In the past few decades, immunotherapy has emerged as a significant breakthrough in cancer treatment. By activating the body's immune system to recognize and attack cancer cells, immunotherapy has provided new hope for patients who do not respond to conventional treatments. Bispecific antibody-drug conjugates (Bispecific ADCs), a novel branch of immunotherapy, are gradually demonstrating their unique advantages and immense potential. This article will delve into the role of bispecific ADCs in modern immunotherapy, including their structure, mechanism of action, comparison with monoclonal antibodies, and their current clinical applications and future prospects.

ADC Immunotherapy

Antibody-drug conjugates (ADCs) are an innovative class of drugs that link monoclonal antibodies to cytotoxic drugs via a linker. The monoclonal antibody component endows ADCs with high specificity, enabling them to precisely recognize and bind to specific antigens. The cytotoxic drug component exerts a potent killing effect, effectively eliminating target cells. The linker acts as a bridge between the two, ensuring stable conjugation while maintaining stability in circulation and releasing the drug efficiently upon reaching target cells.

Fig. 1. Antibody-drug conjugates (ADCs).

The advent of bispecific antibodies has further expanded the application scope of ADCs. Unlike conventional monospecific antibodies, bispecific antibodies can simultaneously target two distinct antigenic sites. This unique structure allows bispecific ADCs to more precisely recognize and attack target cells, reducing off-target effects on normal cells, thereby enhancing therapeutic efficacy and minimizing side effects. Compared to traditional ADCs, the unique dual-epitope/dual-target binding mode of bispecific ADCs not only enables enhanced selectivity by binding to antigens co-expressed in solid tumors but also significantly improves internalization. This holds promise for overcoming current clinical challenges associated with ADCs, particularly issues related to poor internalization, off-target toxicity, and drug resistance.

ADC Products

Bispecific Antibody Therapy

Bispecific antibodies (BsAbs) are a class of antibodies capable of simultaneously binding two different antigens or two distinct epitopes on the same antigen. Compared to conventional monoclonal antibodies (mAbs), bispecific antibodies exhibit a unique "bispecificity," allowing them to recognize and bind to two distinct targets simultaneously. This characteristic grants bispecific antibodies significant advantages in treating complex diseases, particularly cancer.

• Bispecific Antibody Structure

The structures of bispecific antibodies are diverse, with common forms including IgG-like bispecific antibodies and bispecific antibody fragments. IgG-like bispecific antibodies retain the structural framework of traditional IgG antibodies but incorporate two different antigen-binding sites. This structural design enables bispecific antibodies to simultaneously bind two different antigens, thereby facilitating multiple therapeutic mechanisms. For example, bispecific antibodies can target both a tumor-specific antigen and an activation receptor on immune cells, guiding immune cells directly to tumor cells and enhancing their cytotoxic activity. Bispecific antibody fragments, on the other hand, are smaller antibody formats typically composed of two single-chain variable fragments (scFvs) linked by chemical linkers. This structure offers improved tissue penetration, allowing for more efficient entry into tumor tissues. However, due to their smaller molecular weight, bispecific antibody fragments exhibit lower in vivo stability, necessitating optimization of linkers and antibody fragment structures to enhance stability and bioavailability.

• Bispecific Antibody Mechanism of Action

Bispecific antibodies possess powerful targeting capabilities, with one of their core advantages being the ability to simultaneously bind two different antigens for precise therapeutic intervention. In cancer treatment, they can target both a tumor-specific antigen and an activation receptor on immune cells, directing immune cells to the vicinity of tumor cells, thereby enhancing immune recognition and attack on tumor cells. This effectively overcomes tumor heterogeneity and immune evasion. Moreover, bispecific antibodies play a unique role in immune activation by simultaneously binding tumor cells and immune cells, activating immune cell cytotoxic functions, and enhancing immune-mediated tumor cell killing. They can also activate other immune cells to elicit a synergistic immune response. Bispecific ADCs combine the targeting capability of bispecific antibodies with the potent cytotoxic effects of payload drugs. By leveraging the targeting function of bispecific antibodies, bispecific ADCs enable the precise delivery of cytotoxic drugs to tumor cells, enhancing therapeutic efficacy while reducing toxicity to normal tissues and minimizing treatment-related side effects.

Bispecific Antibody ADC

The structural design of bispecific ADCs endows them with unique therapeutic advantages. Currently, bispecific ADCs can be categorized into two major types: dual-epitope ADCs and dual-target ADCs. Dual-epitope ADCs refer to bispecific ADCs that target different epitopes of the same antigen, thereby improving receptor clustering and promoting rapid internalization of the target. In contrast, dual-target ADCs are designed to target two distinct targets, which can lead to enhanced lysosomal accumulation and payload delivery. Generally, bispecific ADCs consist of a bispecific antibody, a chemical linker, and a cytotoxic drug. The targeting capability of the bispecific antibody ensures precise drug delivery to tumor cells, while the potent cytotoxic drug exerts a significant tumor-killing effect. The chemical linker plays a crucial role in the structure of bispecific ADCs, as it must not only conjugate the bispecific antibody and the cytotoxic drug but also maintain stability in vivo to ensure drug safety and efficacy. Currently, researchers are developing novel linkers to enhance the stability and bioavailability of bispecific ADCs.

Bispecific ADC Mechanism of Action

The mechanism of action of bispecific ADCs involves targeted delivery and the release of cytotoxic drugs. In vivo, bispecific ADCs leverage the targeting ability of the bispecific antibody to precisely deliver the cytotoxic drug to tumor cells. Upon binding to specific antigens on the tumor cell surface, the cytotoxic drug is released, leading to a potent tumor-killing effect. This precise drug delivery mechanism not only enhances therapeutic efficacy but also minimizes toxicity to normal tissues, thereby reducing treatment-related side effects. Compared with traditional chemotherapeutic agents, bispecific ADCs exhibit significant advantages in both therapeutic effectiveness and safety. Despite their immense therapeutic potential, the development of bispecific ADCs faces multiple challenges. First, the stability of bispecific antibodies remains a critical issue. Due to the complex structure of bispecific antibodies, their stability in vivo is relatively low, making them prone to degradation and inactivation. Second, the drug payload of cytotoxic agents is a key consideration. The payload must balance drug efficacy while minimizing toxicity. Additionally, the design of the chemical linker is a crucial aspect. The linker must remain stable in vivo while ensuring efficient cytotoxic drug release upon reaching tumor cells. Currently, researchers are working to overcome these challenges by optimizing bispecific antibody structures, enhancing cytotoxic drug loading, and developing novel linkers.

Bispecific ADC Design

The known targets of bispecific ADCs are primarily HER2, EGFR, and c-MET. Each component of ADCs, including the antibody, linker, and payload, requires independent optimization, as even minor modifications to any of these key components can result in substantial changes to clinical characteristics. Therefore, when designing future bispecific ADCs, optimization of antibodies, linker-payload complexes, and conjugation strategies should be regarded as an interconnected network requiring a holistic approach.

• Bispecific Antibodies

The primary consideration in designing bispecific ADCs is the careful selection of appropriate target combinations. Target selection is fundamental to the successful development of ADCs, significantly impacting the therapeutic window and systemic toxicity. Given the common off-target toxicity and clinical resistance challenges faced by conventional ADCs, the following criteria guide target selection:

• Traditional target selection emphasizes favorable internalization properties, low expression in normal tissues, and high expression in tumors.
• Considering the heterogeneity of solid tumors, it is essential to determine target expression levels across various tumor subtypes and sites to facilitate tailored drug delivery rather than relying on a one-size-fits-all approach.
• Given the unique dual-targeting nature of bispecific ADCs, a comprehensive evaluation of the deep effects of antigen combinations is crucial. This includes factors such as internalization, recycling, turnover rate, lysosomal degradation, and intrinsic mechanisms. Integrating these factors is essential for the effective design of bispecific ADCs.

Additionally, one of the primary classification criteria for bispecific ADCs is whether they contain an Fc region. The design of Fc-free bispecific ADCs faces challenges such as low stability, aggregation issues, and a lack of conjugation sites. On the other hand, Fc-containing bispecific ADCs provide additional advantages, including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), immune phagocytosis, and cytokine release, all of which contribute to tumor cell killing. In summary, the strategies for Fc region construction include:

• Fc engineering modifications – Such as amino acid mutations and glycosylation modifications in the Fc region, which can help mitigate off-target toxicity associated with FcγR binding.
• Retention of ADCC and CDC – The dual-target binding mode promotes hexamer formation, enhancing ADCC and CDC effects and increasing tumor-killing efficiency.
• Preservation of FcRn binding or application of antibody engineering – This approach helps to improve the half-life and safety of bispecific ADCs.

• Linker

The linker in BsADCs is a crucial component that connects the antibody and the cytotoxic payload, playing a vital role in payload release and drug stability. An ideal linker should exhibit stability in plasma while enabling effective release within tumors. Currently, ADC linkers can be classified into cleavable and non-cleavable linkers.

• Non-cleavable linkers: Non-cleavable linkers exhibit high stability in plasma and can only be degraded in lysosomes to release the payload. This type of linker results in lower off-target toxicity, increased plasma half-life, and enhanced safety. However, potential resistance may arise due to obstacles in internalization and lysosomal trafficking. BsADCs based on non-cleavable linkers should focus on further optimizing internalization, endosomal trafficking, and lysosomal degradation.
• Cleavable linkers: Compared to non-cleavable linkers, ADCs with cleavable linkers have broader applications. The main challenge of cleavable linkers lies in off-target toxicity caused by non-specific release. To design lysosome-independent BsADCs, certain cleavable linkers, such as Val-Cit linkers, can effectively facilitate payload release in both early and late endosomes. Additionally, in recent years, non-internalizing ADCs have been proposed, where chemical or enzymatic cleavage is triggered extracellularly, targeting antigens in the tumor microenvironment (TME) and vasculature to activate the bystander killing effect.

• Payload

The cytotoxic payload largely determines the overall anti-tumor efficacy and potential adverse effects. Given the low permeability of ADCs, an ideal payload should exhibit high potency at nanomolar to picomolar levels. Additionally, these payloads should possess sufficient plasma stability, low immunogenicity, and appropriate water solubility. Finally, the payload should contain functional groups available for conjugation to the antibody. The bystander killing effect of payloads in BsADCs is a key aspect worth discussing. This effect refers to the ability of the payload to kill adjacent non-targeted cells after release. In terms of the pharmacokinetics/pharmacodynamics (PK/PD) of ADCs, this is a double-edged sword. While the bystander effect enhances the overall efficacy of ADCs in heterogeneous tumor environments, it also poses a risk of off-target cytotoxicity in normal tissues surrounding the tumor. This effect depends on cleavable linkers and hydrophobic payloads. If a target antigen, such as c-Met, is expressed at certain levels in normal tissues, BsADCs should avoid using payloads with bystander effects.

Clinical Trials of Bispecific ADCs

• BsADC Targeting HER2

• ZW49

ZW49 is based on Zanidatamab and employs an interchain disulfide cysteine and a protease-cleavable linker to conjugate an N-acyl sulfonamide auristatin payload, providing good tolerability. The bispecific nature of ZW49 enhances internalization, while its Fc region confers ADCC, ADCP, and CDC effects. This design addresses several unmet clinical needs in HER2-expressing patients. Preclinical data indicate that ZW49 exhibits potent tumor-killing effects and good tolerability without compromising HER2 affinity. ZW49 is currently undergoing a Phase I clinical trial. As of September 2022, disclosed clinical trial data show an objective response rate (ORR) of 31% in patients with advanced HER2-expressing solid tumors. However, ocular toxicity remains a concern, with keratitis observed in 42% of patients.

• MEDI4276

MEDI4276 is a tetravalent HER2-targeting ADC that fuses the scFv of trastuzumab with the N-terminal region of another anti-HER2 IgG1 antibody, 39S. While MEDI4276 demonstrated significant activity in mouse xenograft models of refractory HER2+ cancers, it failed to achieve an optimal efficacy-safety balance in clinical testing. In breast cancer patients, the overall ORR was low (9.4%), with the maximum tolerated dose (MTD) determined at 0.75 mg/kg every three weeks. Compared to ZW49, the lower MTD of MEDI4276 may be influenced by its valency, payload, and antibody configuration, indicating the need for further optimization.

• BsADC Targeting HER2

• BsADC Targeting CD63

CD63 is a member of the tetraspanin superfamily and exhibits broad, though not ubiquitous, expression. It is primarily localized on the cell surface, late endosomes, and lysosomes. The presence of CD63 in these cellular compartments makes it a potential target for BsADCs aimed at enhancing internalization and lysosomal trafficking, ultimately improving drug delivery and therapeutic efficacy. A BsAb targeting HER2×CD63 was generated by combining a low-affinity CD63-binding arm with another Fab arm derived from an anti-HER2 antibody. This design leverages antibody-dependent receptor cross-linking to enhance HER2 internalization and promote lysosomal co-localization. The HER2×CD63 BsAb was subsequently conjugated to the mitotic inhibitor payload duostatin-3 via a VC linker. However, insufficient efficacy was observed in tumors with low HER2 expression, suggesting the need for further optimization, including potential enhancement of the drug-to-antibody ratio (DAR) and addressing tumor heterogeneity.

• BsADC Targeting PRLR

Prolactin receptor (PRLR), an overexpressed target in malignant mammary epithelial cells, effectively mediates clathrin-dependent initial internalization and lysosomal trafficking through self-ubiquitination and recruitment of the AP2 complex. Using the knobs-into-holes (KIH) approach, a BsAb was designed with one arm targeting HER2 and the other targeting PRLR. The BsADC was conjugated to DM1 via a non-cleavable SMCC linker at an average DAR of 3.324 through surface lysine residues. Compared to highly expressed HER2, even low levels of PRLR expression on the cell surface were sufficient to drive constitutive internalization and subsequent lysosomal degradation. This suggests that even at low expression levels, high-turnover surface targets can mediate efficient internalization and lysosomal degradation, improving the efficacy of BsADCs.

• BsADC Targeting EGFR

EGFR is a member of the ERBB receptor tyrosine kinase family and plays a crucial role in regulating the fundamental functions of epithelial malignancies. However, due to acquired genomic alterations induced by therapeutic pressure, EGFR-targeting monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs) often lead to clinical resistance. BsADCs are expected to address mechanisms of resistance to anti-EGFR therapies, including sensitizing mutations and bypass pathway activation.

• BsADC Targeting Dual Epitopes of EGFR

To mitigate resistance, researchers have developed a bispecific antibody targeting two distinct epitopes of EGFR. This bispecific antibody was designed by fusing nanobodies specific to non-overlapping EGFR epitopes (9G8 and 7D12). Among them, 7D12 disrupts the EGFR signaling cascade, while 9G8 stabilizes the locked conformation of the EGFR extracellular domain (ECD), spatially preventing dimerization. Additionally, 7D12 and 9G8 exhibit efficacy against different EGFR-mutant cell lines, inducing a more effective CDC effect in NIH-3T3 cells expressing wild-type EGFR or cetuximab-resistant mutations.

• BsADCs Targeting Other EGFR Antigens

BL-B01D1 is the first bispecific ADC in China to enter Phase I clinical trials, targeting both EGFR and HER3. It employs a proprietary Ac linker, which exhibits better stability, higher hydrophilicity, and lower aggregation propensity compared to Mc linkers. The payload is a proprietary camptothecin analog, ED04. Its Phase I clinical trial demonstrated good safety, with no drug-related patient deaths. Among 10 evaluable late-line NSCLC patients with good safety profiles, the objective response rate (ORR) was 60%, and the disease control rate (DCR) was 90%. Several bispecific antibodies targeting c-MET and EGFR have been reported, demonstrating synergistic effects in inhibiting tumor proliferation and metastasis. In BsADC design, careful selection of appropriate epitope combinations is crucial to avoid complete or partial activation of c-MET. AZD9592, developed by AstraZeneca, is an EGFR/c-MET ADC that conjugates a novel topoisomerase I payload via a cleavable linker, primarily addressing resistance to osimertinib. Compared to EGFR, AZD9592 exhibits higher affinity for c-MET to reduce EGFR-driven normal tissue toxicity. In patient-derived xenograft (PDX) and resistant models, AZD9592, either as monotherapy or in combination with osimertinib, demonstrated promising antitumor activity.

• BsADC Targeting MET

The hepatocyte growth factor (HGF)-mesenchymal-epithelial transition factor (MET) pathway plays a crucial role in cancer development across various stages, from initiation to metastasis. Upregulation and amplification of MET are considered major escape mechanisms during anti-EGFR therapy. C-MET can cross-react with EGFR, leading to resistance to EGFR-targeted therapies, making MET inhibition a viable strategy to overcome EGFR resistance. Compared to conventional MET-targeted ADCs, the dual-epitope MET×MET BsADC design provides an innovative solution to existing challenges. Bispecific antibodies targeting MET can form a 2:2 antigen-antibody complex, facilitating effective MET internalization and lysosomal trafficking. By conjugating the maytansinoid payload M114 to surface lysine residues of MET-targeting BsAb via a protease-cleavable linker, REGN5093-M114 achieves a DAR of 3.12. Preclinical data indicate that REGN5093-M114 significantly inhibits the proliferation of MET-overexpressing NSCLC cells. A Phase I dose-escalation and dose-expansion study has been initiated to evaluate the safety and efficacy of REGN5093-M114 in adult patients with MET-overexpressing advanced cancers (NCT04982224).

In Conclusion

In summary, the emergence of unique bispecific targeting strategies has introduced new innovations in the ADC field, marking the advent of a new generation of ADCs. Although still in early development, BsADCs represent a highly promising novel approach. BsADCs play a crucial role in overcoming existing clinical challenges faced by conventional ADCs and in developing more precise targeted therapies. Beyond BsADCs, a range of next-generation strategies is poised to make groundbreaking contributions to novel ADC design. These include dual-payload ADCs, immunomodulatory ADCs, radionuclide ADCs, prodrug ADCs, ADC combination therapies, and peptide-drug conjugates. Future research should assess the application of these strategies in novel ADC designs, either individually or in combination, which will provide critical prospects for advancing this field.

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