In the early stages of ADC development, researchers often overlooked linker design, thinking that adding a cytotoxin to an antibody was enough. Over time, it became clear that the linker plays a crucial role in ADC performance in two key ways:
- ADC linker chemistry helps optimize the therapeutic index (TI) by ensuring the right amount of cytotoxin reaches the target cell.
- The choice of ADC linker types, such as cleavable or non-cleavable linkers, affects how efficiently the payload is released at the tumor site. Cleavable linkers can improve bystander activity and reduce hydrophobicity.
As ADCs advanced, researchers realized that linker design and stability are key for controlling payload release. Innovations in cleavable linkers and methods to reduce ADC linker hydrophobicity have improved the efficacy and safety of ADC therapies. The best linker design now takes into account tumor targeting, payload potency, and pharmacokinetics (PK), which all contribute to ADC success.
ADC Effectiveness
To create an effective ADC, it’s essential to select the right combination of antibody, linker, and payload. Different cancer types require specific linker designs, which can influence the ADC’s functionality, safety, and manufacturability. Structural elements of the linker, such as the attachment site, release mechanism, and solubilizing portion, are vital for determining the ADC’s therapeutic potential and profile (Table 1). This article provides a brief overview of how linker design impacts ADC performance, aiming to serve as a reference for our future ADC development.
Linker Design and ADC Potency
Payload potency is crucial to the effectiveness of antibody-drug conjugates (ADCs). The level of antigen expression on cancer cells influences how well the ADC delivers the payload to the target cells. For instance, DNA cross-linkers like PBD dimers are potent payloads that show low IC50 values in cell-killing assays, enabling effective treatment at low doses. However, the linker design can also impact ADC efficacy, especially in terms of the bystander effect, which refers to the ability of the ADC’s payload to kill nearby, antigen-negative cells. Cleavable linkers improve this by allowing the drug to diffuse and act on surrounding cells, while non-cleavable linkers limit this effect.
Cleavable vs. Non-Cleavable Linkers
The choice of cleavable or non-cleavable linkers significantly affects an ADC’s effectiveness and safety.
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Cleavable Linkers: These linkers, such as disulfide-based ones, enable the cytotoxic drug to be released once the ADC reaches the tumor. Modifying the steric hindrance around the disulfide bond can enhance cleavage efficiency. For example, in the CanAg-targeting huC242 antibody with maytansinoid payloads, ADCs with less steric hindrance (DM1) demonstrated better efficacy in a Colo 205 xenograft model, despite poorer pharmacokinetics (PK). Cleavable linkers also improve bystander activity, making them more effective in tumors with varying antigen expression levels. In experiments with the FR-α-targeting ADC mirvetuximab soravtansine, cleavable linkers (e.g., SPP-DM1) showed higher efficacy across both high- and low-expressing tumor cell lines compared to non-cleavable linkers (e.g., MCC-DM1).
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Non-Cleavable Linkers: These linkers, such as MCC-DM1, do not release the payload once the ADC reaches the tumor. While effective in targeting cancer cells, their efficacy decreases in tumors with lower antigen expression. For example, in low-expressing Igrov-1 and JEG-3 cell lines, ADCs with non-cleavable linkers showed a reduction in activity, while cleavable linkers maintained higher potency.
Modulating Linker Stability for Improved ADC Efficacy
The stability of the linker also plays a role in ADC efficacy. ADCs using hydrolysable linkers have shown superior in vivo efficacy because they release more payload in the tumor microenvironment (TME), enhancing the bystander effect. The FDA-approved Trodelvy utilizes this type of hydrolysable linker for improved therapeutic outcomes.
Optimizing Drug-to-Antibody Ratio (DAR)
The drug-to-antibody ratio (DAR) is a critical factor in ADC development. Higher DARs can improve potency but may lead to faster clearance and reduced efficacy. Preclinical studies suggest that a DAR of around 4 provides the best balance of efficacy and tolerability. However, some ADCs, such as DS-8201a (DAR 8), may benefit from higher DARs, particularly for HER2-low expressing tumors.
Hydrophobicity and ADC Performance
Hydrophobicity is an important consideration in linker design, as highly hydrophobic payloads can lead to rapid clearance by the liver, reducing ADC effectiveness. One way to reduce hydrophobicity is by incorporating hydrophilic groups such as sulfonates or polyethylene glycol (PEG) into the linker. This modification improves pharmacokinetics (PK) and enhances the safety and efficacy of the ADC, helping to reduce liver toxicity and non-specific cellular uptake.
PK and Safety
The PK profile of ADCs is quite complex in terms of its absorption, distribution, metabolism, and excretion ( ADME ) properties. Several key features of ADCs, including linker stability, binding site, DAR, cytotoxin, and overall hydrophobicity, can influence its PK profile(Table 2).
Reducing hydrophobicity in antibody-drug conjugates (ADCs) improves their pharmacokinetics (PK), safety, and efficacy. High hydrophobicity can lead to rapid liver clearance and unwanted side effects, including hepatotoxicity. To address this, researchers use strategies like adding polar units (e.g., sulfonates or phosphates) to the linker, which improves solubility and reduces non-specific uptake. Quaternary ammonium salt linkers also reduce hydrophobicity and enhance PK.
Incorporating polyethylene glycol (PEG) groups further shields the hydrophobic payload, improving PK and reducing liver clearance. Studies suggest that a PEG length of 8 units strikes an optimal balance between reducing hydrophobicity and maintaining therapeutic efficacy. These modifications enhance the ADC’s therapeutic index (TI) by improving tumor targeting while minimizing toxicity.
Amino esters and MMAE Linkers: