Antibody-oligonucleotide conjugates (AOCs) are an emerging and exciting development in precision medicine. AOCs combine the specificity of antibodies with the gene-modulating potential of oligonucleotides, allowing them to effect gene expression within targeted cells. This dual capability addresses the challenges in oligo drug delivery, opening the door to more precise interventions with reduced off-target effects.
The development and manufacturing of AOCs isn’t as straightforward as it might seem, compared with an antibody drug conjugate (ADC). AOCs integrate two fundamentally distinct and individually complex biomolecular entities: the antibody and the oligo. Achieving this requires expertise in protein engineering, oligo chemistry and bioconjugation strategies. The challenges are substantial, spanning sequence fidelity, conjugation stability and impurity management.
The complexity and distinct chemical differences between the two components is where scientific ingenuity becomes necessary. For drug developers, the goal is to maintain the desired functionality of each component, while ensuring both efficacy and manufacturability. This novel modality has the potential to be transformative for a range of hard-to-treat conditions, but success hinges on understanding and overcoming intricate chemistry and analytical challenges.
The potential of AOCs
An AOC combines the cell targeting precision of an antibody moiety with the gene expression modulation capability of a functional oligo. This ability to directly alter cellular function means they can:
- Block mRNA translation: Antisense oligos (ASOs) bind to target RNA to reduce protein levels.
- Induce RNA interference: Small interfering RNA (siRNA) degrades specific mRNA, preventing protein production.
- Modulate splicing: Splice-switching oligos correct abnormal pre-mRNA splicing, with potential in diseases like Duchenne muscular dystrophy.
- Act as aptamers: The oligo serves as a highly specific 'protein binder' that, when bound to the protein of interest, disrupts pathological interactions. These longer oligos can form tertiary structures capable of binding proteins or enzymes and thereby offer the opportunity to move beyond gene modulation and into protein-inhibition therapies.
AOCs offer the potential of precise delivery across complex barriers such as the blood-brain barrier and are treatment options for neurological diseases. In oncology, they could be deployed to directly target cancer-driving genes. When used to treat rare genetic disorders, AOCs help to overcome the systemic challenges of oligo delivery, in particular when it comes to delivery beyond the liver. Overall, AOCs’ are promising candidates to address many currently intractable diseases, realizing the promise to “drug the undruggable.”
The chemistry behind AOC development
The successful development of AOCs requires precise control of both the oligo chemistry and the bioconjugation process that links the oligo to the antibody. Both areas present different challenges that must be addressed to create a functional and stable conjugate.
Oligonucleotide synthesis
Oligos are typically synthesized using well-established solid-phase chemical methods. These are not perfect and can result in incomplete deprotection of intermediate groups, missed incorporations, or sequence fidelity issues that introduce hard-to-remove impurities.
Engineered modifications such as phosphorothioate (PS) backbones or 2’-O-methyl (2’-OMe) groups are often incorporated to enhance stability against enzymatic degradation. However, PS modifications, while enhancing oligo stability, introduce diastereomers that vary in biological activity, enzymatic resistance, and toxicity. These diastereomers complicate characterization and separation, requiring advanced analytical techniques to isolate the desired forms and ensure product consistency.
Additional engineered developments like locked nucleic acids (LNA) or constrained ethyl (cEt) modifications can be introduced to help increase target affinity and specificity and further minimize off-target effects.
Secondary structures, such as hairpins, can form under specific buffer conditions, which render the oligo unable to perform its intended function. Managing these structures through sequence design and formulation is an important step for maintaining activity.
Bioconjugation strategies
Typically, oligos are coupled to the antibody of interest using maleimide-thiol chemistry, where maleimide-functionalized linkers react with free cysteine residues on the antibody. The maleimide linker is prone to hydrolysis in aqueous environments thus reducing conjugation efficiency. Site-specific conjugation technologies can provide more stable and controlled conjugation.
Linker design is paramount for ensuring the desired functionality of the resulting AOC. The option to use a cleavable and non-cleavable linker, to vary the length of the linker, all impact the therapeutic activity and stability of the resulting conjugate. Cleavable linkers release the oligo upon entering the target cell, while non-cleavable linkers maintain the conjugate's integrity throughout its action.
Managing oligo characteristics
The inherent properties of oligos introduce additional challenges. Self-interactions, for example, can interfere with the intended function. Their negative charge must be carefully managed to prevent undesirable aggregation or binding to non-target structures. These characteristics demand meticulous design and testing to ensure the oligo retains its efficacy while being compatible with the antibody to which it is conjugated.
Analytical considerations during AOC development
While AOCs and the more established antibody drug conjugates both combine antibodies with functional payloads, their development processes differ significantly due to the complexity of oligo payloads in AOCs. These differences present distinct technical and analytical challenges that must be addressed to ensure a smooth regulatory journey.
Impurity profiles
Due to the nature of the component parts, the impurity profile of AOCs are inherently complex. Impurities stem from oligo synthesis, such as truncated sequences, diastereomers, and byproducts often co-elute with the desired product, complicating purification and characterization. Advanced analytical techniques are essential to identify and control these impurities, ensuring the safety and efficacy of the final AOC product.
Analytical techniques
High levels of precision in analytical techniques, including ion-exchange chromatography, capillary electrophoresis, and specialized high-resolution mass spectrometry, are required to fully characterize both the components and the resulting bioconjugate.
Stability and formulation
Oligo payloads are prone to degradation, have diverse secondary structures (e.g., hairpins), and require chemical modifications for stability and function. These characteristics demand more intricate stabilization strategies. The oligo components are vulnerable to enzymatic degradation, so formulations require the use of stabilizing agents such as EDTA and surfactants. The AOC formulations must incorporate stabilizing agents, such as chelating agents or cryoprotectants, and consider factors like pH and ionic strength to maintain long-term structural and ensure the AOC retains its activity throughout its shelf life.
Regulatory considerations
Developing AOCs involves navigating a regulatory landscape that is dynamic and evolving. The unique characteristics of AOCs present challenges that existing frameworks do not fully address. This gap means drug developers have to adopt proactive and scientifically rigorous approaches to de-risk navigation of this challenging landscape, pulling learnings from naked therapeutic oligo strategies in addition to those that can be inferred from ADCs.
Evolving regulatory frameworks
Regulatory agencies like the FDA and EMA classify AOCs differently. In the U.S., they are typically regulated as part of cell and gene therapy products, whereas the EMA has a draft guideline, "Development and Manufacture of Oligonucleotides,” which aims to address some gaps in oligo regulation but remains under review, with final adoption expected in 2025.
Regulatory agencies have yet to standardize expectations for conjugates combining antibodies and oligos. The lack of precedent will require close communication with regulators, sound application of ADC principles and integration of considerations germane to the naked oligo drug submissions.
Impurity characterization and control
One of the most scrutinized aspects of oligo development, and directly applicable to AOCs is characterization of impurities. As discussed above, the complexity of oligos introduces unique challenges that require the employment of advanced analytical techniques to ensure comprehensive impurity profiling. This is specifically important in the area of conjugatable impurities.
Stability and immunogenicity
Oligos’ susceptibility to enzymatic and physico-chemical degradation pose a potential to elicit immunogenic responses and are areas of regulatory focus. AOCs must have proven stability profiles during manufacturing, storage, and administration. Approval demands robust data on immunogenicity, particularly for oligos modified with chemical groups or linkers which can provoke unwanted immune responses.
Target product profiles
Use of Target product profiles (TPPs) will bridge regulatory uncertainties by clearly defining the desired attributes for the AOC, including stability, purity, efficacy and safety. TPPs linked to rational specification selection must be rooted in scientific justification and mechanism. This helps make scientific arguments simpler to articulate and experiments clearer to plan. This approach allows early alignment with regulatory expectations and ensures that every development decision — from oligo sequence design to final formulation — remains focused on achieving a successful product.
The future of AOCs
The intricate chemistry of AOC synthesis demands an informed and nuanced approach. Achieving stability, efficacy and manufacturability requires a deep understanding of all the individual components and their interplay — and balancing these options is a critical part of successful and commercially viable AOC design and development. Using established ADC workflows is a good starting point and these can be adapted through meticulous planning, expertise and scientific understanding of antibody, oligo, and linker subtleties heretofore not required in other treatment modalities.
The regulatory landscape for oligo-based therapies continues to develop and, in the years, ahead, AOCs could become a core component of precision medicine, particularly in oncology, rare genetic disorders, and other challenging therapeutic areas. To get there, drug developers must create sophisticated analytical techniques that precisely define and characterise these novel moieties to give patients and regulators safe and efficacious treatment options.
Author Details
Jeffrey Mocny, PhD- VP of Regulatory Strategy, Abzena
Stephen Verespy, PhD- Scientific Leader, Abzena
Publication Details
This article appeared in American Pharmaceutical Review:
Vol. 28, No. 1
Jan/Feb 2025
Pages: 10-12