Next generation linker chemistry in ADCs

The development of monoclonal antibodies (mAbs) has become a major focus in cancer therapy due to their high specificity and the increasing discovery of cancer-specific antigens. This field began to expand significantly with the first drug[1] approval in 1997. The main advantage of mAbs lies in their ability to be engineered for precise antigen binding, which is achieved by modifying the variable domain, particularly the complementarity-determining regions (CDRs).

Target activities

mAbs alone often lack sufficient cytotoxicity to eliminate cancer cells. This limitation is addressed by antibody-drug conjugates (ADCs), which combine mAbs with potent cytotoxic payloads. However, only a small fraction of ADCs typically reaches solid tumors, so the payloads must be highly potent and stable. Currently, FDA-approved ADC payloads fall into two main categories: tubulin inhibitors, which disrupt cell division, and DNA-damaging agents, which interfere with DNA replication or cause breaks.

Antibody-drug conjugates (ADCs) are an emerging class of targeted cancer therapies that combine a monoclonal antibody with a cytotoxic drug, connected by a chemical linker. Although the concept of ADCs dates back nearly a century, significant progress has been made in the last decade, leading to the approval[2] – by end of 2024 – of more than ten ADCs and at least eighty more in clinical trials.

Antibody-drug conjugates (ADCs) function like precision-guided “biological missiles,” combining the targeting ability of monoclonal antibodies with the potent killing power of cytotoxic drugs. Their main mechanism of action[3] begins when the antibody component binds to specific antigens on the surface of cancer cells. This triggers internalization of the ADC into the cell, where it travels through endosomes and eventually fuses with lysosomes. Inside the lysosome, the cytotoxic payload is released, either chemically or enzymatically, leading to cell death by damaging DNA or disrupting microtubules (Figure 1).

If the released payload is membrane-permeable, it can also diffuse into neighboring cells, producing a bystander effect that enhances the overall therapeutic impact and may even modify the tumor microenvironment to further support cancer cell killing.

Clinical advances

The development of ADCs has evolved through three distinct generations[4], each marked by technological advancements in antibody design, linker chemistry, and payload selection. Conventional (first-generation) antibody-drug conjugation methods rely on naturally abundant amino acids like lysine or cysteine to attach cytotoxic drugs to antibodies. However, this approach often results in heterogeneous mixtures, which can lead to inconsistent therapeutic effects, faster drug clearance, reduced stability, and narrower therapeutic windows.

To address these limitations, next-generation site-specific conjugation techniques have been developed. These methods enable precise attachment of drugs to defined sites on the antibody, improving consistency, stability, and therapeutic performance. There are four main strategies for achieving site-specific conjugation:

• Targeting specific natural amino acids engineered into the antibody;
• Incorporating unnatural amino acids that provide unique reactive sites;
• Using short peptide tags that can be selectively modified;
• Modifying glycans (sugar chains) on the antibody for controlled conjugation.

Newer ADCs are also exploring immunomodulatory payloads, such as TLR and STING agonists, which stimulate the immune system rather than directly killing tumor cells. These are known as immune-stimulating antibody conjugates (ISACs) and show promise for long-term cancer immunity.

Chemical triggers of the linker

The linker, which connects the antibody to the drug, plays a critical role in the effectiveness of ADCs. It must be stable enough to keep the drug intact while circulating in the bloodstream, yet capable of releasing the cytotoxic payload specifically at the tumor site. This dual requirement presents a major challenge in linker design.

Linkers are generally categorized into two types: cleavable and non-cleavable.

• Cleavable linkers, which dominate current clinical use, are designed to respond to specific triggers in the tumor environment.

• Non-cleavable linkers, on the other hand, remain intact and become part of the payload.

Around 80% of approved ADCs use cleavable linkers, which are engineered to respond to specific conditions within or near tumor cells, such as pH, redox potential, glutathione levels, or lysosomal enzymes, ensuring that the drug is released only where needed. Peptide-based cleavable linkers are the most common, but they are vulnerable to premature cleavage by enzymes like serine elastase in the bloodstream, which can lead to off-target toxicity.

To address these issues, recent research has focused on improving linker technology. Advances include refining chemical triggers for better selectivity, developing new methods for attaching linkers to antibodies to enhance stability and uniformity, expanding the range of compatible payloads, and optimizing linkers to improve the pharmacokinetics of ADCs. The design of newer types is being developed such as enzyme-sensitive, photo-sensitive, and bioorthogonal cleavable linkers. Additionally, the limited compatibility between existing linkers and new types of payloads restricts the development of next-generation ADCs.

These innovations aim to overcome current limitations and support the continued evolution of ADCs in cancer therapy and beyond.

For illustration, one such innovation is the legumain linker that exploits the overexpression of the lysosomal enzyme legumain in cancer cells, allowing for highly specific cleavage and reducing systemic toxicity. This linker can also incorporate a hydrophilic peptide cap, known as a “cell trapper”, which restricts drug diffusion and enhances accumulation within tumor cells, improving both efficacy and safety.
Another strategy is the tandem-cleavage linker, which requires two sequential enzymatic steps to release the drug.

This dual-step mechanism adds an extra layer of protection against premature release and has shown improved stability and tolerability in preclinical studies. These linkers often use glucuronide moieties to shield dipeptides until the ADC is internalized and degraded in the lysosome.

Concluding remarks

Over the years, research has led to the successful development of numerous ADC therapies. Besides, extensive studies have shed light on the critical components that determine ADC performance. To advance the next generation of ADCs, it is essential to design novel payloads with optimal potency and safety, and engineering linkers that strike the right balance between stability and controlled drug release.

SEQENS, as a major CDMO for small molecule APIs, supports ADCs development through its small molecule API development and manufacturing capabilities, including for potent molecules, and bioavailability improvement services.
Our industrial expertise in chemistry is backed by +25 years of cGMP production of active ingredients. We have strong expertise in innovative synthesis design and development along with cutting-edge capabilities in analytics and high throughput experimentation.
We cover manufacturing at different scales, from gram to ton scale in cGMP environment. We have also continued investing in OEB4 and OEB5 state-of-the-art capabilities.

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[1] J.Y. Fong et al. Advancements in antibody-drug conjugates as cancer therapeutics, Journal of the National Cancer Center, 2025. Doi: 10.1016/j.jncc.2025.01.007
[2] Gogia P et al. Antibody–drug conjugates: a review of approved drugs and their clinical level of evidence. Cancers (Basel). 2023,15, 15, 3886.
[3] Z. Fu et al. Antibody drug conjugate: the “biological missile” for targeted cancer therapy. Sig Transduct Target Ther, 2022, 7, 93.
[4] Z. Su et al. Antibody–drug conjugates: Recent advances in linker chemistry, Acta Pharmaceutica Sinica B, 2021, 11, 12, 3889-3907.