Drug research and development has evolved from focusing only on small molecule drugs into an era of advanced therapeutics. Among the pathogenic proteins related to human diseases, more than 80% belong to non-druggable targets. Meanwhile, most existing druggable targets are restricted by their structure and are difficult to develop. Unlike traditional small molecule and antibody drugs, which require recognition of the spatial conformation of proteins, Oligonucleotides (ONs) rely on nucleotide sequence information to regulate gene expression or binding to proteins to control cellular biological function. Because its mechanism of action does not require consideration of the localization or structure of the target molecule, it can achieve goals that cannot be achieved by small molecules or antibody drugs, which is expected to break through the limitations of the drug for existing targets.
ONs refer to single-stranded or double-stranded nucleic acid drug molecules with short length and less than 30 bases or base pairs, mainly including antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), small activating RNAs (saRNAs) and RNA aptamers. The ONs that have been approved and are under research are mainly ASOs and siRNAs. ASO is a synthetic single-stranded nucleic acid polymer of about 18-30 nucleotides and possesses a variety of chemical properties. ASOs can directly facilitate the degradation of RNA and can inhibit RNA expression through steric hindrance. siRNA is a class of double-stranded RNA molecules with a length of 20-25 base pairs, which are integrated into other proteins to form an RNA-induced silencing complex (RISC) after entering the cell. As part of the RISC, siRNAs can find complementary mRNAs for degradation.
ON Development
Nucleic acid drugs require a long development process. From the first report by Paul Zamecnik et al. in 1978 that ASOs can inhibit the sarcoma virus to the first ASO drug being approved for market in 1998, it was two decades before ON drugs were introduced into clinical practice. In 2006, the Nobel Prize in Physiology or Medicine was awarded for siRNA, unleashing the development of many siRNA drugs. However, clinical progress was poor due to problems with delivery and immunity. It was not until 2018 that the first siRNA drug, Patisiran, was approved by US regulatory authorities. In recent years, research and development of ONs have continuously broken through technical barriers and become a new wave of drug research and development after recombinant protein drugs and antibody drugs. There are currently 15 ONs on the market covering a variety of disease types, including genetic diseases, ophthalmic diseases, and cardiovascular diseases.
Considerations for Immunogenicity of Oligonucleotide Drugs
Understanding the immunogenicity of ONs has greatly limited their research and development. Guidelines and industry white papers on immunogenicity assessment of antibody drugs and other biologics have been published, but the immunogenicity of ONs in animals or patients is rarely reported.
The key to drug immunogenicity is to consider whether the immune system may perceive and tolerate the drug itself. Other aspects affecting immunogenicity include the route of administration and the specific disease treated, etc.
- Drug properties: ONs are generally considered less immunogenic than typical biologics (e.g., monoclonal antibodies) because they are relatively small and contain fewer potential epitopes than their larger protein counterparts. However, the interaction between ONs and the immune system is complex and has the potential to induce innate immunity. It has been shown that siRNA sequences can affect immunostimulation through toll-like receptors (TLRs), while siRNAs rich in guanosine and uridine motifs tend to stimulate more immunostimulatory activity. Chemical modification is usually performed during research and development of a new generation of ONs to improve the stability, safety, cell absorption, and efficacy of ONs. Modifications of the nucleic acid backbone, the glycosidic part of the ribose, and the nucleobase have been widely used, and these modifications are very likely to increase the immune response. In addition, nucleic acid sequences similar to microorganisms also have the potential for immune stimulation so that novel oligonucleotides may have higher immunogenicity.
- Routes of administration: ONs can act through a variety of routes of administration, such as intravenous and tissue injection and oral and subcutaneous administration. At present, approved ONs mainly act via delivery to immune-restricted sites. However, due to the difficulty of ONs passing through the plasma membrane, effective delivery of nucleic acid drugs into a variety of tissues remains a major challenge. The development of delivery vectors has greatly solved this problem, bringing about immune responses that vary by up to two orders of magnitude by working through multiple mechanisms. Different delivery vectors uniquely escort siRNA molecules across the cell membrane, and siRNAs are exposed to different numbers and types of pattern recognition receptors (PRRs), making them more susceptible to recognition by innate immunity. In addition, currently commonly used N-acetylated galactosamine (GalNAc) conjugated and lipid nanoparticle (LNP) carriers are mainly intravenous and subcutaneous injection. However, subcutaneous injection and other methods have a high immunogenicity risk. In recent years, new ONs delivery methods such as endogenous vesicles, spherical nucleic acids, and nanotechnology applications have also been continuously promoted, which will also bring new challenges to the detection of immunogenicity.
Analytical Strategy for Immunogenicity of Oligonucleotide Drugs
Immunogenicity is highly correlated with the efficacy and safety of drugs, so immunogenicity analysis of ONs is essential. Many bioanalytical principles applied to protein drugs can also be applied to ONs, mainly following a hierarchical immunogenicity analysis strategy. However, the unique structural components of ONs also need to be considered, as well as the administration route and site of action, which add complexity to immune responses and biological analysis methods. Based on the characteristics of ONs, the following should be considered in the detection of their immunogenicity:
- Drug modification components, delivery substances, and delivery that may produce anti-drug antibodies (ADA) need to be comprehensively evaluated.
- The administration routes of oligonucleotide drugs are diverse, and the detection of different immunoglobulin antibody types in various types of samples must be considered.
- Compared with traditional drugs, ON-induced ADA responses sometimes develop more slowly, possibly over several months. Therefore, while performing immunogenicity risk assessment at the early stage of development, it is recommended that samples be preserved at this stage and their immunogenicity investigated based on the data of new clinical exposure, providing information for later critical studies.
A Final Word
When developing siRNA drugs, working with someone who has developed assays for siRNA-generated ADAs is a good idea and can guide you through the method development process. WuXi AppTec, for example, has successfully applied a new labeling method combining biotin and digoxin because, due to various protein antibody structures, the conventional protein labeling method is not suitable for labeling siRNA drugs. Also, despite the weak immunogenicity of siRNA and the poor affinity of the prepared positive control antibody, the method sensitivity has been made to meet the existing regulatory requirements by continuously optimizing the detection steps.
We have established a rapid, accurate, and reliable electrochemiluminescence Meso Scale Discovery (MSD) method to detect anti-siRNA antibodies effectively. The bridging assay of the MSD platform is characterized by high sensitivity, wide effective linear range, homogeneity, high repeatability, small matrix effect, good stability, and high detection throughput, and has been successfully applied to the immunogenicity detection of ONs.
As a global company with operations across Asia, Europe, and North America, WuXi AppTec provides a broad portfolio of R&D and manufacturing services that enable the global pharmaceutical and life sciences industry to advance discoveries and deliver groundbreaking treatments to patients. Through its unique business models, WuXi AppTec’s integrated, end-to-end services include chemistry drug CRDMO (Contract Research, Development and Manufacturing Organization), biology discovery, preclinical testing and clinical research services, helping customers improve the productivity of advancing healthcare products through cost-effective and efficient solutions. WuXi AppTec received an AA ESG rating from MSCI for the fourth consecutive year in 2024 and its open-access platform is enabling more than 6,000 customers from over 30 countries to improve the health of those in need – and to realize the vision that “every drug can be made and every disease can be treated.”
