Antibody-drug conjugates (ADCs) consist of linker, payload, and monoclonal antibodies (mAb). They combine the advantages of high specificity targeting ability and potent cytotoxic effects, achieving precise and efficient destruction of cancer cells, making them one of the hot topics in anticancer drug development.Since the first ADC drug Mylotarg® (gemtuzumab ozogamicin) was approved by the FDA in 2000, as of December 2021, a total of 14 ADC drugs have been approved globally for hematologic malignancies and solid tumors, and currently, over 100 ADC candidates are in various stages of clinical trials.Recently, a comprehensive review published in the journal Signal Transduction and Targeted Therapy by Huazhong University of Science and Technology Tongji Medical College reviewed the history and mechanism of action of ADCs, discussed the key components of ADCs, and their impact on ADC activity mechanisms.Additionally, the review detailed the approved ADC drugs and other promising candidates (in phase III clinical trials), discussing current challenges and future prospects for the next generation of ADC development.
1. Timeline of Important Events in ADC Drug Development and ApprovalAs early as the early 20th century, Paul Ehrlich first proposed the concept of “magic bullet” and hypothesized that certain compounds could directly enter specific targets within cells to cure diseases. Theoretically, these compounds should effectively kill cancer cells while being harmless to normal cells.
In 2000, the US Food and Drug Administration (FDA) first approved the ADC drug Mylotarg® (gemtuzumab ozogamicin) for adult acute myeloid leukemia (AML), marking the beginning of the era of ADC-targeted cancer therapy.
Figure 1 depicts the significant events in ADC drug development from infancy to maturity over the past century.
With the continuous expansion of targets and indications, ADCs are leading a new era of targeted cancer therapy, with the potential to replace traditional chemotherapy drugs.
Figure 1 Important Timeline of ADC Drug Development and Approval (Image Source: Reference [1])
Composition of ADCs
ADCs consist of antibodies, cytotoxic payloads, and chemical linkers. An ideal ADC remains stable in circulation, accurately reaches therapeutic targets, and ultimately releases cytotoxic payloads near the target (e.g., cancer cells).
Each component affects the final efficacy and safety of the ADC. Overall, ADC development requires consideration of all these key components, including targets, antibodies, cytotoxic payloads, linkers, and the choice of conjugation methods.
Figure 2 Structure and Characteristics of ADCs (Image Source: Reference [1])
Target Antigens
The target antigens expressed on tumor cells guide the ADCs in recognizing cancer cells, determining the mechanism by which cytotoxic payloads are delivered into cancer cells (e.g., endocytosis). Therefore, selecting appropriate target antigens is a primary consideration for ADCs.
First, to reduce off-target toxicity, the target antigens should be expressed only or predominantly in tumor cells and not expressed or minimally expressed in normal tissues. For example, compared to normal cells, the expression of HER2 in certain types of tumors is approximately 100 times higher than in normal cells, which lays a solid foundation for the development of ado-trastuzumab emtansine, fam-trastuzumab deruxtecan, and disitamab vedotin.
Second, the target antigens should be non-secretory, as secreted antigens in circulation can lead to ADC binding outside the tumor site, resulting in reduced tumor targeting and safety issues.
Third, ideal target antigens should be internalized after binding to the corresponding antibodies, so that the ADC-antigen complex can enter cancer cells and release cytotoxic payloads through appropriate intracellular transport pathways.
Currently approved ADC drugs target specific proteins that are overexpressed in cancer cells, including HER2, Trop2, Nectin4, and EGFR in solid tumors, as well as CD19, CD22, CD33, CD30, BCMA, and CD79b in hematologic malignancies. Driven by basic research in oncology and immunology, the selection of ADC target antigens has gradually expanded from traditional tumor cell antigens to targets within the tumor microenvironment (e.g., in stroma and vascular systems). New evidence from preclinical and clinical studies suggests that components of the neovasculature, subendothelial extracellular matrix, and tumor stroma may serve as valuable target antigens for ADC drug development.
Figure 3 Available Tumor Cell and Tumor Microenvironment (Vascular System and Stroma) Targets for ADCs (Image Source: Reference [1])
Antibodies
Tumor-targeting antibodies are crucial for the specific binding between target antigens and ADCs. In addition to having high binding affinity for target antigens, an ideal antibody should also promote effective internalization, exhibit low immunogenicity, and maintain a longer plasma half-life.
In the early stages of ADC drug development, mouse-derived antibodies were primarily used, but due to severe immunogenic side effects, the failure rate was high. With the advent of recombinant technology, mouse antibodies have largely been replaced by chimeric antibodies and humanized antibodies. Currently, ADCs increasingly utilize fully humanized antibodies with significantly reduced immunogenicity. Among the 14 approved ADC drugs, only Brentuximab vedotin used a chimeric antibody.
As the main component of immunoglobulins in serum, the antibodies used in ADCs are mostly immunoglobulin G (IgG) antibodies, including four subtypes IgG1, IgG2, IgG3, and IgG4. IgG1 is the most commonly used subtype in ADCs due to its highest serum concentration.
The internalization of the antibody-antigen complex primarily depends on the binding affinity between the antibody and the tumor cell surface antigen, and higher affinity usually leads to faster internalization. However, high-affinity antibodies may reduce the antibody’s penetration into solid tumors. Treating solid tumors is more complex than hematologic malignancies due to the presence of binding site barriers (BSB) in solid tumors, where extremely high affinity between antibodies and antigens can lead to ADCs being trapped near blood vessels after binding but less penetrating into tumor cells away from blood vessels. Therefore, the reasonable affinity between antigens and antibodies should be optimized to balance rapid uptake by target cells and anticancer efficacy.
In addition to binding affinity, another factor affecting tumor penetration is the size of the antibody. The large molecular weight (about 150 kDa) of IgG antibodies usually poses significant barriers to penetrating capillaries and the stroma in tumor tissues. Thus, early ADC drugs primarily targeted hematologic malignancies. To improve the treatment of solid tumors, researchers attempted to miniaturize antibodies by removing the Fc fragment. Miniaturized antibodies not only retain high affinity and specificity but also penetrate blood vessels into solid tumors more easily, significantly enhancing their efficacy against solid tumors. However, it has also been found that this change can lead toreduced half-life. Therefore, various factors should be considered when designing ADCs with miniaturized antibodies.
Figure 4 Changes in the Stroma in Cancer (Image Source: Reference [2])LinkersThe linker in ADCs connects the antibody with the cytotoxic drug. It has a significant impact on the stability of the ADC and the release curve of the payload, which is crucial for the final efficacy of ADC drugs. An ideal linker should not induce ADC aggregation, prevent premature release of the payload in plasma, and effectively release it within cancer cells. Depending on the metabolic fate in cells, most ADC drugs use two types of linkers, including cleavable and non-cleavable linkers.Cleavable linkers utilize the environmental differences between systemic circulation and tumor cells to accurately release free cytotoxic drugs and can be further divided into chemical cleavable linkers (hydrazone bonds and disulfide bonds) and enzyme-cleavable linkers (glucosidic bonds and peptide bonds). Another type is chemically sensitive cleavable linkers that are sensitive to reducing glutathione (GSH). GSH plays a crucial role in maintaining the intracellular redox balance during cell survival, proliferation, and differentiation. The concentration of GSH in the bloodstream is much lower than the intracellular concentration in cancer cells. Therefore, this type of linker can remain stable in the bloodstream while specifically releasing active payloads within cancer cells.Peptide-based linkers are sensitive to lysosomal proteases and have been used in many ADCs. Lysosomal proteases, such as cathepsin B, are often overexpressed in cancer cells, allowing for accurate drug release near tumors. Additionally, due to the presence of protease inhibitors in the bloodstream, enzyme-cleavable linkers are usually stable in systemic circulation, thus reducing various risks associated with premature cleavage. Among the approved ADC drugs, 9 out of 14 use peptide-based linkers.Non-cleavable linkers (e.g., thioether or maleimide-based) are inert to the common chemical and enzymatic environments in vivo. The main advantage of non-cleavable linkers is their lower off-target toxicity due to increased plasma stability, but the bystander effect on the payload is affected.PayloadsThe payloads exert cytotoxic effects after ADC internalization into cancer cells. Since only about2% of ADCs can reach the target tumor site after intravenous administration, the compounds used as payloads in ADCs need to be highly toxic (IC50 in the nM and pM range). Moreover, these compounds should remain stable under physiological conditions and possess functional groups that can covalently bind with antibodies.Currently, the cytotoxic payloads used in ADCs mainly include potent microtubule inhibitors, DNA-damaging agents, and immunomodulators.
Figure 6 Representative Small Molecule Payloads Used in ADCs (Image Source: Reference [1])Conjugation MethodsBesides selecting the antibody, linker, and payload, the method of connecting the small molecule portion (i.e., linker plus payload) to the antibody is also crucial for successfully constructing ADCs. Typically, the presence of lysine and cysteine residues on antibodies provides reactive sites for conjugation. Early ADC drugs usually relied on random conjugation through lysine or cysteine residues. However, antibodies typically contain about 80-90 lysine residues, of which 40 residues are usually reactive. Random conjugation with lysine residues can lead to a wide distribution of drug-antibody ratios (DAR) with different numbers of small molecule toxins attached to antibodies, resulting in highly heterogeneous mixtures of varying DAR. Additionally, since lysine residues are distributed throughout the antibody light and heavy chains, conjugation reactions near the antibody-antigen recognition sites may reduce ADC binding to targets.Cysteine-based reactions provide another conjugation method. Typically, IgG1 antibodies have both interchain and intrachain disulfide bonds. Interchain disulfide bonds are exposed on the antibody’s exterior, making them easily reducible to expose free cysteine residues, thus providing usable sites for linking the payload to the antibody. Due to the limited number of binding sites and the unique reactivity of thiol groups, using cysteine as a conjugation site helps reduce the heterogeneity of ADCs. Depending on the reduction rate, products with DARs of 2, 4, 6, and 8 may exhibit better homogeneity compared to products conjugated with lysine residues. This is the most commonly used conjugation method in commercial products to date. However, it is worth noting that breaking interchain disulfide bonds may compromise the integrity of the antibody.Random conjugation of lysine and cysteine residues can lead to many issues.This conjugation’s stability is sometimes insufficient, which can lead to premature payload release, resulting in off-target toxicity. Furthermore, ensuring that the payload is consistently attached to the same site on the antibody becomes challenging, increasing the difficulty of controlling the quality and uniformity of DAR values. To reduce the heterogeneity of ADCs, several site-specific conjugation strategies have been developed in new ADCs.
Figure 6 Site-Specific Conjugation Strategies for ADCs (Image Source: Reference [1])2. Mechanism of Action of ADCsADCs exert their effects by combining “specific” targeting and “efficient” killing of cancer cells. These drugs act like precision-guided “biological missiles,” capable of accurately destroying cancer cells, improving the therapeutic window, and reducing off-target side effects.
Figure 7 Mechanism of ADC Killing Cancer Cells (Image Source: Reference [1])The anticancer activity of ADCs also involvesADCC, ADCP, and CDC mechanisms.Some ADC antibody Fab fragments can bind to antigen epitopes on virus-infected or tumor cells, while the Fc fragment binds to the surface Fc receptors of effector cells (NK cells, macrophages, etc.), mediating direct killing effects (Figure 7 left). Additionally, the antibody component of ADCs can specifically bind to antigen epitopes on cancer cells, inhibiting downstream signaling transduction of antigen receptors (Figure 7 right).3. Advances in ADC ResearchFrom the perspective of drug composition and technical characteristics, ADCs can be subdivided into three generations.
Figure 8 Evolution of ADC Drug Development (Image Source: Reference [1])First Generation ADCsThe early ADCs were mainly formed by conjugating conventional chemotherapeutic drugs with mouse-derived antibodies through non-cleavable linkers. The efficacy of these ADCs was not superior to that of free cytotoxic drugs and had significant immunogenicity. Later, the combination of more effective cytotoxic agents with humanized mAbs greatly improved efficacy and safety, leading to the market approval of first-generation ADCs (including gemtuzumab ozogamicin and inotuzumab ozogamicin). In both of these products, humanized mAbs of the IgG4 isotype were used and conjugated with effective cytotoxic calicheamicin through acid-labile linkers. However, this system also has significant drawbacks:1) Instability of Linkers: For example, acidic conditions may occur in other parts of the body, and the linkers in first-generation ADCs can be slowly hydrolyzed in systemic circulation (pH 7.4, 37°C), leading to uncontrolled release of toxic payloads and unexpected off-target toxicity.2) Prone to Aggregation:The hydrophobic effective payloads used in the first generation are prone to cause antibody aggregation, leading to several defects such as short half-life, rapid clearance, and immunogenicity.3) Heterogeneity of Drugs:The conjugation of first-generation ADCs was based on random conjugation through lysine and cysteine residues, resulting in highly heterogeneous mixtures with varying DARs. Therefore, first-generation ADCs exhibited suboptimal therapeutic windows and required further improvements.Second Generation ADCsSecond-generation ADCs represented by Brentuximab vedotin and Ado-trastuzumab emtansine were approved after optimizing mAb isotypes, effective payloads, and linkers.These two ADCs have the following characteristics:1) Use of IgG1 isotype mAbs, which are more suitable for bioconjugation with small molecule effective payloads and high cancer cell targeting capability.2) Higher toxic effective payloads that improve solubility and conjugation efficiency.More effective payload molecules can be loaded onto each mAb without inducing antibody aggregation.3) Improvements in linkers achieved better plasma stability and uniform DAR distribution.Overall,improvements in all three factors enhance the clinical efficacy and safety of second-generation ADCs. However, there remain many unmet needs, such as insufficient therapeutic windows due to off-target toxicity, and aggregation or rapid clearance in ADCs with high DARs. When DAR exceeds 6, ADCs exhibit high hydrophobicity and tend to reduce ADC efficacy due to faster clearance in vivo. In such cases, optimization of DAR through site-specific conjugation and continuous optimization of mAbs, linkers, and effective payloads is still needed.Third Generation ADCsThird-generation ADCs represented by polatuzumab vedotin, enfortumab vedotin, fam-trastuzumab deruxtecan, and later approved ADCs.1) Uniform DAR (2 or 4), ADCs with consistent DAR show less off-target toxicity and better pharmacokinetic efficiency.2) Fully humanized antibodies instead of chimeric antibodies to reduce immunogenicity.In addition, antigen-binding fragments (Fab) are being developed to replace full mAbs in many candidate ADCs, as Fab is more stable in systemic circulation and may be more easily internalized by cancer cells. Furthermore, more effective payloads have been developed: such as PBD, microtubule disruptors, and immunomodulators with new mechanisms. Although there are no updates on linker types in the third generation, some new entities have been developed for conjugating various effective payloads.To avoid interference with the immune system and improve retention time in systemic circulation, more hydrophilic linker combinations, such as PEGylation, are adopted in third-generation ADCs. Hydrophilic linkers can also be used to balance the high hydrophobicity of certain cytotoxic payloads (such as PBD), as ADCs with hydrophobic payloads are often prone to aggregation.Overall,third-generation ADCs exhibit lower toxicity and higher anticancer activity and stability, allowing patients to receive better anticancer treatments.4. Current Challenges and Next-Generation ADCsFrom the approved drugs and candidates in development, it is evident that the specificity and cytotoxicity of the next generation of ADCs are increasingly better than those of previous generations. However, there are still many challenges in the development of ADCs, including the complexity of pharmacokinetics, insufficient tumor targeting and payload release, and resistance.Main ChallengesComplex Pharmacokinetic CharacteristicsAfter ADC administration (mainly through intravenous infusion), three major forms may exist in systemic circulation: intact ADCs, naked antibodies, and free effective payloads. In the typical pharmacokinetic characteristics of ADCs, the concentrations of conjugated ADCs and naked antibodies continuously decrease with ADC internalization and antibody clearance. Factors affecting antibody clearance include the mononuclear phagocyte system and Fc receptor (FcRn)-mediated recycling. By binding to internalized ADCs with high affinity, FcRn outputs ADCs to the extracellular compartment for recycling. Therefore, compared to traditional small molecule drugs, antibodies, including conjugated ADCs and naked antibodies, usually have longer half-lives.Free cytotoxic effective payloads are primarily metabolized in the liver and excreted through the kidneys (urine) or feces, which may lead to liver and kidney function impairment.All these factors, combined with high inter-patient variability, make it difficult to establish PK and PD models to describe the clinical characteristics of ADCs and assist in designing new ADCs.Inevitable Side EffectsAmong the 14 approved ADCs, the most common severe side effects (grade 3 or higher) are hematologic toxicities, including neutropenia, thrombocytopenia, leukopenia, and anemia. Hematologic toxicity, as well as liver toxicity and gastrointestinal reactions, may be related to the premature release of cytotoxic effective payloads into systemic circulation. This is consistent with conventional chemotherapeutic agents that primarily affect rapidly proliferating healthy cells.Additionally, the immune response induced by the antibody portion of ADCs may cause secondary damage, leading to renal toxicity. According to recent clinical observations, potential pulmonary toxicity (such as ILD) during ADC treatment should be noted, especially in anti-HER2 ADCs. In clinical trials of T-DM1 and DS-8201, several deaths have been reported related to ILD. However, the detailed mechanism of ILD remains unclear. One possible reason may relate to the adverse uptake of ADCs in healthy lung cells and the release of free effective payloads from ADCs. Due to the highest blood flow and longest residence time in the lungs, adverse uptake of ADCs and free effective payloads in the bloodstream is most likely to occur in the lungs, leading to ILD. This requires corresponding optimizations for the next generation of ADCs to minimize side effects. Adverse reactions should be closely monitored during treatment, and preventive or supportive care should be provided.Tumor Targeting and Effective Payload ReleaseCompared to traditional cytotoxic drugs, ADCs are much larger in molecular weight, resulting in limited efficiency of drug penetration into tumors.Current studies indicate that only a small fraction of ADCs input into patients can reach tumor cells, thus the potency of payloads needs to be considered when designing ADCs.ResistanceAnother challenge in ADC development is resistance. Resistance to tyrosine kinase inhibitors (TKIs) often involves escape mutations of drug targets. However, the resistance mechanisms of ADCs have not been fully characterized. ADC resistance is more complex and diverse. Current evidence suggests that tumors can develop ADC resistance in various ways, such as reducing antigen expression levels, altering intracellular transport pathways, and developing resistance to effective payloads.Next-Generation ADCs1) Using ADCs to Target Mutated ProteinsCurrent research indicates that the internalization and intracellular transport pathways of ADCs have a key impact on their cytotoxic activity. Compared to wild-type proteins, mutated proteins often have higher levels of ubiquitination, making them easier to internalize and degrade. This suggests that using ADCs to target mutated proteins may yield significant clinical responses. It is conceivable that ADCs targeting oncogenic mutated proteins (such as certain EGFR mutants) can maximize the tumor specificity of treatment, reaching levels comparable to selective TKIs.2) Bispecific or Dual-Target ADCsAdvances in bispecific antibody technology offer more possibilities for ADC innovation.These ADC designs can improve antibody internalization and enhance tumor specificity. Therapies currently under development are exploring these possibilities. Bispecific ADCs targeting different epitopes on the same antigen can improve receptor clustering and lead to rapid internalization of targets. Additionally, bispecific ADCs targeting both HER2 and LAMP-3 have shown better lysosomal accumulation and payload delivery in preclinical experiments.3) Using Two Different Effective Payload CombinationsUsing two different cytotoxic agents as payloads in dual-effective payload ADCs can reduce resistance. By accurately controlling the ratio of the two drugs, simultaneous delivery of two synergistic payloads to cancer cells can achieve more effective efficacy. Furthermore, the incidence of resistance will be significantly reduced with the application of two effective payloads with different mechanisms. For example, a uniform anti-HER2 ADC containing both MMAE and MMAF has been designed and demonstrated significantly greater antitumor activity in xenograft mouse models than co-administration of corresponding single effective payload ADCs.4) Peptide-Drug Conjugates (PDCs)Another ADC development strategy is to abandon the traditional structure of mAbs and opt to conjugate payloads with smaller molecular weight peptide fragments. The main goal of these strategies is to reduce the molecular weight of ADCs, thereby improving penetration efficiency and the delivery of effective payloads to tumor tissues. For example, PEN-221 is an ADC composed of DM-1 conjugated with a peptide chain targeting growth hormone receptor 2. Its molecular weight is only 2 kDa, far lower than the 150 kDa IgG molecules in traditional ADCs. Currently, such ADCs face technical challenges of potentially rapid clearance in plasma. However, if we can overcome this barrier, they have the potential to treat hard-to-reach tumors, including poorly vascularized tumors and central nervous system tumors.5) Developing Non-Internalizing ADCsTraditionally, to deliver effective payloads to cancer cells, ADCs require mAbs with high internalization capabilities. However, due to antigen barriers, mAbs often struggle to diffuse into solid tumor masses. Therefore, non-internalizing antibodies can be developed for ADCs. This is based on the principle that effective payloads are released directly into the tumor microenvironment under reducing conditions and then diffuse into cancer cells, leading to cell death.Finally, there are still many innovative opportunities in effective payload selection.5. ConclusionVarious ADC therapies have been successfully developed, benefiting thousands of cancer patients.The approval of 14 ADC drugs and the excellent clinical performance of multiple ADCs have also attracted more attention to this field, which is very important for this relatively young but highly complex domain.With the continuous efforts of researchers in these areas, it is not hard to imagine that future ADCs will showcase even more surprises in targeted cancer therapy.References[1] Antibody drug conjugate: the “biological missile” for targeted cancer therapy. Signal Transduction and Targeted Therapy (2022) 7:93[2] The matrix in cancer. Nat Rev Cancer. 2021 Feb 15. doi:10.1038/s41568-020-00329-7. Epub ahead of print. PMID: 33589810.
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