Here, we introduce 4-azidophenyl glyoxal (APG) as an efficient plug-and-play reagent for the selective functionalisation of arginine residues in native antibodies. The selective reaction between APG and arginines' guanidine groups allowed a facile introduction of azide groups on the monoclonal antibody trastuzumab (plug stage). These pre-functionalised antibody-azide conjugates were then derivatised during the "play stage" via a biorthogonal cycloaddition reaction with different strained alkynes. This afforded antibody-fluorophore and antibody-oligonucleotide conjugates, all showing preserved antigen selectivity and high stability in human plasma. Due to a lower content of arginines compared to lysines in native antibodies, this approach is thus attractive for the preparation of more homogeneous conjugates. This method proved to be orthogonal to classical lysine-based conjugation and allowed straightforward generation of dual-payload antibody.

We report a plug-and-play strategy for the preparation of functionally enhanced antibodies with a defined average degree of conjugation (DoC). The first stage (plug) allows the controllable and efficient installation of azide groups on lysine residues of a native antibody using 4-azidobenzoyl fluoride. The second step (play) allows for versatile antibody functionalization with a single payload or combination of payloads, such as a toxin, a fluorophore, or an oligonucleotide, via copper-free strain-promoted azide-alkyne cycloaddition (SPAAC). It is notable that in comparison to a classical N-hydroxysuccinimide ester (NHS) strategy, benzoyl fluorides show faster and more efficient acylation of lysine residues in a PBS buffer. This translates into better control of the DoC and enables the efficient and fast functionalization of delicate biomolecules at low temperature.

Controlled protein functionalization holds great promise for a wide variety of applications.

Here we present the synthesis and evaluation of antibody-drug conjugates (ADcs), for which antibody and drug are non-covalently connected using complementary DnA linkers. these ADcs are composed of trastuzumab, an antibody targeting HER2 receptors overexpressed on breast cancer cells, and monomethyl auristatin e (MMAe) as a drug payload. in this new ADc format, trastuzumab conjugated to a 37-mer oligonucleotide (ON) was prepared and hybridized with its complementary ON modified at 5-end with MMAE (cON-MMAE) in order to obtain trastuzumab-DNA-MMAE. As an advantage, the con-MMAe was completely soluble in water, which decreases overall hydrophobicity of toxic payload, an important characteristic of ADcs. the stability in the human plasma of these non-engineered onbased linkers was investigated and showed a satisfactory half-life of 5.8 days for the trastuzumab-DnA format. finally, we investigated the in vitro cytotoxicity profile of both the DNA-linked ADC and the on-drug conjugates and compared them with classical covalently linked ADc. interestingly, we found increased cytotoxicity for MMAE compared to cON-MMAE and an EC50 in the nanomolar range for trastuzumab-DNA-MMAE on HER2-positive cells. Although this proved to be less potent than classically linked ADC with picomolar range EC50, the difference in cytotoxicity between naked payload and conjugated payload was significant when an ON linker was used. We also observed an interesting increase in cytotoxicity of trastuzumab-DNA-MMAE on HER2-negative cells. This was attributed to enhanced non-specific interaction triggered by the DNA strand as it could be confirmed using ligand tracer assay. Antibody-drug conjugates (ADCs) are promising therapeutic agents used mainly for cancer indications 1,2. There are currently seven ADCs on the market, including three ADCs approved by the US Food and Drug Administration (FDA) in 2019 3. Conjugation of highly potent cytotoxic drugs with antibodies recognizing the antigens overexpressed on cancer cells affords ADCs, which enable the delivery of the cytotoxic payload into the tumor cells in a controlled and selective manner. Following internalization and proteolytic cleavage, the drug induces tumor cell dysfunction and apoptosis. Classical approaches for the preparation of ADCs from native antibodies are based either on drug conjugation to exposed lysine residues or to cysteine residues generated by a reduction of interchain disulfide bonds 4. Alternatively, protein engineering and enzymatic approaches have been actively used for production of homogeneous ADCs 5,6. For more information about advancements in antibody, linker, and warhead technologies, we refer readers to several excellent reviews on these topics 7-9. We have recently reviewed antibody-oligonucleotide conjugates (AOCs) for applications as therapeutic and detection agents. Interestingly, AOCs have been used for targeted therapy as carriers of doxorubicin drug which was intercalated between the CG base pairs of the AOC's double-strand DNA (Fig. 1a) 10-12. The Gothelf ...

An isatoic anhydride derivative conjugated to a biotin and a disulfide linker was specifically designed for the separation of nucleic acids. Starting from a DNA-RNA mixture, a selective 2'-hydroxyl acylation of RNAs followed by capture with streptavidin-coated magnetic beads and cleavage of the disulfide led to elution of RNAs.