Release date:2023/7/31 17:09:35

Antibody-drug conjugate (ADC) is a monoclonal antibody covalently bound to a cytotoxic chemical substance (payload) through linker. As an emerging class of anticancer therapeutic drugs, ADCs can deliver highly cytotoxic molecules directly to cancer cells to kill them.

Studies have shown that the linker plays a key role in ADC drugs because its properties greatly affect the therapeutic index, efficacy and pharmacokinetics of these drugs. The stable linker can maintain the drug concentration of the ADC in the blood circulation and will not be released before the cytotoxic drug reaches the target, resulting in minimal off-target effects and improving the safety of the ADC drug.

 The structure and function diagram of ADC

Figure 1.The structure and function diagram of ADC

In order for the ADC to be selective and therapeutic, the linker adopted should strive to achieve three key characteristics :

(1) High cycle stability: Payload will not be released before it reaches the target, thus minimizing the off-target effect;

(2) High water solubility: it is helpful for conjugating and avoiding the formation of inactive ADC aggregates;

(3) Efficient release: Allows efficient release of highly cytotoxic linker-payload metabolites.

The development of ADCs has progressed significantly over the past decade due to improvements in payloads, linkers, and conjugation methods. In particular, linker design plays a key role in regulating the stability of ADCs in the systemic circulation and the efficiency of payload release in tumors, thereby affecting the pharmacokinetics (PK), efficacy, and toxicity profiles of ADCs.

1. Antibody in ADCs

An important component of ADCs are monoclonal antibodies. The basic premise for selecting an antibody for ADC design is that it can specifically recognize and bind to tumor antigen receptors, and in the process deliver the payload to tumor cells. In addition, the antibody must also have high binding affinity for the specific target antigen, low immunogenicity, apparent stability in the bloodstream, and low cross-reactivity.

Currently, antibodies used in ADC design are mostly from human immunoglobulin G (IgG) subclasses (IgG1, IgG2, IgG4), consisting of two heavy chains and two light chains. ADC targeting antigens must be highly expressed on tumor cells and should also have internalization properties to enhance receptor-mediated endocytosis of ADCs. Currently, Nectin4, CD79b, CD22, CD33, HER2, CD30, FOLR1, and TROP2 are the most targeted antigens in ADCs. In addition, more than 70 other antigens are in various stages of clinical development.

2. Payload in ADCs

The payload is a potent drug designed to kill cancer cells. In general, the payload needs to have maximal plasma stability and subnanomolar IC50 values on tumor cells in vitro, since only 1–2% of the injected ADC reaches the tumor. Cytotoxic drugs are transported throughout the body through the blood. Currently, auristatins and maytansinoids are the most commonly used drugs in ADC development (Figure 2).

 Common ADC payloads

Figure 2. Cytotoxic drugs used in ADC design
(Source: References[2])

3. Linker in ADCs

Linkers are essential components in ADC design, which link antibodies to cytotoxic payloads through covalent conjugation.

A linker is an essential component of ADC design, which connects antibodies to cytotoxic loads through covalent conjugation. The key requirement for the linker is that it must ensure the chemical stability of the ADC in blood (i.e., a half-life ~10-fold longer than that of the ADC) and allow rapid release of the payload at the target site after internalization. Meanwhile, proper hydrophilicity/lipophilicity of the linker, which can enhance payload coupling and reduce immunogenicity, is also a key aspect of linkers.

The linker used in ADCs is divided into two types: cleavable linker and non-cleavable linker (Figure 3B). These linkers play a major role in determining the pharmacokinetic properties, selectivity, and therapeutic index of ADCs. For the non-cleavable linker, after entering the lysosome, the mAb is metabolized through the proteolytic mechanism, and the released metabolites include payload, linker and amino acid appendages. Substantial modifications to payload can also produce effective ADCs if the key pharmacophore of Payload is not affected, as is the case with Kadcyla. However, due to the lack of cell permeability of the charged amino acid appendages, the non-cleavable linker is usually unable to exert the bystander effect , so the application range of the ADC containing the non-cleavable linker is limited, and it is mainly used for the treatment of hematological cancers or tumors with high antigen expression.

 Common ADC payloads

Figure 3. The role of Linker in ADC (A) and classification (B)
(Source: References[2])

Cleavable linkers are a major class of linkers used in ADC development. Compared with the non-cleavable linkers, the cleavable linkers release the drug at the target cell under specific conditions. Cleavable linkers are less stable and more prone to off-target toxicity, but they are also more versatile allowing its use with a greater number of payloads. Cleavable linkers can be further subdivided as chemically labile linkers and enzyme-cleavable linkers. Although cleavable linkers are generally better than non-cleavable linkers in terms of scope of application, they are more unstable in blood circulation. Thus, the success of cleavable linkers depends on their ability to efficiently discriminate between blood circulation conditions and target cell conditions. Let's mainly introduce the cleavable linkers used in ADC development.

4. Cleavable Linkers

Cleavable linkers can be can be classified into two subclasses, that is chemically labile linkers and enzyme-cleavable linkers. Chemically labile linkers can be further classified as acid-cleavable linkers and reducible or disulfide linkers.

4.1 Chemically Labile Linkers 

4.1.1 Acid-cleavable linkers: Mylotarg, Besponsa, Trodelvy

Acid-cleavable Linkers are designed to utilize the acidity of endosomes (pH 5.5–6.2) and lysosomes (pH 4.5–5.0), while maintaining circulation stability under physiological conditions at pH 7.4. This strategy achieved the earliest clinical success with Pfizer's Mylotarg (AcBut Linker). During its development, testing the stability of a series of hydrazone-containing linkers at pH 4.5 and pH 7.4 and its use as part of an ADC in vitro and in vivo in mice, linkers stable at pH 7.4 and unstable at pH 4.5 provided the most effective ADC. This Linker-payload is also applied to Besponsa.

In addition to the hydrazone bond mentioned above, the carbonate Linker used by Trodelvy is also a kind of acid cleavage Linker (Figure 4B). Although ester bonds are theoretically more stable than carbonates in blood circulation, experimental results show that ADCs constructed from the former are not very stable in human serum. The serum stability of the ADC was significantly improved (t1/2=36 hrs) by the introduction of a p-amino-benzyl (PABC) septer, which showed some selectivity for acidic lysosomal compartment, t1/2 for 10 hours at pH 5.

 Acid cleavage of Mylotarg and Trodelvy

Figure 4. Acid cleavage of Mylotarg and Trodelvy

4.1.2 Reducible or disulfide linkers

Despite the clinical success of Mylotarg, Besponsa, and Trodelvy, acid-cleavable Linkers are no longer used in most ADC ligation techniques. Linker's requirement to strictly distinguish between pH 5 and pH 7.4 environments is inherently very difficult, and research and development is now focused on other approaches that can produce higher tumor selectivity. While in some cases a slow release of payloads can yield beneficial results, this approach usually only works with moderately cytotoxic payloads, and the highly toxic payloads now preferred by ADCs require a more stable linker.

The release of the Payload in Mylotarg and Besponsa not only requires the acid-sensitive hydrazone bond to play a role, but also the disulfide bond in the linker. Disulfide bonds are stable at physiological pH but are vulnerable to nucleophilic attack by thiols (Figure 5A). In plasma, the major thiol species is the reduced form of human serum albumin (HSA, ~422 μM), but its reactivity to macromolecules is hindered because of the limited solvent exposure of free thiol-containing residues. In contrast to the limited reducing capacity of plasma, the cytoplasm contains high levels of glutathione (GSH, 1–10 mM). The reducing conditions of plasma and cytoplasm provide an opportunity for ADCs to selectively release the payload. In addition, tumor-associated oxidative stress often results in elevated GSH levels compared to normal tissue, which adds additional selectivity to cancer cells.

 Hydrolysis mechanism of ADC drugs under reducing conditions

Figure 5. Hydrolysis mechanism of ADC drugs under reducing conditions

Linker-Payloads involving disulfide bond hydrolysis mostly contain maytansinoids (DM1/3/4, Figure 5B) and disulfide carbamate payloads (Figure 5C). Similar to disulfide bonds that can release Payload under reducing conditions, many literatures have reported that Linker can release Payload under strong reducing conditions in tumor tissue.

4.2 Enzyme-cleavable Linkers

In the classic mechanism of action of ADC, ADC is transported to the lysosome of the cell, where there is a high concentration of unique hydrolytic enzymes, thus providing an opportunity for the enzyme-cleavable Linker to be selectively cleaved within the cell. At present, the application of cathepsin B is the most successful.

The currently marketed drugs Adcetris, Polivy, Padcev, Tivdak, Aidixi, Lumoxiti, Zynlonta, and Enhertu all use the polypeptide linker cleaved by cathepsin B. P-aminobenzyl carbamate (PABC) is used as a self-degrading spacer, which is spontaneously eliminated by 1, 6-elimination after proteolysis, releasing payload, CO2 and azaquone methylate (Figure 6). PABC maintains enzyme activity independent of payload, increasing the range of application of linker in this polypeptide. All of these linker combinations have some stability in isolated human plasma.

 The structure and cleavage mechanism of the peptide linker

Figure 6. The structure and cleavage mechanism of the peptide linker

5. Non-cleavable Linkers

Non-cleavable linkers are divided into two classes, namely thioether or maleimidocaproyl (MC). They consist of stable bonds that prevent proteolytic cleavage and ensure greater stability than their cleavable counterparts. ADCs containing this type of linker rely on complete lysosomal enzymatic degradation of the antibody to release the internalized payload, resulting in simultaneous dissociation of the linker.

Currently, Genentech/Immunogen has successfully explored this linker strategy, such as the clinically approved trastuzumab emtansine (Kadcyla/T-DM1). This ADC contains a non-cleavable SMCC (N-succinylidene-4-(maleimethylene)cyclohexane-1-carboxylic acid) linker that connects the DM1 cytotoxin to the Lys residue of anti-HER2 monoclonal trastuzumab.

The advantage of non-cleavable linkers over cleavable linkers is their increased iso-stability. Overall, non-cleavable linkers provide a larger therapeutic window than cleavable linkers, since non-cleavable ADCs can also kill target cells.

6. Conclusion

Linkers often affect the stability, toxicity, pharmacokinetic properties, and pharmacodynamics of ADCs, so great care must be taken when selecting linkers in ADC design. Multiple studies have shown that a suitable linker remains an important pillar of a successful ADC. Typically, linkers must remain stable in circulation and guarantee the safe release of the payload within the cell.

A suitable Linker is an important guarantee for the safety and effectiveness of ADC. Although there are many examples of non-cleavable Linkers, cleavable Linkers are the first choice for most ADC drugs due to the cytotoxicity of free payloads and the importance of the bystander effect. Future research on cleavable Linker is expected to further explore the possibility of combining multiple mechanisms of action, including the field of exogenously induced cleavage, which is still in its infancy.

The development of new cleavable linkers is a long way to go, but as we gain a deeper understanding of the impact of different payloads on tumor biology and the clinical validation of optimal linker-spacer connections, we will eventually develop ideal ADCs with disruptive efficacy and drive personalization drug development to address urgent medical needs in oncology.

PEG linkers are particularly attractive as a linker for ADCs. As a worldwide leader of PEG linker supplier, Biopharma PEG is dedicated to being your most reliable partner to provide a variety of featured PEG linkers such as NH2-PEG24-COOH (CAS No.: 196936-04-6), 2-((Azido-PEG8-carbamoyl)methoxy)acetic acid (CAS No.: 846549-37-9), Mal-NH-PEG8-COOH (CAS No.: 1334177-86-4) etc. to facilitate your ADC development projects.

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[6]. Rady, T., et al., A Novel Family of Acid-Cleavable Linker Based on Cyclic Acetal Motifs for the Production of Antibody-Drug Conjugates with High Potency and Selectivity. Bioconjug Chem, 2022. 33(10): p. 1860-1866.
[7]. Migliorini, F., et al., A pH-responsive crosslinker platform for antibody-drug conjugate (ADC) targeting delivery. Chem Commun (Camb), 2022. 58(75): p. 10532-10535.

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[2].How To Choose The Best ADC Linker?
[3].Antibody Drug Conjugates (ADCs): Novel Linker Design


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