Antibody-drug conjugates (ADCs) consist of an antibody, a linker, and a cytotoxic payload. Compared to traditional chemotherapy, ADCs target tumors with the potential to reduce systemic toxicity, provide a broader therapeutic window, and offer a higher therapeutic index. Over the past decade, ADCs have matured in the treatment of solid tumors and hematologic malignancies, becoming a revolutionary field in anti-tumor drug research.
Although ADC development has achieved significant success, unexpected toxicities and resistance have posed substantial challenges. Various factors contribute to widespread clinical dilemmas, especially in solid tumors. These factors include (1) the heterogeneity of solid tumors limiting the clinical efficacy of targeting a single antigen; (2) treatment pressure inducing antigen downregulation, epitope mutations, and activation of bypass pathways, leading to significant ADC resistance; (3) off-target toxicity due to target antigen expression in normal tissues, linker instability, and other variables; (4) resistance to internalization of the target antigen hindering adequate therapeutic effects. Therefore, optimizing antibodies, linkers, and payloads is a challenge for the next generation of ADCs.
A forward-looking approach to addressing the above clinical challenges is to conjugate bispecific antibodies (BsAbs) with linker-payload complexes, resulting in bispecific antibody-drug conjugates (BsADCs). Compared to traditional ADCs, BsADCs’ unique dual-epitope/target binding mode not only enhances selectivity by binding to co-expressed antigens in solid tumors but also significantly improves internalization. These unique advantages make BsADCs an important force in the next generation of ADCs. Currently, at least 10 BsADCs are undergoing clinical trials. The design of BsADCs is not merely “1+1=2”; the change in binding mode affects overall efficacy, including comprehensive coordination and optimization of BsAbs, linkers, and payloads.
Design of BsADCs
The known targets of BsADCs are mainly focused on HER2, EGFR, and c-MET. Each component of ADCs, including antibodies, linkers, and payloads, requires independent optimization, and minor modifications to any of these key components can lead to substantial changes in clinical characteristics. Therefore, in designing future BsADCs, the optimization and conjugation strategies of antibody, linker-payload complexes should be viewed as interconnected networks, requiring a holistic approach.
Bispecific Antibodies
The primary consideration in constructing BsADCs is the wise selection of appropriate target combinations. Target selection is fundamental to the successful development of ADCs, critically impacting the final therapeutic window and systemic toxicity. Given the challenges of off-target toxicity and clinical resistance faced by traditional ADCs, the following criteria help guide target (target specific linkers ) selection:
(1) Traditional target selection focuses on good internalization characteristics, relatively low expression in normal tissues, and high expression in tumors. (2) Given the heterogeneity of solid tumors, it is essential to determine the expression levels of targets in various tumor subtypes and sites, facilitating the optimal implementation of tailored drug delivery rather than relying on a single “magic bullet.” (3) Due to the unique dual-targeting characteristics of BsADCs, comprehensive consideration of the deep effects of antigen combinations is crucial. This includes factors such as internalization, recycling, turnover rate, lysosomal degradation, and inherent mechanisms. Integrating these factors is essential for the effective design of BsADCs.
Additionally, one of the main classification criteria for BsADCs is whether they contain an Fc region. The design of BsADCs without an Fc region faces challenges such as low stability, aggregation issues, and lack of conjugation sites. On the other hand, BsADCs containing an Fc region bring additional advantages, such as ADCC, CDC, immune phagocytosis, and cytokine release, all contributing to tumor killing. In summary, Fc region construction strategies include (1) Fc engineering modifications, such as amino acid mutations and glycosylation modifications in the Fc region, which can help mitigate off-target toxicity caused by FcγR binding; (2) Retaining ADCC and CDC: the dual-target binding mode favors the formation of hexamers, enhancing ADCC and CDC effects, improving tumor killing; (3) Retaining FcRn binding or applying antibody engineering helps improve half-life and safety.
Linkers
Linkers in BsADCs are the critical connection between the antibody and the cytotoxic payload, playing a crucial role in payload release and drug stability. Ideal linkers should exhibit stability in plasma while facilitating effective release in tumors. Currently, linkers in ADCs can be divided into cleavable and non-cleavable linkers.
Linker related products at AxisPharm:
EY-CBS-A Linker | CAS: 50567-77-6
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 from obstacles in internalization and lysosomal transport. BsADCs based on non-cleavable linkers should focus on further optimizing internalization, subsequent endosomal transport, and lysosomal degradation.
Compared to non-cleavable linkers, ADCs based on 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 promote payload release in early and late endosomes. Additionally, non-internalizing ADCs have been proposed in recent years, where chemical or enzymatic cleavage triggers payload release extracellularly, targeting antigens in the TME and vascular system, and activating bystander-killing effects.
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Payloads
Cytotoxic payloads largely determine the overall anti-tumor effect and potential adverse reactions. Considering the low permeability of ADCs, ideal payloads for ADCs need to exhibit high potency at nanomolar to picomolar levels. Additionally, these payloads should have sufficient plasma stability, low immunogenicity, and appropriate water solubility. Finally, payloads should have available groups for conjugation with antibodies.
The bystander-killing effect of payloads in BsADCs is a key aspect worth discussing. The bystander killing effect refers to the ability of the payload to kill adjacent non-target cells after release. For the PK/PD characteristics of ADCs, this is a double-edged sword. While the bystander-killing effect can enhance the overall efficacy of ADCs in heterogeneous tumor environments, it also poses a risk of off-target killing in normal tissues surrounding the tumor. This effect depends on cleavable linkers and hydrophobic payloads. If one of the target antigens is expressed at certain levels in normal tissues (such as c-Met), BsADCs should avoid using payloads with bystander effects. In summary, the bystander-killing effect of BsADCs holds promise for overcoming tumor heterogeneity, tumor barriers, and poor internalization. However, potential safety issues related to off-target effects need careful consideration.
Furthermore, novel drug payloads are crucial for the development of BsADCs. Emerging payloads, such as PROTACs, ferroptosis inducers, oligonucleotides, etc., contribute to the expansion of drug choices in the BsADC field. The development of new drugs can significantly enrich the types of selective drugs in the BsADC field.
Targeting HER2 BsADC
ZW49
ZW49 is based on Zanidatamab, utilizing interchain disulfide cysteine and protease-cleavable linkers to conjugate N-acyl sulfonamide auristatin, providing good tolerance. The bispecific antibody nature of ZW49 aids in better internalization, and 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 strong tumor-killing effects and good tolerance without compromising HER2 affinity. Currently, ZW49 is undergoing Phase I clinical trials. As of September 2022, disclosed clinical trial data show an objective response rate (ORR) of 31% in patients with advanced HER2-expressing solid tumors, with notable ocular toxicity characteristics (keratitis at 42%).
MEDI4276
MEDI4276 is a tetravalent HER2-targeting ADC that fuses the scFv of trastuzumab with the N-terminus of another anti-HER2 IgG1 antibody, 39S. MEDI4276 demonstrated significant activity in mouse xenograft models of refractory HER2+ cancers but did not show a good efficacy-safety balance in clinical testing. In breast cancer patients, the overall ORR was low (9.4%), and the maximum tolerated dose (MTD) was determined to be 0.75 mg/kg every 3 weeks. Compared to the safety of ZW49, the lower MTD of MEDI4276 may be influenced by its valence, payload, and antibody configuration, indicating the need for further optimization.
Targeting HER2 & CD63 BsADC
CD63 is a member of the tetraspanin superfamily, exhibiting broad but 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, aiming to enhance internalization and lysosomal transport, ultimately improving drug delivery and therapeutic efficacy.
By combining a low-affinity mutant arm of CD63 with another Fab arm from a HER2 antibody, a HER2×CD63 BsAb was generated. This design utilizes antibody-dependent receptor cross-linking to enhance the effective internalization of HER2 and promote lysosomal co-localization. Subsequently, the Her2×CD63 BsAb was conjugated with the anti-mitotic payload duostatin-3 via a VC linker. However, the efficacy observed in low HER2 tumors was insufficient, indicating the need for further optimization, including potential enhancement of DAR and addressing tumor heterogeneity.
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Targeting HER2 & PRLR BsADC
Prolactin receptor (PRLR), as an overexpressed target in malignant breast epithelium, can effectively mediate clathrin-dependent initial internalization and lysosomal transport through self-ubiquitination and recruitment of the AP2 complex.
Using the “KIH” method, a BsAb with HER2 and PRLR arms was designed. The BsADC was conjugated with DM1 via a non-cleavable linker (SMCC) through surface lysine, with an average DAR of 3.324. Compared to highly expressed HER2, the relatively low expression of PRLR on the cell surface was sufficient to induce constitutive internalization and subsequent lysosomal degradation. This indicates that even with low expression, high-turnover surface targets can mediate good internalization and lysosomal degradation to enhance the efficacy of BsADCs.
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Targeting HER2 & APLP2 BsADC
The intracellular tail of APLP2 contains overlapping tyrosine-based NPXY and YXXæ motifs. After clathrin-mediated endocytosis, APLP2 can bind to AP-2, mediating effective internalization and directly leading to lysosomal degradation.
Targeting EGFR BsADC
EGFR is a member of the ERBB receptor tyrosine kinase family, playing a key role in regulating fundamental functions of epithelial malignancies. However, targeted EGFR monoclonal antibodies and TKIs often lead to clinical resistance due to treatment pressure-induced acquired genomic alterations. BsADCs hold promise in addressing anti-EGFR resistance mechanisms, including sensitizing mutations and activation of bypass pathways.
Targeting Dual Epitopes of EGFR BsADC
To mitigate the emergence of resistance, a bispecific antibody targeting two different epitopes of EGFR was developed. This bispecific antibody was designed by fusing nanobodies specific to non-overlapping epitopes (9G8 and 7D12) on EGFR.
Among them, 7D12 disrupts the EGFR signaling cascade, while 9G8 stabilizes the tethered conformation of EGFR-ECD, spatially preventing dimerization. Additionally, 7D12 and 9G8 exhibit efficacy against different EGFR mutant cell lines, inducing more effective CDC effects in NIH-3T3 cells expressing wild-type EGFR or cetuximab-resistant mutations.
Targeting EGFR and Other Antigens BsADCs
BL-B01D1 is the first bispecific ADC in China to enter Phase I clinical trials, targeting EGFR and HER3. The linker used is its proprietary Ac linker, which has better stability and hydrophilicity compared to the Mc linker, making it less prone to aggregation; the toxin is its proprietary camptothecin analog ED04. Its Phase I clinical drug safety is good, with no drug-related patient deaths. Among 10 evaluable end-line NSCLC patients with good safety, the ORR was 60%, and the DCR was 90%.
Currently, several BsAbs targeting c-MET and EGFR have been reported, demonstrating synergistic effects in inhibiting tumor proliferation and metastasis. In the design of BsADCs, careful selection of appropriate epitope combinations is crucial to avoid complete or partial perturbation of c-MET. AZD9592, developed by AstraZeneca, is an EGFR/c-Met ADC conjugated with a novel topoisomerase 1 payload via a cleavable linker, primarily addressing osimertinib resistance. Compared to EGFR, AZD9592 has a higher affinity for c-MET, aiming to reduce normal tissue toxicity driven by EGFR. In PDX and resistant models, single-agent or combination therapy with osimertinib showed good anti-tumor activity.
1231 is a MUC1/EGFR bispecific ADC developed by Sutro and Merck subsidiary EMD Serono, using site-specific conjugation technology with non-natural amino acids, conjugated with hemiasterlin derivatives (microtubule inhibitors) via a cleavable VC linker, with a DAR of 4. Preclinical studies showed strong anti-tumor activity in patient-derived xenograft models of ESCC and NSCLC.
Targeting MET BsADC
The hepatocyte growth factor (HGF)-mesenchymal-epithelial transition factor (MET) pathway plays a crucial role in the development of various types and stages of cancer, from initiation to metastasis. Upregulation and amplification of MET are considered major escape pathways during anti-EGFR therapy. C-MET can cross-react with EGFR, leading to resistance to EGFR-targeted therapies, making inhibition of C-MET a viable strategy to overcome EGFR resistance.
Compared to traditional MET-targeting ADCs, the design of dual-epitope MET×MET-BsADC offers an innovative solution to overcome existing challenges. MET bispecific antibodies have the ability to form 2:2 antigen-antibody complexes, promoting effective MET internalization and lysosomal transport. By conjugating the maytansinoid payload M114 via a protease-cleavable linker to the surface lysine of the MET-targeting BsAb, REGN5093-M114 produced a BsADC with 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).
Novel ADCs
BsADCs significantly expand the range of potential targets and scaffolds beyond traditional paradigms. In fact, BsADCs provide an effective means to convert non-internalizing antigens into internalizing antigens. For example, utilizing EphA2, characterized by rapid internalization, and activated leukocyte cell adhesion molecule (ALCAM) (a non-internalizing or slowly internalizing antigen) to generate BsAbs. Interestingly, when the cell surface ratio of EphA2 to ALCAM exceeds a threshold of 0.2, the bispecific antibody exhibits effective internalization. Conversely, when the ratio is below this threshold, internalization is hindered.
The MC-VC-pab-MMAF payload complex is site-specifically conjugated via cysteine residues. In the context of bispecific binding of BsADCs, the internalization effect can be significantly influenced by simultaneously targeting adjacent antigens, depending on their expression ratios. This capability broadens the range of antigen selection, allowing for the design of BsADCs with poor internalization types. This innovative approach provides a subtle strategy to enhance the internalization potential of certain antigens, contributing to the versatility and efficacy of BsADCs in targeting a broader range of tumor antigens.
The introduction of various new technologies has revolutionized the generation of BsADCs, presenting significant advantages. Another notable advancement is PEG-conjugated BsADCs (P-BsADCs), which not only ensure uniform conjugation but also exhibit high endocytosis efficiency, tissue penetration, and reduced toxicity due to their small molecular weight and absence of Fc fragments.
Know more about PEG:
What is the difference between ADC linker and PEG linker?
PEG related products:
Polyethylene Glycol PEG Linkers
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Additionally, ligand-induced transient binding of multiple antibody domains (LITE) is a cutting-edge technology that combines the half-life extension advantages of biologics with precise time control of small molecule-related activity. These innovative strategies hold great promise in minimizing off-target effects while achieving therapeutic efficacy, representing a significant step forward in the development of BsADC technology.
Challenges BsADCs Facing
BsADCs represent a new therapeutic category that combines the advantages of ADCs and BsAbs. However, challenges remain, primarily due to the complexity of solid tumors, including heterogeneity, histological barriers, and poor permeability. BsADC design strategies must be refined to overcome these challenges.
Broadening Antibody Frameworks
The current target selection for BsADCs remains somewhat limited, mainly focusing on HER2, c-MET, and EGFR. However, the bispecific strategy has the potential to expand the range of targets, including those with poor internalization or low expression. Considering the diversity of BsAbs types, further enriching the antigen selection for BsADCs can diversify anti-tumor mechanisms. Promising avenues include immune-modulatory BsADCs based on T-cell engagement or PD-L1 targeting BsAbs.
Eliminating Heterogeneous Conjugation
Constructing BsADCs through random chemical coupling of payloads based on functional groups carries the risk of heterogeneous conjugation. This heterogeneity can disrupt the bispecific binding mode and alter the physical and PK (PK Study Pricing List) characteristics of BsADCs. Site-specific conjugation strategies can produce BsADCs with uniform DAR, ensuring consistent therapeutic responses and improving drug delivery accuracy. Emphasizing site-specific conjugation is crucial, as the choice of conjugation sites significantly impacts ADC drug efficacy.
Clarifying Parameters Between Two Targets
The multivalent binding mode and parameters of BsADCs impose mutual constraints and dependencies. Design considerations between the two targets need careful examination, particularly regarding affinity magnitude, expression, and valence changes. The bispecific binding mode not only influences each other through valence but also affects overall internalization through cross-arm binding. Besides affinity, the expression threshold of antigens on the surface of cancer cell lines plays a key role in determining overall drug efficacy. Addressing these key points requires comprehensive screening of combinations of binding arms with different affinities to achieve optimal bioactivity.
Transport
BsAbs can navigate by using the initial specificity of the BsAb as a transport mode for the second specificity. One example is the BsAb platform utilizing low-affinity transferrin receptors to carry β-secretase antibodies, promoting effective passage through the blood-brain barrier. BsADCs targeting CD63, PRLR, and APLP2 have been reported to show auxiliary transport capabilities. Based on these strategies, designing BsADCs is expected to proficiently cross biological barriers and evade lysosomal degradation.
Addressing Potential Safety Issues
While BsADCs aim to enhance specificity, thereby reducing off-target toxicity and side effects, early clinical data from examples like ZW49, MEDI4276, and BL-B01D1 indicate that their clinical safety is not as expected. Off-target toxicity is a major characteristic of ADC-related toxicity. However, relying solely on BsAbs may not sufficiently address the reduction of off-target toxicity. In addition to antibody innovation, critical research around linker-payload complexes is also essential, including linker stability, homogeneous conjugation strategies, DAR, and bystander-killing effects.
In conclusion, the emergence of unique bispecific targeting modes has brought new innovations to the ADC field, marking the birth of a new generation of ADCs. Although still in the early stages of development, BsADCs offer a promising new approach. BsADCs are significant for overcoming existing clinical challenges faced by traditional ADCs and for developing more precise targeted drugs.
In addition to BsADCs, a series of next-generation strategies are expected to make groundbreaking contributions to the design of new ADCs. These include ADCs with dual payloads, immune-modulatory ADCs, radionuclide ADCs, precursor ADCs, ADC combination therapies, and peptide-drug conjugates. Future evaluations of these strategies in the design of new ADCs, whether individually or in combination, will provide important prospects for advancing the field.
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Reference:
1. Bispecific antibody-drug conjugates: Making 1+1>2. Acta Pharmaceutica Sinica B. 20 January 2024