Page 73 - Tyrosine-Based Bioconjugations - Jorick Bruins

Monofunctionalization of Knob-in-hole Antibodies
lissamine (Figure 3, lane 8), TCO–IL-2 (Figure 3, lane 9) or TCO–UCHT1 (Figure 3, lane 11). In all cases, the desired mono-functionalized antibody was successfully conjugated on the desired hole-HC, and formation of the fluorophore-, IL-2-, and UCHT1-conjugates on Tras[KiH-HC]MeTz were confirmed by LC-MS (SI). Interestingly, SDS-PAGE shows that the conversion is significantly cleaner when compared to the Tras[KiH-HC]TCO MeTz-protein combination. A potential explanation for the lower conjugation efficiency of TCO-modified antibody may be due to in situ isomerization of the trans-alkene in TCO caused by the cysteine thiol in the active site of sortase A.35 In this light, having the MeTz on the antibody and the TCO/BCN on the reactive protein partner is clearly the preferred strategy towards antibody-protein conjugates.
Finally, we explored the potential of the two-stage protocol of sortagging-tetrazine ligation for the controlled formation of functionally homogeneous antibody dimers. To this end, Tras[KiH- HC]MeTz was incubated with Tras[KiH-HC]TCO in a 1.5:1 ratio. The rationale for the latter strategy was based on our finding that excess tetrazine-modified antibody could be conveniently removed by capture with TCO-modified agarose beads, leading to clean antibody dimers (SI). Utilization of the modularity of these antibodies allows for straightforward production of dimeric antibodies with good conversion and high purity, and offers a viable chemical alternative for known methods that require the expression of fusion proteins36, 37 or are based on chemical approaches yielding highly heterogeneous antibody dimers.38
Direct conjugation based on ortho-quinone chemistry. Besides the two-step method that combines sortagging with MeTz or TCO followed by tetrazine ligation, the possibility of direct labelling of knob-in-hole antibodies with SPOCQ chemistry was investigated next. SPOCQ (strain- promoted oxidation-controlled ortho-quinone cycloaddition) was originally developed based on chemical generation of ortho-quinones.18 Recently, we showed it can be conveniently applied in a one-pot process involving oxidation of a tyrosine side-chain to an ortho-quinone by the action of mushroom tyrosinase (mTyr), followed by in situ reaction with a BCN-bearing probe by (4+2) cycloaddition to form a stable product (Figure 4B).19
Earlier we described that SPOCQ on the symmetrical antibody Tras[HC]G4Y converts to 95% of the desired bis-functionalized adducts when small fluorophores were conjugated.22 However, this level of conversion could not be achieved when protein-protein conjugation was attempted on symmetrical G4Y-tagged heavy chain antibodies (SI). Presumably, the second ligation is sterically hindered, leading to intramolecular side-reactions of the in situ generated, highly reactive ortho-quinone intermediate with nucleophilic side-chains of amino acids such as lysine and histidine.39 However, we anticipated that a single SPOCQ conjugation event on a KiH antibody would not suffer from incomplete conjugation. In addition, direct SPOCQ would offer the opportunity to eliminate the sortase-mediated step as an exposed tyrosine moiety can be readily engineered at the C-terminus of any protein.
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