Polyethylene Glycol in Antibody-Oligonucleotide Conjugates

Polyethylene glycol (PEG), a polymer known for its high biocompatibility and chemical stability, plays a critical role in the design and application of antibody-oligonucleotide conjugates (AOCs). AOCs combine the targeted recognition ability of antibodies with the gene-regulatory functions of oligonucleotides, emerging as a cutting-edge technology for treating cancer, genetic disorders, and other complex diseases. However, AOCs face challenges in practical applications, including low delivery efficiency, suboptimal pharmacokinetics, and high immunogenicity. PEG offers solutions through its unique structure and properties, significantly enhancing the solubility, stability, and circulation time of the conjugates. Furthermore, PEG serves as a critical linker between the antibody and the oligonucleotide, optimizing the spatial configuration of the carrier and regulating the precision and rate of drug release.

Oligonucleotides Meaning

Oligonucleotides are short DNA or RNA oligomers, typically 20 to 60 units in length, which naturally exist in single-stranded (ss) or double-stranded (ds) forms. They can modulate protein function by targeting gene expression, thus achieving therapeutic effects. Following the advent of small-molecule drugs and monoclonal antibodies, oligonucleotide therapeutics have rapidly advanced due to their unique mechanism of regulating disease gene transcription and translation at the RNA level. This positions them to address rare, refractory, and previously untreatable diseases. Currently, oligonucleotides have been developed as gene-targeting therapeutic agents for treating viruses, tumors, and genetic disorders.

Oligonucleotide Therapeutics

Oligonucleotide therapeutics are treatments based on oligonucleotides that regulate gene expression by targeting specific genes or RNA molecules. In recent years, these therapies have shown tremendous potential in addressing rare diseases, cancers, autoimmune disorders, and infectious diseases. Oligonucleotide drugs consist of artificially synthesized single- or double-stranded ribonucleotides, typically 12-30 nucleotides long, which act on target messenger RNA (mRNA) via Watson-Crick base pairing. As a novel class of therapeutic molecules, oligonucleotide drugs are polar, charged, and require chemical modifications and delivery systems to improve drug-like properties, distinguishing them from small-molecule drugs. The primary categories of oligonucleotide drugs include:

• Antisense Oligonucleotides (ASOs): These drugs bind specifically to target mRNA, blocking its translation and inhibiting the production of disease-associated proteins. ASOs can also promote the degradation of specific mRNA or alter splicing patterns to generate alternative protein forms. For example, Nusinersen, used for spinal muscular atrophy (SMA), modifies the splicing of the SMN2 gene to restore protein functionality.
• Small Interfering RNA (siRNA): siRNA induces mRNA degradation through the RNA interference (RNAi) pathway, thereby halting protein expression. siRNA consists of double-stranded structures recognized by the intracellular RISC complex, which facilitates the degradation of complementary mRNA. For example, the FDA-approved drug Patisiran utilizes siRNA to suppress transthyretin (TTR) protein production for the treatment of hereditary TTR amyloidosis.
• MicroRNA (miRNA) and Mimics: miRNAs are small non-coding RNA molecules that regulate gene expression by affecting mRNA stability and translation. miRNA mimics and inhibitors are used to replicate or suppress the biological functions of specific miRNAs.
• Aptamers: These are single-stranded nucleic acid molecules selected through in vitro chemical evolution techniques to bind specifically to target proteins or other molecules. Aptamers can inhibit the activity of target proteins or serve as recognition elements for specific molecules.

Antibody Oligonucleotide Conjugates

Antibody-oligonucleotide conjugates are synthetic bioconjugates composed of monoclonal antibodies and oligonucleotides. They combine the dual properties of antibodies and nucleic acids—offering the precise targeting capability and favorable biodistribution characteristics of antibodies, along with the functional and structural versatility of oligonucleotides. Antibodies in AOCs provide targeting specificity, allowing precise localization to specific cells or tissues. Oligonucleotides contribute therapeutic mechanisms by modulating gene expression, enabling cellular functions or protein synthesis. In essence, AOCs are drugs that regulate specific gene expression by targeting RNA or DNA, thereby interfering with disease-related gene activity. This combination enhances efficacy, minimizes side effects, and offers potential for treating or curing challenging diseases. Currently, AOCs are being explored for indications such as muscular diseases, central nervous system disorders, and cancers. Typically, AOCs consist of three key components:

Fig. 1. Antibody-oligonucleotide conjugates (J Med Chem. 2024, 67(17): 14868-14884).

Antibodies

Antibodies are the core component of AOCs, responsible for recognizing and binding to specific surface antigens on target cells. The selection of antibodies directly determines the targeting specificity of AOCs. Commonly used antibody types include monoclonal antibodies and antibody fragments, such as Fab fragments or single-chain antibodies.

Oligonucleotides

Oligonucleotide molecules provide AOCs with therapeutic functionality. They can be antisense oligonucleotides (ASOs), small interfering RNA (siRNA), or aptamers. Oligonucleotides interact with target RNA or DNA within cells to regulate gene expression or capture target molecules.

Linkers

Linkers are the chemical bridges that conjugate antibodies and oligonucleotides in AOCs. Their design plays a critical role in the stability, delivery efficiency, and controlled release of AOCs at the target site. Linkers can be either cleavable (e.g., enzyme-sensitive or reduction-sensitive linkers) or non-cleavable, depending on the therapeutic application requirements.

Oligonucleotide Linkers

Linkers serve as critical chemical bridges in AOCs, enabling the stable conjugation of antibodies and oligonucleotides while preserving their biological functions. An ideal linker design must fulfill several criteria, including biocompatibility, stability, and efficient release capability within target cells. Linkers are generally categorized into cleavable and non-cleavable types. Cleavable linkers release oligonucleotides precisely in response to acidic, enzymatic, or reductive conditions, while non-cleavable linkers provide enhanced stability during circulation. The chemical structure of the linker must ensure controlled and consistent conjugation while avoiding interference with the activity of either the antibody or oligonucleotide. In recent years, linkers such as PEG derivatives, disulfide bonds, and ester bonds have gained widespread application due to their excellent physicochemical properties and biocompatibility. Optimizing linkers has not only improved the pharmacokinetic properties of AOCs but also laid a solid foundation for precision therapeutics.

Antibody Oligonucleotide Conjugates Preparation Protocol

The preparation of AOCs is a complex and highly precise process involving the chemical modification of antibodies and oligonucleotides and the selection of appropriate conjugation methods. First, antibody modification ensures effective linkage with oligonucleotides while preserving the biological activity of the antibody. Second, chemical modifications of oligonucleotides enhance their stability and improve their conjugation efficiency with antibodies. Furthermore, optimizing the conjugation process directly affects the purity and functionality of the final product. The successful implementation of this process is crucial for advancing precision therapies, particularly in the treatment of cancer, genetic disorders, and other complex diseases. Broadly, AOCs preparation methods can be divided into non-covalent and covalent conjugation techniques.

Non-Covalent Conjugation

Streptavidin-Biotin System

This is one of the earliest methods for generating AOCs. It leverages the strong non-covalent interaction between streptavidin and biotin. In this approach, antibodies are first biotinylated, and the biotinylated monoclonal antibodies are then conjugated with streptavidin. The streptavidin-biotin complex can further bind with biotinylated oligonucleotides. Since streptavidin has four biotin-binding sites, up to three oligonucleotides can be conjugated to a single antibody via this method.

Protein A and Protein G

Amine-modified oligonucleotides react with activated ester-maleimide crosslinkers to generate maleimide-labeled oligonucleotides. These oligonucleotides then conjugate with cysteine-labeled Protein A or Protein G, forming oligonucleotide-modified proteins. These modified proteins exhibit high affinity for the Fc region of antibodies, allowing them to bind monoclonal antibodies and generate the desired AOCs.

Covalent Conjugation

Covalent conjugation relies on chemical methods to link monoclonal antibodies and oligonucleotides. Amino acids commonly used for oligonucleotide attachment on antibodies include lysine (Lys), reducible cysteine (Cys), tyrosine (Tyr), and arginine (Arg). Oligonucleotides can be chemically linked to free thiols or amines, or transformed into more reactive aldehyde structures for conjugation.

Amine-Thiol Conjugation

This is one of the most widely used methods for antibody-oligonucleotide conjugation. It employs heterobifunctional amine-thiol crosslinkers for chemical conjugation. The process involves modifying antibodies into maleimide-activated substrates, which are then mixed with thiol-modified oligonucleotides. Raising the reaction buffer's pH initiates the conjugation. This approach is versatile and suitable for conjugating any oligonucleotide with monoclonal antibodies. However, it may produce heterogeneous mixtures. Derivatives of this method include using photo-cleavable bifunctional maleimide-NHS crosslinkers to create photo-cleavable DNA barcode oligonucleotide-antibody conjugates. Upon UV light exposure, these DNA barcodes can be released for separation, amplification, and detection after binding to targets.

Antibody Thiolation Conjugation

This method uses N-succinimidyl S-acetylthioacetate (SATA) to introduce protected thiol groups into antibodies. DNA fragments are modified with SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate), producing maleimide-labeled DNA. The thiolation process is completed by adding hydroxylamine to SATA-modified antibodies to release free thiols, which can then react with activated DNA-maleimide fragments to achieve conjugation.

Hydrazone Conjugation

In this method, antibodies react with succinimidyl-4-hydrazinonicotinate acetone hydrazone (S-HyNic) to introduce nucleophilic hydrazine groups into the antibody. Oligonucleotides are treated with succinimidyl-4-formylbenzoate (S-4FB) to introduce electrophilic aldehyde groups into their structure. Once purified, the two components react to form hydrazone-linked antibody-oligonucleotide conjugates.

Photo-Crosslinking

Apart from chemical methods, UV light-mediated conjugation can also link monoclonal antibodies and oligonucleotides. This method uses Fc-binding peptides (FcBPs), which specifically recognize and non-covalently bind to the Fc region of IgG. Under UV light, FcBPs with photoactivatable groups covalently bond with antibodies. Various photoactivatable groups, including benzophenone or maleimide-benzophenone (MBP), benzoylbenzoic acid, para-benzoylphenylalanine (BPA), photoleucine (pLeu), and photomethionine (pMet), have been successfully integrated into FcBPs. Photo-crosslinking is a simple and gentle method for conjugating oligonucleotides with commercially available antibodies. UV-induced crosslinking enhances conjugation efficiency. The major advantage of this approach is its high specificity. However, it may produce isomeric AOCs with one or two labels, which could pose challenges.

Antibody Oligonucleotide Conjugates in Clinical Trials

AOCs are rapidly emerging as a promising area for the development of precision medicines. These trials aim to evaluate the safety and efficacy of AOCs in treating cancer, genetic disorders, and infectious diseases. Early clinical studies demonstrate that AOCs can leverage the high specificity of antibodies to deliver therapeutic oligonucleotides to specific cell types or tissues, significantly improving targeting accuracy while reducing off-target effects. For instance, AOCs targeting HER2-positive breast cancer are under clinical evaluation, with preliminary data showing efficient inhibition of related oncogenes. Additionally, clinical studies on AOCs for genetic disorders aim to correct gene function defects by delivering antisense oligonucleotides. While results are encouraging, challenges such as delivery efficiency, immune response, and long-term safety remain. With advancements in technology and ongoing clinical trials, AOCs hold the potential to provide more effective treatments, driving the progress of personalized medicine.

DYNE-101

DYNE-101, developed by Dyne Therapeutics, is an AOC targeting myotonic dystrophy type 1 (DM1) and is currently in a global Phase I/II ACHIEVE clinical trial. DM1 is a rare, progressive genetic disorder affecting skeletal, cardiac, and smooth muscles. DYNE-101 comprises an ASO conjugated to an engineered antibody fragment (Fab) that binds to transferrin receptor 1 (TfR1), which is highly expressed on muscle cells. This design allows targeted delivery of the payload to skeletal, cardiac, and smooth muscles. Compared to monoclonal antibodies (mAbs), Fab offers enhanced tissue penetration, improved tolerability, and reduced immune activation risks. DYNE-101 reduces toxic nuclear DMPK RNA, freeing splicing proteins and allowing normal mRNA processing and protein translation, potentially halting or reversing disease progression. The FDA has granted DYNE-101 Orphan Drug Designation.

DYNE-251

Another AOC candidate from Dyne's pipeline, DYNE-251, targets Duchenne muscular dystrophy (DMD) and is in a Phase I/II DELIVER study. DYNE-251 is an innovative therapy comprising phosphorodiamidate morpholino oligomer (PMO) conjugated to an antibody fragment (Fab). The Fab specifically binds to TfR1, facilitating targeted muscle tissue delivery and promoting exon skipping within nuclei. This mechanism enables muscle cells to produce truncated but functional dystrophin proteins, aiming to halt or reverse DMD progression. Recognizing its innovation and potential, the FDA has granted DYNE-251 Fast Track, Orphan Drug, and Rare Pediatric Disease designations for treating DMD mutations addressable by exon 51 skipping.

AOC 1001

AOC 1001, developed using Avidity Biosciences' AOC platform, comprises a monoclonal antibody targeting TfR1 conjugated to a small interfering RNA (siRNA) aimed at DMPK mRNA. It is designed to reduce abnormal DMPK mRNA levels in fibroblasts of DM1 patients, addressing the root cause of the disease. Preclinical studies demonstrate that AOC 1001 successfully delivers siRNA to muscle cells, resulting in sustained, dose-dependent reductions of DMPK mRNA across various muscle types, including skeletal, cardiac, and smooth muscles.

AOC 1020

AOC 1020, also developed by Avidity Biosciences, combines a proprietary monoclonal antibody targeting TfR1 with siRNA targeting DUX4 mRNA. It aims to reduce the expression of DUX4 mRNA and protein in the muscles of patients with facioscapulohumeral muscular dystrophy (FSHD). AOC 1020 is the first investigational AOC therapy addressing the root cause of FSHD. The FDA and EMA have granted Orphan Drug Designation, and the FDA has also granted Fast Track Designation. Preclinical studies show that intravenous administration of AOC 1020 prevents muscle weakness through functional assays such as treadmill running, in vivo strength, and compound muscle action potential.

TAC-001

TAC-001 is a lead candidate from Tallac's Toll-like Receptor Agonist Antibody Conjugate (TRAAC) platform. In preclinical studies, TAC-001 induces innate and adaptive immune responses, demonstrating potent antitumor activity. It is a systemically administered TRAAC molecule combining a potent TLR9 agonist with a CD22 antibody to selectively activate B cells and drive antitumor immunity. In July 2022, Tallac announced the initiation of a Phase I/II study for TAC-001 in patients with advanced solid tumors. By November 2023, Phase I clinical safety and efficacy data demonstrated that TAC-001, when combined with cancer vaccines, promoted robust and durable vaccine-specific IgG titers, restored vaccine responses, and enhanced vaccine-specific T-cell activation and cytotoxic activity. Phase I/II clinical studies are ongoing.

DNL310

DNL310, the lead candidate from Denali's pipeline, is a fusion protein combining human iduronate-2-sulfatase (IDS) with Denali's enzyme transport vehicle. It is being developed to treat mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome. Phase I/II trial results published in February showed that DNL310 reduced heparan sulfate levels in cerebrospinal fluid to normal levels in patients. Behavioral adaptability and cognitive function scores also indicated potential benefits. DNL310 has entered Phase III clinical trials, with Denali seeking accelerated approval.

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