Unlocking the potential of bispecific ADCs for targeted cancer therapy

Abstract

Antibody–drug conjugates (ADCs) are biologically targeted drugs composed of antibodies and cytotoxic drugs connected by linkers. These innovative compounds enable precise drug delivery to tumor cells, minimizing harm to normal tissues and offering excellent prospects for cancer treatment. However, monoclonal antibody-based ADCs still present challenges, especially in terms of balancing efficacy and safety. Bispecific antibodies are alternatives to monoclonal antibodies and exhibit superior internalization and selectivity, producing ADCs with increased safety and therapeutic efficacy. In this review, we present available evidence and future prospects regarding the use of bispecific ADCs for cancer treatment, including a comprehensive overview of bispecific ADCs that are currently in clinical trials. We offer insights into the future development of bispecific ADCs to provide novel strategies for cancer treatment.

Keywords

antibody–drug conjugate / bispecific antibody / bispecific ADC / cancer

1 Introduction

Antibody–drug conjugates (ADCs) were initially developed according to the concept of a “magic bullet” proposed by Paul Ehrlich, who is the pioneer of chemotherapy [1, 2]. A complete ADC comprises a cytotoxic molecule covalently linked by a chemical linker to a high-specificity monoclonal antibody [3–6]. This innovative design combines the distinct advantages of highly specific targeting and potent cytotoxicity [7, 8]. Mylotarg, the first ADC, was approved as treatment for acute myeloid leukemia by the US Food and Drug Administration (FDA) in 2000 [9]. However, owing to linker instability and intolerable hepatotoxicity, it was withdrawn from the market in 2010 (but was subsequently reapproved by the FDA in 2017) [10, 11]. Second- and third-generation ADCs have been optimized with improved conjugation methods, showing enhanced linker stability and effectiveness and accommodating different antibody formats. These advances have bolstered antitumor efficacy while reducing off-target toxicity [7, 12, 13]. To date, a total of fifteen ADCs have been granted market approval by the FDA EMA or NMPA for solid and hematological malignancies (Tab.1), and hundreds of ADCs are in various stages of clinical trials [37]. Approval for three ADCs (gemtuzumab ozogamicin, moxetumomab pasudotox, and belantamab mafodotin) was withdrawn due to intolerable toxicity, lack of advantages over the current standard of care, and business and commercial considerations [38]. Specifically, belantamab mafodotin failed its phase III clinical trial because the primary endpoint of progression-free survival (PFS) was not achieved [39], whereas moxetumomab pasudotox was delisted for commercial reasons rather than reasons related to safety and efficacy.

Despite their promise in targeted cancer therapy, ADCs still pose toxicity challenges because target antigens might be expressed in healthy tissues, particularly in the case of solid tumors [40, 41]. Bispecific antibodies offer an enticing avenue for cancer therapy because of their ability to target two different antigen sites and decrease off-target toxicity. They can deliver drugs containing isotypes and toxic agents [42, 43]. Compared with monospecific antibodies, bispecific antibodies present several advantages, including the ability to circumvent drug resistance [44–46] and increased internalization and specificity that can reduce toxicity [47–50]. These attributes support the efficacy and safety of ADCs. The use of bispecific ADCs represents a novel strategy in the development of clinical cancer treatment. In this review, we discuss current knowledge regarding the design, mechanism of action, and clinical efficacy of bispecific ADCs. We also discuss prospects for the development of next-generation bispecific ADCs, aiming to provide insights into the research and development of novel cancer therapeutics.

2 Bispecific ADCs

Bispecific antibodies constitute a new generation of antibody-based molecules designed to recognize two different antigen-binding sites [51]. Fourteen bispecific antibodies have been granted market approval, and more than one hundred are in clinical trials (Tab.2). They have multiple applications, including effector cell bridging, receptor cross-linking, cofactor mimicry, and drug delivery efficiency enhancement [67–69]. Furthermore, the advent of bispecific antibody technology has expanded the array of antibody formats available for utilization in ADCs, presenting additional opportunities for innovation [70–73]. Bispecific antibodies can be harnessed to increase tumor specificity and boost antigen internalization [74, 75]. Conjugating payloads to bispecific antibodies to yield bispecific ADCs is an emerging strategy for cancer therapy. Since 2019, an increasing number of bispecific ADCs have entered clinical trials (Fig.1). Bispecific ADCs possess a structure analogous to that of monospecific ADCs. Antibodies with dual specificity attach to distinct molecular targets and tether to cytotoxic drugs by cleavable or noncleavable linkers. Differences between bispecific and monoclonal ADCs are summarized in Tab.3.

2.1 Mechanism of action of bispecific ADCs

Bispecific and traditional monoclonal ADCs differ in several ways. Mechanistically, bispecific ADCs can bind to multiple antigens or different epitopes, counteracting drug resistance by simultaneously interfering with multiple signaling pathways [76–78]. Bispecific ADCs can thus be constructed with selectively combined targets to achieve synergistic function, effectively suppressing tumor cell activity and facilitating receptor internalization and lysosomal transport (Fig.2). In addition, incorporating a dual-targeting strategy increases the selectivity of ADCs, markedly reducing on-target and off-tumor toxicity in healthy tissues [79]. By inducing receptor aggregation and cross-linking, biparatopic ADCs promote drug release in target cells. Moreover, dual-targeting ADCs can selectively target fast-internalizing receptors to promote the internalization of otherwise noninternalizing antigens [80]. After bispecific ADCs are internalized, the antigen–ADC complex is transported to the lysosome, where the linker is processed to release the cytotoxic payload, which then induces tumor cell apoptosis [81, 82]. The free payload can be released to affect adjacent tumor cells, causing a bystander effect [83–85].

2.2 Bispecific strategies and ADCs

2.2.1 Bispecific strategies to increase the internalization of bispecific ADCs

The efficacy of ADCs hinges on multiple factors, including antibody specificity, pharmacokinetics, and the internalization and intracellular trafficking of targeted antigens [86, 87]. Bispecific antibodies designed to target two distinct epitopes on a single antigen can significantly increase the effectiveness of ADCs by accelerating the uptake and lysosomal degradation of antigens. These antibodies can mimic the effects of combination therapy, providing substantial benefits when antigens resistant to internalization, such as HER2, are used [67, 88–90]. This mechanism is particularly suitable for increasing ADC efficacy, especially targets with poor internalization [91].

The effectiveness of ADCs targeting HER2, such as T-DM1, is compromised by suboptimal internalization [92, 93]. Conversely, novel biparatopic anti-HER2 antibodies, which are currently in clinical trials, can suppress tumor growth by promoting HER2 clustering and internalization within cancer cells [94, 95]. To increase the efficacy of anti-HER2 therapy, researchers have developed various biparatopic anti-HER2 antibodies, such as KN026 (NCT04165993), ZW25 (NCT05035836), BCD-147 [96] (NCT03912441), MBS301 [97] (NCT03842085), TQB2930 (NCT05380882), and KM501 (NCT05804864), which are now in clinical trials. These biparatopic antibodies can be designed to function as ADCs, increasing internalization through enhanced receptor cross-linking and clustering (Fig.2).

Bispecific antibodies that target tumor antigens and fast-internalizing receptors can increase the internalization and cytotoxicity of ADCs. Lee et al. [98] studied the relationship between the cell surface antigen density ratio and internalization of bispecific ADCs; they constructed bispecific antibodies that targeted the internalizing antigen EphA2 and the noninternalizing antigen ALCAM; when the EphA2-to-ALCAM ratio on the cell surface exceeded 1:5, the internalized antigen EphA2 promoted ALCAM clearance by the bispecific antibody, which transformed the non-internalizing antigen into an internalizing antigen. A similar strategy was applied to anti-CD63/HER2 bispecific antibodies [99]. CD63, which is a lysosome-associated membrane glycoprotein found mainly in lysosomes and endosomes, regulates the transport of other proteins [100]. The anti-CD63 antibody promotes internalization and lysosomal trafficking, whereas the HER2-binding arm specifically recognizes HER2-positive tumor cells. A bispecific anti-CD63/HER2 ADC coupled with tubulin receptors kills HER2-positive tumor cells more effectively than monovalent anti-HER2 and anti-CD63 ADCs. Another bispecific ADC that targets amyloid precursor-like protein 2 (APLP2) and HER2 successfully increased intracellular trafficking [101].

2.2.2 Bispecific strategies to increase the selectivity of ADCs

Toxicity results when ADCs target normal tissues rather than tumors, and this effect has been observed in clinical trials; gastrointestinal toxicity, skin toxicity, and dysgeusia occur and might be related to the expression of a target in normal tissues [102–104]. Bispecific antibodies have shown efficacy in targeting widely expressed antigens, such as CD47 and FGF21, and increase the precision of treatment [105–107]. Therefore, bispecific strategies might increase the selectivity of ADCs. A pegylated bispecific ADC targeting CD47 and PD-L1 exhibits considerable in vitro efficacy. It selectively targets CD47/PD-L1 dual-positive cells without causing on-target off-tumor toxicity in human red blood cells. Furthermore, in vivo studies in mice revealed a maximum tolerated dose of 50 mg/kg, demonstrating the promising selectivity and safety profiles of the ADC [108].

The expression of EGFR in tumors and normal skin cells often leads to skin toxicity during EGFR-targeted therapy [109, 110], potentially limiting the therapeutic efficacy of ADCs. In a dual-targeting strategy, bispecific ADCs can be designed to target EGFR along with cMet, HER3, MUC1, or other targets, thus distinguishing between tumor and normal tissue for heightened selectivity [111–113].

2.2.3 Bispecific strategies for overcoming drug resistance to ADCs

The development of ADCs must address several obstacles, such as variations in resistance mechanisms, which include tumor heterogeneity, low antigen expression, and tumor target mutations [114, 115].

Dual-targeting antibodies block two disparate signaling pathways concurrently, thereby inhibiting the proliferation and invasion of cancer cells (Fig.2) [51]. This approach not only increases the therapeutic effect but also circumvents potential drug resistance encountered in therapies that block a single signaling pathway. In addition to using the cytotoxicity of the payload and the bystander effect to increase therapeutic efficacy, dual-targeting ADCs can employ dual-signal blockade as a supplementary anticancer mechanism. ADCs with dual-signal blocking capabilities have thus attracted considerable interest. Three ADCs currently in clinical trials bind to dual targets with synergistic effects on tumor proliferation, metastasis, and drug resistance.

Furthermore, biparatopic antibodies can increase the efficacy of ADCs in patients with low antigen expression levels. Anti-HER2 biparatopic antibodies have been used in the formulation of ADCs in clinical trials. In addition, biparatopic ADCs targeting FRα have been formulated to increase response in patients with low FRα expression levels [116]. Moreover, biparatopic antibodies targeting receptors, such as cMet, achieve a higher growth inhibition rate than monospecific antibodies by promoting the downregulation of receptor expression and disrupting recycling, thereby improving upon kinase inhibitors (KIs) [117]. The mechanism of action of anti-cMet biparatopic ADCs involves the targeting tumor antigens for the delivery of drugs to tumor cells. This mode of targeting may result in limited effects in patients with low antigen expression levels [118, 119]. Fan et al. [120] constructed tetravalent biparatopic anti-EGFR ADCs consisting of two tandemly fused anti-EGFR nanoparticles targeting two distinct nonoverlapping epitopes; they fused the Fc domain to the ADCs and introduced the E430G mutation to increase the CDC effect of ADC drugs. Biparatopic ADCs exhibit excellent antitumor activity against drug-resistant EGFR-expressing tumors.

2.2.4 Bispecific strategies for improving the half-life and efficacy of ADCs

Owing to their low molecular weight, small-format ADCs have the advantages over other types of ADCs in terms of penetrating solid tumors. However, a low molecular weight leads to a short half-life and rapid clearance of ADCs. A nonspecific antibody strategy can compensate for the short half-life of small-format antibodies. Albumin-based drug delivery strategies can improve the biodistribution of antibody fragments and facilitate tumor deposition [121]. AB.Fab4D5, which is a bifunctional molecule that targets HER2 and albumin, can be rapidly cleared from the blood, inhibits the exposure of normal tissues, and can accumulate at tumor sites more effectively than trastuzumab-derived Fab (Fab4D5) and trastuzumab. Similar to the traditional antibody trastuzumab, AB.Fab4D5 exhibits tumor deposition and retention [122]. A similar bispecific strategy was used in the construction of diabody–drug conjugates. The albumin-binding domain (ABD)–diabody targeting 5T4 and albumin is coupled with cytotoxic pyrrolobenzodiazepine, which features ABDs, to improve the pharmacokinetics. In vivo, this diabody–drug conjugate exhibits an extended half-life, excellent tumor growth inhibition, and higher tolerability than PEGylation antibody fragment conjugates [123].

2.3 Bispecific ADCs in clinical trials

Although fifteen ADCs are currently available on the market, the development of bispecific ADCs is still in the early stages, and most existing ADCs are in phase I or preclinical trials [124–129]. Fig.3 and Tab.4 offer an overview of bispecific ADCs formulated with a biparatopic or dual-targeting design that are currently in clinical trials.

2.3.1 BL-B01D1

The ADC drug BL-B01D1, which is rapidly advancing toward clinical use, comprises a tetravalent bispecific antibody targeting EGFR and HER3 coupled with a topoisomerase I inhibitor via a cleavable linker [112, 130]. Unlike other EGFR family members, HER3 relies on heterodimerization with other receptors, particularly EGFR and HER2, for downstream signaling activation [131, 132]. This heterodimerization enables HER3 to promote cell proliferation, tumor formation, metastasis, and drug resistance [131]. The mechanism of action of BL-B01D1 involves two processes. It enters cells through endocytosis, releases DNA damage agents, and kills tumor cells; additionally, it blocks the formation of EGFR and HER3 heterodimers, inhibiting downstream cell growth signals. In a phase I clinical trial of 122 patients with locally advanced or metastatic solid tumors, 34 patients had received either third-generation EGFR tyrosine KI (TKI) therapy or platinum-based chemotherapy. BL-B01D1 demonstrated an objective response rate (ORR) of 61.8% and a disease control rate (DCR) of 91.2%, indicating its potential as treatment for EGFR TKI-resistant NSCLC patients [112].

2.3.2 REGN5093-M114

cMet dimerizes and activates downstream signaling pathways in an HGF-dependent or HGF-independent manner [133–135]. In certain cases, mutations (such as METex14) or amplifications of the MET gene have been identified and are recognized as critical contributors to acquired resistance to EGFR or cMet TKIs [136–139]. Achieving durable responses to current TKI therapies is still challenging [140]. Regeneron developed REGN5093-M114, which is a biparatopic ADC that targets the multiple epitopes of cMet.

The anti-cMet/cMet antibody recognizes the unique nonoverlapping epitopes of cMet, more efficiently promoting internalization and degradation than parental antibodies [117]. REGN5093-M114 comprises a protease-clearable linker, a maytansinoid payload (M24), and an anti-cMet/cMet antibody. Once the antibody part of the ADC targets and enters tumor cells, the linker is cleaved in secondary and circulating endosomes, releasing the payload to effectively eliminate tumor cells [141, 142]. In the MET-amplified/MET-mutated Hs746T xenograft model, unconjugated anti-cMet/cMet bispecific antibodies initially lead to complete tumor regression, although tumors reappear subsequently. However, REGN5093-M114 completely inhibits and clears escaped tumors. In a MET-amplified NSCLC patient–derived xenograft (PDX) model, which is resistant to cMet inhibitors, an unconjugated anti-cMet/cMet bispecific antibody only delayed tumor growth, whereas an ADC induced complete and sustained tumor regression [141]. In addition, REGN5093-M114 exhibits a favorable toxicity profile in cynomolgus monkeys and is currently entering phase I clinical trials (NCT04982224).

2.3.3 Anti-HER2 biparatopic ADCs

ZW49 and MEDI4276 are anti-HER2 biparatopic ADCs with different designs. The antibody component of MEDI4276 adopts a tetravalent symmetric design and simultaneously recognizes extracellular domain 2 (ECD2) and extracellular domain 4 (ECD4) of HER2, triggering the cross-linking of receptors and promoting internalization. In vivo, MEDI4276 has shown promising efficacy in tumor models with low expression of HER and has exhibited acceptable safety in cynomolgus monkeys [143]. ZW25 is the antibody component of ZW49 and consists of an anti-HER2-ECD4 single-chain variable fragment (scFv) and an anti-HER2-ECD2 fragment antigen binding (Fab) domain. To develop ZW49, this antibody was conjugated with a novel auristatin payload, resulting in a DAR of 2. This ADC with an asymmetric bivalent format was developed by Zymeworks using proprietary Azymetric and ZymeLink platforms [144]. The internalization and lysosomal trafficking of ZW49 are superior to those of anti-HER2 monospecific ADCs. Preclinical studies have suggested its effectiveness in low- and high-HER2 expression models and in a brain metastasis model. Currently, ZW49 is under evaluation in a phase I trial for patients with metastatic HER2-expressing cancer that have not responded to standard treatments, including those targeting HER2.

In a phase I dose escalation study of another anti-HER2 biparatopic ADC, MEDI4276, when the dose exceeded 0.9 mg/kg, two patients experienced grade 3 dose-limiting toxicity in the liver function test, limiting the therapeutic window. In addition, in 47 patients with HER2-positive breast and gastric cancer and on treatment, the most common grade 3/4 drug-related adverse events were increased AST (21.3%), and five patients discontinued treatment due to drug-related adverse events; thus, the authors speculated that the lower gastrointestinal toxicity and hepatotoxicity in clinical trials may be related to the “on-target” effect of ADCs [145]. Whether MEDI4276-related hepatotoxicity is HER dependent remains unclear. By contrast, ZW49 showed high safety, and most patients showed grade 1 or 2 severity only. However, in patients treated with ZW49 at a dose of 2.5 mg/kg Q3W, the objective response rate was 28%, and the DCR was 72%, which suggested that ZW49 had a less pronounced effect than anticipated [146]. These different clinical outcomes might stem from differences in the valency of the antibody moiety, the DAR, and the payload despite that MEDI4276 and ZW49 target the ECD4 and ECD2 epitopes of HER2 (Tab.4 and Fig.3). To reduce the risk of on-target toxicity in ADCs, it is crucial at the preclinical stage to consider the differences in expression profiles among target antigens, organ or cell types, the regenerative potential of healthy cells, and mechanism of action of the payload [147].

Other anti-HER2 biparatopic ADCs, such as JSKN003 and TQB2102 [148, 149], are also in clinical trials. JSKN003 is an ADC comprising KN026, which is a bispecific antibody targeting the extracellular domains II and IV of HER2 and is conjugated to a topoisomerase I inhibitor via a specialized linker. This ADC demonstrates high-affinity HER2 binding, efficient internalization by HER2-positive cancer cells, and tumor growth inhibition in preclinical models. A pharmacokinetic analysis in cynomolgus monkeys revealed linear dynamics, and the highest nonseverely toxic dose was 30 mg/kg. TQB2102 adopts an scFv–Fab configuration similar to that of ZW49, mitigating the risk of light chain mispairing during bispecific antibody production. Notably, deuterated Dxd was utilized as the payload, and the ADC outperformed Dxd alone in terms of antitumor efficacy in NCI-N87 cells. When evaluated against a trastuzumab-based ADC, this bispecific ADC demonstrated increased endocytosis efficiency in HER2-positive NCI-N87 and SK-BR-3 tumor cells.

2.3.4 M1231

The interaction between MUC1 and EGFR is related to the activation of EGFR and EGFR-mediated signaling, and targeting MUC1-C can help to overcome resistance to EGFR TKIs [150, 151]. M1231, a bivalent asymmetric bispecific anti-MUC1/EGFR ADC, can deliver microtubule inhibitors to tumor cells expressing MUC1 and EGFR [111]. Dual targeting can improve the efficacy of ADCs and reduce off-target toxicity. This drug is based on a strand-exchange engineered domain and the Xpress CF+ platform for the preparation of bispecific antibodies and the incorporation of nonnatural amino acids [111, 152]. Anti-MUC1/EGFR bispecific antibodies exhibited higher internalization, lysosomal trafficking, and antitumor activity in the PDX model than monospecific bivalent antibodies. In vivo, a single injection of M1231 resulted in complete tumor regression in NSCLC and ESCC PDX models [111]. Another novel bispecific anti-MUC1/EGFR ADC, BSA01, demonstrated potent antitumor activity in preclinical evaluation and reduced the binding and internalization of the EGFR arm, thereby decreasing skin toxicity caused by nonspecific targeting [153].

2.3.5 AZD9592

EGFR and cMet, two crucial targets in tumor immunotherapy, exhibit crosstalk between signaling pathways that can lead to tumor resistance to EGFR and cMet TKIs [154–156]. Dual-targeting inhibitors have been developed to mitigate this effect [157–159]. AZD9592 is a dual-targeting ADC that selectively binds to the tumor cell surface receptors EGFR and cMet and delivers topoisomerase I inhibitors to tumor cells. This design increases the therapeutic window and overcomes tumor cell resistance. The AZD9592 antibody is designed with a knob-into-hole bispecific antibody structure, and a lower-affinity antigen binding domain is used to minimize EGFR-related skin toxicity [110]. Preclinical experiments revealed that AZD9592 mainly induces DNA double-strand breaks by delivering and releasing topoisomerase I, leading to tumor cell death. In EGFR-mutated PDX models, a single dose of 8 mg/kg AZD9592 inhibited tumor growth in 73% (16/22) of patients [113].

2.3.6 IMGN151

FRα is a folate-binding protein in cell membranes and is associated with tumor progression. It is overexpressed in various solid tumors, such as triple-negative breast cancer, lung cancer, and particularly ovarian cancer, demonstrating its potential as a therapeutic target [160, 161]. IMGN853 (mirvetuximab soravtansine in Tab.1) is an FRα-targeting ADC that comprises the anti-FRα monoclonal antibody M9346A conjugated to the tubulin inhibitor DM4 via a cleavable linker. IMGN853 demonstrates antitumor activity in preclinical models [162, 163]. In a phase II SORAYA study of high-FRα platinum-resistant epithelial ovarian cancer, IMGN853 exhibited effective antitumor activity, with an ORR of 32.4%; complete responses were obtained from five patients, and partial responses from 29 patients [164]. Phase II and III trial results led to FDA approval [36, 164, 165]. However, IMGN853 provided few benefits to patients with low FRα expression in the phase III clinical trials [119], prompting the development of IMGN151 (a biparatopic ADC). The bispecific antibody part of IMGN151 adopts an asymmetric form, targeting different FRα epitopes. In addition, the linker–drug complex includes novel and potent maytansine-derived DM21-LG [116, 163]. In vitro, IMGN151 exhibits stronger killing activity than IMGN853 against tumor cells with medium or low FRα expression. IMGN151 achieved complete tumor regression in human tumor xenograft models with varying FRα expression levels [116]. Currently, IMGN151 is in a phase I clinical trial (NCT05527184), with its safety and antitumor activity in patients with recurrent endometrial cancer and recurrent, high-grade serous epithelial ovarian, primary peritoneal, or fallopian tube cancer, currently being evaluated. Similarly, CBP-1008, a bispecific ADC that targets FRα and TRV6, is in a phase I clinical trial (NCT04740398) for patients with advanced malignant solid tumors [166].

Bispecific ADC drugs in clinical applications have a few targets, which are mainly the epidermal growth factor receptor family (EGFR, HER2, and HER3), cMet, and MUC1. The main goals of designing bispecific ADCs are to increase therapeutic efficacy in patients with low antigen expression, reduce off-target normal tissue toxicity, and overcome drug resistance.

3 Design principles and future prospects of bispecific ADCs

Five key aspects should be considered in the development of ADCs: target antigen selection, linker, payload, conjugation strategy, and antibody moiety [7]. Another challenge in the development of bispecific ADCs is the mispairing of heavy and light chains during the production of IgG-like bispecific antibodies. Currently, numerous technologies can be employed to resolve this issue. Additionally, the application of small-format antibodies has facilitated the construction of bispecific ADCs. Therefore, researchers can focus on the optimization of payloads, linkers, conjugation techniques, and antibody affinity.

3.1 Selection of target antigens

To mitigate off-target toxicity, the target antigen should be nonsecretory, tumor specific, and uniquely expressed on tumor cells, distinguishing them from normal tissues. Additionally, an effective ADC requires a target that is conducive to internalization and has a low rate of endocytic recycling [167, 168].

The specificity and expression intensity of tumor antigens markedly influence target selection for ADCs. These tumor antigens can be classified into tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs) [169]. Antigens such as carcinoembryonic or leukocyte differentiation fall into the TAA category and are overexpressed or dysregulated in tumor cells compared to normal cells. However, insufficient specificity of TAAs can result in off-target effects. Considering the aforementioned factors, the two TAAs of a dual-targeting ADC must present analogous expression specificity patterns in the tumor. Concurrently, TAA expression within normal tissue should be minimal and has no overlap.

Neoantigens are tumor-specific mutated peptides produced by tumors through gene mutation or other mechanisms and can be ideal targets for cancer immunotherapy [170–172]. Neoantigens can be presented on the surface of tumor cells as peptide/MHC complexes, which can be recognized by antibodies [173–176]. For example, a bispecific single-chain diabody targeting the most common mutation in TP53, R175H, can effectively activate T cells. Even when the density of peptide–HLA complex on the surfaces of cancer cells is extremely low, the mutation can still be targeted. Activated T cells can lyse cancer cells harboring neoantigens in vitro and in mice [177]. Neoantigens can distinguish explicitly between tumor and normal cells, increasing the selectivity of ADCs. Relevant studies have confirmed this effect [178]. KRAS G12V peptides can bind to MHC I molecules, resulting in their presentation on the tumor cell surface. This peptide-MHC I complex, regarded as a TSA, can be recognized by TCR-mimic antibodies. Researchers have coupled TCR-mimic antibodies with the microtubule protein inhibitor MMAE to devise a TCRm–ADC that targets the KRAS G12V peptide–HLA complex; the obtained ADC showed substantial antitumor activity in a xenograft model. After treatment was administered at a dose of 20 mg/kg every four days four times (q4d × 4), no significant changes in either the weight of the treated mice or the histomorphology of their organs were observed, supporting the safety and tolerability of the TSA-targeting approach.

The design of noninternalizing ADCs may broaden the target range for bispecific ADCs. This strategy bypasses strict requirements for the internalization properties of an antigen and enables the targeting of cell surface antigens with weak internalization, such as CD20 [179, 180], CEACAM5 [181], and others [179, 182, 183]. Activated platelets and some specific proteins of the tumor stroma, such as MMP9 and Gal3BP, can serve as noninternalizing targets for ADCs [184–186].

A vascular-targeting ADC that specifically targets the tumor neovasculature obviates the need for internalization, relying on the release of glutathione and cysteine resulting from tumor cell death during the delivery of cytotoxic drugs. This approach enables the targeting of most cancer types. Bispecific ADCs constructed by combining this target with TSAs might inhibit tumors through multiple mechanisms [187].

Another way to broaden the range of target selection is to construct a probody drug conjugate [188, 189]. CytomX contains proposed probes that can be activated by protease cleavage. A masking peptide is connected to the N terminus of the antibody light chain through a cleavable spacer [190, 191]. A reaction in a tumor microenvironment removes the masking peptide, allowing the antibody to bind to a target on the tumor cell surface without binding to normal tissues. CX2029 is an anti-CD71 probody drug conjugate coupling the tubulin inhibitor MMAE with a DAR of 2 [192, 193]. As in the probody strategy, the pH-dependent or ATP-dependent conditional activation of antibodies can increase selectivity and may contribute to ADC design [194–196].

3.2 Linker

ADC linkers are classified into two types: cleavable and noncleavable. Cleavable linkers can undergo specific types of degradation, such as acid hydrolysis, glutathione reduction, or tumor cell-specific protease degradation, whereas noncleavable linkers, such as those involving thioether bonds, are stable in plasma. Different types of ADC linkers have their own advantages and disadvantages. Although cleavable linkers allow specific release and bystander effects, they may lead to systemic toxicity and affect drug safety; noncleavable linkers are potentially safer but could limit ADC activity and cannot fully elicit bystander effects [7, 197].

Peptide linkers are extensively utilized in the development of third-generation ADCs. These linkers undergo proteolytic cleavage by lysosomal enzymes, enhancing stability in plasma more effectively than chemically responsive linkers [198]. Bispecific ADCs commonly employ protease-cleavable peptide linkers and exhibit no significant difference in linker technology from monospecific ADCs.

ADCs with elevated DARs exhibit accelerated plasma clearance as a result of increased hydrophobicity, potentially compromising their in vivo efficacy [199]. Moreover, the heightened hydrophobicity undermines the stability of ADCs with high DARs, predisposing them to aggregation. Thus, ADCs commonly sustain average DARs ranging from 2 to 4, with an optimized trade-off between stability and efficacy [12, 197].

Novel linkers optimized for hydrophilicity can mitigate plasma clearance and aggregation that arise from low hydrophilicity in high-DAR ADCs, thereby increasing the exposure and in vivo efficacy of ADCs. This technology has been applied to bispecific ADCs. TQB2102 employs a hydrophilic linker similar to the GGFG linker used in DS8201, facilitating greater payload loading onto the antibody [148, 200].

3.3 Payload

Given the low percentage of administered ADCs that reach the tumor site [12], the selection of a potent and stable cytotoxic drug payload with minimal toxic side effects is integral to the design of bispecific ADCs.

Bispecific ADCs in clinical trials predominantly contain two classes of payloads: microtubule and DNA inhibitors. A prime example of a topoisomerase I inhibitor is Dxd. This exatecan derivative has been deployed in DS-8201a (trastuzumab deruxtecan in Tab.1), which has a DAR value of 8 and has been approved by the FDA because of its high effectiveness [201]. Topoisomerase I inhibitors serve as alternative therapeutic options to microtubule inhibitors and DNA-damaging agents. Several bispecific ADCs, such as JSKN003 and TQB2102, are constructed from Dxd by further modification.

Numerous bispecific ADCs utilize microtubule inhibitors; different platforms, such as REGN5093-M114 and M1231, include diverse modifications on the basis of the original microtubule inhibitors. In some designs, polyethylene glycol is connected to MMAE, which, after site-specific coupling with the antibody, extends the half-life and reduces the immunogenicity of small-format bispecific antibodies [108].

3.4 Conjugation method

Another crucial characteristic of ADCs is uniformity, which is primarily determined by the conjugation method. Conjugation methods involving the random conjugation of the lysine residues of antibodies can produce ADCs with wide DAR distributions. Alternatively, given that IgG1 contains four interchain disulfide bridges, cysteine-based coupling can generate ADCs with two, four, six, or eight uniform DARs. However, in ADCs produced by these random conjugation methods, the payload might not be consistently linked to the same antibody site, which may compromise the stability and uniformity of the ADC. Encouragingly, however, the advent of site-specific conjugation techniques can increase the homogeneity of ADCs [7, 202, 203].

Conjugation strategies differ across various platforms. The Xpress CF+ platform, as employed by M1231, integrates nonnatural amino acids into the native antibody sequence to engineer precise conjugation sites for targeted coupling [204]. ZW49 utilizes ZymeLink Auristatin technology, in which cysteine-based conjugation is used to attach a linker and a drug to the bispecific antibody’s interchain disulfide bonds [205]. Furthermore, lysine-based random conjugation, exemplified by REGN5093-M114 [141], remains a conservative approach for the conjugation of bispecific ADCs.

3.5 Antibody moiety

3.5.1 Affinity and valency

Affinity and valency must be considered in the selection of appropriate antibody moiety. The affinity between the two arms of a bispecific antibody and its target does not maintain a strictly positive correlation with target selectivity. During the preclinical evaluation of bispecific ADCs, fine-tuning the affinity of different binders affiliated with bispecific antibodies can be beneficial. Such adjustments can establish equilibrium between the potency and selectivity of dual-targeting ADCs, facilitating the design of a bispecific ADC with high target selectivity and uncompromised antitumor efficacy.

Mazor et al. [206] engineered a series of anti-HER2/EGFR bispecific antibodies (1 + 1) that exhibited a 10–250-fold reduction in EGFR affinity. The authors quantified the target selectivity of these bispecific antibodies by computing target selectivity parameter (TSP), the ratio of the IC₅₀ values for inhibiting EGFR⁺/HER2⁺ NCI-H358 and EGFR⁺/HER2^– NCI-H358-ko cells in vitro. The results highlighted an increase in the target selectivity of bispecific antibodies as EGFR affinity decreased more than 50-fold, which corresponded to a reduction in the TSP from 0.09 to 0.006. Concurrent in vivo experiments substantiated these findings; employing NCI-H358-ko cells as a surrogate for normal skin tissues and NCI-H358 cells as a target tumor, they demonstrated that bispecific antibodies with an EGFR affinity reduced by more than 10-fold could distinguish between tumor and normal tissue while retaining the antitumor ability of the parent bispecific antibodies. Sellmann et al. [207] constructed an affinity-optimized anti-EGFR/cMet bispecific antibody coupled to MMAE via a protease-clearable valine–citrulline (vc) linker and created a bispecific ADC with a DAR of 2. Reducing the affinity of an antibody for EGFR can reduce the toxicity of normal tissue cells to human keratinocytes, whereas tumor cells overexpressing EGFR and cMet can be selectively killed by the ADC, thereby improving the safety profile of the ADC. In vitro studies revealed that the TD20/ED80 ratio for the anti-EGFR/cMet bispecific ADC variant, characterized by a moderate EGFR affinity, is threefold that of its counterpart, which is the high-affinity variant (6 ± 0.5 vs. 2 ± 0.06). Furthermore, the TD20/ED80 ratio was sixfold that of the monospecific ADC (6 ± 0.5 vs. 1 ± 0.2). These findings suggest that alterations in the affinity of bispecific antibodies can expand the therapeutic window of ADCs.

The selection of the valency of bispecific ADCs requires careful consideration of the intended targets and objectives. Increasing the valency of an antibody can fortify receptor cross-reactions, boost binding stability, and optimize the efficiency of drug delivery. Conversely, when the objective is to increase target selectivity, particularly for antigens that have low expression in normal tissues, the valency of the corresponding binding arm within a bispecific ADC should be reduced. Andreev et al. [208] constructed an anti-HER2/PRLR bispecific ADC. Compared with HER2, PRLR can be efficiently internalized and transported to lysosomes. In vivo, the bivalent anti-HER2 ADC with a DAR of 4 is more effective than the anti-APLP2/HER2 ADC with the same DAR. The interaction between HER2 dimers is an important factor affecting the efficacy of HER2 ADCs, and the efficacy of anti-APLP2/HER2 ADCs is limited because they cannot mediate HER2 dimerization. Thus, research into anti-HER2/HER2/APLP2 bispecific ADCs can be considered a future direction for ADC design. According to the principle of dual-targeting, multivalent quadruple-specific antibodies that are more effective than bispecific antibodies have been developed [209]. However, increasing the valency of bispecific antibodies potentially diminishes target selectivity [206]. Despite exhibiting reduced affinity for EGFR, the tetravalent anti-HER2/EGFR bispecific antibody (2 + 2) cannot distinguish between simulated normal tissues and target tumors, in contrast to its bivalent counterpart, the anti-HER2/EGFR bispecific antibody (1 + 1).

3.5.2 Molecular weight

In the development of bispecific ADCs, the tumor penetration ability of ADCs engineered to improve internalization, including biparatopic or dual-targeting ADCs, may be constrained by the binding site barrier inherent in solid tumors [210]. Extensive experimentation is needed to optimize the affinity and internalization capacity of bispecific antibodies and to ensure a balance between tumor absorption and penetration of bispecific ADCs. A viable alternative is decreasing the molecular weight of antibodies in the design of small-format bispecific ADCs. Owing to their molecular weight, small-format targeted therapies offer increased penetration into solid tumors and can overcome the biological barrier of solid tumors [211]. Furthermore, compared with traditional monoclonal antibodies equipped with Fc fragments, small-format therapeutics exhibit reduced cross-reactivity with Fc receptors, decreased immunogenicity, and diminished interaction with normal tissues [212]. These attributes facilitate their preferential accumulation at tumor sites, suggesting a promising avenue for the efficient delivery of cytotoxic drugs to solid tumors [211, 213].

Wu et al. [214] reported a fully human single-domain antibody (UdAb) targeting the tumor antigen 5T4. They generated a small-format ADC, n501-SN38, with a DAR of 1 through site-specific conjugation with the DNA transcription inhibitor SN38. Compared with the traditional ADC m603-SN38, n501-SN38 exhibited excellent tumor penetration. At 1 h, n501-SN38 achieved peak accumulation at the tumor site, exhibiting an uptake rate 11-fold that of m603-SN38.

Beyond UdAb, researchers have constructed bispecific nanobody drug conjugates (NDCs) incorporating biparatopic nanobodies against EGFR, gadolinium binding domains, and a C3-tag. The gadolinium-binding domain can incorporate Gd³⁺ into magnetic resonance imaging (MRI), while the C3-tag optimizes the conjugation of the Pt(iv) prodrug. A bispecific NDC facilitates the precise targeting of EGFR-positive cancer cells. After its integration with the nanobody, cisplatin accumulates within EGFR-positive tumors, and its distribution within renal and hepatic systems is significantly restricted. The incorporation of an anti-albumin nanobody considerably improves the specificity and therapeutic outcomes of this NDC [215].

Small-format bispecific toxin conjugates had been explored before the development of small-format bispecific ADC strategies [216, 217]. To date, small-format bispecific ADCs have not yet entered clinical trials.

3.5.3 Multiple scaffolds for the design of bispecific ADCs

To increase the antitumor efficacy of ADCs, formulating a strategy for engineering scaffold-based multivalent and multispecific ADCs is necessary. The development and application of multivalent and multispecific ADCs can extend the fundamental concept of bispecific ADCs by enabling binding to multiple antigens and delivering a multitude of drugs to target cells. Scaffolds based on GFP, streptavidin, and self-assembling oligonucleotides can be used in the construction of multivalent and multispecific ADCs, facilitating the oligomerization of ligands [218, 219]. Kim et al. [220] introduced a series of GFP oligomers that autonomously formed oligomeric assemblies in cells; this GFP-based scaffold allowed the precise control of the number, arrangement, and orientation of multivalent proteins. When functional proteins, such as maltose-binding protein and fluorescent mCherry, were N- and C-terminally fused to GFP polygons, the fused proteins retained their binding and fluorescence capabilities during uninterrupted polymerization. The internalization of the anti-EGFR antibody Erbitux into the lung carcinoma epithelial cell line A549 can be increased by protein G-functionalized GFP polygons, as the scaffold of the G-functionalized GFP polygons facilitates antibody-mediated clustering of EGFR on cell surfaces. Another GFP-based trimeric cytotoxic conjugate with a DAR of 3 was prepared by coupling the cytotoxic drug MMAE to a trivalent FGF1 oligomer, which can selectively and effectively kill FGFR1-overproducing cancer cells [221].

Streptavidin exhibits unique binding properties and robust stability [222, 223] and can serve as a scaffold for assembling ADCs in three types of components: a strep-tagged antibody fragment, multivalent protein adapter, and strep-tagged linker drug. This modular design enables the assembly of diverse antibodies and linker drugs [224]. In a parallel approach, the human noncollagenous trimerization domain, which forms a powerful trimeric immunotoxin with carcinoembryonic antigen–targeting capability, offers diverse payload delivery options for antibodies [225]. Linkers can function as scaffolds for the construction of bispecific ADCs. One type of bifunctional branched linker can be subjected to click chemistry reactions with a payload and tumor-targeting ligands and conjugated to a monoclonal antibody. This process produces dual-targeting ADCs that may be effective against tumors with heterogeneous antigen expression [226].

The use of oligomerization scaffolds in the design of multivalent ADCs offers the following advantages: the scaffold allows the assembly of multiple antibody fragments that recognize different epitopes or antigens; multivalent antibodies can bind more tightly to receptors, enabling precise drug targeting and reducing off-target toxicity; and the clustering of receptors on the surface of tumor cells can be induced, leading to the formation of large immune complexes and facilitating the internalization of ADCs into tumor cells and their trafficking to lysosomes. In addition, multiple drugs can be attached to scaffolds to overcome drug resistance in tumor cells. Different scaffolds, including the Fc fragment of IgG, may generate a variety of novel bispecific ADCs and further increase the targeting selectivity and effectiveness of these therapeutics [218].

4 Conclusions

To date, ADCs have emerged as pivotal cancer therapeutics. Fifteen approved ADCs are currently available to treat various cancer types, and over one hundred ADCs are currently undergoing clinical trials. However, the efficacy and safety of monoclonal antibody ADCs remain limited. In addition to refining ADCs through conjugation techniques and the selection of cytotoxic drugs, bispecific antibodies provide an alternative strategy that can increase the drug delivery efficiency of ADCs and overcome drug resistance in tumor cells, offering higher performance than monoclonal antibodies. The precise adjustment of the affinity of the distinct arms of bispecific antibodies can mitigate the on-target and off-tumor toxicity of ADCs.

According to different combinations of targets, bispecific ADCs can be divided into biparatopic and dual-targeting ADCs. Biparatopic ADCs drive receptor cross-linking and aggregation, form large immune complexes, and increase the internalization of ADCs. This approach not only blocks the activation of receptor signaling pathways but also heightens the effectiveness of treatment in patients with low antigen expression. Dual-targeting ADCs are designed to specifically target distinct TAAs. This specific targeting approach minimizes nonspecific interactions with normal tissues and enhances the safety profile of ADCs. Another type of dual-targeting ADC targets a combination of TAAs and rapid internalization receptors, facilitating the internalization of ADCs and lysosomal trafficking. Nine bispecific ADCs based on the above design are undergoing clinical trials, but available targets are still limited. Specific strategies that can broaden the pool of target options for bispecific ADCs include the use of noninternalizing or conditionally activated ADCs and the targeting of tumor-specific antigens or neoantigens in the development of next-generation bispecific ADCs. These approaches can reduce toxicity and side effects related to targeting. In addition, through the use of the avidity principle to antibody-based cancer therapy, multivalent and multitargeting ADCs with enhanced selectivity and safety have emerged. Various scaffolds can be used in the design of new-generation ADCs.

Research on bispecific ADC has considerably advanced preclinical and clinical settings, but challenges persist, especially with regard to the balance between efficacy and safety. Bispecific ADCs can offset existing limitations, and further research can lead to unprecedented advances.

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