Page 115 - Tyrosine-Based Bioconjugations - Jorick Bruins

ortho-quinone cycloaddition (SPOCQ). Thus, by performing the tyrosine oxidation in the presence of the strained alkyne bicyclo[6.1.0]nonyne (BCN, 3),6 fast cycloaddition takes place to the in situ generated quinone (Figure 1A), leading to a stable conjugate (4) in high yield.7 This protocol was first proven on the enzyme laminarinase A (LamA), and subsequently on various monoclonal antibodies for the generation of antibody-drug conjugates (Figure 1B, chapter 2).
Scheme 1. SPOCQ with cycloalkenes TCO (5) and cpTCO (7).
The initial protocol was optimized to increase reaction rate and purity of the products. Cycloaddition chemistry of the intermediate quinone was then extended to SPOCQ with trans- cyclooctene (TCO, 5) and cyclopropanated trans-cyclooctene (cpTCO, 7), and using the cpTCO we were able to perform bioconjugation in an orthogonal fashion to strain-promoted alkyne– azide cycloaddition (SPAAC) for tandem dual labeling of trastuzumab (Figure 2A, chapter 3). By application of knob-in-hole technology, trastuzumab variants were generated with two different heavy chain termini, thereby introducing only a single tag for SPOCQ labeling or sortase ligation, and we demonstrate the use of either strategy for rapid and highly efficient mono- functionalization (Figure 2B, chapter 4). We then developed a two-step labeling method for highly efficient protein-protein coupling using SPOCQ and IEDDA. Combining this two-step labeling strategy with sortase ligation, trifunctional antibodies were created by engineering a G4Y-tag on light chains and sortase tag (LPETG) on heavy chains. This allowed for the introduction of small molecules, cytokines, scFv fragments or oligonucleotides in high-yielding and selective fashion. We also demonstrate that a knob-in-hole antibody with two distinct heavy chains, one with G4Y-tag and one with sortase tag, allows for two consecutive modifications (Figure 2C, chapter 5). Finally, we demonstrate that SPOCQ on internal tyrosine residues is also feasible, by either expressing a tyrosine in a small cystine knob on the CDR or in a solvent-exposed loop in de CH2 region (Figure 2D, chapter 6).
Summary and General Discussion
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