Since 2000, with the FDA approval of Mylotarg™ for the treatment of acute myeloid leukemia (AML), antibody-drug conjugates (ADCs) have gradually been introduced into oncology clinical practice. ADCs combine the precision of antibody-mediated targeting of tumor antigens with the potency of cytotoxic drugs, providing a targeted delivery vehicle for malignant tumors. In this way, ADCs offer a method to reduce off-target toxicity by limiting exposure of the active payload to normal tissues. As of December 31, 2023, there are 15 ADC drugs approved globally, mainly indicated for hematologic malignancies, breast cancer, gastric cancer, and other cancers.

Antibody Drug Conjugate

Antibody-drug conjugates (ADCs) are an innovative class of targeted anticancer drugs that combine the specificity of monoclonal antibodies with the high cytotoxicity of chemotherapy agents to achieve precise tumor treatment. ADCs aim to deliver cytotoxic drugs directly to cancer cells, thereby reducing toxicity to non-target cells and enhancing the therapeutic index. ADCs consist of three main components: a monoclonal antibody that targets a specific antigen on cancer cells, a cytotoxic drug that induces cell death, and a chemical linker that connects the antibody and drug.

Fig. 1. Factors governing ADC activity (MAbs. 2023, 15(1): 2229101).

The linker plays a crucial role in ADCs, ensuring stability in the bloodstream and releasing the payload only within the target cells. The release mechanism typically depends on enzymatic cleavage or specific triggers within the tumor microenvironment. ADC synthesis involves conjugation methods that link the drug payload to specific sites on the antibody, typically via cysteine or lysine residues, to control the drug-to-antibody ratio (DAR). This targeted delivery mechanism makes ADCs highly effective in treating cancers with specific biomarkers, offering a promising strategy to improve treatment selectivity and efficacy while reducing systemic side effects.

Factors Affecting Activity of ADCs

Effective analysis of ADC molecules in clinical testing requires a fundamental understanding of the factors that regulate their biological activity. Three key steps are involved in the basic cellular process of ADC cytotoxin delivery. First, the antibody binds to the target antigen on the surface of antigen-positive cells. Second, the antigen-ADC complex is internalized into the target cell through receptor-mediated endocytosis. Third, the antigen-ADC complex is digested by lysosomal enzymes, releasing the cytotoxic payload that triggers cell death. Compared to standard chemotherapy, ADCs offer the following advantages by delivering the cytotoxic payload directly to tumor tissues, thereby reducing the minimum effective dose (MED) and minimizing on-target/off-tumor adverse events:

• Precisely delivering the cytotoxic payload to cells expressing the selected target antigen;
• Achieving more effective cytotoxic payload utilization than systemic administration;
• Minimizing on-target/off-tumor toxicity.

ADC Clinical Trials

In the 26 years since the first ADC clinical trial in 1997, a total of 266 ADC drugs have been tested in more than 1,200 clinical trials. During this period, 54 ADC projects have been officially terminated, and 38 ADCs have been removed from company pipelines. As of August 2023, a total of 15 ADC drugs have been approved globally.

ADC Targets in Clinical Trials

When selecting ADC targets, it is crucial to focus on the biological characteristics and expression profile of the target protein, as these factors determine the choice of antibody binding affinity—whether high-affinity or low-affinity antibodies are more appropriate. These considerations also influence the selection of payload potency and mechanism of action. True tumor-specific antigens are rare, and many clinically targeted ADCs focus on tumor-associated antigens that may also be distributed or even highly expressed in normal tissues. In such cases, it may be necessary to choose a lower-toxicity payload and avoid antibodies with excessively high affinity. Additionally, increasing antibody binding specificity and enhancing internalization can improve the likelihood of successful ADC development. For example, using bispecific antibodies can enhance tumor-specific recognition and improve payload uptake efficiency within cells.

Fig. 2. Antigen targets of the clinically tested ADCs (MAbs. 2023, 15(1): 2229101).

To date, ADC candidates have targeted 106 tumor antigens. The 15 approved ADCs target 10 unique cancer antigens: six target hematologic cancer antigens, seven target solid tumors, and two are TDM-1 biosimilars. The majority of ADCs target antigens such as HER2 (41 candidate antibodies), Trop-2, CLDN18.2, and EGFR, with fewer than 2% of clinical ADC candidates targeting more than one epitope of a cancer antigen. Among the 267 ADCs in clinical testing, 260 have known antigens (with seven undisclosed). The 15 approved ADC targets include BCMA, CD30, CD33, CD22, CD19, CD79b, EGFR, HER-2, Nectin-4, TROP-2, and TF.

ADC Linkers in Clinical Trials

ADC linkers are primarily divided into two categories: cleavable linkers and non-cleavable linkers. Cleavable linkers can enhance the bystander effect but may cause systemic side effects due to premature release of the toxin in circulation; non-cleavable linkers, conversely, reduce systemic toxicity but also decrease the ADC's bystander effect on tumor cells, potentially reducing efficacy.

Fig. 3. Linkers used in clinically tested ADCs (MAbs. 2023, 15(1): 2229101).

Among ADCs in clinical trials, 54% use cleavable linkers, 16% use non-cleavable linkers, and 30% have undisclosed linker information. Of the 15 FDA-approved ADCs, eight use protease-cleavable linkers. Blenrep and Kadcyla utilize non-cleavable linkers, reducing their systemic toxicity and limiting bystander killing of HER2-low-expressing cells. Enhertu, which also targets HER2, uses a cleavable linker, and in a comparative clinical study between Enhertu and Kadcyla, Enhertu showed significantly better efficacy (median progression-free survival of 28.8 months vs. 6.8 months). This outcome is also influenced by their choice of payloads, as Enhertu uses the topoisomerase inhibitor Dxd, while Kadcyla uses the microtubule inhibitor DM1.

ADC Payloads in Clinical Trials

Data indicate that simply transferring widely used payloads, such as auristatin and maytansinoid, onto different antibodies without considering the target or tumor biology makes it challenging to develop successful ADC drugs. Many ADC development pipelines that attempted to replicate Adcetris (which uses auristatin as a payload) or Kadcyla (which uses maytansinoid as a payload) have failed for this reason. An ideal ADC payload should possess sufficient tumor cell-killing potency, adequate water solubility, a relatively short half-life, and a bystander effect to enhance efficacy while minimizing systemic toxicity.

Fig. 4. Payloads used in clinically tested ADCs (MAbs. 2023, 15(1): 2229101).

The payloads used in clinical trial ADCs can be grouped into four main categories: (1) microtubule inhibitors, (2) DNA-damaging agents, (3) topoisomerase I inhibitors, and (4) targeted small molecules (SM). Microtubule inhibitors are the most commonly used payloads in clinical trials (57%). Of the 15 approved ADCs, eight use microtubule inhibitors, three use DNA-damaging agents, and two use topoisomerase I inhibitors. DNA-damaging agents comprise the second largest category of ADC payloads (17%). In addition to these traditional chemotherapy payloads, about 5% of ADCs include targeted small molecules such as Bcl-xL inhibitors and immunomodulators like TLR and STING agonists. No candidates using non-chemotherapy payloads have yet been FDA-approved. The payloads of 15% of clinical trial ADCs remain undisclosed.

Conjugation Methods in Clinical Trials

The choice of linker and conjugation method should align with the payload's potency, solubility, metabolism, and mechanism of action. More than half of approved ADCs use cleavable peptide linkers, with the payload randomly conjugated to cysteine or lysine residues on the antibody. Although site-specific conjugation methods have been developed, no ADCs using these methods have yet received approval. Among the 267 ADCs in clinical trials, 111 candidates use non-specific amino acid conjugation, 72 use site-specific conjugation, and 84 have undisclosed conjugation methods. Of those using site-specific ADC conjugation, two are approved (Enhertu™ and Trodelvy™), and 26 have been discontinued from clinical trials. Site-specific conjugation methods allow for control over the DAR, typically preserving four interchain disulfide bonds or replacing them with chemical covalent bonds.

In Conclusion

Although several ADC drugs have received regulatory approval, the need to develop next-generation ADCs with higher therapeutic indices, greater efficacy, and improved safety profiles is urgent. Most ADCs fail due to excessive toxicity and insufficient efficacy. Toxicity issues largely stem from on-target toxicity, where ADCs bind to antigens on normal tissues, while efficacy limitations are evident even at the maximum tolerated dose (MTD) where the drug remains ineffective. An ideal ADC should maintain stability and integrity in the bloodstream, accurately reach the therapeutic target (tumor cells), and ultimately release the payload within the tumor cells. Optimizing ADCs requires a comprehensive design and consideration of each component, including the target, antibody, linker, conjugation method, and payload.

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