Page 75 - Tyrosine-Based Bioconjugations - Jorick Bruins

Monofunctionalization of Knob-in-hole Antibodies
oxidation by mTyr and addition of BCN–UCHT1 (Calc. Mw: 27987.0 Da), and the second peak is 53448 Da, corresponding to the same conjugate with incomplete deglycosylation by endo S2, corresponding to a G0F glycan (calc. Mw: 53446 Da).
4.3. Conclusion
We have successfully developed a modular approach to site-selectively mono-functionalize one heavy chain of an antibody. Using asymmetrical knob-in-hole antibodies, a single peptide tag was readily introduced for chemoenzymatic functionalization of one heavy chain by an indirect approach using sortase or by direct labelling with SPOCQ. Subsequently, the method was shown to allow for rapid and selective introduction of a single fluorophore, a cytokine, or an scFv on the antibody. Additionally, highly efficient dimerization of monoclonal antibodies was achieved, which would allow for the production of clean bispecific antibodies in a modular fashion and with high conversions. The interest in these bispecific antibodies have increased significantly over the last years, since bispecific antibodies can directly target immune cells to tumours,40 with many of them in clinical trials.41 With SPOCQ, near-quantitative conversions are obtained after only 20 minutes of reaction time. We envision this modular approach may open up possibilities for new and potent therapeutic agents. Comparison of our technology with established fusion formats for making such bioconjugates should reveal if our current method is, indeed, a viable chemical alternative to the more established biological methods.
4.4. Supporting information
This work can be found at https://pubs.acs.org/doi/full/10.1021/acsomega.9b01727. Further permissions related to the material excerpted should be directed to the ACS
The Supporting Information is available free of charge.
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