Page 89 - Tyrosine-Based Bioconjugations - Jorick Bruins

Preparation of Trifunctional Protein-antibody Conjugates
When time-resolved SPOCQ was performed on all three trastuzumab variants with TCO–AF568 as reactant, significant differences in reaction rates were observed (Figure 5). Tras[LC]G4Y shows the slowest reaction rate, with about 90% conversion after eight hours. Tras[LC]G4SG4Y shows over fourfold enhanced reaction rate, with the reaction nearing completion after two hours and showing full completion after three hours. Finally, SPOCQ on Tras[LC]G4SG4SG4Y is nearly complete after one hour, and full completion is detected after two hours.
Figure 5. Structures of Tras[LC]G4Y, Tras[LC]G4SG4Y, and Tras[LC]G4SG4SG4Y and time-resolved SPOCQ with TCO-AF568.
The significant increase of reactivity by insertion of even a single G4S-spacer is most likely due to the LC-HC disulfide bridge, which for the light chain is located at the very C-terminus (C214). In Tras[LC]G4Y the tyrosine residue is only four amino acids removed from the disulfide bridge, whereas in Tras[LC]G4SG4Y the residue is nine amino acids removed. As anticipated, having the tyrosine residue more exposed allows it to enter the active site of mTyr more easily, resulting in faster oxidation and subsequent cycloaddition. By increasing the linker to G4SG4SG4SG4Y one would expect even faster conjugation. While this might be true, increasing the length of flexible linkers such as the (G4S)n linker can lead to significant drops in expression yields and/or loss of biological activity, including reduced antibody-antigen or scFv-antigen binding.23, 25 Therefore, the significant increase in reaction rate that comes with one or two G4S-spacers should be sufficient for this approach.
Finally, a time-resolved SPOCQ on Tras[LC]G4SG4SG4Y with BCN–lis was performed. This reaction yielded identical results compared to the reaction with TCO-AF568. The lack of difference in the
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