The linker is not only the molecular part of the covalent connection between the antibody and the small molecular payload but also a key element with design properties in targeted drug therapy. The addition of linkers should not induce aggregation, and it is necessary to ensure acceptable competitive characteristics, while limiting the premature release of payloads in plasma (stability) and the effective release of active molecules at targeted sites. Linkers are divided into two types: cleavable and non-cleavable linkers.
- Non-cleavable linker
The ADC 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 in the development of ADC, the representatives of which are N-maleimide methyl and cyclohexane-1-carboxylate (SMCC). Kadcyla uses this type of linker.
The catabolism of this structure causes Lys-SMC-DM1 to become the main tumor metabolite. In addition, drugs linked to such linkers usually do not have a bystander effect due to the poor permeability of the catabolite released. Currently, the research is mainly focused on cleavable linkers.
The cleavable linkers are both feasible for designs with and without internalized ADC, as the release is triggered by the nature of the cleavage site (lysosome and/or tumor environment). Linkers can be divided into two categories: enzyme-dependent and chemical (non-enzyme) -dependent linkers.
- Chemical-dependent linker
The linkers containing disulfide bonds are attacked by mercaptan to release the active load. Although the most abundant mercaptan is the reduced form of human serum albumin (HSA) in plasma, it is poorly responsive to macromolecules. The cytoplasm also contains high levels of glutathione (GSH), a tripeptide containing sulfhydryl groups that easily reacts with S-nucleophilic proteins. The difference in GSH concentration between blood (micromoles) and cytoplasm (millimoles) and oxidative stress caused by cancer cells contribute to the preferential release of drugs in cancer cells. The linkers with disulfide bonds are mainly related to maytansinoid payloads. The reactivity of disulfide bonds can be regulated by steric hindrance: α-methyl substitution significantly affects the reduction rate and resistance to mercaptan-disulfide bond exchange, such as SAR-3419 linkers through dimethyl substitution to obtain the best antitumor activity of SPDB-DM4.
Hydrazone linkers show pH-dependent stability, are stable at neutral pH, and are hydrolyzed in an acidic medium (pH < 6 in the endosome, pH < 5 in the lysosome) to form corresponding ketones and hydrazine.
The method has been successfully applied to IMMU-110, including a cleavable acyl Hydrazone linker, which is formed by the reaction of 4-maleimide methyl cyclohexane-1-carboxylate (MCC) hydrazide with ketones in adriamycin.
Hydrazone linkers are also often used as payloads of the kaliomycin family. In this case, the release is triggered by two steps of activation: the first step is the hydrolysis of acid-sensitive hydrazone, and the second step is the reduction of disulfide bonds by GSH, resulting in the cyclization of sulfhydryl intermediates. These linkers have been used in the marketed Mylotarg and Besponsa, but they are not as stable in plasma as expected and are not as attractive as other cleavable linkers.
- Enzyme-dependent linker
In order to limit the release of the payload before internalization, preventing or minimizing the extracellular degradation of the target, the protein components of lysosomes become a reasonable place to find enzymes that can degrade ADC and exist at high concentrations.
- Cathepsin-B
Cathepsin B is a kind of cysteine protease that exists in the late endosome and lysosome of mammals, overexpressed in many cancer cells. Initially, a cleavable dipeptide used as a substrate for cathepsin B was used as a prodrug to doxorubicin, which established the dipeptide part of SAR: the P1 site requires a hydrophilic residue (citrulline or arginine), while the P2 position lipophilic residue enhances plasma stability (phenylalanine, valine, or alanine).
In addition, a self-degradable spacer was introduced to promote the entry of the enzyme, thus limiting the steric hindrance of the payload: p-aminobenzyl carbamate (PABA) was spontaneously eliminated in acidic media, releasing carbon dioxide, p-azaquinone formamide, and doxorubicin. Finally, this discovery transferred from prodrugs to the field of ADC, proving the antigen-driven cellular activity of Val-Cit and Phe-Lys dipeptide linkers.
The Val-Cit dipeptide is the most commonly used cleavable linker in ADCs. At present, 25 molecules are in the clinical stage, which may be due to their good plasma stability, release behavior, and chemical tractability. Two approved ADC drugs (Adcetris and Polivy) use the same linker mc-VC-PABC, including a Maleimide spacer, a standard Val Cit dipeptide sequence as a cathepsin substrate, and a PABC self-degradable spacer.
The Val-Ala dipeptides are also widely used, with seven molecules in the clinical stage. Loncastuximab tesirine is developed fast, which includes a polyethylene glycol spacer to balance the lipophilicity of the payload SG3199, belonging to 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, Val-Ala linkers allow DAR up to 7.4 with limited aggregation (less than 10%). Compared with Val-Cit, Val-Ala has lower hydrophobicity, which explains why this linker performs well in lipophilic payloads (such as PBD dimer). The Val-Ala linkers of 7 clinical candidate ADCs are connected to PBD.
Some studies have compared the dipeptide structures of Val-Cit and Val-Ala with the payload connection of MMAE. In the case of non-internalized antibodies, Val-Cit and Val-Ala linkers bound to engineered cysteine showed similar characteristics and showed better performance than Val-Lys and Val-Arg analogs. In the case of random cysteine-bound anti-Her2 ADC, Val-Ala showed less aggregation in the high DAR structure than Val-Cit. On the other hand, the two linkers showed similar buffering stability, cathepsin B release efficiency, cell activity, and histopathological characteristics.
The tetrapeptide gly-Gly-Phe-Gly shows all the properties of stable and effective cleavable linkers, which are used in the marketed ADC drug Enhertu. The first triad, Enhertu, is a plasma stable ADC with a DAR of 7.7, which is degraded by protease in lysosomes and releases DX-8951f, which is an effective topoisomerase I inhibitor derived from exatecan. Since the linkers do not contain solubilizers, it is considerable to achieve such a high DAR because it contradicts the widely established principle that high DAR conjugates may have poor pharmacokinetic characteristics. The self-degradation spacer used here is a simple and compact hemiamination rather than the PABC used by Val-Cit linkers.
- Phosphatase and pyrophosphatase
Like cathepsin, pyrophosphatase and phosphatase are hydrolases selectively expressed in lysosomes. In 2016, Merck researchers designed linkers containing phosphate and pyrophosphate to match cathepsin B-sensitive Val-Cit-PABA to transmit glucocorticoids: phosphate and pyrophosphate partially bind between the self-degradable spacer PABA and the payload. After internalization, the payload can be released sequentially by cathepsin B, self-degradable spacer, and phosphatase (naphth1). Another step involving pyrophosphatase may be required for pyrophosphate (naphthyl 2).
The advantage of this hydrophilic and permanently charged group is its solubility, which can not only be biologically coupled with lipophilic glucocorticoid derivatives but also promote the purification of ADC, with less than 0.1% residual linkers in ADC. An ADC containing phosphoric acid and pyrophosphate is active in vitro.
- β-glucuronidase
β-glucuronidase is a kind of glucosidase that catalyzes the hydrolysis of β-glucuronic acid residues. It is highly expressed in lysosomes and tumor stroma. Seattle Genetics researchers published groundbreaking work in 2006 in which anti-CD70 ADC uses linkers containing glucuronic acid, which attaches to self-degraded spacers. This linker exhibits low levels of aggregation, high plasma stability, and strong in vivo efficacy.
The linker is also applied to other amine-containing payloads through additional dimethylethylenediamine (DMED) self-degradable spacers, such as camptothecin analogs, SN38, doxycycline, and matrine. From the hydrolysis of β-glucuronic acid to the self-degradation spacer, another cyclization of DMED occurred spontaneously to form 1, 3-dimethylImidazoline-2-one, and finally released the drug containing hydroxyl. Due to the hydrophilicity of linkers, this technique makes the preparation of ADC DAR=8 easier compared with cathepsin-sensitive linkers.
- β-galactosidase
Recently, it has been reported that an ADC containing a PEG10 spacer is used to cleave the linker with β-galactosidase. The spacer was replaced by nitro to increase the rate of self-degradation. Compared with β-glucuronidase linkers, the dissociation mechanism of β-glucuronidase involves the hydrolysis of β-galactosidase, which gives hydrophilicity to chemical precursors. Another advantage is that β-galactosidase exists only in lysosomes, while β-glucuronidase is expressed in lysosomes and in the microenvironment of solid tumors. In the context of anti-HER2-ADCs that release MMAE, studies have shown that ADCs with β-galactosidase linkers are more effective than T-DM1 in vitro and in vivo.
- sulfatase
Recently, sulfatase-cleavable linkers have emerged. Sulfatase is overexpressed in several cancer types, showing potential selectivity. The study mainly focused on anti-Her2 antibodies with MMAE as the payload. Compared with the classical cleavable Val-Cit and Val-Ala linkers, the sulfatase linkers showed a similar effect on Her2+ cell lines.