In recent years, the development of various forms of conjugated drugs has flourished, with antibody-drug conjugates (ADCs) leading the way in achieving significant success in cancer treatment. ADCs are a targeted therapy platform that combines monoclonal antibodies with cytotoxic drugs, designed to specifically recognize tumor cells through antibodies and deliver drugs precisely to the lesion site. SPDB (N-Succinimidyl 4-(2-pyridyldithio)butyrate) is a bifunctional chemical linker that facilitates stable drug conjugation and targeted release through its unique disulfide bond structure. Owing to its reduction sensitivity, stability, and broad chemical compatibility, SPDB has been widely employed in ADC research.

ADC Linkers

Linkers are not only the molecules establishing covalent connections between antibodies and payloads but also critical considerations in the design of targeted drugs. Linkers should not induce aggregation, ensure acceptable pharmacokinetic (PK) properties of the conjugate, possess appropriate stability to prevent premature payload release in plasma, and enable effective release of the active molecule at the target site. Structurally, linkers are categorized into non-cleavable and cleavable linkers.

Fig. 1. Structure of ADC (Molecules. 2020, 25(20): 4764).

• Non-Cleavable Linkers

ADCs with non-cleavable linkers must undergo internalization, with the antibody component degraded by lysosomal proteases to release the toxic payload. Many non-cleavable linkers have been explored in ADC development, the most representative being N-Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), used in trastuzumab emtansine. The primary metabolite of this conjugate after decomposition is Lys-SMC-DM1. Furthermore, drugs linked to such linkers generally cannot exhibit a bystander effect due to the poor membrane permeability of the metabolites. Currently, most ADC research focuses on cleavable linkers.

• Cleavable Linkers

Cleavable linker designs are applicable to both internalizing and non-internalizing ADCs, as payload release is triggered by the nature of the cleavage site, whether within the lysosome or the tumor microenvironment. Cleavable linkers are broadly categorized into enzyme-sensitive and chemically-sensitive (or non-enzyme-sensitive) types. Enzyme-sensitive linkers rely on specific enzymes present in the tumor microenvironment to achieve cleavage. For example, peptide-based linkers can be selectively cleaved by proteases, such as cathepsin B, which are overexpressed in tumor cells, thereby releasing the drug. The design of such linkers often involves optimizing peptide sequences to enhance enzymatic efficiency and tumor specificity. Chemically-sensitive linkers, on the other hand, exploit the unique chemical characteristics of the tumor microenvironment, such as reductive or acidic conditions, to enable cleavage. For instance, disulfide-based linkers are cleaved in the presence of high glutathione concentrations within tumor cells, while acid-sensitive linkers break down in low-pH environments, releasing the active payload. These cleavable linkers allow precise drug release while minimizing off-target toxicity, significantly improving the therapeutic efficacy and safety of ADCs.

ADC Linker Design

In the early stages of ADC drug development, the importance of linker design was often underestimated, as it was initially believed that simply attaching a cytotoxin to an antibody would suffice for ADC creation. However, with the evolution of ADC development and lessons learned from clinical successes and failures, it has become increasingly evident that linkers are critical elements in ADC design for at least two reasons: first, linkers can be structurally modified to optimize the therapeutic index (TI); second, linkers must ensure the precise delivery of the correct amount of cytotoxin to the target cells. To develop a successful ADC, it is essential to identify the optimal combination of antibody, linker, and payload, as different cancer treatment targets require distinct linker structures. The structural elements of linkers, including the conjugation site, release mechanism, and solubilizing components, must be carefully designed to regulate the functionality, safety, and manufacturability of the ADC, thereby establishing an appropriate target product profile. The following summarizes the key considerations in ADC linker design:

• Selection of Release Mechanism: Linkers should employ a release mechanism aligned with the characteristics of the tumor microenvironment (TME) or target cells. Cleavable linkers (e.g., disulfide or enzyme-sensitive linkers) enable payload release in response to the reductive conditions of the TME or specific enzymatic activity, enhancing therapeutic efficacy. Non-cleavable linkers, in contrast, strictly release the payload within target cells, minimizing bystander effects and ensuring precise targeting.
• Regulating Bystander Effects: Bystander effects occur when cleavable linkers release diffusible payloads that can kill neighboring antigen-negative cells, a critical feature for treating heterogeneous tumors. Non-cleavable linkers suppress this effect, focusing drug action on target cells and reducing off-target toxicity.
• Hydrophobicity and Hydrophilicity Balance: The hydrophobicity of linkers significantly influences ADC stability and pharmacokinetics (PK). Excessive hydrophobicity may cause ADC aggregation and rapid clearance. Incorporating hydrophilic groups (e.g., sulfonate or PEG segments) can enhance solubility, improve PK performance, and boost in vivo efficacy.
• Optimizing Drug-to-Antibody Ratio (DAR): The ideal DAR is critical for balancing ADC efficacy and safety. High DAR often correlates with poor PK and increased toxicity, while low DAR may result in insufficient therapeutic activity. The optimal DAR should be determined based on the payload type and target antigen expression. For instance, the Brentuximab ADC demonstrates peak performance at DAR4.
• Controlling Linker Stability: Linkers must be stable in circulation while ensuring efficient payload release under specific conditions. Stable linkers, such as enzyme-sensitive ones, release payloads precisely within target cells, whereas easily hydrolyzable linkers are suited for applications requiring stronger bystander effects.
• Strategies to Enhance PK and In Vivo Efficacy: Introducing hydrophilic linkers or amphiphilic polymers like PEG can significantly improve ADC PK by reducing renal and hepatic clearance rates, thereby extending the half-life. Linkers combined with functional units like β-glucuronidase can further enhance efficacy.
• Diversity and Customization of Linker Designs: Tailored linker designs are necessary to maximize efficacy based on payload properties, target antigen expression levels, and tumor microenvironment characteristics. For example, HydraSpace technology incorporates polar spacer units to enhance solubility and PK, offering personalized therapeutic solutions.
• Synergistic Design of Antibodies and Linkers: The interplay between antibody structure and linker design must account for specificity, affinity, and compatibility with payload release. The natural shielding effect of antibodies can modulate linker hydrophobicity, reducing aggregation and clearance while optimizing efficacy and safety.

What is SPDB Linker?

SPDB (N-Succinimidyl 4-(2-pyridyldithio)butyrate) is a widely used bifunctional chemical linker commonly employed in the preparation of ADCs. As a cleavable linker, SPDB enables drug payload release through reduction mechanisms in the tumor microenvironment, achieving precise drug delivery. In ADCs, SPDB not only ensures the stable conjugation of the antibody and drug but also facilitates targeted drug release under specific conditions, making it a critical component of modern ADC technology. SPDB is composed of several key chemical modules, designed to balance stability and controllability. Firstly, it features a succinimidyl ester (NHS ester) group that reacts with lysine residues on the antibody to form a stable amide bond, ensuring robust attachment to the antibody. Secondly, its central structure includes a butyl chain, providing molecular flexibility and minimizing steric hindrance between the antibody and drug. The core functional unit is a disulfide bond (pyridyldithiol), which is readily cleaved in a reductive environment to release the drug payload. Finally, the pyridylthio group released upon cleavage allows for monitoring of the drug release process.

SPDB Linkers from BOC Sciences

Advantages of SPDB Linker

The SPDB linker, as an advanced design, offers several significant advantages in ADC development:

• Reduction Sensitivity: The disulfide bond in SPDB is highly susceptible to cleavage in reductive environments. This characteristic allows selective drug release in the reductive microenvironment of tumor cells, enhancing therapeutic specificity while minimizing systemic toxicity.
• Stability and Controllability: SPDB exhibits exceptional stability under non-reductive conditions in blood circulation, preventing nonspecific drug release and protecting healthy tissues from toxic effects.
• Broad Chemical Compatibility: The structural design of SPDB enables its efficient conjugation with various antibodies and small-molecule drugs without the need for complex chemical modifications.
• Multifunctionality: Beyond conventional drug delivery, SPDB supports multi-drug conjugation or dual-functional ADC designs, thereby enhancing therapeutic outcomes.

SPDB Linker in ADC

The SPDB linker holds a pivotal role in ADC applications, especially in designs utilizing reduction-sensitive release mechanisms. A notable example is the ADC huC242-SPDB-DM4 (IMGN242) reported by Kellogg et al. in 2011. In this design, SPDB acts as the linker connecting the antibody huC242 to the cytotoxic drug DM4. SPDB uses its disulfide bond to attach DM4, while spatial hindrance around the disulfide bond is optimized to ensure stable and precise drug release. During ADC delivery, huC242-SPDB-DM4 transports the drug to tumor cells via the antibody's targeting capability. Subsequently, protease-mediated cleavage removes the antibody portion, and the disulfide bond in SPDB cleaves within the highly reductive tumor cell environment. This releases the active drug DM4, triggering a cytotoxic response. The reduction-sensitive release mechanism enabled by the SPDB linker not only improves drug delivery efficiency and therapeutic efficacy but also significantly reduces off-target toxicity in healthy tissues. The successful application of the SPDB linker in ADCs lays a strong foundation for the development of highly efficient and safe anti-tumor therapies in the future.

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