Page 90 - Tyrosine-Based Bioconjugations - Jorick Bruins
P. 90

Chapter 5
conversion over time for two compounds that have significantly different reaction rates for the cycloaddition indicate that the rate-limiting step of SPOCQ is the oxidation by mTyr. This is in line with all our findings so far, where the reaction rate of SPOCQ on proteins is based on the accessibility of the tyrosine residue.
5.4. Protein-protein conjugation with BCN-PEG3-TCO
To ability of the two-step conjugation method to introduce proteins on trastuzumab was investigated next. Using BCN-PEG3-TCO 6, TCO moieties were introduced on Tras[LC]G4SG4Y and Tras[LC]G4SG4SG4Y and the products were subsequently purified with protein A spin columns. Next, the obtained TCO-bearing antibodies were subjected to conjugation with one of the following constructs: the fluorophores azido–lissamine (N3–lis) or MeTz–TAMRA, the scFv MeTz– UCHT1, cytokine MeTz–IL-2, or the oligodeoxynucleotide (MeTz–ODN1826).26 The products of these reactions were analyzed by SDS-PAGE (Figure 6).
SDS-PAGE analysis of the TCO-bearing antibodies show a clean product after protein A purification (Figure 6, lanes 1 and 7), while no reaction was observed when incubated overnight with N3–lis (Figure 6, lanes 2 and 8). This further confirms the selectivity of BCN over TCO for the initial conjugation with 6, leaving only free TCO on the antibody. Since reaction of TCO with azides is highly sluggish (0.0064 M-1 s-1),27 it is too slow for efficient labelling with azido-lissamine under relatively high dilution conditions. However, fluorescence is clearly visible when incubated with MeTz–TAMRA (Figure 6, lanes 3 and 9), confirming that TCO is present on the light chains. These findings were confirmed by LC-MS (SI S5.5).
Analogously, conjugation of MeTz–UCHT1, MeTz–IL-2 and MeTz–ODN1826 with TCO-bearing antibodies were analyzed. The mass of MeTz-UCHT1 (27740 Da) is similar to the light chains, and SDS-PAGE analysis does not depict the conjugation properly (Figure 6, lanes 4 and 10). LC-MS analysis, however, shows the expected mass increase of the Tras[LC]G4SG4Y light chain from 24679 Da to 52392 Da (SI Figure S10), corresponding to the addition of MeTz-UCHT1.28 The same observations are made for Tras[LC]G4SG4SG4Y conjugation, with the mass of the TCO-bearing light chain shifting from 24994 Da to 52705 Da (SI Figure S16). Finally, MeTz–IL-2 and MeTz– ODN1826 were conjugated to the TCO-bearing antibodies, both showed high-yielding conversion on SDS-PAGE (Figure 6, lanes 5 and 11, and lanes 6 and 12, respectively).
5.5. Combining SPOCQ and sortase ligation for the generation of trifunctional antibodies
Having successfully developed the two-step SPOCQ ligation, we next considered the generation of trifunctional antibodies by combination of SPOCQ with sortase A. To this end, antibody AT1002 was modified to contain a sortase tag (ST) on both heavy chains and a G4SG4SG4Y-tag on
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