Cancer therapy remains a difficult task. Chemotherapy has a significant clinical benefit for many tumours, but it has low selectivity and high toxic effects, result in devastating effects and decreased therapeutic efficacy [1]. Antibody–drug conjugates (ADCs) are a promising cancer treatment that includes delivering toxic drugs to specific tumor cells that exhibit specific antigens connected to malignancy. The antibody, cytotoxic agent, and linker are the three primary structural units of an ADC. ADCs are expected to provide powerful therapeutic modalities against various cancers by combining the selectivity of monoclonal antibodies (mAbs) and the efficacy of various chemotherapeutics [2]. Together, the three components comprise a highly effective anti—tumour agent directly and selectively providing chemotherapy drugs to cancer cells, directed by antibodies with exceptional specificity and affinity.

Cleavage of the ADCs linker components by certain tumor-associated enzymes (i.e. matrix metalloproteinases) or by lower pH encountered in the tumour microenvironment results in the release of the active component [3]. These non-internalizing ADCs did not increase drug selectivity and, as a result, did not reduce toxicity considerably [4]. Despite the fact that ADCs have been studied for many years, we have only just recognized their true potential, thanks to significant advancements in linker and conjugation technology, as well as very powerful cytotoxic drugs [5]. ADCs are intended to broaden the therapeutic window of these medications by only delivering them to tumour cells that express a specific antigen targeted by the ADC’s mAb antigen [6, 7]. The properties of the antibody, therapeutic payload, and linker are critical in the overall efficacy of ADCs, which is dependent on intricate interactions between the ADCs and numerous tumour cell and tumour microenvironment (TME) targeting components [8].

Despite promising ADC-induced therapeutic activity against resistant and recurrent cancers, several barriers remain to their widespread use, including unidentified drug resistance mechanisms, toxicity, the lack of predictive prognostic biomarkers, and their clinical advantages over standard therapies. The development of new ADCs is a continual process that relies on advancements in several technologies such as biosynthesis of novel linkers, mAb synthesis and manufacturing, and the introduction of new payloads that are more powerful against tumour cells with fewer systemic side effects.

History, design, construction and mechanism of action of ADC

In the twenty-first century, the development of ADCs has reached significant milestones. Since the early 1900s, efforts have been made to improve the safety and efficacy of Paul Ehrlich’s “magic bullet”, which was the first therapeutic technique to convey lethal drugs to selected cancer cells depending on the presence of cell specific antigen(s) [9, 10]. Leukemia cells were targeted after the successful chemical linkage of polyclonal rodent immunoglobulins and methotrexate [11]. Hybridoma technology permitted the manufacturing of mAbs in 1950, and by the early 1970s, it had sparked important breakthroughs in the field of ADCs, both in vitro and in vivo [12].

The use of ADCs in animal models was described in the literature in the 1960s, and clinical trials with ADCs based on mouse immunoglobulin G (IgG) molecules were conducted in the 1980s [13].

The first ADC to be approved by the US Food and Drug Administration (FDA) for the treatment of patients with acute myeloid leukaemia was gemtuzumab ozogamicin (developed by Wyeth).

This was followed by the approval of two second generation ADCs: brentuximab vedotin (developed by Seattle Genetics) in 2011 [14, 15] and trastuzumab emtansine (also known as T-DM1 and ado-trastuzumab emtansine; developed by Roche) in 2013 [16], both of which target the cancer antigens CD30 (also known TNFRSF8) and human. Since 2013, the field has changed dramatically. More than 30 new ADCs have entered clinical development (all for oncological indications), and more than 60 ADCs are currently in clinical trials [17]. A list of the most recent ADCs currently approved by the US FDA is provided by Drago el al [18, 19].

Clinical trials for treating cancer patients with ADCs began in 1980, however the trials’ clinical usefulness was hampered by the development of medication toxicity without a significant clinical benefit [20,21,22]. Gemtuzumab ozogamicin, an FDA approved CD33-targeted medication for the treatment of relapsed and/or refractory acute myeloid leukemia (R/R) has been withdrawn from the market due to unfavorable adverse effects (AE) [23,24,25]. Brentuximab vedotin, a CD30-targeted ADC, and ado-trastuzumab emtansine (T-DM1), a HER2-targeted ADC, were approved in 2011 and 2013, respectively, for the treatment of R/R classical Hodgkin lymphoma and trastuzumab-resistant metastatic breast cancer [15, 26, 27]. Several ADCs are currently being studied in preclinical and clinical development, and the FDA has fully approved nine of them [28].

Components of ADC: mAb, linker, and payloads

ADCs are primarily composed of three major components: a drug, a linker, and an antibody. The efficacy of each ADC is largely determined by differences in the three fundamental components of ADCs. The development and purification of mAbs utilizing proper cell culture techniques are among the phases in the production of antibody–drug conjugates. Chemically generated and refined cytotoxic payloads. After being functionalized using a specific linker, the mAbs are finally attached to the cytotoxic drug payload (Fig. 1).

Fig. 1

The development, purification, and production of antibody-drug conjugates

Antibodies selection

In addition to cancer treatment, antibody-based therapies have made significant advances in the treatment of other diseases including autoimmune diseases, and cardiovascular and bone diseases [29]. One of the most crucial parts of ADC design is antibody selection,and high antigen specificity [30]. Antibodies with low specificity that cross-react with other antigens might have unpredictable effects, by interacting with healthy tissues, it might cause off-target toxicities or cause premature clearance from the body before it reaches the tumor site [30]. Immunoglobulin M (IgM), A, D, IgE, and IgG are the five types of antibodies. Among the five immunotherapy classes, IgG is the most commonly used [31]. Despite the enormous potential for innovation offered by antibody fragments and bispecific antibodies, immunoglobulin G (IgG) is currently the most common used in in ADCs [32,33,34]. The classical complement system is activated by IgG subclasses, particularly IgG1 and IgG3. The membrane attack complex (MAC) forms pores on the tumour cell surface leading to cancer cell lysis [35]. IgG1 antibodies have similar serum half-lives to their IgG2 and IgG4 counterparts, but higher complement-fixation and FcR-binding efficiency. Although IgG3 antibodies are the most immunogenic, they are often avoided in ADC design due to their short circulation half-lives [36]. The immunogenicity degree of an ADC is a critical aspect that influences circulatory half-life [7]. mAbs can penetrate tumor after being administered into the bloodstream [2]. The antibody’s size, which typically accounts for roughly 95% of an ADC's bulk, prevents ADCs from spreading into tumor tissue.

mAb target selection

Searching for cell-surface proteins expressed in tumours rather than non-malignant tissues has been one guiding methodology in selecting the right mAb target [37]. HER2, TROP2, and Nectin 4 are effective targets for ADCs now approved for the treatment of solid malignancies [38,39,40]. With the exception of a tiny fraction of lymphocytes, CD30 is a target of brentuximab vedotin and is expressed by malignant lymphoid cells in Hodgkin lymphoma and ALCL in the setting of haematological malignancies [41]. Similarly, inotuzumab ozogamicin, polatuzumab vedotin, and belantamab mafodotin are highly specific for hematological malignancies lineages [42, 43].

Different mAbs may have different Fc-dependent effector activities [44]. As a result, mAbs designed for other therapeutic uses may not be the optimal ADC backbones, in particular when considering mounting evidence that internalization and intracellular trafficking of ADC are critical to ADC cytotoxicity. Pertuzumab’s affinity for HER2 is pH-dependent, unlike trastuzumab, resulting in rapid dissociation of the Ab–Ag complex in a low-pH environment. As a result of this discovery, a preclinical recombinant pertuzumab-based ADC with increased cytotoxicity was developed [45]. Variant proteins are more prone to ubiquitylation, absorption, and/or instability than wild-type counterparts when targeted by ADCs [46, 47]. ADCs based on mAbs that target proteins with mutations of truncal oncogenic driver (for instance some mutant versions of EGFR) could attain tumour specificity levels hitherto only achieved with extremely selective inhibitors of small-molecule tyrosine kinase [48, 49]. Bispecific antibodies have opened up new research and development opportunities. Antibody absorption and/or processing, but also tumor selectivity, could all benefit from such compounds [50].

Linker design and technologies

The specificity, efficacy, and safety of an ADC are determined by the design, structure, and chemistry of the linker that connects the cytotoxic payload to the antibody. Linkers are typically designed to be constant in the blood system (enabling for a prolonged timeframe of bloodstream), but labile enough to efficiently deliver the cytotoxic payload to the tumour [51].

Cleavable and non-cleavable linkers are the two types of linkers. Following exposure to acidic or reducing environments or proteolytic enzymes, cleavable linkers are cut and release the ADC’s cytotoxic payload (for example, cathepsins). pH-sensitive hydrazone (found in brentuximab vedotin, enfortumab vedotin, polatuzumab vedotin, trastuzumab deruxtecan, and sacituzumab govitecan) is another enzyme-cleavable peptide-based linker (T-DXd) [51, 52].

Non-cleavable linkers are becoming more appealing than cleavable linkers due to the advantage of greater plasma stability. Furthermore, studies show that non-cleavable linkers perform far better in vivo, with payload release occurring primarily in the lysosome following ADC internalisation and destruction of both the antibody and the linker (Fig. 2). As a result, the danger of systemic toxicity from premature payload release is reduced. As a result, non-cleavable linkers may offer a wider therapeutic window, as well as increased stability and tolerability [53]. T-DM1 with mafodotin belantamab is one of two FDA-approved ADCs with non-cleavable linkers [18].

Fig. 2

ADC internalization and destruction of both the antibody and the linker

The systemic stability of ADCs post administration is one of the crucial issues to ensure the efficacy of ADCs, and several strategic approaches are taken for overcoming this issue. ADCs should ideally remain stable or intact in the circulation before entering target cells, but there are cases where ADC catabolites are still biologically active [54, 55]. ADC stability refers primarily to metabolic stability or integrity. To improve ADC stability, several approaches involving conjugation site selection and linker modification have been developed [54]. In general, modifications to each component (e.g., antibody, linker, and payload) can be performed for this purpose. The conjugation site, linker length, and linker steric hindrance are effective general approaches for site-specific ADCs and should be more broadly applicable to a variety of ADC platforms [54]. By choosing a more sterically hindered conjugation or attachment site, the antibody can provide the desired steric shield. On the other hand, introducing proximal steric hindrance around the cleavable or labile site of the linker has been shown to be an effective method of improving stability [56]. ADC biotransformation and drug-antibody-ratio (DAR) profiling have evolved into critical integrated data for assessing and comprehending ADC stability [54, 57].

Payloads

Monoclonal antibodies (mAbs) are well-known therapeutic agents used to treat a wide range of illnesses, including cancer [58]. Because of the limitations of mAbs’ anticancer activity, researchers are working to improve their potential efficacy. These efforts include mAb conjugation to radionuclides, fusion with immunotoxins, and coupling to ADCs. Payload [59] is the combination of a mAb with a cytotoxic agent or a small molecule. Methotrexate, doxorubicin, and vinca alkaloids are examples of traditional chemotherapy drugs with proven anticancer activity [21, 60, 61], were initially carried by ADCs. ADCs sometimes required high dosages to be effective, as result of increasing systemic toxicities [62]. Currently, optimizing ADCs is a never-ending problem, with most research and development activities focusing on the mAb or chemical linker, on small-scale endeavors, aimed to optimize the cytotoxic payload. There is a dearth of diversity in the medicinal payloads used in the 114 finished or continuing human trials, with only 7 payload formulations described (4 additional ongoing clinical studies with undetailed structures). Natural products account for six of the seven payload mixes, emphasizing the importance of natural materials as cytotoxic payloads for ADC in research investigations [63]. Furthermore, the findings demonstrate that a small part of the mAbs targeting the tumor (on the order of 0.1 percent) penetrates tumor tissue, emphasizing the significance of larger cytotoxicity payloads for treatment response [64, 65]. These discoveries contributed to the growth of ADCs, which include highly effective chemotherapeutic medications like as auristatins, calicheamicins, camptothecin, and maytansinoids analogs that can be lethal even at sub-nanomolar quantities [66, 67]. Nine cytotoxins were generated from plants, and 21 were natural product formulations from 79 anticancer and antiviral approved medications, according to FDA investigation from 1983 to 2002 [68]. Furthermore, 13 of the 39 anticancer compounds were based on natural chemicals. Sixty percent of contemporary pharmaceuticals are bioengineered from natural sources [68, 69].

For determining ADC efficacy, the drug–antibody ratio (DAR), or the amount of drug molecules attached to a single ADC, is critical. DAR varies a lot and is influenced by other ADC variables [70]. The DAR values are also affected by the conjugation site and whether light or heavy conjugated chains are used [70]. The DAR value affects the medicine’s effectiveness since low drug loading reduces potency, whereas high drug loading can affect toxicity and pharmacokinetics (PK) [71, 72]. In general, there are two types of payloads that are commonly utilized in ADC design, as listed below.

Rapid plasma clearance may limit the ability of small-molecule drug conjugates to reach tumor cells or poorly vascularized tumors or the central nervous system [73,74,75]. Other innovative ADCs include immunostimulatory agents such Toll-like receptor agonists, chemokines, or STING agonists to attract and/or activate immune effector cells to tumours [76, 77]. Several ADCs containing cytotoxic radioisotopes, notably the CD20-targeted drugs ibritumomab tiuxetan, 131I-tositumomab, and 131I-rituximab [78], have shown clinical activity against lymphomas. Prostate cancer (trying to target prostate-specific membrane antigen), glioblastoma (directly attacking EGFR), and gastrointestinal tumors (designed to attack carcinoembryonic antigen) are all being studied with similar treatments [79]. Antibodies can transport oligonucleotides, allowing for in vivo selective modification of signal transduction pathways [31].

Microtubule-disrupting agents

The synthetic antineoplastic agent auristatin is produced from dolastatin 10, a natural substance [80]. Because dolastatin 10 is a nonspecific toxic chemical, it is not used as a cytotoxic warhead in ADCs. In this class of drugs, synthetic analogues including MMAE and MMAF are currently being employed in ADCs as a cytotoxic payload [81]. MMAE is an antimitotic drug that works by preventing tubulin polymerization, which causes cell cycle arrest and apoptosis [82].

Maytansinoids are a second significant family of microtubule-disrupting drugs derived from the benzoansamacrolide maytansine. Tubulin polymerization is inhibited by these medications, resulting in mitotic arrest then cell death [83]. Maytansinoids perform the same action as Vinca alkaloids. The cytotoxicity of the maytansinoids, on the other hand, was over 100 times that of the Vinca alkaloids [84]. Maytansinoids have failed in human trials as anticancer treatment due to a lack of tumour selectivity and substantial systemic toxicity. Maytansinoids’ potent cytotoxicity can be used as a targeted delivery vehicle, notably in the form of antibody–maytansinoid conjugates (AMC).

DNA-damaging agents

Calicheamicins are a kind of enediyne antitumor antibiotic produced from the Micromonospora echinospora bacterium [85]. Calicheamicin binds to the minor groove of the TCCTAGGA DNA sequence and prevents it from replicating [86]. The payload in the ADC design is N-acetyl-calicheamicin, a calicheamicin derivative [87]. Gemtuzumab ozogamicin, sometimes known as Mylotarg, is the name of this ADC. It consists of a humanised IgG4 mAb conjugated to a calicheamicin payload that targets the CD33 surface antigen, which is present in 85–90% of individuals with acute myeloid leukaemia [88].

Duocarmycin is a natural chemical generated from bacterium strains of the Streptomyces genus [89]. Duocarmycin is another DNA minor groove–binding alkylating agent. By binding to the minor groove of DNA and causing persistent alkylation of DNA, this family of medicines affects nucleic acid architecture and thus structural integrity [90]. Yu and colleagues' work recently highlighted an example of duocarmycin application in ADC setting [91]. Promiximab-DUBA, a new ADC against CD56, was described in this work. In this ADC, an anti-CD56 hIgG1 antibody is linked to the payload duocarmycin via a reduced interchain disulfide linker. In vitro and in vivo, this novel ADC showed significant cytotoxic effect against cancer cells.

Doxorubicin works by intercalating DNA, which prevents DNA synthesis [