Oligonucleotide-Conjugated Antibodies for Diagnostics and Therapeutics

What are Oligonucleotide-antibody Conjugates?

Oligonucleotide-antibody conjugates (oligo-Ab) are a new class of synthetic chimeric biomolecules. After the oligonucleotide-conjugated antibody binds to its target, the protein level can be converted to the oligonucleotide level, which greatly improves the sensitivity of protein detection and allows the use of multiplexed methods. Oligonucleotide-antibody conjugates have been used in numerous applications ranging from diagnostics to therapeutics and have played an important role in enhancing a wide range of biotechnology, including immunological and proteomic research, biomarker discovery, clinical Diagnostics (including point-of-care) and other new serial technologies. Similar to antibody-drug conjugation, oligonucleotides can be attached to different parts of the antibody using different conjugation chemistries. Various endogenous amino acids can be used as potential binding sites. Antibodies and oligonucleotides can be coupled by different covalent and non-covalent conjugation strategies.

Amine Conjugation

Antibodies contain several amino groups (NH2) that can be distributed as lysine side chain epsilon-amine and N-terminus α-amino groups. These residues are most often targeted for conjugation as they are easily modified due to their stereospecific accessibility. In addition to amino groups, glutamate and aspartate residues as well as carboxylic acids can also be conjugated.

Sulfhydryl Conjugation

Oligonucleotide-antibody conjugation can also take place via the reactive thiol (sulfhydryl) group. Antibodies contain oxidized thiol groups (-SH) in the form of disulfide (S-S) bridges, which must first be reduced by a reducing agent to expose their reactive groups.

Fig. 1 Direct conjugate method to oligonucleotide amine (Dugal-Tessier J, 2021)

Azide-Alkyne Click Chemistry-based Conjugation

Strain-promoted alkyne–azide conjugation (SPAAC) was one of the first click reactions in biological conjugation that allowed stoichiometric linkage of any two reaction partners and was compatible with complex proteins.

The antibody is chemically labeled with a strained alkyne and the resulting DBCO-antibody intermediate is then reacted with an azide-modified antisense oligomer to form a conjugate. The advantage of this approach is that a batch of antibodies with the same linker-antibody ratio can be functionalized and different identical oligonucleotides can be orthogonally conjugated. Due to its orthogonality and its compatibility with oligonucleotides, click chemistry is a robust and popular method of conjugation. The hydrophobicity of DBCO and BCN reagents does not pose a challenge due to the water solubility of the oligonucleotides.

Fig. 2 SPAAC functionalization of azide-labeled antibodies prepared using lysine or arginine residues of native antibodies (Dovgan I, 2019)

Conjugation Based on Streptavidin-biotin System

By chemically linking thiol-modified DNA to maleimide-activated streptavidin, researchers can generate a streptavidin DNA substrate that further binds to various biotinylated proteins Non-covalent linkage, which is based on the strong non-covalent interaction between streptavidin and biotin. Alternatively, biotinylated antibodies can be attached to biotinylated DNA strands using the four biotinylated binding sites present in the streptavidin molecule.

Fig. 3 (a) Covalently linked oligos-streptavidin conjugate and biotinylated antibody; (b) streptavidin linkage with two biotinylated biomolecules (Dovgan I, 2019)

Hydrazone Conjugation

Antibodies are first reacted with succinimidyl 4-hydronicotinic acid acetone hydrazine (S-HyNic) to introduce nucleophilic hydrazine residues into IgG. The oligonucleotide is reacted with sulfo-S-4-formylbenzoate (4-FB) to introduce an aromatic aldehyde functional group into the oligonucleotide structure. After purification of the two reaction partners, they readily react with each other to yield IgG-4FB-oligonucleotides containing hydrazone bonds.

The Hnatowich team used this chemistry to generate antibody conjugates containing unnatural oligonucleotides such as PMO and morpholine oligomers to form radiolabeled constructs for quantitative antibody internalization assays and pretargeted cancer therapy body.

Inverse Electron-Demand Diels–Alder Reaction (iEDDA)

Tetrazine-alkene inverse Diels–Alder reactions are among the most recent in the pool of biorthogonal reactions. They have recently been shown to possess many advantages over SPAAC in terms of kinetics and were subsequently applied to functionalize antibodies for their use in qPCR and cell phenotyping using barcoding. The latter technique combines antibody-based protein detection with a pool of oligo-Ab conjugates, in which the oligo fragment is used as a barcode. The prepared oligo-Ab conjugates with tetrazine-modified antibodies containing disulfide cleavable splices and trans-cyclooctene (TCO) modified-DNA tags can be sequenced through the reduction of disulfide bonds between Ab and DNA components after being incubated with cells and thoroughly washed.

Fig. 4 The application of iEDDA reaction (Dovgan I, 2019)

Oligonucleotide-Conjugated Antibodies for Diagnostics and Therapeutics

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