Page 92 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 5
addition of sortase to any antibody-protein conjugate featuring the LPET-GGG linker would lead to sortase-mediated hydrolysis of peptide bond and deconjugation of the earlier attached functional groups. The alternative order of events, i.e. sortagging prior to SPOCQ, was therefore explored instead.
First, sortase A was used to introduce a methyltetrazine-tag on both heavy chains as reported.10 The methyltetrazine-tagged antibody was incubated with TCO–UCHT1 to produce two UCHT1- modified heavy chains. SDS-PAGE analysis indicated a clear conversion for the heavy chain by showing a protein band a 75 kDa product (Figure 7C, lane 1-3), which corresponds to the combined mass of the heavy chain and the UCHT1. Attention was then focused on conjugation of another protein, IL-2, to the antibody light chain based on SPOCQ chemistry. To this end, BCN- TCO bifunctional reagent 6 was incubated in the presence of tyrosinase to introduce a TCO- moiety on the G4Y-terminated light chain, followed by Protein A purification and subsequent incubation with MeTz–IL-2 for subsequent tetrazine ligation with the TCO-moiety (Figure 7A). Gratifyingly, the modified light chain showed up as a band at 45 kDa (Figure 7C, Lane 3-5), which corresponds to the combined mass of the light chain and the IL-2 cytokine.
Having successfully achieved the conversion of an antibody to the trifunctional construct with full loading of heavy and light chains mAb-to-protein ratio [2]:2:2, we investigated if it would be possible to obtain a trifunctional mAb-protein conjugate in [2]:1:1 ratio by execution of two mono-conjugation reactions in sequence. To this end, a AT1002 mutant was designed and transiently expressed based on knob-in-hole technology with two differentiated heavy chain C- termini: one bearing a LPETG sortase tag and one bearing a G4SG4Y-tag. The generated antibody, AT1002[KiH-HC]ST/G4Y, was subjected to the same sequence of events as described above, i.e. the LPETG-fused heavy chains was subjected to sortase-mediated introduction of methyltetrazine, followed by IEDDA with TCO–UCHT,9 whereas the C-terminal tyrosine on the other heavy chain was modified based on the reaction sequence by SPOCQ-mediated introduction of TCO followed by IEDDA with MeTz–IL-2 (Figure 7B). Whilst performing the reactions, samples were taken and analyzed with SDS-PAGE (Figure 7D). The starting antibody (lane 1) and the sortase-ligated product (lane 2) do not significantly shift on SDS-PAGE, however a 75 kDa side-product becomes apparent, as well as a band at 27 kDa (sortase A). However, after conjugation with TCO–UCHT1 (28 kDa) and subsequent protein A purification (lane 3), a clear shift to a 75 kDa band was observed, in analogy with the expected mass increase. Furthermore, the 75 kDa band of the modified heavy chain has roughly the same intensity as the 50 kDa band corresponding to the G4Y-bearing unmodified heavy chain, indicating that the generation of a mono-functionalized antibody is efficiently performed. When subsequent SPOCQ with 6 is
90