Page 100 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 6
6.1. Introduction
The pharmacokinetic profile of an antibody conjugate depends not only on the homogeneity of the conjugation method,1 but also on the selected site of conjugation.2 First of all, highly solvent- accessible conjugation sites may be susceptible to increased deconjugation through various mechanisms, such as reverse Michael reaction of thiol-maleimide conjugate,3 or valine-citrulline- p-aminobenzylcarbamate (vc-PABC) linker cleavage by endogenous esterases.4 Generally, more readily accessible linker-drugs on a protein display compromised pharmacokinetics, which can be improved by optimizing conjugation site and the type of linker.5 The enhanced clearance of ADCs obtained by C-terminal conjugation suggests that our previously reported SPOCQ method, although highly stable, is based on labeling of exposed tyrosine tags engineered at either terminus, and might therefore generate antibody conjugates with non-optimal in vivo efficacy.
While functionalization of non-terminal tyrosine residues has been demonstrated, these methods rely either on non-specific oxidation conditions (Fremy’s salt6) or involve the use of non-natural amino acids bearing a catechol moiety in the form of L-DOPA.7 While the latter approach allows for mild oxidation conditions (e.g. sodium periodate), it also requires the rather complicated and low-yielding genetic encoding of L-DOPA during protein expression. We8 and others9 have reported earlier that selective tyrosine oxidation and subsequent conjugation under the action of mushroom tyrosinase (mTyr) is feasible in case the tyrosine residue resides at (or close to) the protein’s N- or C-terminus, leaving all other tyrosine residues in the protein sequence unmodified. Based on the premise of potentially improved pharmacokinetics, we explored the conjugation of non-terminal tyrosine residues based on selective oxidation and conjugation. We here report two strategies for non-terminal SPOCQ, either based on the use of an endogenous, naturally exposed tyrosine or based on the engineering of specific tyrosine- containing peptide loop structures.
6.2. Cystine knobs
Disulfide bridges in CDR regions are rare, but in some cases are naturally encoded in the third heavy chain loop of the CDR (CDR-H3): approximately 6% of human CDR-H3 sequences contain two additional cysteine residues separated by 0-12 amino acid residues,10 with the vast majority (91%) separated by 3 or 4 amino acids, forming a short loop, also known as a cystine knob.10 A recent study of an antibody containing a cystine knob showed that mutagenesis of the two cysteine residues severely hampered binding efficiency.11 By mutating one or two cysteines to alanines, antigen binding would be decreased 102-104 fold, demonstrating the importance of the rigid cystine loop. Another antibody known to feature a cystine knob is AT1413. This patient- derived antibody specifically recognizes a sialylated epitope on CD43,12 a transmembrane cell surface protein expressed on all cell lines of acute myeloid lymphoma (AML).13, 14
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