Release date:2022/11/11 17:30:15

Antibody drug conjugate (ADC) therapies have developed rapidly in recent decades. Currently, 15 ADC drugs have been approved worldwide and more than 140 ADCs are in clinical trials. By 2030, the ADC market will reach more than $15 billion. 

Although the principle of ADC drugs is simple, there are many factors that need to be considered in the actual research and development and industrial production process. The design of an ideal ADC drug needs to consider the target, antibody, linker, payload, conjugation technology and their reasonable combination. Many elements need to be considered in the design of ADC drugs. The success of complex ADC drugs usually depends on the design of five elements.

Design Elements for ADC Drugs

ADC is a complex structure composed of three parts: antibody, linker and payload, which determines that its preparation process will be quite complicated. During the preparation and production process, small molecule toxins need to go through multiple synthetic steps, be dissolved in various solvents, and maintain their chemical structure and properties during these processes. In order for ADC drugs to play an effective role and achieve the expected efficacy and safety, their design and development should focus on the study of targets, antibodies, linkers, payloads, conjugation methods and their reasonable combination. The success of ADC drugs largely depends on the design of the drug.

1. Target

Target selection is a critical part of ADC drug design and a major consideration in ADC development. An ideal target antigen should have the following characteristics:

Tissue specificity: the antigen should be expressed at a high level in tumor cells, but not expressed or expressed at a low level in normal tissues;

Stability: The target antigen should not easily fall off from the target tissue, so as to prevent the combination of antigen and ADC drug in the internal circulation system, reduce the amount of aggregation to the target site, and affect the efficacy and safety of the drug;

Efficient induction of internalization: After the antibody binds to the tumor cell surface antigen, the ADC-antigen complex must be able to effectively induce the internalization process and enter the tumor cell to achieve a rapid release of the payload.

In theory, ADC drugs can release toxins outside tumor cells and kill tumor cells through the "bystander effect" without cellular internalization. But in fact, the effectiveness of most ADC drugs is based on the internalized release of the drug. Therefore, after the antibody in the ADC drug binds to the tumor cell surface antigen, the ADC-antigen complex must be able to effectively induce the internalization process, enter the tumor cell, and achieve the effective release of the small molecule drug through the appropriate intracellular transport and degradation process .

The targets of currently approved ADC drugs include: CD33, CD30, HER2, CD22, CD79b, Nectin-4, BCMA, EGFR, CD19, tissue factor and FRa. From the perspective of development trends, the most popular target in the ADC field is HER2, followed by Trop-2. At the same time, Claudin 18.2, a popular target in the field of gastric cancer, is also another popular target currently being developed. For Claudin 18.2, in addition to ADCs, there have also been many new attempts, such as CAR-T.

2. Antibody

The antibody moiety in the ADC can specifically bind the target antigen on the surface of tumor cells and be highly internalized into the cell, which requires the following characteristics. The first is the targeting specificity and high endocytosis. Antibodies should have high antigen specificity and affinity, and achieve efficient internalization, releasing the payload inside the cell. The second is low immunogenicity. It is necessary to select humanized or fully human antibodies to achieve the lowest immunogenicity. The third is the long circulation half-life. ADC drugs should have a long circulation time in the blood to smoothly enter tumor cells.

The antibodies of ADC drugs are mainly immunoglobulin G (IgG) antibodies, including four subtypes of IgG1, IgG2, IgG3, and IgG4. IgG1 is currently the most mainstream ADC antibody. After it binds to target cells, it can induce various immune responses such as ADCC and CDC, and has excellent characteristics such as strong stability and long half-life.

The advent of antibody-based drugs has enabled substantial progress in the treatment of a variety of diseases, including cancer, autoimmune diseases, cardiovascular diseases, benign blood disorders, and bone diseases. Antibody fragments and bispecific antibodies offer great therapeutic promise for innovative therapies. Antibodies need to meet high specificity, strong target binding ability, low immunogenicity, and low cross-reactivity, so that tumor cells can more effectively take up ADC drugs and prolong the half-life of ADC drugs in serum.

3. Linker

Linkers are used to link mAbs and payloads, and their chemical properties and conjugation sites are critical to the stability of ADC drugs and the release of payloads. The role of the linker: 1. Prevent the premature release of the payload in the blood circulation; 2. After the ADC enters the tumor cells, ensure the effective release of the payload.

Linkers can be roughly divided into two categories: cleavable linkers and non-cleavable linkers. The former has become the mainstream trend. Cleavable linkers can be divided into two categories: enzyme-dependent and chemical-dependent. They have a bystander effect, killing cells with low antigen expression in heterogeneous tumors. Most of the approved drugs use degradable linkers. Drugs such as Enhertu are cleavable linkers whose breakdown products can cross cell membranes and exert bystander effects.

4. Payload

Payload is one of the most critical components of ADC drugs, and it is the decisive factor for ADC drugs to exert the killing effect on target cells. The payload should have the following characteristics.

Firstly, it needs to be highly cytotoxic. Since the amount of toxin ingested at the tumor site is very low, the toxin molecules should exert the highest cytotoxic effect at low concentrations and effectively kill tumor cells. Only about 2% of the ADC can reach the target tumor site after intravenous administration, so the payload needs to be highly toxic (IC50 in the nM and pM range).

The second is modifiability, which can modify the structure of the drug to ensure that it can be coupled with the linker.

The third is high stability. The target of the toxin molecule is mainly located in the cell, and it cannot be degraded and inactivated in the biochemical environment after being released into the cell.

In addition, it needs to have a certain degree of hydrophobicity and membrane permeability. After the toxin with these characteristics is released into the cell, it can penetrate the cell membrane to exert a bystander effect and kill cells with low expression of surrounding antigens.

5. Conjugation method

Conjugation technology connects antibodies to small molecule toxins through linkers, which includes chemical reactions, antibody modification and transformation and other related technologies. The conjugation technology adopted by ADC drugs is closely related to its final drug-antibody ratio (DAR), and the value and distribution of DAR will significantly affect the performance of ADC drugs. Excessively high DAR may lead to the accumulation of ADC drugs, which are then cleared in the circulation. Too low a DAR may lead to suboptimal therapeutic effects of ADC drugs. It is generally believed that a DAR between 2 and 4 is the best choice for ADC drugs.

In addition to traditional structural design, with the continuous advancement of technology, the industry has put forward more "creative" ideas for ADCs, such as Peptide-Drug Conjugates (PDC), Small Molecule-Drug Conjugates (SMDC), and Immune Stimulating Antibody Conjugate (ISAC), Antibody Oligonucleotide Conjugates (AOC), Radionuclide Drug Conjugates (RDC), Fragment-Drug Conjugates (FDC), Aptamer Drug Conjugates (ApDC), etc. The indications of these new drugs are no longer limited to tumor treatment, but have begun to expand to fields such as autoimmune diseases, further broadening the application of ADC.

However, although the FDA has approved multiple ADC drugs, the failure rate of ADCs during clinical development remains high. The inherent complexity of ADCs is a double-edged sword, providing opportunities to provide better treatment while also increasing confounding factors for treatment failure. ADC design drives its pharmacokinetics and pharmacodynamics, and requires more in-depth analysis than the commonly used Cmax and area under curve (AUC) metrics to obtain the optimal dose for clinical application. Current FDA-approved ADCs targeting solid tumors share some common characteristics, including the humanized IgG1 antibody domain, high expression of tumor receptors, and high doses of antibodies. These common features have potential implications for clinical pharmacokinetics and mechanisms of action and provide a reference for ADC design at all stages of clinical development.

Design Criteria For ADC Drugs

Three recently approved solid tumor ADCs highlights important design criteria. Although several components of ADCs show significant differences, their shared characteristics are noteworthy. The structures of the four FDA-approved ADCs for solid tumors are very different, including different linker types (cleavable vs. non-cleavable, different release mechanisms, different stability), specific and non-specific binding, different targets , cancer types and drug-to-antibody ratio (DAR). Interestingly, these ADCs share three common characteristics: (i) a high expression target (105–10receptors/cells), (ii) a high antibody dose (3.6 mg/kg or greater over 3 weeks), and (iii) an IgG1 allotype antibody backbone.

 structural components for ADCs

These three common features have a significant impact on drug delivery and distribution. In fact, because ADCs use known cytotoxic payloads (such as microtubule inhibitors) and known targeting antibodies, a key feature of their clinical success is delivery -- payloads that target every tumor cell at a tolerated dose. These shared design features have their own implications for the targeted delivery of the payload to the tumor.

1. High Expression Target

HER2, Nectin-4, and Trop-2 are highly expressed tumor antigens with over 105 receptors per tumor cell and significantly reduced expression in healthy tissues. Because of the large expression differences, high expression targets can provide a larger window of treatment. Because drug delivery to healthy tissues is usually more effective than delivery to tumors, high antibody doses and high expression tumor targets can quickly saturate uptake in low-expression healthy tissues while still maximizing tumor uptake. The payload toxicity and/or DAR can then be modified to ensure delivery to tumor cells above the therapeutic threshold, while maintaining the subtherapeutic threshold in healthy tissue (to avoid target-mediated healthy tissue toxicity). In contrast, targeting lower expressed tumor antigens requires more potent payloads to achieve therapeutic concentrations in targeted cells. Increasing payload potency usually results in higher toxicity and a lower ADC-tolerated dose. While a lower ADC dose reduces tumor uptake, it may not reduce healthy tissue uptake by the same amount (for example, if target-mediated healthy tissue uptake remains saturated), potentially reducing the therapeutic index. It is important to note that this trade-off is very different from small-molecule drugs, which are usually balanced with plasma concentrations so that low doses result in lower exposure to healthy tissue. In contrast to small molecules, lower doses of more effective ADCs can limit tumor penetration, thereby reducing efficacy rather than toxicity.

2. High Antibody Dose

Developing ADCs for solid tumors is challenging because of poor vascular leakage, tortuosity, and poor lymphatic drainage in solid tumors, resulting in poor convection and increased interstitial pressure. These characteristics combine to create an unfavorable environment for the delivery of ADC drugs. The most direct way to increase antibody delivery and tissue penetration is to give higher doses of antibodies, a second common feature among currently approved ADCs.

Kadcyla is the first ADC to receive FDA approval for solid tumors in many years, and was approved in 2013 through the use of a human IgG1 skeleton, a medium DAR (3.5), a non-cleavable linker, and a potent microtubule inhibitor. Many current ADCs targeting solid tumors have higher doses (3.75-20mg/kg) than Kadcyla over a 3-week period, significantly exceeding many approved ADCs targeting hematoma (for example, Besponsa and Zynlonta have doses of approximately 0.02 and 0.15 mg/kg, respectively). These doses are necessary to overcome high expression and effective internalization to deliver the payload to the cells.

3. Other Design Criteria

Payload selection is critical to ADC development. Today, most payloads fall into one of three categories: (i) DNA damage inducers, (ii) microtubule inhibitors, and (iii) topoisomerase inhibitors. DNA damage payloads are usually very potent (e.g. PBD), while microtubule (DM4, MMAE) and topoisomerase (exatecan, SN-38) inhibitors are more modest. Determining the optimal payload requires a case-by-case analysis. Lower payloads provide greater maximum tolerated dose (MTD). However, for indications with low antigen presentation, payload delivery may not exceed the therapeutic threshold, and a higher potency payload is required. In addition to potency, recent studies have identified multiple payloads as inducers of immunogenic cell death (ICD). The ability of some payloads to induce an immune response after cell death is a new avenue of research that may have broad implications for the next generation of ADC payload selection.

The selection of linkers is also critical, as non-cleavable linkers are generally more stable in plasma. At present, linker chemistry has also made remarkable progress. For example, the Kadcyla non-cleavable linkers lost 18.4% of its payload in four days, while the Enhertu cleavable linkers lost 2.1% of its payload in 21 days. However, even if the linker can reduce the ADC payload loss in the loop, it faces a more difficult challenge after systemic absorption and degradation of the ADC itself. Since most ADC doses do not reach the tumor, the ADC will be metabolized elsewhere in the body, releasing the payload at an undesired location. Payload, linkers, and binding sites can all influence where non-specific release occurs in vivo and dose-limiting toxicity (DLT).

Current FDA-approved drugs exhibit significant variability in these design features, suggesting the need to personalize ADCs for their specific targets. However, the similarity of high-dose antibodies and highly expressed targets, combined with preclinical evidence, suggest that tissue penetration and tumor saturation are key components of efficacy in solid tumors. Therefore, we need to go beyond Cmax and AUC to consider tumor tissue penetration and tumor saturation to design next-generation ADCs.

 tissue penetration & tumor saturation

Tumor Penetration 

Systemic pharmacokinetics, toxicity and efficacy are key metrics to be measured during drug development. Toxicity and pharmacokinetics can quickly screen out poor drug candidates at an early stage. In contrast, testing efficacy requires greater effort, and tissue penetration plays an important role in this process.

The permeability of tumor tissue that affects the efficacy cannot be considered in isolation, but toxicity is equally important. For example, at a clinical dose of 3.6mg/kg, 43.1% of patients treated with Kadcyla showed grade 3 or greater adverse events. Investigators are investigating alternative delivery methods using ADCs to potentially increase tolerability, such as batch dosing. However, when the efficacy of Kadcyla was reviewed for single versus batch administration, the clinical benefit of batch administration was reduced. Thus, a reduced dose and increased frequency of administration may be more tolerable, but smaller doses generally result in lower plasma concentrations, reduced tumor penetration, and lower targeting of tumor cells.

Tumor Saturation

Another key aspect is tumor saturation. This depends on a variety of conditions, including dose (Cmax), expression (receptor/cell), internalization rate, and plasma clearance. The importance of tumor saturation is reflected in two ways: saturation dosage are more likely to be used in preclinical models; and saturation dosage may have opposite results to subsaturation dosage. First, the dosage administered to mice did not always correspond to clinically tolerated dosage. Dosages are sometimes increased in response to faster clearance or less responsive tumor models in mice, or because they are better tolerated in mice. This may lead to saturation in preclinical models, while clinically tolerated dosages may be subsaturated. Because mouse cells typically do not respond to ADC payloads as well as human cells and require high dosages, these high dosages may mask delivery problems in the clinic.

Second, the results of preclinical studies at saturation dosage may produce results opposite to those at subsaturation dosage in the clinic. For example, if a saturation dosage is given, increasing the DAR is more effective, while when a supersaturation dosage is given, lowering the DAR may be more effective. Typically, doses are limited by payload toxicity, so comparisons are made at constant payload doses. When the tumor is supersaturated, the cancer cells receive the largest number of antibodies, so more payloads per antibody will yield greater efficacy. For supersaturated doses, the opposite is true. In this case, the ADC cannot reach all cancer cells, and increasing the DAR (at a constant payload dose) will reduce the amount of antibodies delivered, thereby reducing the number of cells targeted and killed. Conversely, reducing DAR and/or increasing the total antibody dose under these conditions improves tissue permeability and overall efficacy. Therefore, the payload MTD should be associated with the saturation dosage of the antibody to achieve maximum tissue penetration and efficacy.  

In addition, the three recently approved ADCs for solid tumors all use payloads that can have a bystander effect. The bystander effect allows the payload to diffuse out of the target cell and into adjacent cells after release. In theory, bystander payloads could also increase tissue penetration beyond what the antibodies themselves could achieve. This could explain the increased efficacy exhibited by Enhertu compared to Kadcyla in the NCI-N87 mouse model, despite similar cellular potency. However, increased tissue penetration at higher antibody doses would still improve efficacy even when using ADCs with bystander payloads. Although bystander payload improves distribution, direct antibody delivery is more efficient than bystander killing, which explains the greater effect of higher antibody doses even with bystander payload.


Target selection, linker stability, and payload toxicity have been major considerations in ADC drug design over the past few decades. However, many ADCs that looked promising in preclinical studies ended up failing in clinical trials due to toxicity and/or poor therapeutic windows.

By analyzing the common characteristics of recently FDA-approved ADC drugs, it is clear that tissue penetration and tumor saturation are equally critical for successful ADC design. Therefore, data on the most important parameters of efficacy, including tumor penetration and saturation, need to be given special attention in preclinical studies. In an ideal design scenario, at least two pieces of information are known: the tolerable payload dose in humans and the antibody dose required to saturate the tumor target. With this information, we can maximize efficacy at clinically tolerated doses by modifying the DAR to deliver the highest tolerated payload to all tumor cells.

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Key metrics to expanding the pipeline of successful antibody-drug conjugates. Trends Pharmacol Sci.2021 Oct;42(10):803-812
Antibody-Drug Conjugates: A Comprehensive Review

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