ADC Linkers And Research Progress In Detail

Linkers are not only the molecular parts that form covalent connections between antibodies and small molecular payloads but also the key components with design properties in targeted drug therapy. The addition of the linkers should not induce aggregation, and it is necessary to ensure acceptable PK characteristics while limiting the premature release (stability) of the payload in plasma and enabling the effective release of active molecules at the target site of action. Linkers are divided into two categories: non-cuttable and cuttable.

Non-cleavable Linker

ADCs based on non-cleavable linkers must be internalized, and the antibody portion needs to be degraded by lysosomal proteases to release active molecules. Many non-cleavable linkers have been explored during the ADC development process, the most representative of which is N-succinimidyl-4-(N-maleimide methyl) cyclohexane-1-carboxy Salt (SMCC), Kadcyla is the type of linker used.

The catabolism of this structure leads Lys-SMC-DM1 to become the main tumor metabolite. In addition, drugs linked to this linker usually cannot exert a bystander effect because the released catabolites are less permeable. Current research mainly focuses on cleavable linkers.

The use of cleavable linkers is equally feasible for both internalized and non-internalized ADC designs because the release is triggered by the nature of the cleavage site (lysosome/tumor environment). Linkers can be divided into two categories: enzyme-dependent Sexual and chemical (non-enzymatic) dependence.

Chemically Dependent Linker

The disulfide bond-containing linkers are nucleophilically attacked by thiols to release the active load. Although the reduced form of human serum albumin (HSA) in plasma is the most abundant thiol, it has very poor reactivity to macromolecules. The cytoplasm also contains high levels of glutathione (GSH), a tripeptide containing sulfhydryl groups that readily reacts with S-nucleophilic proteins. This kind of tripeptide containing sulfhydryl groups can easily react with S-nucleophilic protein.

The difference in GSH concentration in blood (micromolar range) and cytoplasm (millimolar range) and the oxidative stress caused by cancer cells contribute to the preferential release of drugs in cancer cells. Linkers containing disulfide bonds are mainly related to maytansinoid-like payloads. The disulfide bond reactivity can be adjusted by steric hindrance: α-methyl substitution significantly affects the reduction rate and resistance to thiol-disulfide bond exchange. For example, the linkers of SAR-3419 were replaced by Thorpe-Ingold to obtain the best antitumor activity of SPDB-DM4.

The hydrazone linker shows pH-dependent stability, is stable at neutral pH, and is hydrolyzed in acidic media (endosomal pH<6, lysosome pH<5) to form corresponding ketones and hydrazine.

This method has been successfully applied to IMMU-110, which contains a cleavable acyl hydrazone linker, which is composed of 4-maleimide methyl cyclohexane-1-carboxylate (MCC) hydrazide and doxorubicin. The keto group is formed during the reaction.

The hydrazone-linked body fluid is often used for the payload of the capreomycin family. In this case, the release is triggered by a two-step activation process: in the first step, the acid-sensitive hydrazone is hydrolyzed, and in the second step, the disulfide bond is GSH Reduction to cyclize the sulfhydryl intermediate. This linker has been used in the marketed Mylotarg and Besponsa, but their stability in plasma is not as expected and not as attractive as other cleavable linkers.

Enzyme-dependent Linker

In order to limit the release of the payload before internalization, thereby preventing or minimizing the degradation outside the target cell, the protein component of the lysosome becomes a reasonable place to find enzymes that can degrade ADC and exist in high concentrations.


Cathepsin B is a cysteine ​​protease that exists in the late endosomes and lysosomes of mammals and is also overexpressed in many cancer cells. Initially, a cleavable dipeptide was used as a cathepsin B substrate as a prodrug of doxorubicin. This work established the dipeptide portion of SAR: the P1 position requires a hydrophilic residue (citrulline or arginine), while the lipophilic residue at the P2 position enhances plasma stability (phenylalanine/valine/alanine).

In addition, a self-degrading spacer is introduced to promote the entry of enzymes, thereby limiting the steric hindrance of the payload: p-amino benzyl carbamate (PABA) spontaneously 1,6-eliminates in acidic medium and releases carbon dioxide, P- aza-quinone carboxamide, and doxorubicin. Finally, this discovery transferred from the prodrug to the ADC field, proving the antigen-driven cellular activity of Val-Cit and Phe-Lys dipeptide linkers.

Val-Cit dipeptide is the most commonly used cleavable linker in ADCs. There are currently as many as 25 molecules in the clinical stage, possibly due to its overall good plasma stability, release behavior. The two approved ADC drugs (Adcetris/ Polivy) both use the same linker mc-VC-PABC, which contains a maleimide spacer, the standard Val Cit dipeptide sequence as a cathepsin substrate, and the PABC self Degradation of the spacer.

The Val-Ala dipeptide is also widely used, with seven molecules in the clinical stage, the most advanced being Loncastuximab tesirine, which includes a PEGylation spacer to balance the lipophilic property of the load SG3199 in the PBD dimer family.

Studies have shown that it is difficult for Val-Cit to achieve high DAR due to precipitation and aggregation. In contrast, the Val-Ala linkers allow DAR up to 7.4, and limited aggregation (<10%). The lower hydrophobicity of Val-Ala compared to Val-Cit explains why this linker performs well in lipophilic payloads (such as PBD dimer), with the Val-Ala linker attached to PBD in all seven clinical candidates ADCs.

Some studies have compared the Val-Cit and Val-Ala dipeptide structures with the payload ligations of MMAE. In the case of non-internalized antibodies, the Val-Cit and Val-Ala Ligons bound to engineered cysteine both exhibit similar characteristics and exhibit better performance than the Val-Lys and Val-Arg analogs. In the case of using a random cysteine-conjugated anti-Her2 ADC, Val-Ala shows less aggregation in the high DAR structure compared to Val-Cit. On the other hand, the two linkers showed similar buffer stability, cathepsin B release efficiency, cell activity, and histopathological characteristics.

The tetrapeptide Gly-Gly-Phe-Gly shows all the characteristics of a stable and effective cleavable linker, which is used in the ADC drug Enhertu, which is already on the market. Since the linker does not contain a solubilizer, achieving such a high DAR is very substantial because it contradicts the widely established principle. That is, high DAR conjugates may have poor pharmacokinetic characteristics. The self-degradable spacer used here is a simple and compact semi-amination instead of the PABC used in the Val-Cit linker.

Phosphatase & Pyrophosphatase

Like cathepsins, pyrophosphatase and phosphatase are also hydrolases that are selectively expressed in lysosomes. In 2016, Merck’s researchers designed a linker containing phosphoric acid and pyrophosphate to match the cathepsin B-sensitive Val-Cit-PABA to deliver glucocorticoids: the phosphate/pyrophosphate part binds to the self Degradation spacer between PABA and the payload. After internalization, the payload can be released sequentially by cathepsin B from the degrading spacer and phosphatase (n=1). For pyrophosphate (n=2), another step involving pyrophosphatase may be required.

The advantage of this hydrophilic and permanently charged group is solubility, which can not only be coupled with lipophilic glucocorticoid derivatives but also promote the purification of ADC. The residual linker in ADC is less than 0.10%. Contains phosphoric acid The ADCs of pyrophosphate and pyrophosphate are both active in vitro.

The same team of researchers at Merck also developed a unique pyrophosphatase-based linker for releasing hydroxyl-containing payloads of dexamethasone and fluticasone propionate.

In addition, the nature of the hydroxyl attachment point is critical for effective release. The primary alcohol of dexamethasone works well, while the more hindered secondary alcohol of fluticasone requires an acetal spacer to achieve acceptable release. Both ADCs show good stability in vitro and have strong activity on tumor cell lines.


β-glucuronidase is a type of glycosidase that catalyzes the hydrolysis of β-glucuronic acid residues. It is highly expressed in lysosomes and tumor stroma. Researchers of Seattle Genetics published a pioneering work in 2006 The work of anti-CD70 ADC uses a linker containing glucuronic acid, which is attached to the self-degrading spacer. This linker exhibits low levels of aggregation, high plasma stability, and powerful in vivo efficacy.

The linker is also applied to other amine-containing payloads through an additional DMED self-degrading spacer, such as camptothecin analogs, SN38, duocarmycin, and matrine. The release sequence is from hydrolysis of β-glucuronic acid to self Degrading the spacer, another cyclization reaction of DMED occurs spontaneously, forming 1,3-dimethyl Imidazolidine-2-one, and finally releasing the hydroxyl-containing drug. Due to the hydrophilic nature of the linker, this technology makes it easier to prepare ADC DAR=8 compared with cathepsin-sensitive linkers.


Recently, an ADC using β-galactosidase to cleave the linker was reported, which contains a PEG10 spacer. The spacer is replaced by a nitro group to increase the self-degradation rate. Analogous to the β-glucuronidase linker, its dissociation mechanism involves the hydrolysis of the β-galactosidase moiety, which imparts hydrophilicity to the chemical precursor. Another advantage is that β-galactosidase only exists in lysosomes, while β-glucuronidase is expressed in lysosomes and also expressed in the microenvironment of solid tumors. Studies have shown that it is resistant to HER2-ADCs In the context of releasing MMAE, ADC containing β-galactosidase linker is more effective than T-DM1 in vitro and in vivo.


Recently, linkers cleaved by sulfatase have appeared, and sulfatase is overexpressed in several cancer types, showing potential selectivity. The research involved anti-Her2 antibodies with MMAE as the payload. Compared with the classic cleavable Val-Cit and Val-Ala linkers, the sulfatase linker showed similar efficacy to Her2+ cell lines.