Antibody-oligonucleotide conjugates (AOCs) may be synthesized using a wide range of chemical approaches. These include stochastic methods where protein lysines provide a large number of potential sites of oligonucleotide attachment. A second category includes site-directed methods which allow the oligonucleotides to be attached at predetermined locations on the antibody molecule, or at least away from the antigen binding domains. Very precise control of the site of attachment generally requires genetic engineering of the heavy or light chains to insert motifs which can be acted upon by transferase enzymes. Some of the more popular approaches are outlined in the table, which will be expanded periodically.
Conjugation approach | Oligo reactive group | Antibody reactive group | Oligo distribution | Original reference or Aboligo review |
Disuccinimidyl substrate (homobifunctional) | NHS ester | Lysine | Stochastic | Smart application of an old conjugation reagent in AOC development |
Click (strain-promoted) | DBCO | Azide (via STFP ester reaction with lysine) | Stochastic | |
Click (strain-promoted) | Azide | DBCO (via DBCO-NHS ester reaction with lysine) | Stochastic | |
Thiol-maleimide | Maleimide (via SH /bismaleimidohexane) | Thiol (via DTT reduction) | Site directed | |
Thiol-maleimide | Protected thiol (SATA) + hydroxylamine | Maleimide (via lysine modification) | Stochastic | |
mTransglutaminase/antibody engineering | DBCO | Azide introduced by mTGase at LLQG motif | Site directed | |
Sortase/antibody engineering | Gly-Gly-Gly | LPETGG motif | Site directed | |
Glycan oxidation | Hydrazine | Aldehyde | Site directed | |
Click (strain promoted) | TCO (trans-cyclooctene) | Tetrazine (via cleavable NHS ester) | Stochastic |
References
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