The linker used in ADC is divided into two types: cleavable linker and non-cleavable linker. This artile mainly introduced the cleavable linkers used in ADC development.

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Cleavable Linkers Used In ADC Development
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.
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;

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(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).

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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

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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.
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

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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.
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

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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.
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.

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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.
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.

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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.
References:
[1]. Bargh, J.D., et al., Cleavable linkers in antibody-drug conjugates. Chem Soc Rev,
2019. 48(16): p. 4361-4374
[2]. Linkers: An Assurance for Controlled Delivery of Antibody-Drug Conjugate.

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Pharmaceutics 2022, 14, 396.
[3]. Sheyi, R., B.G. de la Torre, and F. Albericio, Linkers: An Assurance for Controlled
Delivery of Antibody-Drug Conjugate. Pharmaceutics, 2022. 14(2).
[4]. Antibody-drugconjugates: Recent advances in linker chemistry. Acta Pharm Sin B.
2021Dec;11(12):3889-3907.
[5]. Chari, R.V., M.L. Miller, and W.C. Widdison, Antibody-drug conjugates: an emerging
concept in cancer therapy. Angew Chem Int Ed Engl, 2014. 53(15): p. 3796-827.
[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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