkers in ADC Development

Antibody-drug conjugates (ADCs) are a revolutionary class of targeted cancer therapies that combine the specificity of monoclonal antibodies with the potent cytotoxicity of small-molecule drugs. A critical component of ADCs is the linker, which connects the antibody to the drug. Cleavable linkers are designed to release the drug selectively within the target cancer cells, exploiting specific intracellular conditions or enzymatic activities. Cleavable linkers are usually classified into chemically labile linkers and enzyme-cleavable linkers.

Chemically Labile Linkers

Chemically labile linkers are designed to remain stable under normal physiological conditions but undergo cleavage in response to changes in pH, redox potential, or the presence of specific ions or molecules. The versatility and precision of chemically labile linkers make them an essential tool in the development of targeted cancer therapies, offering a promising approach to improve the safety and effectiveness of ADCs. Acid-cleavable linkers and reducible or disulfide linkers are primarily classified as chemically labile linkers. In addition, Dde cleavable linkers, –S-S- cleavable linkers, and some other cleavable linkers are also included.

Acid-Cleavable Linkers

Acid-cleavable linkers are designed to utilize the acidic environment of endosomes (pH 5.5–6.2) and lysosomes (pH 4.5–5.0), while maintaining stability in the bloodstream at physiological pH (7.4). This strategy achieved early clinical success with Pfizer’s Mylotarg (AcBut Linker). During its development, a series of hydrazone-containing linkers were tested for stability at pH 4.5 and pH 7.4. Cleavable linkers that were stable at pH 7.4 but unstable at pH 4.5 provided the most effective ADCs. This linker-payload strategy is also applied to Besponsa.

Reducible or Disulfide Linkers

Reducible or disulfide linkers are a pivotal component in the design of antibody-drug conjugates (ADCs), offering a strategic mechanism for the controlled release of therapeutic payloads within target cells. These cleavable linkers exploit the redox potential differences between the extracellular environment and the intracellular milieu, particularly the elevated levels of reducing agents such as glutathione (GSH) found within cells. Disulfide bonds within these cleavable linkers remain stable in the relatively oxidizing conditions of the bloodstream but are cleaved in the reducing environment of the cytoplasm, thereby releasing the cytotoxic drug specifically within cancer cells. This selective release mechanism enhances the therapeutic efficacy of ADCs while minimizing off-target effects and systemic toxicity. The unique properties of reducible linkers make them an attractive choice for delivering highly potent drugs, ensuring that the payload is released precisely where it is needed most, thereby maximizing the therapeutic index of ADCs.

Dde Cleavable Linkers

The Dde (1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) group is known for its unique property of being cleavable under mild hydrazine conditions, which allows for the selective release of therapeutic agents. Dde cleavable linkers are stable under physiological conditions, ensuring that the drug remains intact during circulation and only releases its payload upon encountering the specific chemical trigger. This makes them a valuable tool in the development of advanced drug delivery systems aimed at improving the safety and effectiveness of treatments for various diseases, including cancer.

Enzyme-Cleavable Linkers

Enzyme-cleavable linkers are a sophisticated and highly effective strategy in the design of antibody-drug conjugates (ADCs), enabling the targeted release of therapeutic agents within specific cellular environments. These cleavable linkers are engineered to be stable in the bloodstream but are selectively cleaved by enzymes that are overexpressed in the tumor microenvironment or within cancer cells. Commonly used enzymes include proteases, glycosidases, and esterases, which recognize and hydrolyze specific peptide or chemical bonds within the linker. The precision and reliability of enzyme-cleavable linkers make them a crucial component in the development of next-generation ADCs, offering a promising approach to improve the selectivity and potency of cancer treatments. Enzyme-cleavable linkers can be divided into several types such as protease-cleavable linkers and glycosidase-cleavable linkers based on the specific enzymes that cleave them.

Protease-Cleavable Linkers

Protease-cleavable linkers are selectively cleaved by proteases, a class of enzymes that break down proteins by hydrolyzing peptide bonds. These cleavable linkers are particularly valuable in targeted drug delivery systems, where they enable the controlled release of therapeutic agents in specific biological environments characterized by elevated protease activity, such as tumor tissues or sites of inflammation. By incorporating protease-cleavable linkers into drug conjugates, it is possible to achieve a high degree of specificity, minimizing off-target effects and enhancing the therapeutic index of the drug. Common examples of protease-cleavable linkers include sequences that are recognized and cleaved by matrix metalloproteinases (MMPs) or cathepsins, which are often overexpressed in cancerous tissues.

Protease-Cleavable Linkers
Protease-Cleavable Linkers

Glycosidase-Cleavable Linkers

Glycosidase-cleavable linkers are selectively cleaved by glycosidases, enzymes that hydrolyze glycosidic bonds within carbohydrates. These cleavable linkers are particularly useful in targeted drug delivery and diagnostic applications, where they enable the controlled release of therapeutic agents or imaging probes in environments with specific glycosidase activity. Glycosidases are often overexpressed in certain pathological conditions, such as cancer or lysosomal storage diseases, making glycosidase-cleavable linkers an effective tool for achieving site-specific drug activation. Examples of glycosidase-cleavable linkers include β-Glucuronide Linkers with enzymes that are often upregulated in tumor microenvironments. This targeted approach allows for more precise and effective treatments, improving patient outcomes and reducing side effects.

Glucuronide linker structure
Glucuronide linker structure
Glycosidase-Cleavable Linkers

Cleavable linkers are a major class of ADC linkers, designed to be cleaved by environmental differences (such as redox potential, pH) and specific lysosomal enzymes in response to extracellular and intracellular environments. The choice of linker can significantly impact the efficacy and safety profile of ADCs. Axispharm, for instance, provides several kinds of cleavable linker products, including disulfide linkers, photocleavable biotin, and enzymatic cleavable linkers and other mentioned linkers. At the same time, ongoing research aims to develop novel cleavable linkers that improve the precision and effectiveness of the targeted cancer therapies.

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