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 (Oligos), allowing them to act directly on gene expression within targeted cells while simultaneously addressing longstanding challenges in oligo drug delivery. This dual capability opens doors to interventions with reduced off-target effects, providing a pathway for highly precise treatments.
However, the development of AOCs is far from straightforward. Unlike Antibody-drug conjugates (ADCs), which involve relatively stable small molecule payloads, AOCs demand a seamless integration of two fundamentally distinct biomolecular entities: the antibody and the oligo. Achieving this synthesis requires expertise in proteins, oligo chemistry and bioconjugation strategies. The challenges are substantial, spanning sequence fidelity, conjugation stability, and impurity management.
This complexity is where scientific ingenuity becomes necessary. For researchers and developers, the goal is not just to create these molecules but to do so in a way that maintains the desired functionality of each component, ensuring both efficacy and manufacturability. AOCs have the potential to transform therapies, but their success hinges on understanding and overcoming the intricate chemistry and technical challenges of their creation.
What are Antibody-Oligonucleotide Conjugates (AOCs)?
AOCs are hybrid molecules that combine the targeting precision of antibodies with the functional versatility of therapeutic oligos. The antibody component homes in on targeted cells, while the oligo modulates gene expression once delivered. This mechanism allows for precise intervention at the molecular level in diseases that are challenging to treat with conventional approaches.
Unlike ADCs, which deliver cytotoxic drugs to kill target cells, AOCs alter cellular function by influencing gene expression. 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: Oligos serve as ‘binders,’ that target proteins, binding them with high specificity to disrupt pathological interactions. While not yet widely explored in AOCs, aptamers — longer oligos that form tertiary structures capable of binding proteins or enzymes — could be a unique area for AOCs to expand beyond gene modulation into protein-inhibition therapies.
AOCs are particularly suited for diseases requiring targeted modulation of gene expression, including:
- Neurological diseases: precise delivery across complex barriers like the blood-brain barrier.
- Oncology: directly addressing cancer-driving genes.
- Rare genetic disorders: overcoming systemic challenges with oligo delivery.
- AOCs’ ability to mitigate off-target effects and provide selective gene therapy marks them as promising candidates for diseases previously thought untreatable.
The Chemistry Behind AOC Development
AOC development is rooted in the complex interplay between oligo chemistry and the bioconjugation process that links oligos to antibodies. Both areas present different challenges that must be addressed to create a functional and stable conjugate.
Oligonucleotide Synthesis
Oligos are typically synthesized using solid-phase chemical methods. This process, while well-established, is not without complications. Incomplete deprotection of intermediate groups, missed incorporations, or sequence fidelity issues can introduce impurities. Moreover, modifications such as phosphorothioate (PS) backbones or 2’-O-methyl (2’-OMe) groups are often incorporated to enhance stability against enzymatic degradation.
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 therapeutic consistency. Additional developments like locked nucleic acids (LNA) or constrained ethyl (cEt) modifications can help to 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
Coupling oligos to antibodies typically involves maleimide-thiol chemistry, where maleimide-functionalized linkers react with free cysteine residues on the antibody. The maleimide linker, commonly used for conjugating oligos to antibodies, is prone to hydrolysis in aqueous environments, reducing conjugation efficiency over time. Alternatives like ThioBridge™, Abzena’s site-specific conjugation technology, can provide more stable and controlled conjugation.
Linker design is paramount for ensuring the functionality of AOCs. The choice between cleavable and non-cleavable linkers, as well as their length, affects the conjugate’s stability and activity. Cleavable linkers release the oligo upon entering the target cell, while non-cleavable linkers maintain the conjugate’s integrity throughout its action. Balancing these features with the inherent electrostatic and steric challenges posed by the negatively charged oligo and the bulky antibody is a critical part of development.
Managing Oligo Characteristics
The inherent properties of oligos introduce additional challenges. Self-interactions, for example, can interfere with their intended function. Additionally, 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.
The intricate chemistry of AOC synthesis and conjugation needs a nuanced approach. Achieving stability, efficacy, and manufacturability requires a deep understanding of both the individual components and their interplay – a challenge that continues to drive innovation in the field.
AOC vs ADC Development
While AOCs and ADCs share structural similarities – both combining 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 developers must address.
Payload Complexity
In ADCs, the payload is a small molecule which is typically more stable and can be more straightforward to characterize. AOCs, in contrast, use oligo payloads, which are inherently more complex. Oligos 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 than those needed for small molecule drugs.
Impurity Profiles
The impurity profile of ADCs typically revolves around the small molecule drug and its degradation products, which are easier to identify and quantify. AOCs, however, must contend with impurities stemming from oligo synthesis, such as truncated sequences, diastereomers, and byproducts. These impurities 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
A well-established method for ADC characterization – such as Hydrophobic Interaction Chromatography (HIC)– is often insufficient for AOCs. Specialized techniques, including ion-exchange chromatography, capillary electrophoresis, and high-resolution mass spectrometry, are required to fully characterize the oligo component and the conjugate. Regulators demand high levels of precision in these analyses, especially for oligo-based therapies, which are subject to increased scrutiny due to their complexity.
Stability and Formulation
ADCs benefit from the relative stability of small molecule payloads, whereas AOCs require careful formulation to prevent oligo degradation. Oligos are vulnerable to enzymatic degradation and must maintain structural integrity during storage and delivery. To counteract the enzymatic degradation of oligos, formulations often include stabilizing agents like EDTA and surfactants. These agents prevent degradation and aggregation, ensuring the oligo remains functional throughout its shelf life. AOC formulations must incorporate stabilizing agents, such as chelating agents or cryoprotectants, while considering factors like pH and ionic strength. Long-term stability studies are needed here to ensure the AOC retains its activity throughout its shelf life.
In addition, need for further purification steps, such as anion-exchange chromatography, can significantly reduce material yield during AOC production. These losses represent a major difference from ADC workflows, where such steps are less frequently required.
Regulatory Considerations for AOCs
Developing AOCs involves navigating a regulatory landscape that is both changing and incomplete. While guidance for small molecules, biologics, and ADCs is well-established, the unique characteristics of AOCs – particularly their oligo payloads – present challenges that existing frameworks do not fully address. Implementation of prior knowledge and platform manufacturing strategies to expedite development programs can be difficult. This gap requires developers to adopt proactive and scientifically rigorous approaches, including partnering with experienced suppliers and consultants, and global regulatory bodies to help navigate this challenging landscape.
Evolving Regulatory Frameworks
Regulatory agencies like the FDA and EMA classify oligos differently. In the U.S., oligos are typically regulated as part of cell and gene therapy products, whereas in Europe, manufacturing of synthetic oligos are excluded from the scope of ICH Q3A/B (impurities), ICH Q6A/B (specifications) and ICH M7 (mutagenic impurities) to some degree. The EMA’s draft guideline on the “Development and Manufacture of Oligonucleotides aims to address some gaps in oligo regulation but remains under review, with final adoption expected in 2025.
For AOCs, the challenge lies in their hybrid nature. Regulatory agencies have yet to standardize expectations for conjugates combining antibodies and oligos. This lack of precedent can complicate submissions, meaning developers have to build comprehensive datasets to support product safety, efficacy, and manufacturability.
Impurity Characterization and Control
One of the most scrutinized aspects of AOC development is the characterization of impurities. The complexity of oligos introduces unique challenges, such as controlling diastereomeric purity and managing co-eluting impurities. Regulators are particularly concerned about impurities that could impact the conjugate’s safety or function. Developers must employ advanced analytical techniques to ensure comprehensive impurity profiling, including ion-exchange chromatography, capillary electrophoresis, and advanced mass spectrometry techniques.
Stability and Immunogenicity
Oligos’ susceptibility to enzymatic degradation and their immunogenic potential are areas of regulatory focus. Developers must demonstrate that AOCs remain stable during manufacturing, storage, and administration. Additionally, regulators demand robust data on immunogenicity, particularly for oligos modified with chemical groups or linkers, which can provoke unwanted immune responses, making implementation of platform approaches difficult for developers.
Target Product Profiles
The use of target product profiles (TPPs) helps bridge regulatory uncertainties by clearly defining the desired attributes for AOCs, including stability, purity, efficacy, and safety. This approach allows early alignment with regulatory expectations and smooths the pathway to approval.
Abzena’s Approach to AOC Development
AOC development is uniquely challenging, requiring expertise across oligo chemistry, bioconjugation, and biologics manufacturing. At Abzena, we have extensive experience with ADC development, coupled with expertise in antibody engineering and conjugation, to gives us a solid foundation to deal with the complexities of AOCs. We can build on established ADC workflows and adapt them to the nuances of AOCs, bridging the gap between these two conjugate types.
Ultimately, AOCs require a more sophisticated approach to development than ADCs. From payload stability to regulatory considerations, the path to a successful AOC product demands innovation, meticulous planning, and a deep understanding of both antibody and oligo subtleties.
Collaborative, Scientist-to-Scientist Engagement
Abzena takes a “scientist-to-scientist” approach, acting as a service provider and a scientific partner. This collaboration means customers receive candid, data-driven insights into their projects. Our scientists frequently challenge assumptions, drawing on their extensive experience to guide clients toward more robust and scalable solutions.
Expertise Across Key Development Areas
- Oligo optimization: While clients often bring pre-designed oligo sequences, we offer critical input to optimize these sequences for stability, efficacy, and manufacturability. This includes identifying stabilizing modifications to enhance resistance to enzymatic degradation without compromising target affinity.
- Conjugation efficiency: Our team excels in designing and executing bioconjugation strategies that maximize stability and reduce byproducts. This includes selecting optimal linker chemistries and evaluating conjugation sites to maintain antibody function while ensuring efficient oligo delivery.
- Analytical characterization: Advanced analytical capabilities let us profile impurities, assess conjugate stability, and verify batch-to-batch consistency. Advanced, in-house techniques are used to meet stringent regulatory standards.
- Formulation development: Our formulation scientists design the best approach to maintain AOC stability during storage and administration. This includes identifying excipients, adjusting pH, and testing for resistance to aggregation or enzymatic degradation.
Tailored TTPs
It’s important to us to be able to work closely with clients to develop detailed TPPs, outlining critical quality attributes and aligning the development process with regulatory and clinical goals. This strategic framework ensures that every decision – from oligo sequence design to final formulation – is focused on achieving a successful product.
Above all else, we focus on scientific rigor, transparency, and tailored options. By addressing the complexities of these advanced therapies, we empower our clients to bring their innovations to market with confidence.
The Future of AOCs
AOCs are redefining the boundaries of therapies to tackle diseases rooted in genetic dysfunction. The combination of oligos’ ability to modulate gene expression alongside antibodies’ cell-specific targeting creates opportunities to treat conditions previously considered intractable. However, the complexity of AOC development demands a level of expertise and innovation that few organizations can provide. As the regulatory landscape for oligo-based therapies continues to develop, having an experienced partner, like Abzena, is increasingly beneficial.
In the years ahead, AOCs could become a core component of precision medicine, particularly in oncology, rare genetic disorders, and other challenging therapeutic areas. Partnering with experienced service providers and consultants can help navigate the challenging path towards regulatory approval and help to de-risk and streamline these innovations to patients in need.
