The Mechanism of Action of Various Linkers in ADCs | Antibody-Drug Conjugates

In recent years, three types of innovative drugs (targets), namely ADCs, GLP-1 and PROTACs, have attracted much attention in the field of drug research and development. Whether through pipeline acquisitions or independent research and development, major pharmaceutical companies have been actively making arrangements. Especially the first two types of drugs have entered the "harvest period", with new drugs being approved from time to time.

Antibody-Drug Conjugates (ADCs) are products that combine monoclonal antibodies with potent cytotoxic drugs. The basic principle is to utilize the specific recognition ability of antibodies to directly deliver cytotoxic drugs to tumor cells expressing specific antigens, thereby achieving the goal of precisely targeting cancer cells. They mainly consist of three key components: Antibody, Linker, and Payload.

Fig.1: Composition of Adcetris®, one ofthe frst FDA-approved ADCs.

Adcetris was approved in 2011 for the treatment of Hodgkin's lymphoma and systemic anaplastic large cell lymphoma (ALCL), and its sales reached 476.9 million US dollars in 2018. The drug consists of a monoclonal antibody targeting CD30, which is linked to four monomethyl auristatin E (a microtubule inhibitor MMAE) through a self-immolative linker. Adcetris holds an extremely important position in the development history of ADCs. It is one of the first ADCs approved by the US Food and Drug Administration (FDA) for first-line treatment, marking a milestone in the treatment of lymphoma and also promoting the development of the entire ADC drug field.

Fig. 2: Mode of action of ADCs.

The typical mechanism of action of ADCs: ADCs first circulate in the plasma and upon reaching the target cells, the antibody portion of the ADC binds to antigens that are only present on the surface of cancer cells. Subsequently, the ADC-antigen protein complex is endocytosed into the interior of the cancer cells. Once the complex is degraded, cytotoxic substances are released, which then interact with the target to induce apoptosis of the cancer cells. The linker between the antibody and the highly active small molecule is typically classified as either uncleavable or cleavable, with the latter being cleaved by enzymes such as cathepsin B, β-glucuronidase, or glutathione.

Most off-target adverse events are not related to the antibodies. Regarding the antibodies, the sulfhydryl-forming cysteine, the amino group formed by lysine, the azide group produced by non-standard amino acids, and the specific sequence on the antibody surface, can all be modified through different chemical or enzymatic coupling methods. The drug-antibody ratio (DAR) is an important parameter for evaluating ADCs. A lower DAR value may reduce the anti-tumor efficacy, while a higher DAR may affect the structure, stability, and antigen binding ability of the antibody, thereby leading to loss of activity. The DAR value is also important for the therapeutic index of ADCs. In most ADC drug candidates, the DAR value is usually maintained at approximately 2-4. Therefore, controlling the DAR during ADC preparation is a key step.

2. The principle of cleavage

ADC-linker is divided into degradable and non-degradable types. The reasons and specific locations for cleavage are worth exploring.

The common method involves connecting the antibody using an activated ester that forms an amide bond with lysine. However, since lysine is usually exposed on the antibody surface, different amounts of drug loading attach to different positions, resulting in a higher degree of heterogeneity of the conjugate. To achieve a more uniform DAR value, the disulfide bond between the heavy chain and light chain of the antibody can also be used for connection.

2.1 Cleavage mechanism

ADC drugs circulate in the plasma until they reach the target cells. After endocytosis into the cell, even if the ADC-linker is stable, the complex will be degraded and the drug released. However, by introducing groups that can be cleaved under specific conditions, the release process can be made controllable. The most commonly used group is the bifunctional p-aminobenzaldehyde (PABA), which forms an amide bond with the peptide through the amino group, while the Payload containing the amino group is connected to the benzaldehyde group of the Linker through a carbonate group. The resulting prodrug is activated during the protease-mediated hydrolysis and the cleavage of the amide bond of citrulline to the p-aminobenzyl fragment, resulting in a 1,6 elimination reaction, releasing the unmodified drug, one molecule of carbon dioxide, and the residue of the Linker.

Similar strategies for developing linker-like structures can also be applied to the design of linkers containing groups such as β-glucuronic acid and trimethylquinone.

In addition, apart from the hydrolytic enzyme system, the redox enzyme systems within lysosomes in cells (such as NQO1, GSH, etc.) can also be used for the design of degradable linkers.

2.2 The Disulfide Bond Linker Mechanism

After the disulfide linker enters the cell, it is degraded in the lysosome, generating cysteine (Cys)-disulfide metabolite. Subsequently, the disulfide bonds are reduced in the cytoplasm by GSH and other substances. By designing one to four methyl groups at the adjacent positions of the two sulfur atoms, the kinetic rate of the reduction reaction can be regulated.

2.3 Achieving a multi-Payload structure through a single cleavable linker

Introducing N,N'-dimethylethyl between the carbonate of Linker and the payload can facilitate the release of the drug. After the release of carbon dioxide, N,N'-dimethylethyl will cyclize and form 1,3-dimethylimidazolidin-2-one, which will release the toxin molecules from the linker structure.

3. Non-fragmentable Linker

3.1 Based on PEG (Polyethylene Glycol) type fragments

The cleavage of the non-fragmentable Linker and the release of the drug do not depend on the differences between plasma and cytoplasm. Instead, the release of cytotoxic drugs occurs in the lysosomes. In the lysosomes, the antibody is degraded by intracellular proteases to the amino acid level, during which a drug derivative is released. This derivative consists of the drug, the Linker, and amino acid residues. The drug usually has strong hydrophobicity, and the water solubility of the PEG fragment helps to increase the solubility of the linker-drug complex in physiological media and improve the pharmacokinetic properties of the entire ADC.

For example, Trodelvy and Zynlonta, two ADC drugs approved by the FDA, inserted PEG into the Linker to improve the solubility and stability within the cells.

3.2 Based on hydrophobic segments (alkyl-dominated long chains)

3.3 Based on Maleimides

The Michael addition between mercapto groups and malonyl imides is commonly used in various biological coupling reactions.

3.4 Photocatalytic Class

Use of photo-phenylalanine for the identification of angiotensin, lreceptor binding sites；¹²⁵I is used as a radiotracer.

The design and optimization of Linker are crucial for enhancing the effectiveness of ADC and reducing side effects. Currently, the research on Linker is a popular direction in drug development, and Linker technology further expands the application scope of ADC in cancer treatment.

More Linker products: Click here (https://www.glpbio.com/research-area/antibody-drug-conjugate-adc-related/adc-linker.html)