Polyethylene Glycol in Antibody-Drug Conjugates

Polyethylene glycol (PEG) has emerged as a crucial component in the development of antibody-drug conjugates (ADCs), a novel class of therapeutics that offers targeted cancer treatment. This article provides an in-depth exploration of PEG, its role in ADCs, and the various types of PEG linkers utilized in these advanced drug delivery systems.

What is Polyethylene Glycol?

Polyethylene glycol (PEG) is a hydrophilic polymer composed of repetitive ethylene oxide units with linear or branched structure. Due to its structure, the molecular weight and other properties of PEG can be customized, which makes it a useful material for various applications, especially in the fields of biochemistry and medicine. Its excellent qualities, including high water solubility, biocompatibility and hydrophilicity, increase its practicality in the biological environment. PEG has good tolerance in biological systems because it is non-toxic and non-immunogenic, which makes it useful in drug compositions. In the context of ADCs, PEG linkers improve solubility, reduce immunogenicity, and enhance pharmacokinetic profiles. By addressing the challenges posed by hydrophobic drugs, PEG plays a critical role in the development of more effective and targeted therapeutic strategies.

Antibody Drug Conjugate

ADCs are novel therapeutic agents created especially for targeted cancer treatment, combining the cytotoxic potency of medications with the targeting power of monoclonal antibodies. A powerful cytotoxic medication, a monoclonal antibody, and a linker that joins the two make up an ADC. The linker's characteristics are crucial since they affect important factors like stability, pharmacokinetics, and overall therapeutic efficacy. There are numerous kinds of linkers, including as hydrophilic and hydrophobic linkers, and they all have varied effects on how well a medicine works. Currently, ADC linkers can be divided into:

Fig. 1. PEG linkers improve the antitumor efficacy and safety of drug conjugates (Int J Mol Sci. 2021, 22(4): 1540).

• Cleavable Linkers: These linkers are designed to release the drug in response to specific conditions within the tumor microenvironment, such as pH changes or enzymatic activity.
• Non-Cleavable Linkers: These linkers maintain their integrity throughout the circulation and are only cleaved after the ADC is internalized by the target cell.

What are PEG Linkers?

The PEG linker is a hydrophilic linker that combines the polyethylene glycol portion to enhance the solubility and stability of biomolecules, especially in the development of ADCs. These linkers play a crucial role in biological binding, helping to attach cytotoxic drugs to antibodies while minimizing immunogenic reactions and aggregation. Because of their unique properties, such as water solubility, biocompatibility, and the ability to form a three-dimensional shielding around the binding drug, PEG linkers are widely used in a variety of drug applications, including drug delivery systems, protein stability, bioconjugation, and biolabeling. Generally, PEG linkers can be divided into:

• Linear PEG Linkers: These linkers consist of straight chains of PEG and are commonly used for their simplicity and ease of synthesis.
• Branched PEG Linkers: These linkers feature a branched structure, allowing for multiple drug attachments and enhancing the drug-to-antibody ratio (DAR).
• Multi-arm PEG Linkers: These complex structures can accommodate several cytotoxic agents, improving the delivery capacity of ADCs.

PEG Linkers from BOC Sciences

PEG Linker for ADC Drugs

PEGs are widely used in many applications and industries as they are generally considered safe, biologically inert and very soluble in water. After synthetic activation by adding functional groups to one or both ends of the polymer, PEG can be conjugated to proteins, peptides or small molecules to produce PEGylated products. Monodisperse PEG is produced by purification of polydisperse PEG or specific synthesis of uniform PEG units to produce defined molecules. Using PEG as a linker between the antibody and the payload molecule can achieve higher loaded ADC species. PEG can create a shield that isolates the ADC payload from its microenvironment, thereby improving solubility and stability. Other benefits include reduced aggregation, thereby reducing immunogenicity, improved pharmacokinetics, increased circulation time and reduced toxicity, etc.

Addressing Solubility Challenges

The solubility and aggregation of cytotoxic payloads represent one of the primary challenges in the development of effective ADCs. High drug-to-antibody ratios (DAR) and increased lipophilicity can result in rapid clearance of the ADC from circulation, which narrows its therapeutic window and limits its effectiveness. PEG plays a vital role in overcoming these solubility challenges by forming a protective layer around the drug, effectively enhancing both solubility and stability.

Research has consistently shown that increasing the length of PEG chains can significantly improve the plasma concentration of ADCs, leading to better tolerability and enhanced efficacy. For instance, in a pivotal study by Burke et al., the use of maleimide-PEG-Glucuronide-MMAE with varying PEG lengths enabled the identification of an optimal PEG size to stabilize a fully conjugated ADC with a DAR of 8. The findings indicated that ADCs with longer PEG chains (8, 12, and 24 units) maintained plasma concentrations comparable to those of naked antibodies, while ADCs with shorter PEG chains or no PEG exhibited significantly higher clearance rates. This compelling evidence underscores the importance of PEG in enhancing the stability and overall therapeutic efficacy of ADCs.

Drug Linkers and the Incorporation of PEG

The choice of drug linker in ADC development is critical, as it can influence the overall stability, solubility, and therapeutic effectiveness of the conjugate. The incorporation of PEG linkers has proven to be a successful strategy for improving and controlling the solubility of cytotoxic agents. For example, linear PEG chains can serve both as spacers and solubilizing agents, facilitating the conjugation between the antibody and the drug. This flexibility allows researchers to fine-tune the properties of ADCs to optimize their performance.

Moreover, the use of branched PEGs is gaining traction in ADC constructs to enable higher DAR species. By incorporating branched PEG linkers, researchers can achieve a more favorable balance between drug loading capacity and stability. Numerous studies have validated the application of PEG linkers in ADC development, demonstrating their ability to enhance solubility and improve stability. By adjusting the molecular weight and structure of PEG, researchers can optimize the performance of ADCs, tailoring them to meet specific therapeutic needs.

Analytical Features of PEG

The synthesis and characterization of PEGs are critical to their functionality in ADC constructs. Monodisperse PEGs, which are composed of uniform molecular units, are preferred over polydisperse PEGs to ensure consistent performance and easier analytical characterization. The synthesis of monodisperse PEGs involves precise polymerization processes to produce defined molecular weights, which can be confirmed through techniques such as MALDI-TOF mass spectrometry. When incorporating PEGs into ADCs, factors such as PEG length and functional end groups (e.g., maleimide and azide) are meticulously considered to achieve optimal conjugation and performance. Additionally, the purity of monodisperse PEGs, usually targeted between 90-95%, is essential to meet cGMP requirements and regulatory standards for clinical applications.

Analytical Challenges and Solutions

While PEGs offer numerous benefits for ADC development, their analytical characterization poses certain challenges. The absence of double bonds in PEG molecules makes traditional UV light detectors in HPLC systems less effective. Instead, evaporative light scattering detectors (ELSD) and charged aerosol detectors (CAD) are commonly employed. However, both detectors have their limitations: ELSD may underestimate low-level signals, while CAD might overestimate them. Accurate analytical methods are crucial for distinguishing between pure PEG forms and impurities, which impact the final quality and efficacy of the ADC. To address these analytical challenges, researchers must employ a combination of detection techniques and thoroughly understand each detector's characteristics. For instance, gas chromatography can be used for volatile, smaller PEG units. The combination of advanced analytical technologies ensures that PEG-modified ADCs meet stringent purity and performance standards essential for their clinical success.

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Reference

1. Li, Q. et al. PEG Linker Improves Antitumor Efficacy and Safety of Affibody-Based Drug Conjugates. Int J Mol Sci. 2021, 22(4): 1540.