Nucleic acid drugs are a frontier field in the development of biomedicine, and the targeted delivery system of nucleic acid drugs is both a difficult and important point. At present, small nucleic acid drugs have made significant progress in targeting liver cells and tissues, but they cannot solve the problem of drug delivery outside the liver. There are also issues with dosage, concentration, and time dependence. At the same time, antibody drugs have experienced explosive development in recent years, occupying an increasingly high proportion of the disease market. On the one hand, the high specific affinity of antibodies for tumor cell antigens is an ideal carrier for drug targeting delivery, which can be used to deliver drugs to tumor lesions. On the other hand, the anti-tumor efficacy of currently used antibody drugs used alone is limited, and many patients who initially respond well to antibody treatment easily develop drug resistance.
Therefore, the strategy of combining the current antibody drugs with other drugs to form novel drug combinations, such as antibody-oligonucleotide conjugates (AOCs), is increasingly being adopted. These new delivery methods, like AOC, have garnered widespread attention as effective developmental means for targeting specific tissues outside of the liver.
The Principles of AOC Drug Conjugation Technology
Small nucleic acid drugs usually have more bioconjugation methods than small molecule drugs. Below are four common methods for preparing AOCs:
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Electrostatic Interaction
The main chain of oligonucleotides is negatively charged, and protamine is positively charged. Antibodies or antibody fragments (Fab or scFv) are constructed into fusion proteins with spermine, which couples the negatively charged oligonucleotides through its positive charge to form antibody-oligonucleotide conjugates. The advantage is that the process is simple and flexible, and when oligonucleotides enter the cell, the polycationic complex can act as a lyosomal escape agent. In the lysosome, the polycationic complex acts as a proton sponge, where chloride ions diffuse inside to compensate for charge imbalance, leading to osmotic swelling and the formation of leaky membranes. This lysosomal escape is very important, as oligonucleotides do not have strong membrane permeability. The disadvantage is that ionic interactions are reversible and unstable, and it can be difficult to determine oligonucleotide-to-antibody ratio values (OAR).
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Affinity Connection
Conjugation through Avidin/Streptavidin Biotin: Thiol-modified DNA is chemically connected to maleimide-activated streptavidin to produce streptavidin-DNA, which is further non-covalently connected to various biotinylated proteins. This is mainly applied in the development of immunoassay methods. Further development involves directly using the four biotin binding sites of streptavidin to directly link biotinylated antibodies and biotinylated oligonucleotides.
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Direct Conjugation
Direct conjugation methods are more similar to the coupling strategies of ADC drugs. A connectable group is added to the oligonucleotide and directly coupled to the lysine or cysteine of the antibody. Linkers can be used that are cleavable or non-cleavable through site-specific coupling and other methods.
The advantages of this method are that the linker is smaller and more stable; additionally, oligonucleotides carry a handhold needed for the linker, which can be chemically modified to connect to the linker. The linker has to be present in DNA or RNA and stable during the process of annealing the double strand.
The disadvantage of this method is that it does not contain a lysosomal escape agent, which may lead to a slow escape of oligonucleotides from the lysosome, thereby affecting the activity of the AOC.
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Nucleic Acid Double-strand Hybridization Binding
A single-stranded oligonucleotide is first conjugated to an antibody, and another complementary strand is conjugated via hybridization to form a double-stranded AOC. In the double-stranded hybridization method, length is very important. Too short, the hybrid chain is not stable; too long, it is easy to form a secondary structure. This hybrid technology has great application prospects in diagnosis, but there are major challenges in the manufacturing process for therapeutic drugs.
Combining the tissue specificity advantage of antibody drugs with the target specificity advantage of small nucleic acids can solve the current problem of small nucleic acid drugs only being able to target the liver through LNP (lipid nanoparticle) and GalNAc (N-acetyl-galactosamine) delivery systems. Hence, it has broad development space. The advantages can be summarized as follows: 1. It has specificity, which improves bioavailability and reduces toxicity; 2. It enhances stability, extends the ideal half-life, and increases efficacy.