Page 70 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 4
By changing only a few key amino acids in the CH3 domain that are pivotal to the pairing of the heavy chains, i.e. replacing a small side-chain with a large side-chain on one heavy chain (knob) and vice versa for the other heavy chain (hole), heterodimerization can be promoted.29, 30 While KiH technology has been applied to create immunocytokines based on genetic encoding of a C- terminal fusion protein on one of the heavy chains,25 a modular approach derived from a single engineered antibody scaffold would allow for easy access to a wide range of mono-functional conjugates.
Here we show that knob-in-hole technology can be readily applied for the generation of [2]:1 antibody-conjugates by selective strain-promoted cycloaddition of a single heavy chain (HC) based on ortho-quinone chemistry or tetrazine ligation (Figure 1B). We found that this strategy is suitable for the formation of antibody-small molecule, antibody-protein and antibody- antibody conjugates.
4.2. Results and discussion
Two-stage antibody conjugation based on tetrazine ligation. A knob-in-hole variant of trastuzumab was generated by introduction of a single mutation in the ‘knob’ heavy chain (T366W; knob-HC) and three mutations in the ‘hole’ heavy chain (T366S, L368A and Y407V; hole- HC).31 For conjugation, the hole-HC contained a C-terminal sortase LPETG-tag.14, 31 After transient expression and subsequent purification by protein A affinity chromatography, analysis by SDS- PAGE, HPLC and LC-MS confirmed the existence of two distinct heavy chains (Figure 2A).
Figure 2. HPLC traces of DTT denatured (A) Tras[KiH-HC]ST, (B) Tras[KiH-HC]TCO, and (C) Tras[KiH-HC]MeTz.
68