Antibody-drug conjugates (ADCs) are a class of emerging anticancer drugs that deliver highly cytotoxic molecules directly to cancer cells. To date, 15 ADC drugs have been approved for the market. ADCs are composed of monoclonal antibodies and cytotoxic drugs covalently linked via a linker. Research has shown that the linker plays a crucial role in ADC drugs, as its properties significantly impact the therapeutic indices, efficacy, and pharmacokinetics of these drugs. A stable linker can maintain the drug concentration in the bloodstream and prevent premature drug release before the cytotoxic agent reaches its target, resulting in minimal off-target effects and improved safety of the ADC drug.

Antibody Drug Conjugate

An antibody-drug conjugate is a more complex molecule composed of a targeting antibody and a cytotoxic active payload (cytotoxin) linked together by a chemical linker. In recent years, there has been significant progress in the research of the payload and linker, leading to breakthroughs in the field. ADCs like Enhertu (DS-8201) have redefined the conventional understanding of ADCs and significantly improved clinical outcomes, becoming blockbuster products. In the past two years, ADC drugs have experienced a surge in market approvals, becoming one of the hottest areas in innovative drug development.

Fig. 1. ADC structure (Molecules. 2021, 26(19): 5847).

The successful development of ADCs depends on two key factors. First, the selection of the linker is crucial, and this is reflected in the following aspects: 1) High plasma circulation stability to prevent premature drug release; 2) Maintaining the properties of the monoclonal antibody and the cytotoxic drug's cell-killing ability while reducing systemic toxicity; 3) High water solubility to allow the bioconjugation of lipophilic drugs and prevent antibody aggregation; 4) Drug release at the appropriate time to maximize therapeutic efficacy. Second, the successful development of ADCs also relies on the optimal drug-antibody ratio (DAR). A low DAR reduces the efficacy of ADCs, while a high DAR can cause instability, increase systemic effects, shorten the half-life, and alter the pharmacokinetic properties of the molecule.

ADC Linker

The linker is an essential component in ADC design, connecting the antibody with the cytotoxic payload through covalent bonding. A linker typically consists of four components: the antibody attachment region, a spacer segment, an enzyme-cleavable segment, and a self-cleaving segment, which together covalently link the antibody and the payload. Proper linker design can optimize the drug structure, improve ADC solubility and pharmacokinetics (PK), enhance cleavage efficiency, and increase ADC activity. Generally, a successful ADC construct must meet the following criteria:

• Stability in Plasma: The linker must have sufficient stability in plasma so that the ADC molecule can circulate in the bloodstream and target the tumor site without premature cleavage. Instability of the linker can lead to the early release of toxic payloads, causing unintended damage to non-target healthy cells, potentially leading to systemic toxicity and adverse reactions. However, a clinical study has shown that the stability of the linker in a Herceptin-based ADC is inversely correlated with adverse toxicities. Therefore, identifying ADC linkers with optimal stability for each antigen, target tumor type, and payload combination is critical.
• Cleavage upon Internalization: The linker must have the ability to be rapidly cleaved upon internalization of the ADC into the target tumor cell, releasing the free and toxic payload.
• Hydrophobicity: Another important property to consider in linker design is hydrophobicity. Hydrophobic linkers combined with hydrophobic payloads tend to promote ADC aggregation. For example, King et al. observed that conjugating the monoclonal antibody BR96 with doxorubicin using a hydrophobic dipeptide linker led to non-covalent dimerization. Such molecules are undesirable in therapeutic ADCs, as aggregated proteins tend to be rapidly sequestered in the liver and cleared via the reticuloendothelial system, leading to hepatotoxicity. Additionally, aggregated proteins may act as immunogenic substances, triggering unwanted immune reactions while circulating in the bloodstream. This issue can be mitigated by using hydrophilic linkers containing negatively charged sulfonate groups, polyethylene glycol (PEG) groups, or phosphodiester groups.

ADC Linker Types

Based on the above criteria, significant efforts have been made to develop coupling methods and ADC linker structures. Chemical and enzymatic coupling are currently the two main methods used to link the antibody and payload components. Linker structures can be classified into two major types based on the payload release mechanism: cleavable or non-cleavable linkers.

• Cleavable Linker

Cleavable linkers contain chemical triggers that allow the linker to cleave and release the payload, offering high flexibility. The released payload can diffuse to surrounding cells and exert a bystander effect. Non-cleavable linkers, on the other hand, lack such chemical triggers and cannot exert a bystander effect, making them mainly suitable for blood cancers and tumors with high antigen expression. These linkers generally have less off-target toxicity but may lead to the development of resistance. Given that cleavable linkers effectively distinguish between the circulatory system and target cells, over 80% of currently marketed ADCs use cleavable linkers. Examples include AcBut acyl hydrazone-disulfide, Valine-citrulline, Glycine-glycine-phenylalanine-glycine (GGFG), CL2A, MP-PEG8-VA-PABC, and Sulfo-SPDB, while only two ADCs use non-cleavable linkers, such as SMCC and MC.

• Non Cleavable Linker

Non-cleavable linkers are composed of stable bonds that can resist proteolytic degradation, ensuring greater stability compared to cleavable linkers. Non-cleavable linkers rely on the complete degradation of the ADC's antibody portion by cytoplasmic and lysosomal proteases, ultimately releasing the payload molecule that is linked to the amino acid residues derived from the degraded antibody. Therefore, when associated with non-cleavable linkers, the payload structure must be carefully selected and designed so that the payload exerts comparable or even superior antitumor efficacy in this modified form. To achieve this, it may be necessary to evaluate the pharmacokinetics (PK), pharmacodynamics (PD), and toxicity profiles of all potential metabolites of ADCs containing non-cleavable linkers. Currently, non-cleavable linkers mainly fall into two categories: thioether-based and maleimide propyl (MC)-based linkers.

ADC Linker Chemistry

The structural optimization of ADC linkers can focus on improving stability, enhancing water solubility, increasing payload efficacy, and reducing drug resistance. In terms of stability, the regulatory and amino acid fragments of the linker play crucial roles. For example, a linker composed of valine-citrulline amino acid fragments and a cyclohexyl-based regulatory fragment is more stable than one composed of hexyl or phenyl-based fragments; similarly, a linker with a glycine-phenylalanine-leucine-glycine amino acid fragment and a cyclohexyl regulatory fragment is less stable than other amino acid-based linkers. Regarding water solubility, adding polar groups (such as polyethylene glycol or sulfonic acid) to the regulatory fragment can enhance drug solubility and reduce aggregation. Additionally, designing linkers with multiple payload conjugation sites can increase the number or variety of linked payloads, improving efficacy and reducing resistance. The functional groups in the linker directly affect the drug release efficiency and selectivity. Different functional groups can influence the linker's chemical stability, hydrophilicity, drug payload capacity, and the mode of attachment to the antibody. Some common functional groups include:

• PEG Linker

The polyethylene glycol (PEG) linker connects the cytotoxic drug and antibody through a PEG chain. PEG, as a linker, provides good water solubility and biocompatibility, enhancing the drug's stability in vivo and reducing degradation in the bloodstream. Additionally, the PEG chain can increase the half-life of the ADC, extend its circulation time, and enhance the drug's targeting ability. PEG linkers are commonly used to optimize the antibody and drug payload, reducing unwanted immune responses or toxic reactions.

• Maleimide Linker

The maleimide (MC) linker connects the drug to the antibody by forming a stable covalent bond with the thiol group on the antibody. Maleimide linkers have high chemical reactivity, enabling selective reaction with cysteine residues on the antibody surface, ensuring precise drug loading. These linkers are often used in targeted therapies, as they enhance drug stability and control the release of the drug within the target cells.

• Peptide Linker

The ADC peptide linker consists of short peptide chains that connect the drug and antibody via peptide bonds. This linker exhibits high specificity and can be cleaved under specific enzymatic or environmental conditions (e.g., enzymes inside tumor cells), releasing the drug. Peptide linkers are typically used in ADCs that require precise drug release within the target cells, effectively enhancing drug targeting and selectivity. The flexibility in structural design also allows customization of different drug types through various enzyme cleavage sites.

• SPDB Linker

The SPDB (N-Succinimidyl 4-(2-pyridyldithio)butyrate) linker is a chemical structure that connects the drug to the antibody via a disulfide bond. The SPDB linker contains a thiol-protecting group with a strong affinity, which reacts with cysteine residues on the antibody to form stable disulfide bonds. This linker has high in vivo stability, preventing premature drug release when targeting cells, and can release the drug through a reductive reaction inside tumor cells, resulting in better therapeutic effects.

• Val-Cit Linker

The valine-citrulline (Val-Cit) linker is a specialized linker that connects the drug to the antibody using a peptide bond. It contains a valine-citrulline dipeptide unit, which can be cleaved by specific enzymes (such as aminopeptidases) inside tumor cells, effectively releasing the cytotoxic drug. The VC linker is often used in ADC designs requiring high selectivity and efficiency, ensuring drug release within the target cells and minimizing toxicity to normal cells.

• SMCC Linker

The SMCC (Succinimidyl 4-([2-sulfo-3-nitro-4-(trifluoromethyl)phenyl]thio)butyrate) linker is a chemical structure that connects the drug and antibody via an active ester group. The SMCC linker can react with amino or thiol groups on the antibody, forming stable covalent bonds. This linker has high chemical stability and is suitable for ADC designs requiring efficient drug loading and precise drug release. The SMCC linker provides good in vivo stability and effectively enhances the targeting of ADC, ensuring that the drug is released in the specific environment of tumor cells.

• Amino Acid Linker

Amino acid linkers are chemical structures that connect cytotoxic drugs to antibodies through amino acid residues. These linkers typically consist of one or more amino acid units, such as valine, glutamic acid, and phenylalanine. Amino acid linkers not only establish a stable covalent bond between the antibody and the drug but can also break under specific enzymatic conditions or in a low pH environment, thereby releasing the drug. Since amino acids are widely present in the human body and have good biocompatibility, amino acid linkers are generally biocompatible and less likely to trigger immune reactions. The advantage of amino acid linkers lies in their versatility, allowing the design of linkers with varying amino acid chain lengths, structures, and cleavage enzyme sites based on the drug's properties and targeting mechanisms. Additionally, amino acid linkers exhibit good water solubility and stability, effectively enhancing the efficacy and selectivity of ADCs.