Page 88 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 5
under SPOCQ conditions, but does result in a difference in molecular weight and subsequent shift of the protein band on SDS-PAGE gel (Figure 4C). Interestingly, two-step conjugation with 2.5 or 5 equivalents of BCN–BCN linker 7 on AT1005[LC]G4Y gives rise to a fluorescently labelled light chain, but also generates high-molecular weight side products (Figure 5, lane C and D). In contrast, two-step conjugation with 5 eq. of BCN–TCO linker 6 results in efficient labelling of the light chain without any visible high-molecular weight side products (Figure 4C, lane E). Furthermore, the lack of any reaction on the heavy chain of the antibody confirms the orthogonality of SPOCQ with the LPETG moiety of the sortase tag.
When the two-step conjugation method was attempted on the antibody heavy chain as in AT1005[HC]G4Y however, neither 6 nor 7 gave full conversion. Even with 10 eq. of 6, the heavy chains of AT1005[HC]G4Y seem to dimerize significantly (Figure 4C, lane H). One possible explanation is that the first generation of quinone and conjugation with BCN proceeds according to expectation, however after the quinone is subsequently formed on the other heavy chain, a pseudo-intramolecular reaction takes place with TCO-moiety on the firstly introduced linker. The suggestion of such heavy chain dimerization is corroborated by the fact that this effect is enhanced with bis-BCN 7, where >300 eq. is not enough to prevent full dimerization (Figure 4C, lane K).
From these results we conclude that BCN-PEG3-TCO 6 gives rise to significantly cleaner conjugates than bis-BCN 7. However, the proposed two-step conjugation method does not yield satisfactory results with tyrosines on the C-terminus of antibody heavy chains. One obvious approach to circumvent heavy chain dimerization would be by avoiding a chemical moiety that undergoes cycloaddition with quinone altogether. However, another likely option we followed is to investigate a similar approach on the antibody light chains.
5.3. Accelerating SPOCQ by increasing linker length
While the light chain C-termini are structurally significantly more distant than the heavy chain C- termini, thereby naturally avoiding issues related to dimerization, SPOCQ on the light chain of antibodies is significantly slower than the heavy chain: while various trastuzumab G4Y heavy chain mutant undergo complete SPOCQ in 0.25–1.5 h,9, 20 Tras[LC]G4Y required overnight incubation to obtain high yields.22 Therefore, we reasoned that increasing the availability of the tyrosine residue for oxidation, by elongating the peptide linker, would increase the reaction rate. Whilst there are many peptide linkers with varying functions and properties,23 G4S-peptide linkers were investigated due to their inert nature, flexibility and stability.24 To this end, trastuzumab was expressed with either 0, 1 or 2 additional G4S spacers inserted before G4Y, resulting in Tras[LC]G4Y, Tras[LC]G4SG4Y, Tras[LC]G4SG4SG4Y (Figure 5).
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