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Antibody-Oligonucleotide Conjugates: Precision Targeting for Therapeutics & Diagnostics
Biointron2025-03-24Read time: 9 mins
DOI: 10.1016/j.phrs.2024.107469
Introduction to Antibody-Oligonucleotide Conjugates (AOCs)
The increasing prevalence of cancer and the need for precise treatment modalities have accelerated the development of targeted therapies. Among these, antibody-oligonucleotide conjugates (AOCs) represent a convergence of two established technologies: monoclonal antibodies (mAbs) for cellular targeting and oligonucleotides for gene modulation. AOCs build on the framework of antibody-drug conjugates (ADCs), extending therapeutic utility beyond cytotoxic payloads by enabling gene-level regulation of disease-associated proteins.
AOCs are engineered to selectively deliver oligonucleotide therapeutics, such as siRNAs, antisense oligonucleotides (ASOs), or aptamers, to disease-relevant cells via antibody-mediated targeting. This dual functionality allows for modulation of gene expression while maintaining spatial specificity, offering significant advantages in oncology, rare genetic diseases, and neurological disorders. Moreover, their use in diagnostics through platforms such as immuno-PCR and proximity ligation assays further extends their utility in precision medicine. An article by Jiao et al. (2024) describes this well.
Structural Design and Chemistry of AOCs
AOCs are composed of three primary components: the antibody, the oligonucleotide payload, and the linker that connects the two. The design of these components impacts therapeutic efficacy, stability, and manufacturability.
Antibody Component Monoclonal antibodies used in AOCs are selected for their high affinity to disease-specific antigens. Full-length IgGs, Fabs, single-chain variable fragments (scFvs), and nanobodies have all been explored. The use of antibody fragments can enhance tissue penetration and reduce immunogenicity, especially in solid tumors or central nervous system applications.
Oligonucleotide Component The payload includes siRNA, ASO, or other modified nucleic acids. These oligonucleotides can silence gene expression via RNA interference or modulate splicing and translation through steric hindrance. Chemical modifications such as phosphorothioate backbones and 2′-O-methyl substitutions improve serum stability and cellular uptake while reducing immunogenicity.
Linker Chemistry Linkers can be cleavable or non-cleavable. Cleavable linkers respond to environmental triggers (e.g., pH, redox conditions, or enzymatic activity), allowing payload release in targeted cells. Non-cleavable linkers rely on lysosomal degradation post-internalization. The choice of linker affects drug-to-antibody ratio (DAR), pharmacokinetics, and therapeutic index.
Site-Specific vs. Random Conjugation Random conjugation (e.g., through lysine residues) leads to heterogeneous products, whereas site-specific methods such as THIOMAB engineering or click chemistry enable defined stoichiometry and better reproducibility.
Cancer Therapy AOCs have demonstrated significant potential in tumor-specific gene silencing. Antibody-siRNA conjugates targeting EGFR, HER2, PD-L1, and CD22 have been employed in multiple cancer models. These constructs facilitate RNA interference at target sites, inhibiting oncogene expression and overcoming resistance to small-molecule inhibitors.
For example, a Her2(scFv)-protamine-siRNA complex targeting PLK1 reduced tumor volume in HER2+ breast cancer xenografts. Similarly, an anti-EGFR antibody conjugated with KRAS-siRNA via a protamine linker halted tumor growth in colorectal cancer models resistant to cetuximab.
Neuromuscular and Rare Genetic Disorders AOCs are being clinically tested for myotonic dystrophy (DM1), facioscapulohumeral muscular dystrophy (FSHD), and Duchenne muscular dystrophy (DMD). AOC1001, targeting transferrin receptor 1 (TfR1) for delivery of DMPK siRNA in DM1 patients, has reached Phase III trials. These clinical milestones support the feasibility of systemic delivery using receptor-mediated transcytosis.
Advantages Over ADCs Compared to traditional ADCs, AOCs offer reduced off-target toxicity and can silence undruggable targets at the transcript level. While ADCs rely on cytotoxicity, AOCs manipulate the genetic basis of disease, potentially providing longer-lasting therapeutic effects.
Challenges The therapeutic application of AOCs is limited by endosomal escape, off-target effects, and variability in antigen expression. Delivery efficiency depends heavily on intracellular trafficking and antigen internalization dynamics. Studies show that AOCs often accumulate near vasculature, impeding penetration into tumor cores.
Applications in Diagnostics
Molecular Barcoding and High-Plex Analysis AOCs have been integrated into high-sensitivity diagnostic assays. Techniques such as immuno-PCR, proximity ligation assay (PLA), and DNA-PAINT utilize antibody-oligonucleotide conjugates to amplify detection signals. These methods have enabled the quantification of low-abundance proteins with enhanced sensitivity and specificity.
Spatial Omics and Single-Cell Analysis In spatial transcriptomics, AOCs tagged with DNA barcodes enable simultaneous protein and mRNA profiling at single-cell resolution. This capability is essential for biomarker discovery and therapeutic monitoring in complex tissues like tumors or the brain.
Exosome Targeting and miRNA Tracking Anti-CD63 AOC complexes, such as ExomiR-Trackers, deliver anti-miRNA sequences into exosomes to track and modulate intercellular signaling. In vivo studies demonstrate their capacity to reduce tumor formation by regulating oncogenic miRNAs like miR-21.
Commercial Implementation Companies are actively developing diagnostic AOC platforms for research and clinical applications. The proximity extension assay (PEA) and electrochemical proximity assay (ECPA) have been adapted for multiplex biomarker detection in clinical samples.
Expression Systems and Purification AOCs are typically produced using mammalian expression systems for the antibody component, followed by chemical or enzymatic conjugation of oligonucleotides. The heterogeneity introduced during conjugation poses challenges in downstream purification. Techniques such as size-exclusion chromatography (SEC), capillary electrophoresis (CE), and liquid chromatography–mass spectrometry (LC-MS) are employed for quality assessment.
Analytical Characterization Rigorous characterization is essential to confirm DAR, linker stability, and oligonucleotide integrity. Hybridization-based assays are used to validate oligo functionality post-conjugation. Ensuring oligonucleotide resistance to nucleases and maintaining antibody-antigen binding affinity are critical for therapeutic viability.
Scale-Up and CMC Translating AOCs from bench to clinical-grade production requires addressing scalability, consistency, and regulatory compliance. The complexity of the molecule demands thorough documentation of Chemistry, Manufacturing, and Controls (CMC). Aggregation, incomplete conjugation, and residual solvents or reagents are common pitfalls.
Cost and Production Bottlenecks Compared to traditional mAbs or siRNA drugs, AOCs are expensive to manufacture due to multi-step synthesis and purification. Use of engineered antibodies and complex linker chemistries further raises production costs.
Limitations and Future Directions
Endosomal Escape and Payload Release A major limitation of AOCs is inefficient cytosolic release. The payload, if trapped in endosomes or degraded in lysosomes, cannot reach its site of action. Strategies using pH-sensitive or enzymatically cleavable linkers are being explored to enhance endosomal escape.
Steric Hindrance and Tissue Penetration Steric interference between the antibody and payload can impair intracellular trafficking and RISC loading. Site-specific conjugation and modular linker systems can mitigate these effects. Nanobodies and Fab fragments are also being used to improve tissue distribution and reduce molecular size.
Immunogenicity and Off-Target Effects Immunogenic responses can be triggered by both the antibody and the oligonucleotide. Modified oligos and humanized antibodies help mitigate this risk. Off-target gene silencing remains a concern with siRNAs and miRNAs due to partial complementarity tolerated by the RISC complex.
Clinical Translation Barriers Mouse models often exhibit reduced sensitivity to AOCs, leading to underestimation of effective dosages for human therapy. Furthermore, few robust methods exist to quantitatively assess intracellular oligonucleotide delivery and release in vivo.
Emerging Trends and Technologies
Photo- and ROS-responsive linkers: Enable spatiotemporal control over payload release.
Multivalent AOCs: Incorporating polyspecific antibodies (e.g., DVD-IgG) for dual-targeting of tumor cells and the microenvironment.
Gene editing payloads: CRISPR-Cas9 gRNAs delivered via AOCs for gene knockout or correction.
AI-assisted design: Optimization of sequence and conjugation sites to enhance target selectivity and reduce immunogenicity.
Clinical Trials and Commercial Outlook Multiple AOC-based drugs have entered clinical trials, including AOC1001 (DMPK siRNA for DM1) and TAC-001 (CpG oligonucleotide for cancer). These developments signal growing regulatory and industrial interest. As delivery challenges are addressed, AOCs are poised to complement or replace traditional antibody or oligonucleotide therapies.
Jiao, J., Qian, Y., Lv, Y., Wei, W., Long, Y., Guo, X., Buerliesi, A., Ye, J., Han, H., Li, J., Zhu, Y., & Zhang, W. (2024). Overcoming limitations and advancing the therapeutic potential of antibody-oligonucleotide conjugates (AOCs): Current status and future perspectives. Pharmacological Research, 209, 107469. https://doi.org/10.1016/j.phrs.2024.107469
