Schematic structure of an antibody-oligonucleotide conjugate (AOC)
Antibody–oligonucleotide conjugates orAOCs belong to a class of chimeric molecules combining in their structure two important families of biomolecules: monoclonalantibodies andoligonucleotides.[1]
Combination of exceptional targeting capabilities ofmonoclonal antibodies with numerous functional modalities ofoligonucleotides has been fruitful for a variety of applications with AOC including imaging, detection and targeted therapeutics.[1][2][3]
Cell uptake/internalisation still represents the biggest hurdle towards successfulON therapeutics. A straightforward uptake, like for most small-molecule drugs, is hindered by the polyanionic backbone and the molecular size of ONs. Being adapted from the broad and successful class ofAntibody-Drug conjugates, antibodies and antibody analogues are more and more used in research in order to overcome hurdles related to delivery and internalisation of ON therapeutics. By exploitingbioconjugation methodology several conjugates have been obtained.
The first AOC was reported in 1995 where thelysines of atransferrin-antibody were connected using a SMCC bifunctional linker (NHS ester andmaleimide moiety) to radiolabelled and cys-bearingASOs targetingHIVmRNA.[4]Marcin and his colleagues developed a different construct using the same chemistry, but they utilized siRNA instead of an ASO in 2011.[5] In 2013, MYERS and coworkers then unspecifically labelled an anti-CD19 antibody with N-succinimidyl 3-(2-pyridyl-dithio) propionate to form disulphide bonds with cys-modified ASO targeting the mRNA of oncoprotein E2A–PBX1.[6] Ultimately, they could prove in-vivo antitumour effects which in contrast were not obtained with the single entities.[7] In the same timeframe, several antibodies were exploited for ON delivery in combination withnanoparticles and in non-covalent strategies.[8][9][10]
Only recently the first examples for a site-selective conjugation between an ON therapeutic and a mAb was published: in 2015Genentech exploited the SMCC linker to conjugate siRNA to several engineered mAb based on their proprietary Thiomab technology, which allows site-specific introduction of a cysteine into the antibody sequence[32].[11] They could prove the functionality of both entities in the construct and by screening different antibodies, they validated their importance for an effective antisense effect.[11] The main obstacle encountered was a limitedendosomal escape but ultimately a functional construct which showsantisense effect in-vivo was reported.[11] After development of the SMCC based conjugates, there were two constructs reported in literature based onstrain-promoted alkyne-azide cycloadditions: anMXD3 mRNA targetinggapmer (cEt and PS modified) linked to an anti-CD22 antibody targeting preB cells leads to in-vitroapoptosis of targeted cells and in-vivo increased length of mouse survival inxenograft models. Notably, the dose required for the same therapeutic effect was 20 times lower for the developed conjugate (vs. naked mAb).[12] Another reported conjugate, exploiting the same unselective conjugation chemistry, employs anCD44 respectivelyEphA2 targeting antibody which covalently carries a therapeutically irrelevant "sense-carrier" oligonucleotide.[13] This oligonucleotide base pairs with the actual antisense oligonucleotide (gapmer bearingphosphorothioate linkages and 2’-deoxy-2’-fluoro-beta-D-arabinonucleic acid modifications and a terminal fluorophor) aiming for an increased RNaseH activity.[14][15][16]
Despite their tremendous potential,ADCs and AOCs suffer from the physical size of the antibody (mAb) entity (150 kDa) which limits solidtumour penetration (at least at low concentrations). Moreover, thesite-selective modification of theantibody is hardly achievable: due to the difficult production of mAbs the selective introduction of anunnatural amino acid into the protein is not easily possible.[17]
Thats why there is intensive research to exploit antibody analogues and antibody fragments which retain a high target specificity but combined with a smaller size and a greater possibility of modification.Nanobodies for example are natural single-domain antibodies found in camelids with an average mass of 15kDa. They bear an increased stability, solubility and tissue penetration compared to mAbs.[18][19][20]
One conjugate, consisting out of anEGFRNanobody and asiRNA being combined throughmaleimidebioconjugation, proves the possibility of successful delivery of ONs by nanobodies.[21]
Another example consists out of an anti-CD71Fab fragment which was conjugated to a maleimide bearing siRNA (itself having2’OMe/2’F modifications and phosphorothioate linkages). Several (cleavable and uncleavable) linkers between themaleimide moiety and thesiRNA were screened revealing only a small influence onsilencing efficacy (uncleavable linkers leading to the best results). To play out the small size of the Fab fragment,subcutaneous administration was investigated in mouse models leading to equivalent silencing results compared tointravenous administration. By comparison with other mAb–siRNA conjugates the authors even speculate thatendosomal escape is largely facilitated by the smaller size of the Fab (vs. mAb).[22]
Moreover, Nanobody–ON conjugates are intensively used for imaging purposes exploiting the small nanobody size to reduce imaging displacement.[23][24]
