Advancements in antibody-drug conjugates as cancer therapeutics.

Introduction

Cancer is the second leading cause of death worldwide, claiming the lives of 10 million people each year. 1 Despite the availability of a wide range of cancer therapies, including chemotherapy, immunotherapy, radiotherapy, cell-based therapy and surgery, conventional chemotherapy remains the main therapeutic option for cancer treatment. 2 , 3 While the central paradigm of chemotherapy involves the killing of rapidly dividing tumour cells while minimally affecting normal-growing healthy cells, several associated limitations, such as poor selectivity, systemic toxicity, and drug resistance, cannot be refuted. These limitations alone can narrow the therapeutic window and limit the efficacy of the drug itself. Thus, researchers are continuously exploring ways to reduce or eliminate the off-target side effects of chemotherapy and significantly improve the patients’ health-related quality of life (hrQoL).

The idea of antibody-drug conjugates (ADCs) was first proposed by a German Nobel laureate and the founder of chemotherapy, Paul Ehrlich, about 100 years ago. He described the use of antibodies as “magic bullets” due to their ability to specifically target diseased cells without harming healthy cells. 4 , 5 Ehrlich envisioned arming antibodies with toxins for selective destruction. His idea soon came to light when methotrexate was linked to an antibody against leukemic cells years later. 6 This eventually led to the design and development of ADCs based on chimeric and humanised mAbs in the 1990s, 7 and subsequently, the first anti-cancer antibody, rituximab, was approved by the Food and Drug Administration (FDA) in 1997. 8

ADCs are a class of targeted cancer therapies that address the limitation of cytotoxic drugs due to their lack of non-specific toxicity, 9 and the insufficient potency of mAbs alone. 10 Even though the development of ADCs has constantly presented new challenges to researchers, especially when it comes to finding the right combination of antibody and cytotoxic drug, selecting the clinically relevant targets, and deciding on the type, position and number of linkages, global efforts by researchers have continuously advanced our understanding of these technologies. As of November 2024, a total of 15 ADCs have been approved by the FDA. 11

ADCs are akin to “homing missiles” and are composed of three key components: (1) a mAb that binds to tumour-specific or tumour-associated target antigens on the surface of a cancer cell; (2) a cytotoxic drug; and (3) a cleavable or non-cleavable linker that binds both antibody and drug moieties together. The linkers that adjoin the antibodies and cytotoxic drugs are designed to be highly stable to prevent unwanted cleavage before the ADC molecule/complex enters the cancer cell through receptor-mediated endocytosis. 12 Unlike conventional chemotherapeutics, ADCs are designed to deliver cytotoxic drugs selectively to cancer cells that express the target antigen while minimising exposure to healthy cells, thus minimising the chance of off-target effects associated with systemic administration. Since antibodies bind specifically to cancer cells, this subsequently leads to the release of the cytotoxic payload right after it is internalised by cancer cells, causing apoptotic cell death. Depending on the class of cytotoxic payload, ADCs could cause cell death by either damaging the DNA of cancer cells or by preventing the formation and propagation of new cancer cells. 12

This review aims to highlight the transformative potential of ADCs in the field of cancer treatment, emphasising the need for continued innovation and addressing the challenges in ADC development to fully realise its therapeutic promise.

Advances in ADC technology

There has been a growing emphasis towards the development of mAbs in cancer treatment due to their specificity and the growing number of newly discovered cancer-specific or associated antigens. The expansion of mAbs in the realm of cancer treatment began with the approval of Rituximab in 1997 for the treatment of B-cell lymphoma. Subsequently, other newer generation mAbs for cancer, such as Avastin Ⓡ , Cetuximab Ⓡ , Rituximab Ⓡ , Trastuzumab Ⓡ and many others have emerged.

Modulation of the binding specificity of mAbs is exceptionally essential, as it confers a significant advantage over alternative cancer therapies. 13 Such modifications can be carried out within the variable domain of the antibody molecule, which contains the complementary binding regions (CDRs) that govern the interaction between an antibody and its corresponding antigen. 14 To engineer the variable domain and CDRs, a library comprising a huge number of variants generated by targeted or random mutagenesis must be screened. Targeted or structure-guided mutagenesis, where screening is performed based on the structural knowledge of the antibody-antigen complex, is a more effective alternative due to fewer variants being tested. 15 Typically, epitope-paratope structural data obtained from X-ray crystallography can be used to predict the suitable variants in combination with in silico tools. 16 However, a de novo , computational-based design of the molecular structure and docking of the antibody-antigen complex could be used instead of X-ray crystallography. Potential antibody variants screened may then undergo wet lab testing via mutagenesis to confirm their efficacies. 16

The humanisation of mAbs is another essential step in antibody engineering, particularly when the therapeutic target involves humans. This process reduces the cross-reactivity that may result from the exposure of foreign materials, such as antibodies derived from non-human sources. Since most antibodies are derived from non-human origins, the human immune system may perceive these mABs as threats and, in turn, react with unwanted immune responses, which negatively affects the therapeutic intent of mAbs. 17 As illustrated in Fig. 1 , one method of humanising antibodies is the CDR grafting technique, where CDRs from synthesised non-human mAbs are grafted into a human Ig molecule. This renders the molecule less “foreign” to the immune system without compromising its specificity. 18 Further reduction of immunogenicity can be achieved by applying specificity-determining residue (SDR) grafting after grafting of CDR. The addition of SDRs conserves the CDR’s loop structure and can be easily identified from the 3D structure of an antibody-antigen complex. 19

The utilisation of a phage-display approach can also be employed for the synthesis of a fully functional human antibody. The present methodology involves the integration of multiple genes into a bacteriophage with the purpose of constructing a comprehensive library. Subsequently, the phage will proceed to express the genes in the form of surface proteins, which can be subjected to selection through biopanning and screened via enzyme-linked immunosorbent assays (ELISA). 20 , 21 Another technique for generating human mAbs from murine variable region sequences can also be adopted by utilising information from the repertoire of human germline and employing a computational approach to derive murine variable region sequences by rationally engineering the residues within and proximal to the CDRs and the V H /V L interface. 22 This iteration involves the exploration of successive substitutions guided by the sequence of the nearest human germline sequences. The antibody will, therefore, undergo a gradual transformation, progressively acquiring human characteristics until it reaches a state of complete or acceptable humanisation while simultaneously retaining its functional activity. Three fully humanised mAbs were successfully generated using this technique to target CD25, vascular endothelial growth factor, and TNF-⍺ with 59, 46 and 45 substitutions, respectively. 23

Despite their specificity and exceptional targeting ability, most mAbs alone are deemed not cytotoxic enough to effectively eradicate cancer cells. In these situations, ADCs can be the solution, as the cytotoxicity of ADC comes primarily from the drug payload. At present, approximately only 2% of ADC molecules can be expected to reach and penetrate targeted solid tumour cells; hence, highly potent cytotoxic drugs, which are also able to retain their stability inside the body, are essential to be selected as payloads. 24 , 25

Currently, FDA-approved drug payloads of marketed ADCs can generally be categorised as either tubulin inhibitors or DNA-damaging agents. Tubulin is the protein component of microtubules, which plays a crucial role in cell mitosis; hence, inhibiting them effectively impairs the tumour's ability to divide and metastasise. 26 Meanwhile, DNA-damaging agents induce cytotoxicity by inhibiting DNA replicating machinery, inducing DNA alkylation or double-strand breaks, and so on. This classification of drugs may be desired when a cytotoxic mechanism independent of the cell cycle is required since tubulin inhibitors primarily act on mitotic cells. 24 , 27 Interestingly, some experimental payloads used in ADC technology are immunotoxins derived from marine sources, such as monomethyl auristatin E, which is derived from the shell-less mollusc Dolabella Auricularia, as well as the HALT-1 toxin from Hydra magnippapilata . Cnidarian actinoporins have demonstrated great potential as an immunotoxin drug moiety due to their stability and small size (18.5–20 kDa), thus enabling them to have higher penetration and lower immunogenicity. 28 Their mechanism of action works by forming pores in the cell membrane upon recognition by specific sphingomyelins, effectively disrupting the ion gradient that will lead to osmotic imbalance and cell lysis. 28 Newer generation ADCs that are being developed also incorporate an immunomodulator as a payload to stimulate the immune system instead of directly causing senescence in the tumours. A few examples include toll-like receptor (TLR) agonists and stimulator of interferon genes (STING) agonists. 24 , 29 These immunomodulating ADCs are termed immune-stimulating antibody conjugates (ISACs), which have shown promising potential as cancer immunotherapeutic agents with longer-lasting immunological memory.

Linker is another integral part of ADC technology that is constantly evolving since it governs the release of the drug payload from the antibody moiety as well as the overall stability of the ADC. Linkers can be divided into cleavable and non-cleavable linkers, each with its own purposes. Cleavable linkers refer to those equipped with a chemical trigger in their structure which will induce cleavage and payload release in a designated condition, whereas non-cleavable linkers are a part of the drug payload itself. 30 Hence, selecting an appropriate linker must be done in accordance with the target’s profile and location. Classical linkers still need some improvements with regards to limiting non-specific release, improving linker-payload attachment, and reactions that may reduce the ADC’s efficacy, such as the retro-Michael elimination reaction in maleimide-based linkers. Some more recently developed ADC linkers include new kinds of enzyme-sensitive linkers, photo-sensitive linkers, and bio-orthogonal cleavable linkers, 30 which will be further discussed.

Next generation linker chemistry

Linkers play a crucial role in ADC design by stably connecting the cytotoxic drug to the mAb. Approximately 80% of approved ADCs utilise a cleavable linker for the purpose of facilitating the transportation of therapeutic agents to the tumour site. 30 The linkers are designed to be cleaved in response to differences in the extracellular and intracellular environments, such as redox potential, pH, GSH concentration, and the presence of specific lysosomal enzymes. 31 , 32 This allows the cytotoxic payload to be released specifically within or in close proximity to the target tumour cell. Peptide motifs represent the predominant form of cleavable linkers and have been extensively employed in ADCs at the clinical stage. Nevertheless, an inherent drawback of these cleavable linkers lies in their susceptibility to enzymatic cleavage by extracellular enzymes, such as serine elastase. 33 This susceptibility inadvertently leads to the systemic release of the cytotoxic payload, resulting in off-target toxicities. 33 Thus, an ideal linker must possess sufficient stability to prevent the premature release of the cytotoxic drug in off-target tissues or the induction of systemic toxicity. Simultaneously, the linker must maintain the conjugate in an inactive, non-toxic state while binding to the antibody. Researchers have consistently devoted significant efforts to developing cleavable linkers that are more stable in the circulatory system. Among these approaches include the utilisation of peptide sequences that are more selective substrates for specific proteases, 34 , 35 , 36 , 37 and exploring the use of alternative enzyme classes as release mechanisms. 38 , 39

Traditional methods for releasing cleavable linkers include protonolysis, disulfide reduction, and proteolytic degradation, such as the valine-citrulline (Val-Cit)-PABC linker technology. 33 An ideal linker for ADCs should possess the capability to effectively prevent the premature release of the drug payload in the bloodstream by means of a robust and stable linker to the mAb. Furthermore, it should also offer a specific trigger mechanism for the efficient release of the drug payload at the intended site. While most cleavable linkers manage to tick the former criteria, a novel linker chemistry such as the legumain linker has ticked both boxes as an ideal linker. Legumain linker, which is the proprietary linker innovated by Vincerx Pharma, leverages the overexpression of a lysosomal protease called legumain in cancer cells, which is associated with a poor prognosis. 40 , 41 Legumain is a highly specific lysosomal asparaginyl endopeptidase that cleaves at a specific asparagine residue in its target proteins, thereby enhancing selectivity for cancer cells. Therefore, the unintentional release of the cytotoxic drug payload into the systemic circulation and healthy cell target can be greatly reduced. To further enhance the therapeutic range of ADCs, the prodrug principle is employed by incorporating a hydrophilic peptide cap, also known as a cell trapper, on the cytotoxic drug payload that can be cleaved by legumain. The activation of the prodrug occurs once it is internalised, and the peptide cap is cleaved by the legumain enzyme in tumour cells. The cell trapper effectively restrains the permeability of the cell membrane, resulting in the entrapment and accumulation of drugs within tumour cells, thereby improving efficacy and safety. 42 , 43

Another advancement in linker technology involves the use of tandem-cleavage linkers, where two sequential enzymatic cleavage events are required to facilitate payload release. In tandem enzymatic cleavage events, the second cleavage is halted until the first cleavage occurs, hence reducing the occurrence of premature release of the drug payload during circulation and off-target toxicities. The tandem-cleavage linker strategy was inspired by prodrug approaches utilising hydrophilic glucuronide moieties, 44 and by glucuronide cleavable linkers, which both are responsive to the lysosomal enzyme that is upregulated in cancer cells, β-glucuronidase. 33 The β-glucuronide moiety was utilised to protect dipeptides as an extra layer of protection from unintended cleavage in the circulation. The monosaccharide is only removed after ADC internalisation and lysosomal degradation, which leads to dipeptides being available for degradation and liberation of payloads inside the cell. Tandem-cleavage linkers have been proven to be superior in terms of plasma stability and tolerability while retaining their efficacy in rat studies, thus improving the therapeutic index of ADCs. 33

Several limitations of traditional ADC drugs include limited selection of antibody candidates for ADC since antibodies must be taken up via lysosomes, the occurrence of drug resistance as the release of the drug payload in ADCs rely on multiple processes such as tumour cell endocytosis and lysosomal lysis, and the size of ADCs which significantly impact tumour penetration and permeability rates. The novel tumour microenvironment activable linker (TMALIN) technology platform directly addresses the drawbacks of current ADC technology in tumour antigen expression and payload release mechanisms. The special characteristics of enzyme digestion enable the extracellular lysis of tumours in the microenvironment, allowing ADCs to exhibit high anti-tumour activity regardless of the antibody's endocytosis capability, thus significantly expanding the range of antibody options. The distinctive structure of TMALIN ADCs facilitates their accumulation in the tumour microenvironment, resulting in a higher ratio of payload concentration in tumours compared to blood, which contributes to a higher therapeutic index. Consequently, the combination of the unique enzyme digestion characteristics and tumour enrichment capabilities of TMALIN ADCs leads to a significant accumulation of the payload in tumour tissues, resulting in a potent bystander effect. This translates into effective anti-tumour effects even in tumours with low or no antigen expression. On top of that, ADCs developed using TMALIN technology exhibit exceptional systemic circulation stability, minimising the detachment of the payload in non-target tissues and drastically minimising the off-target toxicity. Lastly, excellent solubility and chemical stability of ADC developed via the TMALIN platform were observed, eliminating the reversible addition reactions caused by the maleimide connection mode in traditional ADCs. This allows for the production of ADCs with high uniformity (DAR=8.0) and precise quantitative coupling. 45 , 46

On top of the linker technologies discussed above, another novel enzyme-sensitive linker approach employs new enzymes that are overexpressed in tumour cells, such as the β-galactosidase-, sulfatase-, and pyrophosphatase-cleavable linkers. 30 Photo-sensitive linkers are useful in the sense that their cleavage can be controlled, thus reducing the risk of off-target release. Just like the name suggests, these linkers are responsive to stimuli from lights with a certain range of wavelength, meaning that the payload will only be released at the site that is irradiated. A linker that will release the drug payload upon irradiation with NIR light. 47 The mechanism is based on a heptamine cyanine fluorophore. Next, a linker that responds to UV light irradiation ( λ =365 nm) employing an o-nitrobenzyl group as the underlying trigger. 48 This ADC was found to have a half-life similar to the regular antibody molecule. More recently, a type of ADC whose cleavage is controlled by light ( λ =365 nm) and a reaction with a self-contained N-terminal amine, implying that the ADC will only be cleaved when both factors are present. 49 Lastly, bioorthogonal-cleavable linkers respond to bioorthogonal reactions, which refer to chemical reactions that can proceed in a living system without causing interference with the endogenous biochemical processes. 50 Based on this, another innovative ADC with a “traceless linker” containing dual-substituted propargyloxycarbonyl (dsProc) and dual-substituted propargyl (dsPra), which are triggers of bioorthogonal reactions upon interaction with Cu(I)-BTTAA, thus the drug payload, in this case, will be released extracellularly at the tumour site. 51

Site-specific conjugation

Conventional conjugations (first generation) leverage lysine or cysteine residues that are abundantly found in antibodies. On the other hand, site-specific conjugation (next generation) allows a couple more antibodies to conjugate with cytotoxins via unique and defined sites based on antibody engineering. Site-specific conjugation can overcome heterogeneity with a suboptimal therapeutic index, higher clearance rates for high drug-to-antibody species, narrower therapeutic windows, and poor stability. There are four methods for site-specific conjugations, including (1) specific amino acids, (2) unnatural amino acids, (3) short peptide tags, and (4) glycans, 52 which are listed in detail in Table 1 .

Prodrug payload

An ideal prodrug needs to exhibit the following characteristics: (1) adequate high cytotoxicity; (2) low immunogenicity; (3) high stability; (4) modifiable functional group without affecting the potency; (5) bystander killing effects; (6) proper water solubility; and (7) intracellular properties of payloads’ target, allowing ADCs entering tumour cells, and release their payloads. 61 The first generation of ADC drugs are mainly traditional chemotherapeutics such as methotrexate, vinblastine, and doxorubicin. They are commonly associated with certain drawbacks, such as the lack of cytotoxicity against cancer cells, low tumour selectivity, low drug accumulations at target sites, and poor efficacy. 61 , 62 , 63 Hence, the search for novel prodrugs is imperative. There are a few selections, as tabulated in Table 2 , that can be developed as potential prodrugs for next-generation ADCs.

Dual-payload ADCs and bispecific ADCs

Intratumour heterogeneity is one of the major causes of treatment resistance and cancer relapse. 76 Even ADCs with a single chemotherapeutic agent were reported to be ineffective in treating heterogenous tumour-containing cells with differential drug sensitivities. 77 Thus, co-delivery of two or more therapeutic agents could be a potential way of overcoming it. Using a two-dimensional hybrid agent-based model that represents tumours, co-administration of ADC ado-trastuzumab emtansine (T-DM1) with its unconjugated antibody, trastuzumab, was demonstrated to enhance the treatment efficacy, especially in tumours with intrinsically resistant cells compared to ADC monotherapy. The co-administration also showed the selection of resistant cells, improved distribution, and minimised heterogenous delivery. 78

Besides that, Yamazaki research team generated an ADC with two payloads, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) ( Fig. 2 ). 79 MMAE diffuses through the cell membrane to kill targeted and neighbouring cells, while cell-membrane impermeable MMAF acts as an inhibitor for drug efflux pumps to combat drug resistance. 80 When evaluated using HER-2-positive breast cancer cell lines, the dual-loaded ADC with MMAE and MMAF exhibited a significant cytotoxic effect compared to a single-drug ADC. Treatment of this dual-loaded ADC in a xenograft model of a human breast tumour consisting of HER2-positive JIMT-1 cells and HER2-negative MDA-MB-231 cells showed better antitumour activity with complete remission and no tumour regrowth as compared to single-drug ADC ( Fig. 2 ). 79 Besides MMAF, Mckertish and Kayser developed another dual-payload ADC, where trastuzumab was conjugated with MMAE via the Val-Cit linker, followed by another conjugation with Mertansine (DM1) via the non-cleavable linker Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) to form a dual conjugate. This dual-payload ADC exhibited cytotoxic effects in SK-BR-3 (highly expressing HER2 + cells) breast cancer cells and DLD-1 (lowly expressing HER2 + cells) colorectal cancer cells. 81 This evidence supports the idea that a dual conjugate approach results in high cytotoxicity and anti-tumour effects against heterogeneous tumours, treatment resistance and tumour recurrences.

In recent years, bispecific antibodies have gained tremendous interest as they are superior to mAbs and have broad applications in tumour immunotherapy and treatment for other diseases. The characteristic of bispecific antibodies is the presence of two binding sites directed at two different antigens or epitopes on the same antigen. 82 Bispecific antibodies have been reported to show promising cancer therapeutic efficacy through various mechanisms, including the activation of immune cells, blocking of immune checkpoints, inflammatory factors, and dual signalling pathways. These mechanisms of action and their respective examples of bispecific antibodies have been summarised in Table 3 .

It is evident that bispecific antibodies are effective against cancer and can be developed as potential ADCs to improve the specificity, affinity, and internalisation of mAb ADCs. Indeed, several bispecific antibodies for ADC have been developed and are currently under investigation. Zong and research team fabricated a bispecific antibody targeting prolactin receptor (PRLR) and HER2 using protein trans-splicing (BAPTS), with MMAE as payloads via the maleimidocaproyl linker ( Fig. 3 ). 87 When compared to HER2 ADC, PRLR x HER2 bispecific ADC was shown to have higher internalisation efficiency with superior antitumour activity against human breast cancer in vitro. 87 Moreover, BIO-201, a novel bispecific ADC targeting both HER2 and Trop-2 conjugated with a potent DNA topoisomerase I inhibitor via a cleavable linker has generated. 87 This ADC was demonstrated to have enhanced cell binding and internalisation with potent cytotoxicity against cancer cells that co-expressed HER2 and Trop-2. The tumour regression effect was also presented in xenograft models with HER2 or Trop-2-positive tumours, suggesting broad coverage of tumour types as compared to mAb ADCs. 88

Apart from antigens in breast cancer, mesenchymal-to-epithelial transition factor (MET)-selective tyrosine kinase inhibitors (TKI) are effective against lung cancers expressing MET genetic mutations. ADC targeting MET has also shown promising treatment efficacy against MET mutation such as MET exon 14 skipping mutation or MET overexpression in non-small cell lung cancer. 89 However, MET genetic alterations are only present in a small population of non-small cell lung cancer. Together with tumour resistance, the treatment efficacy of MET-targeted ADC may have been greatly impacted, which urged the development of combination treatment. 90 , 91 A biparatopic METxMET antibody conjugated to maytansinoid payload was generated to form the bispecific METxMET ADC. This biparatopic ADC was demonstrated to induce tumour regression in xenografts with moderate to high MET expression, including the models that exhibited innate or acquired resistance to MET blockers. 92 A phase I/II study of REGN5093-M114 (METxMET ADC) has been initiated among patients with MET-overexpressed advanced cancer ( NCT04982224 ).

Besides targeting tumours, biparatopic METxMET ADC can also be used to study trans-endosomal trafficking. Recently, a biosensor is conjugated to a biparatopic METxMET antibody to form a cleavable bispecific ADC, 93 and the internalisation was observed in both in vitro and in vivo. 31 , 32 It was demonstrated that the METxMET biparatopic antibody could internalise into sorting endosomes, followed by rapid trafficking to the recycling endosome and matures slowly into the late endosomes, which were the processing sites of MET, EGFR and PRLR ADCs. This evidence not only provides new insights on the catalytic activity of the recycling endosome and the relationship between trans-endosomal trafficking and ADC processing, but also suggests that recycling receptors can be developed as potential targets for ADC as they can efficiently deliver ADC payloads into tumour cells. 93

ADCs targeting the tumour microenvironment, “The Bystander Effect”

As mentioned previously, traditional ADCs are limited in the selection of antibodies as it depends on the choice of target antigen exposed on the cell surface, which tends to be highly expressed in cancerous cells but is found to be lowly expressed in healthy cells. In short, the bystander effect refers to the phenomenon where the cytotoxic payload released from an ADC can diffuse into and kill neighbouring cells, even if those cells do not express the target antigen recognised by the antibody. 94 Current ADCs in preclinical and clinical development mainly target either tumour-associated antigens (TAAs) or tumour-specific antigens (TSAs). 31 , 95 , 96 TAAs are proteins that are highly expressed in tumours but present minimally in healthy tissue, while TSAs are only found in cancer cell types.

In contrast to hematologic malignancies, solid tumours flourish within a complex and ever-evolving ecosystem known as the tumour microenvironment (TME), and its composition exhibits remarkable heterogeneity across tumour types. The TME is a multifaceted and dynamic ecosystem that plays a critical role in tumour development, progression, and response to therapy. Key components of the TME worth highlighting include (1) an abundant extracellular matrix that provides structural support; (2) stromal cells such as cancer-associated fibroblasts (CAFs) that promote tumour growth and invasion; (3) a network of newly formed and abnormal blood vessels that facilitate tumour angiogenesis; (4) immune cells that exhibit a complex interplay of pro- and anti-tumour functions. 31 , 97 , 98 Owing to the close association of tumour cells with the TME, TME-associated antigens (TMAs), aberrantly expressed on non-malignant cells within the TME, emerge as promising targets for solid tumours, offering a distinct approach compared to traditional tumour antigens. Prominent TMA targets include chemokines and cytokines, transcription factors, metabolic enzymes, and checkpoint molecules. 99 The prominent benefits of these targets lie in their elevated expression on endothelial, stromal, and immune cells, contrasting with their rarity or minimal presence in healthy tissues. Apart from that, TMAs exhibit enhanced accessibility to ADCs upon systemic administration, particularly for antigens expressed on neo-vessels or stromal cells.

Currently, several prominent TMA-targeting ADCs are currently being tested in clinical trials: STRO-001, which targets CD74 in advanced B-cell malignancies ( NCT03424603 ), 100 and JBH492, which targets CCR7 in non-Hodgkin lymphoma and chronic lymphocytic leukaemia patients ( NCT04240704 ). 101 Moreover, Camidanlumab tesirine, also known as ADCT-301 , is an ADC in phase I/II for the treatment of classical Hodgkin’s lymphoma (cHL) and non-HL ( NCT02432235 ). 102 On top of that, DS-7300, MGC018, and Mirzotamab clezutoclax are ADCs targeting CD276, mainly for the treatment of advanced solid tumours ( NCT04145622 , NCT03729596 and NCT03595059 ). 103 , 104 , 105 The profiles of TME-associated targeting ADCs, which are currently in clinical trials are summarised in Table 4 below.

Anti-senescence or senescence effects of ADCs

Cellular senescence in cancer cells is characterised by a state of permanent growth arrest and the production of a senescence-associated secretory phenotype (SASP), which includes cytokines, chemokines, and growth factors that can create a pro-inflammatory and tumour-promoting microenvironment. This SASP can paradoxically stimulate tumour progression by supporting surrounding malignant cells, aiding metastasis, and potentially reducing the efficacy of certain therapies. 106 , 107 , 108

Unlike conventional chemotherapy, which has the potential to induce senescence in non-target cells and contribute to SASP, 109 ADCs deliver cytotoxic agents directly to cancer cells, expressing specific antigens without targeting healthy cells. This specificity not only reduces overall toxicity but may also prevent conditions where SASP from senescent cells lead to chronic tissue damage and the development of secondary malignancies. By limiting senescence to targeted cells or inducing direct cytotoxicity, ADCs may lower the potential for secondary tumour-supporting effects, offering a clear therapeutic advantage.

Since senescent cells can contribute to a pro-tumorigenic environment, the ability of ADCs to minimise unintended senescence in normal cells might reduce the likelihood of tumour relapse. 110 By maintaining healthy tissue integrity and avoiding SASP-associated inflammation, ADCs could have implications for long-term patient outcomes and potentially lower the risks of cancer recurrence and therapy resistance.

While ADCs are traditionally designed to kill target cells, there is an emerging interest in designing ADCs that can selectively induce senescence within cancer cells. 111 , 112 , 113 The concept is that by pushing tumour cells into a state of controlled senescence, their proliferation is halted. Moreover, senescent cancer cells could be potentially cleared by the immune system if they express “senescence-associated immune ligands” that stimulate immune cell recognition. 114 Senescence-inducing ADCs may enhance immune surveillance, aiding immune cells to recognise and eliminate these non-dividing damaged cells. This dual approach of inducing senescence in some cells while killing others could maximise tumour suppression. For instance, ADCs targeting proteins associated with cellular ageing mechanisms could promote a state of ‘therapeutic senescence’ as part of combination therapies with immune checkpoint inhibitors or SASP-modulating agents, improving overall treatment efficacy. 115

ADC design should be optimized to minimise unwanted senescence. The rational choice of payload, linker stability, and antigen target specificity are crucial when it comes to minimising unintended senescence in healthy cells. Modifying ADCs to ensure high specificity for tumour cells, especially those with unique antigens, helps in reducing off-target senescence induction.

Surprisingly, senescence has significant implications not only for tumour development but also for the responses to anticancer therapy. Anticancer therapies have been shown to induce the accumulation of senescent cells in both malignant and non-malignant tissues. This is primarily due to their mechanism of causing DNA damage, coupled with the systemic administration of most anticancer drugs, which can result in senescence being triggered across multiple tissues and compartments. 116 Various targeted therapies are known to induce premature senescence in both malignant and non-malignant cells. CDK4/6 inhibitors, such as palbociclib, ribociclib, and abemaciclib, mimic p16 INK4a activity, causing stable cell-cycle arrest indicative of senescence, potentially through p53-dependent pathways. 117 , 118 , 119 , 120 The pan-HDAC inhibitor vorinostat and the DNA methyltransferase inhibitor decitabine have been shown to induce senescence through distinct mechanisms, including CpG methylation modulation and SA-β-gal activity. 121 , 122 , 123 Antiangiogenic agents like VEGF inhibitors can elicit senescence in preclinical cancer models and elevate cytokine levels, suggesting an SASP-like effect. 124 Additionally, non-cytotoxic therapeutic antibodies, such as rituximab, have been implicated in promoting senescence in lymphoma cells. 125 Hence, payloads with mechanisms that are less prone to causing cellular arrest in a senescent state may be preferable in designs aimed at avoiding chronic SASP production. Microtubule-targeting agents: auristatins and maytansinoids disrupt microtubules and induce apoptosis without directly causing DNA damage, 126 which is less likely to result in senescence compared to DNA-damaging agents like alkylators or topoisomerase inhibitors. Non-DNA-damaging apoptosis inducers that activate intrinsic apoptosis pathways, such as Bcl-2 inhibitors, can promote cell death without initiating senescence. 127 Other engineering examples to avoid SASP include the introduction of site-specific conjugation strategy to enhance targeted payload delivery, Fc engineering to modulate interactions with the immune system, and selective use of cleavable linkers that only activate within the tumour microenvironment. 128 , 129

Synergistic actions of ADCs with other therapies

Due to the unique characteristic of ADCs in selectively targeting the cancer cell, ADCs can be combined with other cancer therapies to achieve synergistic effects, especially in patients with complex, multi-condition disease profiles. Several preclinical and clinical studies have shown that ADCs can work in concert with immune checkpoint inhibitors (such as anti-PD-1/PD-L1 therapies), kinase inhibitors, or even low-dose chemotherapy, resulting in enhanced tumour cell killing. 130 , 131 For instance, immune checkpoint inhibitors, when paired with ADCs, may help to overcome immune evasion mechanisms by stimulating an anti-tumour immune response in the patient while the ADC specifically targets cancer cells.

One of the most promising examples of combination therapy is the pairing of ADCs with immune checkpoint inhibitors (PD-1 or PD-L1 inhibitors). These drugs work by promoting the immune system to recognise and attack cancer cells, and it has shown promise in reducing tumour size and improving overall response rates in solid tumours. 131 The synergistic anti-tumour effect happens when the cancer cells are killed by ADCs; tumour antigens may be released into the tumour microenvironment, stimulating the immune system and making the tumour more visible to immune cells. This strategy holds promise for improving outcomes in patients who may not fully respond to either therapy alone. In preclinical studies, the combination of ADCs with nivolumab has been proven to improve outcomes in patients with refractory cancers. 132

When used in combination with other therapies, ADCs may allow for dose reductions in traditional chemotherapy agents, potentially lessening the side effects that patients experience. Some ADCs are designed with highly potent cytotoxic agents, allowing for therapeutic efficacy at lower systemic doses. Additionally, innovations in linker chemistry and antibody engineering have further reduced systemic toxicity by improving the stability and selectivity of ADCs in circulation, ensuring that the drug is released primarily within the tumour microenvironment.

Given the ability of ADCs to specifically target tumour cells and limit off-target effects, they are well-suited for combination with other treatments that may otherwise pose toxicity challenges. In the future, the strategic combination of ADCs with other targeted therapies or immunotherapies could offer a potent, lower-toxicity alternative to traditional chemoradiation approaches, especially for patients with comorbid conditions or those who are more vulnerable to the harsh side effects of conventional treatments.

FDA-approved ADCs for cancer therapy

ADCs represent an innovative class of biopharmaceuticals that leverage mABs to deliver cytotoxic agents with high potency to tumour sites in a selective manner. As of November 2024, the FDA has approved 15 distinct ADCs, as seen in Table 5 , while over 164 additional clinical trials of various phases related to ADCs are currently ongoing.

By utilising the Clinicaltrials.gov database, 164 active clinical trials related to ADC were identified ( Table 6 ). There are 75 distinct ADCs within active clinical trials, as seen in Fig. 4 . The predominant target for ADCs is currently HER2, with TROP2 closely following. 106 , 107

Challenges in ADC development and its systemic stability after administration

While ADCs offer immense potential for targeted cancer therapy, their development and application still face numerous challenges. Firstly, the choice of a suitable linker to conjugate the payload and antibody is critical for ADC development. Various types of linkers, including cleavable and non-cleavable linkers, come with their own set of advantages and disadvantages. Hence, the selection of the appropriate linker largely depends on the specific linker chemistry for conjugating both antibody and payload. Ideally, the linker should only be cleaved and release the drug payload after it is internalised by targeted cancer cells. Premature release due to an unstable linker can lead to off-target toxicity, which is one of the main downsides of ADC technology that needs to be circumvented. 133

Based on the optimal number of drug molecules conjugated to each antibody molecule, ADCs can be categorised by their drug-to-antibody ratio (DAR). DAR is a critical parameter that influences ADC properties, including its potency, stability, and pharmacokinetics. 134 Theoretically, ADCs with higher DARs are expected to exhibit enhanced potency due to an increased payload of cytotoxic drugs. However, several studies have shown that ADCs with excessively high DARs may display increased hepatic clearance, potentially limiting their efficacy in action. This is due to the bulky structure of ADCs with high DARs, which hinders their renal clearance, leading to their elimination via the liver instead. 134 , 135 The optimal DAR for different ADCs varies and depends on factors such as the specific antibody moiety, payload, and target tumour type. A careful evaluation of DAR and its impact on ADC properties is essential for optimising ADC design and maximising its therapeutic efficacy.

Another challenge in ADCs for cancer treatment is the resistance towards ADCs that may occur due to a variety of mechanisms. One such mechanism is attributed to decreased antigen expression, which can occur due to intratumoural heterogeneity, treatment-induced downregulation of the target antigen, or the loss of the target antigen. 135 Dimerisation of the target antigen with another cell surface receptor is another ADC-related drug resistance mechanism, which can block the binding and cytotoxic activity of ADCs. In response, several strategies have been proposed to overcome ADC resistance, including the use of bispecific ADCs, combination therapies that increase antigen expression, and ADCs with structural modifications that enhance their ability to penetrate tumours and exert their cytotoxic action. 135 However, further research is still needed to fully understand the underlying mechanisms of ADC resistance and, therefore, to develop effective strategies to overcome it.

Due to their molecular complexity, ADCs need to surpass a series of intracellular pathways before releasing their payload, thus increasing their susceptibility to an array of different resistance mechanisms in cells, some of which are still poorly characterised. ADCs undergo intricate intracellular trafficking involving endocytosis, endosomal sorting, lysosomal processing, and payload release. These intricate steps are highly dynamic and often cell-type specific. Although few research has been conducted, pre-clinical studies often fail to capture the complexities of intracellular processing, making it difficult to pinpoint the exact driver of such ADC resistance. Few hypotheses have been proposed and suggested on alterations in intracellular uptake and processing. Decreased penetration and abnormal endosomal transit have been suggested, as increased cellular barriers, and altered uptake pathways can impede ADCs from entering the cell. 136 , 137 A different mechanism of uptake from the common clathrin-mediated pathway was found, as they discovered that ADC was delivered in caveolin-1 (CAV1)-coated vesicles, which may lead to inefficient uptake. 138 Also, the changes in lysosomal pH were found to inhibit lysosomal processing and disrupt payload release. 135

Besides the challenges regarding each component of ADC discussed above, manufacturing ADCs on a large scale for commercialisation is a laborious and complicated task. The complexity of the process mainly roots from drug payload synthesis, which is derived from natural sources and possesses a complicated molecular structure, adding to the fact that the manufacturing process must be done in highly contained facilities due to high toxicity of the molecules. 139 Of course, the complicated process also corresponds to a higher cost of production. Another obstacle is the conjugation process, which traditionally is done by coupling the antibody moiety with the drugs via exposed surface amino acid residues after the reduction process, typically lysine and cysteine residues. 11 However, this method poses a disadvantage in that the drug may conjugate at several possible sites randomly, meaning that the conjugation is less specific and batch-to-batch variation of ADC may happen. 52 Moreover, random conjugation of the payloads will result in a varying degree of drug-antibody ratio, which lowers the consistency and homogeneity of the product. 11 Hence, it has been a major challenge for product chemists all around the world to develop strategies to overcome these challenges.

The systemic stability of ADCs post-administration is crucial in determining therapeutic efficacy. To address challenges related to metabolic stability, or integrity, various strategies are in development to enhance ADC stability and prolong systemic ADC half-life. Systemic clearance mechanisms, including renal filtration and reticuloendothelial system uptake, can reduce the active time of ADCs in the bloodstream. 140 An ideal ADC balances specificity, efficacy, and safety, which largely depend on the design, chemistry, and structure of the linker between the antibody and the cytotoxic drug. 141

The choice of linker is pivotal. An ideal linker should be stable in circulation and only release the drug upon reaching the tumour. Cleavable linkers, while designed to release the payload within the tumour microenvironment, may suffer from premature cleavage in circulation, leading to systemic toxicity. In contrast, non-cleavable linkers tend to exhibit higher plasma stability, as they release the cytotoxic payload within the lysosome following ADC internalisation and subsequent degradation of the antibody and linker. This feature reduces systemic toxicity and offers a broader therapeutic window. 141

The antibody component also plays a crucial role in ADC stability and specificity. Antibodies with low antigen specificity may cross-react with healthy cells, causing off-target toxicities and premature clearance. 142 Among immunoglobulin classes, IgG (particularly IgG1) is most used in ADC design due to its favourable half-life compared to antibody fragments. Antibody fragments, though beneficial for tumour penetration, often require modifications like PEGylation to enhance circulation time due to shorter half-lives. 141 The use of bispecific antibodies also holds potential to overcome challenges like tumour specificity, antibody absorption, and processing. 143 Optimised ADC designs that ensure prolonged circulation, targeted drug release, and minimal off-target effects can significantly improve therapeutic outcomes in cancer treatment. 141

Meanwhile, DAR is an essential parameter that influences ADC properties, including its potency, stability, and pharmacokinetics. While higher DARs generally correlate with increased potency due to a greater payload of cytotoxic drugs, few studies have shown that ADCs with excessively high DARs may display increased hepatic clearance, a lower tolerability, and a narrower therapeutic index than ADCs with a lower DAR, potentially reducing the therapeutic efficacy. 144 This is due to the bulky structure of ADCs with high DARs, which hinders renal clearance, leading to elimination via the liver. A higher DAR can also increase the hydrophobicity and the tendency of ADC interchain cysteine aggregation. 145 In addition, a high DAR can substantially influence systemic toxicity, as an increased payload distributed throughout the body may harm healthy tissues in addition to the targeted cancer cells. In essence, a higher DAR typically correlates with a greater risk of off-target effects and toxicity.

Modifications such as selecting optimal conjugation sites and incorporating steric hindrance around labile linker regions have also proven effective in enhancing ADC stability. Choosing conjugation sites with steric hindrance enables antibodies to provide the steric shielding effect, reducing premature release. 141 Approaches like Fc engineering and site-specific conjugation are promising for optimising ADC pharmacokinetics. 146 Fc engineering improves the half-life by extending circulation, while site-specific conjugation enhances stability by precisely controlling drug attachment points.

Lastly, selecting appropriate tumour-specific antigens is essential for ADC efficacy. Common targets include HER2, Nectin-4, and TROP2 in solid tumours. 141 ADCs targeting mutant antigens, such as specific EGFR mutations, offer greater specificity as these mutated proteins are less stable due to ubiquitinylation and degradation. Engineering ADCs with antibodies that target proteins containing oncogenic driver mutations is also likely to improve tumour specificity and efficacy, comparable to selective small molecules such as tyrosine kinase inhibitors. 141

Clinical success of ADCs and their impact on existing cancer therapies

ADCs have achieved remarkable clinical success in the pharmaceutical industry, particularly in hematologic malignancies and specific solid tumours due to their ability to selectively target and eliminate cancer cells with minimal systemic toxicity compared to conventional chemotherapies. With FDA-approved ADCs like trastuzumab, emtansine and brentuximab vedotin demonstrating prolonged survival in patients, ADCs are increasingly viewed as transformative agents in oncology.

ADCs have several advantages over traditional therapies. Their high specificity minimises off-target effects, sparing healthy cells and reducing common side effects like myelosuppression and systemic inflammation, which are prevalent in chemotherapy. Furthermore, ADCs' modular design offers flexibility in optimising each component (antibody, linker, and cytotoxic payload), allowing for highly targeted and customisable treatments. The use of potent cytotoxic agents such as auristatins and maytansinoids that could not be used in systemic chemotherapy due to toxicity concerns have been overcome through ADC technology. 141 This approach has brought new hope for treating resistant cancers and offers a promising route for managing diseases with unmet therapeutic needs.

Despite these advantages, challenges remain in achieving the full therapeutic potential of ADCs. Issues such as drug resistance, variability in antigen expression on tumour cells, and challenges in maintaining stability and efficacy in circulation have limited the success of certain ADC candidates. 147 , 148 While the development of more stable linkers and optimised antibodies have mitigated some of these issues, ensuring consistent efficacy across a broad range of patients and cancer types remains a significant hurdle.

The potential of ADCs to revolutionise cancer treatment lies in their ability to be combined with other therapies, such as immune checkpoint inhibitors, and to enhance immune-mediated tumour cell destruction. For example, combining ADCs with PD-1/PD-L1 inhibitors has shown synergistic effects in preclinical models, paving the way for combination therapies that leverage the benefits of targeted cell killing and immune activation. 149 , 150 Additionally, advances in bispecific or multi-specific ADCs, which can target two or more different antigens on tumour cells, are anticipated to further augment specificity and reduce the risk of resistance. 151

Given the continued advancements in ADC technology, the next generation of ADCs is likely to offer even greater precision, reduced off-target toxicity, and broader applicability across cancer types. In our opinion, ADCs are poised to become a cornerstone of targeted oncology therapies, potentially replacing or complementing traditional chemotherapies. As ADCs continue to evolve, their integration into multimodal cancer treatment regimens could substantially improve patient outcomes, providing a balance of efficacy and tolerability that few current therapies can achieve. Overall, while ADCs are not without their limitations, their unique mechanism of action and the ongoing technological progress in their design suggest they will play an increasingly significant role in modern oncology, likely reshaping the landscape of cancer treatment over the coming decades. This impact underscores the importance of continued research and clinical development in the ADC field to fully harness their therapeutic potential.

Conclusions

An innovative class of treatment against cancer termed ADC, which is the combination of an antibody moiety and drug payload, promises great potential to be the next generation of anti-cancer agents. It possesses a prominent advantage over conventional cancer drugs in terms of its significantly heightened specificity. More and more new bispecific and multivalent ADCs are being developed at a rapid pace and have expanded the range of ADC applications. Although some ADCs have been successfully marketed with many more in the clinical trial pipeline, this technology still has room for a lot of improvements to increase its efficacy. These improvements can be made in terms of innovative linker technologies, novel antibody moiety designs, as well as broadening the targeting capabilities of ADC to include the tumour microenvironment.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) used ChatGPT and Bard AI in order to enhance consistency and objectivity in language. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

Acknowledgements

This work was supported by the UCSI University under the Research Excellence and Innovation Grant (REIG) (grant numbers: REIG-FAS-2023/006, REIG-FAS-2024/001).

Author contributions

The study was conceived by J.Y.F., L.L.A.I, and M.Y.M.T. The manuscript draft was prepared by J.Y.F., Z.P., D.Y.C., C.M.H., and Y.H.L. M.Y.M.T., A.W.R.N., and L.L.A.I. supervised and reviewed the drafted manuscript, as well as approved the final draft. J.Y.F., D.Y.C., Z.P., C.M.H., Y.S.L., and K.C.O. made the revisions.

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