Oligonucleotide drugs have been developed for more than 40 years since the first one was born in 1978. However, their development has been slower than other types of drugs due to their inherent instability and low cellular uptake efficiency. The emergence of new delivery systems and chemical modification techniques have greatly improved these problems, and oligonucleotide drugs have shown rapid development in recent years. Currently, there are already guidelines and industry white papers on the immunogenicity evaluation of antibody drugs and other biologics, but there are still few reports on the immunogenicity of oligonucleotide drugs in animals or patients, and there is no systematic guidance. The limited understanding of the immunogenicity of oligonucleotide drugs also greatly restricts the development of this class of drugs.
The key to drug immunogenicity is to consider whether the immune system may recognize the drug as self and tolerate it, and other aspects that affect immunogenicity include the route of administration and the specific disease being treated (Figure 1).
Figure 1 Ways to affect immunogenicity (Front. Immunol., 18 August 2020 Sec. Vaccines and Molecular Therapeutics)
Drug characteristics: Compared with most typical biologics (such as monoclonal antibodies), oligonucleotide drugs are relatively small, containing fewer potential epitopes than larger protein counterparts, so they are generally considered to have low immunogenicity. However, the interaction between oligonucleotide molecules and the immune system is complex, with the potential to induce innate immunity. Previous studies have shown that siRNA sequences can affect immune stimulation through toll-like receptors (TLRs), and siRNA sequences rich in guanine and uracil bases tend to elicit more immune stimulatory activity. In order to improve the stability, safety, cellular uptake and efficacy of oligonucleotide drugs, chemical modifications are usually applied to them in the development process of new generation oligonucleotide drugs. Modifications of the nucleic acid backbone, the glycosyl part of the ribose and the nucleobase have been widely used, and these modifications are likely to increase immune responses. In addition, nucleic acid sequences similar to microorganisms may also have immune stimulatory potential, so novel oligonucleotide sequences may have higher immunogenicity.
Route of administration: Oligonucleotide drugs can act through various routes of administration, such as intravenous and tissue injections, oral and subcutaneous injections (Figure 2).
Figure 2 Oligonucleotide drug routes of administration and delivery methods (Nat. Nanotechnol. 16, 630–643 (2021).)
At present, approved oligonucleotide drugs mainly act by delivering to immune-limited sites, but delivering oligonucleotide drugs effectively to various tissues is still a major challenge for oligonucleotide drug development due to the difficulty of oligonucleotides passing through membranes. The development of delivery carriers has greatly solved this problem, and delivery carriers can bring about immune responses that vary by two orders of magnitude and can act through various mechanisms. Different delivery carriers deliver siRNA molecules across cell membranes in unique ways (Figure 2), exposing siRNA to different amounts and types of pattern recognition receptors (PRRs), making them more susceptible to innate immune recognition . In addition, the current common delivery methods of GalNAc (N-acetylated galactosamine) conjugation and lipid nanoparticle (LNP) carriers are mainly intravenous and subcutaneous injections, which have a higher risk of immunogenicity . In recent years, new oligonucleotide drug delivery methods such as endogenous vesicles, spherical nucleic acids, and nanotechnology applications are also advancing, which will also bring new challenges to immunogenicity testing.
Analysis Strategy for Immunogenicity of Oligonucleotide Drugs
Considering that immunogenicity is highly related to the efficacy and safety of drugs, immunogenicity analysis is needed for oligonucleotide drugs to support their development and clinical trials. Many bioanalytical principles applied to protein drugs are also applicable to oligonucleotide drugs, mainly following a hierarchical immunogenicity analysis strategy, but we also need to consider the unique structural components of oligonucleotide drugs, routes of administration and sites of action that increase the complexity of immune responses and bioanalytical methods.
Based on the characteristics of oligonucleotide drugs, some attention should be paid when testing their immunogenicity: 1) Anti-drug antibodies (ADAs) that may be produced by drug modification components, delivery substances and delivery media need to be fully evaluated; 2) Oligonucleotide drugs have a variety of routes of administration, we need to pay attention to the detection of different types of immunoglobulin antibodies in different types of samples; 3) According to the current knowledge, compared with traditional drugs, the ADA response induced by oligonucleotide drugs sometimes develops slowly, possibly for several months. Therefore, while conducting immunogenicity risk assessment in the early stage of development, it is recommended to save the samples at this stage and investigate their immunogenicity based on the new data exposed in the clinic, providing information for later and key studies.
To support the development and clinical trials of siRNA drugs, Pharmaron has developed a detection method for ADAs produced by siRNA, and has some considerations and suggestions in the process of method establishment:
Due to the different structure from protein antibodies, the conventional protein labeling methods are not applicable to siRNA drug labeling, and a new labeling method combining biotin and digoxin has been successfully applied;
Due to the weak immunogenicity of siRNA, the affinity of the prepared positive control antibody is poor. By continuously optimizing the detection steps, the method sensitivity has met the current regulatory requirements.
