Page 102 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 6
In a previous chapter, we reported that engineering of a C-terminal G4Y tag on an antibody light or heavy chain enables selective tyrosine oxidation followed by in situ conjugation based on SPOCQ with a BCN-modified fluorophore. SPOCQ with BCN-lissamine was attempted on an AT1413 mutant bearing the C-terminal G4Y tag on its light chain, as well as a heavy chain sortase tag (i.e. AT1413[HC]ST-[LC]G4Y). SDS-PAGE revealed conjugation on the light chain went in identical fashion to earlier findings; however, fluorescence was also detected on the antibody heavy chain with similar efficiency (Figure 1A). This was also the case for the AT1413 mutant AT1413[HC]ST (Fc0), in which Fc0 represents two single point mutations (G236R and L328R) to abolish interaction with other, non T-cell, immune effector cells.15 Variation of pH (pH 7.0 and pH 5.5) had little influence on conjugation efficiency, with SPOCQ performed at pH 5.5 yielding slightly cleaner conjugates as reported before.16 Similarly, prior sortagging at the heavy chain C- terminus (with scFv UCHT1), led to identical conjugation on both chains (Figure 1B). In order to exclude any influence of the sortase recognition sequence of the antibody or fusion of UCHT1 to the antibody, SPOCQ was also performed on the similar conjugate based on control antibody AT1002[HC]UCHT1-[LC]G4Y, which showed exclusive labeling on the light chain, corroborated by SDS-PAGE and HPLC analysis (Figure 1). Since we had not earlier observed any labeling of native antibody heavy chain in the past, it was reasoned that the addition reactivity had to be attributed to the presence of the CDR cysteine loop in AT1413.
Earlier, we found that AT1413 is no longer able to recognized to CD43 when tyrosine was mutated (unpublished results), strongly indicating an essential role for the tyrosine residue in target binding. This in turn would suggest that the tyrosine residue, presented in a cysteine loop “...GCGYSSCF...” in the CDR-H3 (HC-Y106), might be the tyrosine oxidized by mushroom tyrosinase. However, this could not be concluded based on SDS-PAGE and HPLC experiments only. While a logical next step could involve expression of an AT1413 mutant at position Y106 or tryptic digest and MS analysis of the peptide fragment before and after SPOCQ labeling, we considered an entirely new approach to confirm the labeling of Y106. We reasoned that the addition to AT1413 of a stoichiometric quantity of an anti-idiotypic antibody, which binds to the CDR by definition, would sterically block to accessibility for mushroom tyrosinase and thereby effectively preventing oxidation of Y106. Indeed, this hypothesis was confirmed when SPOCQ was attempted in the presence of various stoichiometries of an anti-idiotypic antibody (AIA) for AT1413:17 addition of AT1413-AIA to AT1413[HC]ST under SPOCQ labeling conditions fully blocked oxidation and subsequent cycloaddition (Figure S1). To the best of our knowledge, no such transient ‘protection’ of native amino acid functionality with monoclonal antibodies during a biochemical processing step has been reported to date and warrants further investigation. At the same time, while these findings clearly provide a clear proof-of-concept for the feasibility of SPOCQ for labeling of non-terminal tyrosine residues, it is also apparent that conjugation on tyrosine in the CDR of an antibody would impede the essential binding of the antibody to its target antigen and was therefore not further explored here.
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