Covalent Approaches for AOC Preparation

Covalent coupling is based on the formation of a single covalent bond between two molecules, one molecule can be modified by certain chemical linker that reacts with specific chemical groups on the other molecule to achieve the conjugation. This method is more specific and controllable than the noncovalent method and the characteristics of covalently conjugated linkers/payloads can be optimized for a particular application. Creative Biolabs has years of experience in antibody conjugation and now provides our clients a comprehensive suite of covalent conjugation services for antibody-oligonucleotide conjugates (AOCs) development. Different noncovalent approaches are also available upon request.

The main residues used for monoclonal antibody (mAb) conjugation have been Lys, reduced Cys, Tyr, and Arg residues. The oligonucleotide (ON) fragment can either be chemically coupled with free thiol or amine or be transformed into a more reactive aldehyde moiety. The choice of the appropriate bifunctional chemical linkers is usually defined by the accessibility of ON derivatives and the reactive group present in the antibody structure. Creative Biolabs provides numerous covalent conjugation methods for AOC development, including but not limited to the following approaches:

• Conjugation on Cysteine Residues of Antibodies

One possibility in the generation of free thiol-containing mAbs is the genetic incorporation of additional protected Cys residues into the mAb structure. Such antibodies can be used for linking mAb with thiol-reactive ON obtained using SMCC or SPDB crosslinkers. Normally, a preliminary deprotection step is required to generate free thiolates from protected Cys residues. Besides incorporating additional cys, free thiolates could also be generated using the partial reduction of interchain disulfide bonds in mAb. This approach was the main approach used for antibody conjugation, it is also suitable for generating AOCs.

Fig.1 Preparation of cysteine-linked AOC using (a) engineered mAbs; or (b) the reduction of interchain disulfide bonds. (Dovgan, 2019)

• Conjugation on Lysine Residues of Antibodies

ONs contain ribose residues, they can be readily converted into dialdehydes using oxidation reaction with sodium periodate. These electrophilic can then react with the nucleophilic Lys residues of a protein, yielding the corresponding Schiff bases in a reversible condensation reaction, which can be reduced with sodium borohydride to yield stable adducts. Please note that the usage of relatively harsh and unselective chemical reagents in this method may cause targeting abilities and functions losing.

Besides, Lys residues of antibodies can be modified with various heterobifunctional linkers and used for the conjugation with ON, which including but not limited to thiolation of antibodies, conjugation based on click chemistry, hydrazone couplingamine-to-thiol coupling, and inverse electron-demand diels-alder reaction (iEDDA).

• Thiolation of Antibodies

This methodology is applicable to add latent thiolates onto mAbs. For instance, the mAb is first modified with SATA to introduce protected thiol residues. The DNA fragment is modified using SMCC, thus yielding maleimide-containing DNA. The conjugation reaction can then be started by adding the hydroxylamine into the SATA-modified mAb followed by the addition of the activated DNA-maleimide fragment.

Fig.2 Thiolation of antibodies using the SATA reagent. (Dovgan, 2019)

• Conjugation based on Click Chemistry

Click chemistry is a versatile reaction that can be used for the synthesis of a variety of conjugates including ADCs and AOCs. It takes place between two components: azide and alkyne yielding covalent product-1,5-disubstituted 1,2,3-triazole. Since both azido and alkyne groups are nearly never encountered in natural biomolecules, the reaction is highly bioorthogonal and specific. Click Chemistry is based on copper catalysis. The catalyst is often introduced as Cu-TBTA complex. Azide-functionalized antibodies are “clicked” to alkyne-modified ONs and the resulting AOCs are purified and characterized for further analysis.

Sometimes, the click reaction is not suitable for applications involving functional biomolecules because copper ions may be detrimental to these functional biomolecules molecules. For instance, copper ions can cause protein denaturation. To circumvent this issue, a Cu-free click reaction based on strain-promoted alkyne-azide cycloaddition (SPAAC) has been developed. There are mainly two approaches for the introduction of both strained alkynes and azides into antibodies, classical chemical approaches based on lysine modification with heterobifunctional linkers and the introduction of unnatural amino acids into antibodies.

Fig.3 Schematic representation of the conjugation process using click chemistry. (Gong, 2016)

• Hydrazone Coupling

Hydrazone chemistry is used to covalently crosslink any two amine-containing biomolecules based on the reaction of a succinmidyl 4-hydrozinonicotinate acetone hydrazine (S-HyNic) linker with a succinmidyl 4-formylbenzoate (4-FB) linker to form a stable hydrazone bond. The antibody is first reacted with an S-HyNic to introduce nucleophilic hydrazine residues. The ON is reacted with a 4-FB linker to introduce an electrophilic aldehyde residue. After the purification of two reaction partners, they readily react with each other yielding an AOC containing a hydrazone bond. The bond created is both stable and UV-traceable.

Fig.4 An example of hydrazone coupling. (Dovgan, 2019)

• Amine-to-Thiol Coupling

The most often applied chemical bioconjugation approaches for linking ONs with mAb make use of heterobifunctional cross-linking reagents, such as SMCC. The thiol-modified ON can be directly prepared on an automated DNA synthesizer using standard phosphoramidite chemistry. The mAb is converted into a maleimide-modified substrate using a reaction with cross-linkers containing activated ester and maleimide residues in their structure. The two modified parts are mixed, and the reaction is initiated by increasing the pH of the reaction buffer. Gel filtration is usually performed to purify the obtained AOC. The main advantage of direct chemical coupling is its universal applicability to any mAb and ON without reengineering.

Fig.5 Amine-to-thiol coupling approach for the preparation of AOCs. (Dovgan, 2019)

• Inverse Electron-Demand Diels-Alder Reaction (iEDDA)

The iEDDA is an organic chemical reaction, in which two new chemical bonds and a six-membered ring are formed. This reaction was demonstrated to have some advantages over SPAAC in terms of kinetics and was subsequently applied to AOC generation. Based on iEDDA, AOCs can be generated by coupling tetrazine-modified antibodies containing a disulfide cleavable linker with trans-cyclooctene (TCO)-modified DNA tags.

Fig.6 Application of iEDDA reaction for AOC generation. (Dovgan, 2019)

• DNA-Templated Protein Conjugation (DTPC)

DTPC is based on the principle by which the local concentration of reactive components is increased through site-selective non-covalent preorganization. This method relies on three components: a protein to be labeled that possesses an affinity for a metal ion, an ON modified with a ligand functionality (NTA groups) and a complementary ON that carries an activated ester capable of reacting with the ɛ-amine on lysines. As Fig.6 shown, a chemically activated ON (red) is site-selectively conjugated to a protein (green) by a template-facilitated (blue) reaction.

Fig.6 Expanding the concept of DTPC. (Rosen, 2014)

Our custom antibody covalent conjugation services combine years of expertise with a wide selection of approaches. We provide covalent conjugation services for both research and commercial use. As a well-recognized leader in AOC preparation, Creative Biolabs is dedicated to the development of AOC agents. If you are interested in developing AOCs by our covalent approaches, please feel free to contact us for more information.

References

1. Dovgan, I.; et al. Antibody-oligonucleotide conjugates as therapeutic, imaging, and detection agents. Bioconjugate chemistry. 2019, 30(10), 2483-2501.
2. Gong, H.; et al. Simple method to prepare oligonucleotide-conjugated antibodies and its application in multiplex protein detection in single cells. Bioconjugate chemistry. 2016, 27(1): 217-225.
3. Rosen, C. B.; et al. Template-directed covalent conjugation of DNA to native antibodies, transferrin and other metal-binding proteins. Nature chemistry. 2014, 6(9): 804-809.

For Research Use Only. NOT FOR CLINICAL USE.