لخّصلي

Antibody-drug conjugates (ADCs) are a promising cancer treatment modality that enables the selective delivery of highly cytotoxic payloads to tumours.Upon reaching the tumour microenvironment (TME), either the masking moieties are removed or the antigen-binding sites change conformation in response to certain tumour-associated factors such as the abundance of proteases and acidic conditions, resulting in the localized restoration of the original target binding affinity of the antibody and payload release.Notable examples of homogeneous conjugation technologies include full alkylation of interchain disulfides (used in T-DXd and sacituzumab govitecan), THIOMAB80 (a conjugation method that involves genetically incorporated cysteine residues), incorporation of non-naturally occur- ring reactive amino acids81-83, cysteine rebridging84-89, Fc-affinity tags90 and site-specific conjugation using various enzymes (such as engineered glycosidases91-94, transglutaminases95-98, formylglycine-generating enzymes99,100 and sortases101-103) (Fig.By contrast, homogeneous ADCs with defined DARs are generated by full alkylation of interchain disulfides (as used in the manufacture of trastuzumab deruxtecan and sacituzumab govitecan) or site-specific conjugation through cysteine engineering techniques (such as THIOMAB), incorporation of reactive unnatural amino acids and following orthogonal coupling or enzymatic reactions.Subsequently, we highlight and discuss selected examples of novel ADC designs that are currently in the early stages of preclini- cal and clinical development as next-generation cancer therapeu- tics, including bispecific ADCs, probody-drug conjugates (PDCs), immune-stimulating antibody conjugates (ISACs), protein degrader- antibody conjugates (DACs) and dual-payload ADCs.To further enhance the tumour specificity of an ADC, antibodies capable of recog- nizing tumour-specific antigen variants with structural variations such as truncation, nicking (peptide bond cleavage caused by tumour-associated proteases) and other unique post-translational modifications have been explored (for example, EGFR variant III24,25, nicked TROP2 (refs.Resistance to ADCs is intricately linked to the tumour heterogeneity; aggressive ADC therapy can create selective pressures that favour small subpopulations of resistant clones that harbour specific traits, including alterations in drug metabolism, mutations in the target proteins or their downstream signalling pathways, activation of alternative signalling pathways and/or the presence of cancer stem-like cells8,9.Affinity-attenuated bispecific ADCs equipped with an MMAE payload showed a five- to sixfold greater therapeutic index than a cetuximab-MMAE ADC, based on differ- ences in in vitro cytotoxic potency against EGFR and MET-expressing tumour cells and nonmalignant keratinocytes.Certain ADCs involve antibodies of the IgG4 subclass (such as gemtuzumab ozogamicin and inotuzumab ozogamicin)11; nonetheless, IgG1 anti- bodies are now preferentially used because of their general stability in the systemic circulation (reflecting an elimination half-life of 14-21 days) and robust engagement of innate immune cells, such as natural killer (NK) cells and macrophages, through interactions with Fc?The pay- loads of current FDA-approved ADCs include anti-mitotic agents such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and the maytansine derivatives DM1 and DM4, DNA-damaging agents such as calicheamicins and pyrrolobenzodiazepine dimers (PBDs) and topoisomerase I inhibitors such as SN-38 and DXd.An interim analysis of the phase III MARIPOSA study (NCT04487080) supports the poten- tial of this approach; the combination of the EGFR-MET bispecific antibody amivantamab with the third-generation small-molecule EGFR inhibitor lazertinib resulted in a median progression-free survival (PFS) of 23.7 months, compared with 16.6 months with osimertinib, another third-generation inhibitor, as monotherapy128. In this Review, we highlight advances in each ADC component (the monoclonal antibody, payload, linker and conjugation chemistry) and provide more-detailed discussions on selected examples of emerging novel ADCs of each format, enabled by engineering of one or more of these components.Several emerging ADC formats exist, including bispecific ADCs, conditionally active ADCs (also known as probody-drug conjugates), immune-stimulating ADCs, protein-degrader ADCs and dual-drug ADCs, and each offers unique capabilities for tackling these various challenges.Multiple factors might contribute to the lack of improvement in antitumour activity, although the authors speculated that monovalent binding did not effectively induce HER2 dimerization, a key process for HER2 endocytosis, thus offsetting any potential benefits from accelerated lysosomal trafficking.Aglycosylated antibodies can be vulnerable to structural distortion owing to thermal instability; nonetheless, data published in 2022 indicate that attaching small-molecule payloads to the CH2 domain of the Fc region can compensate for this instability15.ADCs with other payloads including tubulysins (anti-mitotics)35-39, duocarmycins (DNA alkylators)40, PNU-159682 (a topoisomerase II inhibitor)41-43 and amani- tin (an RNA polymerase II inhibitor)44-46 are currently being evaluated in preclinical and clinical studies.Research efforts from the past decade have focused on developing more-stable cleavable linkers, such as the GGFG tetrapeptide linker employed in T-DXd70, cathepsin-responsive trip- eptide linkers71-73, as well as linkers cleaved by ?-glucuronidase37,74,75, sulfatase76, phosphatase77 and legumain78,79.Second, if appropriate targets are chosen, bispe- cific ADCs might be capable of more tumour-specific binding owing to limited expression of both target antigens by nonmalignant cells

and/or promoted payload uptake, thereby minimizing the risk of toxici- ties in nonmalignant tissues.Data from a preclinical study demonstrate superior internalization, lysosomal trafficking and improved thera- peutic activity in PDX models of NSCLC and oesophageal squamous cell carcinoma (ESCC) compared with monospecific bivalent ADCs.receptors (Fc?Rs)12.18), XMT-1522 (refs. 6). 10).29)).1 |2).115,118). 123). 133).141).

Antibody–drug conjugates (ADCs) are a promising cancer treatment modality that enables the selective delivery of highly cytotoxic payloads to tumours. However, realizing the full potential of this platform necessitates innovative molecular designs to tackle several clinical challenges such as drug resistance, tumour heterogeneity and treatment-related adverse effects. Several emerging ADC formats exist, including bispecific ADCs, conditionally active ADCs (also
known as probody–drug conjugates), immune-stimulating ADCs, protein-degrader ADCs and dual-drug ADCs, and each offers unique capabilities for tackling these various challenges. For example, probody–drug conjugates can enhance tumour specificity, whereas bispecific ADCs and dual-drug ADCs can address resistance and heterogeneity with enhanced activity. The incorporation of immune-stimulating and protein-degrader ADCs, which have distinct mechanisms of action, into existing treatment strategies could enable multimodal cancer treatment. Despite the promising outlook, the importance of patient stratification and biomarker identification cannot be overstated for these emerging ADCs, as these factors are crucial to identify patients who are most likely to derive benefit. As we continue
to deepen our understanding of tumour biology and refine ADC design, we will edge closer to developing truly effective and safe ADCs for patients with treatment-refractory cancers. In this Review, we highlight advances in each ADC component (the monoclonal antibody, payload, linker
and conjugation chemistry) and provide more-detailed discussions on selected examples of emerging novel ADCs of each format, enabled by engineering of one or more of these components.

Key points
• Antibody–drug conjugates (ADCs) are an effective cancer therapy, although responses to these agents are often limited by acquired resistance and treatment-related adverse effects.
• Advances in the various ADC components (namely the antibody, linker, payload and conjugation chemistry) will be key to improving both the efficacy and safety of these agents.
• To address these challenges, several novel ADC formats have been developed, including bispecific ADCs, probody–drug conjugates, immune-stimulating ADCs, protein-degrader ADCs and dual-drug ADCs.
• Probody–drug conjugates are expected to have improved tumour specificity, whereas bispecific ADCs and dual-drug ADCs have the potential to combat drug resistance and tumour heterogeneity.
• Integrating immune-stimulating ADCs and protein-degrader ADCs with current treatment regimens might enable multimodal treatment, potentially through several distinct mechanisms of action.
• Patient stratification and biomarker identification will be crucial to maximize the clinical benefits of these emerging ADCs.
Introduction
Antibody–drug conjugates (ADCs) have emerged as a promising class of cancer therapeutics. An ADC consists of a monoclonal antibody and a potent cytotoxic payload connected through a chemical linker. This molecular design combines the target specificity and long circulation half-life of an antibody with the high cytotoxic potency of antitumour agents that are too toxic for standalone use. Consequently, compared with conventional chemotherapies, ADCs can have enhanced antitu- mour efficacy, leading to improved clinical benefit and quality of life outcomes1–4. The considerable success of this emerging drug modality in patients with various types of cancer is demonstrated by the availabil- ity of 11 FDA-approved ADCs for at least 20 specific indications (Table 1). The level of interest in this modality has increased exponentially over the past few years, as demonstrated by the approval of four new ADCs within the past 3 years with more than 100 different ADCs currently being tested in clinical trials5. An economic analysis has revealed the rapidly growing size of the global market for ADCs: US $7.82 billion in 2022 with a projected compound annual growth rate of 11.2% from 2023 to 2030 (ref. 6).
Despite major progress in ADC development, the clinical potential of these drugs in patients with treatment-refractory cancers is often limited by various factors. Intratumour and intertumour heterogeneity is a major obstacle leading to poor therapeutic outcomes7,8. Tumour heterogeneity refers to the variability in genetic and phenotypic char- acteristics within a tumour (intratumour heterogeneity) or between tumours present within the same or different patients (intertumour heterogeneity), and both can cause variations in treatment response and lead to the emergence of ADC-resistant clones. Resistance to ADCs is intricately linked to the tumour heterogeneity; aggressive ADC therapy can create selective pressures that favour small subpopulations of resistant clones that harbour specific traits, including alterations in
drug metabolism, mutations in the target proteins or their downstream signalling pathways, activation of alternative signalling pathways and/or the presence of cancer stem-like cells8,9. In addition to having activity across multiple tumour cell subpopulations, which minimizes the development of resistant clones, tolerability is a crucial parameter for the successful development of an ADC3. Indeed, many ADCs have been withdrawn either from clinical studies or from the market after initial approval owing to unacceptable toxicities and/or an overly narrow therapeutic window. An example includes the decision to withdraw the FDA-approved ADC gemtuzumab ozogamicin for CD33+ acute myeloid lymphoma (AML) from the market in 2010, and the subsequent re-approval of this agent at a lower dose in 2017 (ref. 10). Even successful ADCs that have been shown to provide clear clinical benefits to most patients can come with certain toxicity risks, as exemplified by inter- stitial lung disease and pneumonitis, which can be clinically serious and occasionally fatal, in patients receiving trastuzumab deruxtecan (T-DXd)3. Ingenious payload and linker designs, along with the identi- fication of tumour-specific target antigens and effective biomarkers, will be crucial for the development of next-generation ADCs capable of overcoming these clinical challenges.
In this Review, we first provide a brief overview of the basic molecu- lar design of an ADC and how each component (the antibody, linker, payload and conjugation chemistry) can affect the physicochemical and biophysical properties of the final product, including intracellular payload trafficking and metabolism, antitumour activity and safety profiles. Subsequently, we highlight and discuss selected examples of novel ADC designs that are currently in the early stages of preclini- cal and clinical development as next-generation cancer therapeu- tics, including bispecific ADCs, probody–drug conjugates (PDCs), immune-stimulating antibody conjugates (ISACs), protein degrader– antibody conjugates (DACs) and dual-payload ADCs. We aim to inform readers about the key design features necessary to generate effective and safe ADCs, as well as to provide an update on the extensive ongo- ing efforts to develop these agents and thus provide more and better treatment options for patients with cancer.
ADC design
The principles of ADC design have evolved through the optimization of each structural component: namely the antibody, the cytotoxic pay- load and the chemical linker that connects these components (Fig. 1a). Extensive research efforts have provided insights into the implications of target selection and conjugation chemistries for the physicochemical properties as well as efficacy and safety of the ADC.
Antibody and target selection
Among the many types of antibody available, humanized and fully human IgGs are most commonly used as the ADC backbone. Certain ADCs involve antibodies of the IgG4 subclass (such as gemtuzumab ozogamicin and inotuzumab ozogamicin)11; nonetheless, IgG1 anti- bodies are now preferentially used because of their general stability in the systemic circulation (reflecting an elimination half-life of 14–21 days) and robust engagement of innate immune cells, such as natural killer (NK) cells and macrophages, through interactions with Fcγ receptors (FcγRs)12. The use of human IgG1 also helps to reduce the overall immunogenicity of the ADC, which minimizes the risk of hyper- sensitivity reactions and the formation of anti-drug antibodies (ADAs)13. Most current ADCs maintain an N-linked glycan in N297 of the constant heavy chains to enable FcγR binding. However, interactions between such glycans and mannose receptors could drive nonspecific uptake

of the ADC by hepatocytes14. Thus, using aglycosylated monoclonal antibodies might, in future, be a reasonable approach, depending on the specific pharmacokinetic or pharmacodynamic parameters of each ADC, such as payload potency, durability and biodistribution. Aglycosylation could be especially advantageous when the priority is to minimize the incidence of liver toxicities and inflammatory responses over enhancing potency. Aglycosylated antibodies can be vulnerable to structural distortion owing to thermal instability; nonetheless, data published in 2022 indicate that attaching small-molecule payloads to the CH2 domain of the Fc region can compensate for this instability15.
Antibodies that recognize antigens that are specifically expressed on cancer cell surfaces and are entirely absent from nonmalignant tissues are the ideal backbones for ADC construction, enabling the tumour-specific delivery of potent payloads. However, most ADC targets, including
several that have already been successfully targeted (such as HER2 and TROP2), are also expressed by nonmalignant tissues to some extent16,17. Thus, even if ADCs are designed to target such validated molecules, both target-dependent and target-independent toxicities can still occur and might result in clinical holds or even the discontinuation of early phase testing (such as with MEDI4276 (ref. 18), XMT-1522 (refs. 19,20) and XMT- 2056 (refs. 21,22) for patients with HER2+ breast cancer and PF-06664178 (ref. 23) for patients with TROP2-expressing solid tumours). To further enhance the tumour specificity of an ADC, antibodies capable of recog- nizing tumour-specific antigen variants with structural variations such as truncation, nicking (peptide bond cleavage caused by tumour-associated proteases) and other unique post-translational modifications have been explored (for example, EGFR variant III24,25, nicked TROP2 (refs. 26–28) and glycosylated PD-L1 (ref. 29)).

Fig. 1 | Components, molecular properties and novel designs of antibody– drug conjugates. a, Schematic representation of an antibody–drug conjugate (ADC); each component (the antibody, payload, linker and conjugation chemistries) can all have important implications for the properties of the
ADC. b, Chemical structures of non-cleavable and cleavable linkers. Although non-cleavable linkers remain attached to the payload structure upon intracellular release, cleavable linkers facilitate efficient release of the attached payloads
in response to acidic pH, a reducing environment or degradation mediated by enzymes present within tumour cells or the tumour microenvironment (TME). c, Heterogeneous and homogeneous ADCs. Stochastic conjugation of payloads

via lysine coupling or partial cysteine alkylation results in heterogeneous ADCs with variable drug to antibody ratios (DARs). By contrast, homogeneous
ADCs with defined DARs are generated by full alkylation of interchain disulfides (as used in the manufacture of trastuzumab deruxtecan and sacituzumab govitecan) or site-specific conjugation through cysteine engineering techniques (such as THIOMAB), incorporation of reactive unnatural amino acids and following orthogonal coupling or enzymatic reactions. DAC, protein degrader– antibody conjugate; FcγR; Fcγ receptor; ISAC, immune-stimulating antibody conjugate; NK, natural killer; PDC, probody–drug conjugate; w/o, without.

In addition to the antigen expression profile, ADC internalization and turnover rates can have important implications for efficacy30. Opti- mization of the binding affinity is also a crucial step towards maximiz- ing ADC efficacy. Paradoxically, excessively strong antigen binding can lead to the retention of ADC molecules on the surfaces of tumour cells, thereby limiting the extent of tissue penetration (a phenomenon known as the binding-site barrier effect31,32). Thus, the antibody backbone of an ADC must be carefully selected, taking into account these various parameters to ensure optimal performance.

Payloads
ThepayloadsusedforADCsaretypicallymuchmoretoxicthanconven- tional chemotherapies, with sub-nanomolar or even picomolar levels of in vitro cytotoxicity observed as opposed to the micromolar levels of such activity seen with several common chemotherapies33,34. The pay- loads of current FDA-approved ADCs include anti-mitotic agents such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and the maytansine derivatives DM1 and DM4, DNA-damaging agents such as calicheamicins and pyrrolobenzodiazepine dimers (PBDs) and topoisomerase I inhibitors such as SN-38 and DXd. ADCs with other payloads including tubulysins (anti-mitotics)35–39, duocarmycins (DNA alkylators)40, PNU-159682 (a topoisomerase II inhibitor)41–43 and amani- tin (an RNA polymerase II inhibitor)44–46 are currently being evaluated in preclinical and clinical studies. Beyond these cytotoxic payloads, immunomodulators47 and protein-degrader-recruiting molecules48 have emerged as promising novel payloads.
Most payload molecules have moderate to high levels of hydropho- bicity, which is crucial in determining both the efficacy and the toxicity of the ADC. With several exceptions, such as MMAF, DM1 and amanitin, hydrophobic payloads can diffuse from target-expressing tumour cells into adjacent cells that might have limited or even no target expression, a phenomenon known as the bystander effect49,50. This effect is crucial for the successful eradication of heterogeneous tumours, in which both antigen-expressing and antigen-negative cells coexist. Despite this notable benefit, a hydrophobic payload can also have negative implications for ADC effectiveness. First, hydrophobic payloads can serve as good substrates for multidrug resistance proteins (such as MDR1, MRP1 and BCRP)51, thus diminishing the potency of certain ADCs against tumours that express these transporters. Second, hydrophobic ADCs tend to form aggregates, which can be rapidly cleared from the body52,53 and might be immunogenic54. Last, excessive hydrophobicity has been shown to facilitate liver uptake and cause hepatotoxicity55–57. ADC hydrophobicity is also a factor promoting nonspecific uptake through macropinocytosis, which might lead to ocular toxicities58 and thrombocytopenia59,60. As such, fine-tuning of the payload and ADC hydrophobicity are of paramount importance to overcome such issues while ensuring that the potential for bystander killing is retained. One

approach to address this issue is to lower the number of conjugated payloads (the drug to antibody ratio (DAR)). However, lowering the DAR entails a reduction in antitumour activity, highlighting the importance of fine-tuning the DAR for each payload class to balance efficacy and toxicity. Installing hydrophilic masking groups such as long polyethyl- ene glycol (PEG)53,61,62 or polysarcosine63–66 is another approach to this end, which enables the construction of high-DAR ADCs while avoiding some of the unwanted effects of high hydrophobicity.
Linkers
Chemical linkers have a pivotal role in enabling the cytotoxic pay- loadstoremainattachedtotheantibodyuntilthetargetisreached67,68 (Fig. 1b). Two primary linker types exist: non-cleavable and cleavable. Non-cleavable linkers are composed of stable bonds that resist pro- teolytic degradation, affording excellent stability in the systemic circulation. However, the release of the cytotoxic payloads bound by such non-cleavable linkers necessitates complete endocytosis and digestion of the antibody. This process is facilitated by cytosolic and lysosomal proteases, and results in the liberation of payload molecules that remain linked to a remnant amino acid residue from the degraded antibody (typically a cysteine or lysine). By contrast, cleavable link- ers, which are preferentially used in current ADCs, are designed to be degraded by tumour-associated factors (such as the acidic and/or reducing conditions associated with most tumours or intracellular proteases). These linkers enable the efficient release of active payloads upon internalization into cancer cells, which results in cytotoxicity, thereby maximizing ADC potency and facilitating the bystander effect. Disulfide linkage and cathepsin-sensitive valine–citrulline dipeptides are commonly used for this purpose. However, cleavable linkers come with the risk of premature release of the payload into the circulation, which results in systemic toxicities and less-efficient payload delivery69. Therefore, careful linker design that strikes a balance between stabil- ity and efficacy is crucial. Research efforts from the past decade have focused on developing more-stable cleavable linkers, such as the GGFG tetrapeptide linker employed in T-DXd70, cathepsin-responsive trip- eptide linkers71–73, as well as linkers cleaved by β-glucuronidase37,74,75, sulfatase76, phosphatase77 and legumain78,79.
Conjugation and homogeneity
In addition to the structural components discussed above, achieving high levels of homogeneity in bioconjugation is crucial to maximize the therapeutic window of an ADC. Most ADCs have traditionally been constructed using cysteine–maleimide alkylation or, less commonly, lysine–amide coupling (Fig. 1c). With some exceptions (such as T-DXd and sacituzumab govitecan), these stochastic conjugation methods result in a heterogeneous mixture of ADCs with variations in the payload attachment site and DAR. ADC heterogeneity often leads to less-efficient

payload delivery owing to the rapid clearance of hydrophobic high-DAR components, necessitating strict production control to minimize such variations. To overcome these limitations, considerable efforts have been devoted to the development of site-specific conjugation methods for the production of homogeneous batches of ADCs with defined DARs. Notable examples of homogeneous conjugation technologies include full alkylation of interchain disulfides (used in T-DXd and sacituzumab govitecan), THIOMAB80 (a conjugation method that involves genetically incorporated cysteine residues), incorporation of non-naturally occur- ring reactive amino acids81–83, cysteine rebridging84–89, Fc-affinity tags90 and site-specific conjugation using various enzymes (such as engineered glycosidases91–94, transglutaminases95–98, formylglycine-generating enzymes99,100 and sortases101–103) (Fig. 1c).
Bispecific ADCs
As described previously, tumour heterogeneity and drug resistance often limit the antitumour activity of therapies directed towards a single target7,8. To address this challenge, bispecific antibodies have emerged as a method that enables simultaneous binding to two distinct target molecules and/or cells104,105. This approach has featured most prominently in bispecific T cell engagers, which elicit robust antitumour immune responses by tethering target-expressing tumour cells to T cells106,107. Bispecific ADCs that leverage this technology have received some attention as a potential avenue towards enhanced antitumour effi- cacy (Fig. 2). Of the various bispecific antibody formats developed1

human IgG1-based scaffolds are the most commonly used in the bispe- cific ADCs currently in development. The designs explored to date can be categorized into two types: bispecific ADCs that target different epitopes of the same antigen, which are also known as biparatopic ADCs; and bispecific ADCs that target two different antigens.
Biparatopic ADCs
Data from previous studies indicate that the use of two anti-HER2 anti- bodies that bind to distinct epitopes can induce the formation of large receptor–antibody clusters on the cell surface, leading to endocytosis, lysosomaltraffickinganddownregulationofthistargetreceptor109,110. Based on this observation, a multivalent biparatopic ADC that tar- gets two different epitopes within HER2 hypothetically could have improved binding affinity, potentially resulting in more-efficient pay- load delivery, in particular to HER2low cancer cell populations111. To test this hypothesis, investigators generated a tetravalent HER2-targeting ADC named MEDI4276 by fusing a single-chain variable fragment (scFv) from trastuzumab with the N terminus of 39S, another anti-HER2 IgG1 antibody (Fig. 2a). A tubulysin derivative (AZ13599185), which is an anti-mitotic agent with low-picomolar levels of potency capable of bystander effects112, was then conjugated to this construct at a DAR of 4 using a stable linker via a site-specific conjugation method. As antici- pated, this homogeneous biparatopic ADC had faster internalization kinetics and lysosomal trafficking via HER2 receptor clustering than was seen with either trastuzumab or the parent 39S antibody111. A later study

revealed that the parent bispecific antibody targets HER2 extracellular domain 2 (ECD2) via the 39S Fab moiety and HER2 ECD4 by the trastu- zumab scFv moiety113. Notably, MEDI4276 showed remarkable activity in mouse xenograft models of treatment-refractory HER2+ breast cancer, such as those featuring JIMT-1 cells, or the T-DM1-resistant NCI-N87 cell line, as well as in a panel of HER2low patient-derived xenograft (PDX) models characterized by intratumour heterogeneity111. Despite high levels of activity in preclinical models, MEDI4276 did not demonstrate a good efficacy–safety balance when tested clinically18. In patients with breast cancer, the overall response rate (ORR) was low (9.4%, 3 of 32 patients)18, which does not compare favourably with T-DXd (ORR 37% in patients with HER2low advanced-stage breast cancer in a sepa- rate study)114. The maximum tolerated dose (MTD) of MEDI4276 was determined to be 0.75 mg/kg every 3 weeks, although 7 of 12 patients who received this dose had one or more clinically serious and/or grade ≥3 adverse events, necessitating dose reduction18.
Another biparatopic HER2-targeting ADC, zanidatamab zovodotin (also known as ZW49), has been developed over the past few years115,116. ZW49 consists of a heterodimerized Fc region fused with an scFv that targets HER2 ECD4 and an Fab that targets ECD2 of the same protein, conjugated to an auristatin payload with an average DAR of 2 (ref. 117) (Fig. 2a). This asymmetrical structure enables bivalent HER2 binding, in contrast to the tetravalent binding accomplished by MEDI4276. Despite these differences in binding mode, ZW49 also induces recep- tor clustering and rapid internalization of HER2 (refs. 115,118). In a phase I dose-finding study testing ZW49, a recommended phase II dose (RP2D) of 2.5 mg/kg every 3 weeks was established119, which is comparable to the RP2Ds of other auristatin-based ADCs120–122. Among the 29 response-evaluable patients who received ZW49 under this dos- ing regimen, the confirmed ORR across multiple HER2+ advanced-stage cancer types was 28%, and the disease control rate was 72%. Only 9% of patients had grade ≥3 treatment-related adverse events (TRAEs), with clinically serious events in a further three patients. These results suggest that ZW49 has a manageable safety profile and promising antitumour activity in heavily pretreated patients. Both MEDI4276 and ZW49 are designed to recognize HER2 and promote receptor clustering, internalization and lysosomal trafficking. However, these biparatopic ADCs differ in terms of payload potency (tubulysin is more potent than auristatin in vitro), binding mode (tetravalent versus biva- lent) and dissociation constant (137 pM111 versus 830 pM117). Although further investigations will be needed, these parameters might all be crucial in determining the therapeutic index of this novel class of ADCs.
REGN5093-M114, another biparatopic ADC, is designed to target two distinct epitopes of MET and is equipped with a maytansine deriva- tive payload named M24 (ref. 123). REGN5093-M114 can be rapidly traf- ficked to recycling endosomes. However, in contrast to the biparatopic anti-HER2 ADCs described previously, the accumulation of this agent in late endosomes or lysosomes was not accelerated124,125. After multiple preclinical assessments, REGN5093-M114 is currently being tested in a phase I/II trial involving patients with advanced-stage non-small-cell lung cancer (NSCLC)126.
Bispecific ADCs targeting two different antigens
Simultaneous targeting of two different antigens using a bispecific ADC could offer multiple advantages. First, bispecific ADCs can recognize and kill a broader spectrum of tumour cells, including those from het- erogeneous tumours. Second, if appropriate targets are chosen, bispe- cific ADCs might be capable of more tumour-specific binding owing to limited expression of both target antigens by nonmalignant cells

and/or promoted payload uptake, thereby minimizing the risk of toxici- ties in nonmalignant tissues. Furthermore, engaging multiple antigens and/or cells simultaneously could elicit a synergistic effect that might not be feasible by targeting either antigen individually. Although these potential advantages have not yet been validated clinically, the use of bispecific ADCs could provide opportunities to improve efficacy and bypass the mechanisms of resistance that can arise during treatment with therapies directed towards a single target.
EGFR has been a focal point for the development of targeted thera- pies owing to the overexpression of this cell-surface receptor by various solid tumours. One promising approach that prolongs the clinical activ- ity of EGFR-targeted therapy involves simultaneous inhibition of com- mon resistance pathways, such as MET signalling127. An interim analysis of the phase III MARIPOSA study (NCT04487080) supports the poten- tial of this approach; the combination of the EGFR–MET bispecific antibody amivantamab with the third-generation small-molecule EGFR inhibitor lazertinib resulted in a median progression-free survival (PFS) of 23.7 months, compared with 16.6 months with osimertinib, another third-generation inhibitor, as monotherapy128. Leveraging these find- ings, researchers have created bispecific ADCs that target EGFR and one other molecule that is preferentially co-expressed with EGFR on the tumour-cell surface129–133 (Fig. 2b). One group of investigators designed and evaluated a panel of EGFR–MET-targeted bispecific ADCs with varying binding affinities for EGFR129. Affinity-attenuated bispecific ADCs equipped with an MMAE payload showed a five- to sixfold greater therapeutic index than a cetuximab–MMAE ADC, based on differ- ences in in vitro cytotoxic potency against EGFR and MET-expressing tumour cells and nonmalignant keratinocytes. Another line of research following the same concept led to the development of AZD9592, an EGFR–MET-targeted bispecific ADC equipped with a topoisomerase I inhibitor payload130,131. This agent demonstrated promising activity as monotherapy or in combination with osimertinib in PDX models of both EGFR-mutant and wild-type NSCLC, as well as head and neck squamous cell carcinoma (HNSCC). Importantly, AZD9592 was well tolerated in monkeys130. These promising preclinical results prompted the initiation of a phase I trial (NCT05647122).
Tumour-associated antigens other than MET have also been explored as combination targets for EGFR-targeting bispecific ADCs. For example, M1231 is a MUC1–EGFR-targeted bispecific ADC con- structed from a heterodimeric antibody with an anti-MUC1 scFv and an anti-EGFR Fab domain132. The payload comprises SC209, a hemiasterlin derivative with anti-microtubule activity, conjugated through a cleav- able valine–citrulline linker. Data from a preclinical study demonstrate superior internalization, lysosomal trafficking and improved thera- peutic activity in PDX models of NSCLC and oesophageal squamous cell carcinoma (ESCC) compared with monospecific bivalent ADCs. A phase I dose-escalation study to evaluate the safety, pharmacokinet- ics and preliminary efficacy of M1231 (NCT04695847) was reportedly completed in June 2023, although detailed results do not appear to have been publicly disclosed thus far. Another example is BL-B01D1, a tetravalent bispecific ADC targeting EGFR and HER3 (ref. 133). This con- struct features a camptothecin derivative payload named ED04, which is attached by full cysteine conjugation and has a DAR of 8. Preclinical assessments confirmed the antitumour activity of this compound in mouse xenograft models of pancreatic or colorectal cancers com- pared with the corresponding ADCs targeting EGFR or HER3 only133. A phase I clinical trial has been initiated to test this agent in patients with unresectable locally advanced or metastatic solid tumours, including gastrointestinal and breast cancers

The bispecific ADC format has also been used to modulate intracel- lular processing and payload release kinetics. Internalized HER2 predomi- nantly recycles back to the cell surface and is not efficiently trafficked to lysosomes134,135, thus hampering effective payload release following lyso- somal degradation. Conversely, the prolactin receptor (PRLR) and amy- loid precursor-like protein 2 (APLP2) have been shown to undergo rapid lysosomal trafficking upon endocytosis136–139. To capitalize on this feature, researchers have developed monovalent bispecific ADCs that target HER2 in combination with either PRLR140 or APLP2 (ref. 141). Both conjugates showed improved trafficking of ADC–HER2 complexes to lysosomes and comparable or slightly enhanced levels of in vitro potency relative to those of ADCs that target HER2 only. However, the HER2–APLP2-targeted ADC did not have improved activity in mouse xenograft models of breast cancer compared with its monospecific variant141. Multiple factors might contribute to the lack of improvement in antitumour activity, although the authors speculated that monovalent binding did not effectively induce HER2 dimerization, a key process for HER2 endocytosis, thus offsetting any potential benefits from accelerated lysosomal trafficking. This observation suggests that the efficacy of this construct might be improved by transforming the monovalent structure into a bivalent one.
In summary, bispecific ADCs offer promising therapeutic oppor- tunities with the ability to target a wider spectrum of antigens, with improved activity, and possibly improved safety. However, this format also presents potential pitfalls that require careful consideration. One such example is the risk of unintended receptor activation and ago- nistic activity, as observed with certain anti-MET antibodies142–144 and EGFR–MET-directed bispecific antibodies145. Additionally, the expres- sion ratios of the two target antigens can differ between tumours and patients, thus complicating the selection of potential responders. Care- ful evaluation of epitopes, binding modes and the underlying biology will therefore be crucial to achieve favourable treatment outcomes.
Probody–drug conjugates
Traditional ADCs that target receptors expressed not only on tumour cells but also on certain nonmalignant tissues (such as EGFR and TROP2) are often associated with unavoidable on-target off-tumour toxici- ties, leading to dose reductions or treatment discontinuation23,146–148. To address this issue, novel ADC designs that feature conditionally active antibodies (often referred to as probodies) have been developed (Fig. 3a,b). This design concept is inspired by prodrug formulations of small molecules, whereby pharmacologically inactive forms of the drugs are administered and then metabolized to their active forms in the circulation or in certain organs, leading to improved in vivo stabil- ity and/or specificity149–151. Probodies are IgG molecules that are either fused with self-masking moieties at the N terminus via cleavable spacers or designed with antigen-binding sites that undergo pH-dependent conformational changes, which reduces the target binding affinity of the IgG152–159. Upon reaching the tumour microenvironment (TME), either the masking moieties are removed or the antigen-binding sites change conformation in response to certain tumour-associated factors such as the abundance of proteases and acidic conditions, resulting in the localized restoration of the original target binding affinity of the antibody and payload release. This novel approach has the potential to enhance the therapeutic index of ADCs that might otherwise have excessive crossreactivity with nonmalignant tissues