Page 17 - Tyrosine-Based Bioconjugations - Jorick Bruins

version of chemotherapy. By coupling a highly potent cytotoxic agent to an antibody, it can be delivered directly to the tumor after binding and internalization. This approach may significantly increases the therapeutic index of the chemotherapy.i Release of the drug is important in this matter, which can be achieved via a cleavable linker between the drug and antibody,31-33 by using bispecific antibodies to increase internalization,19, 20 and using click-chemistry to release the drug by adding a second chemical probe.34
The conjugation of radionuclides or cytotoxic molecules to an antibody of choice is not trivial. Although ADCs can be readily obtained by acylation of the reactive amino group side-chain of lysine residues, random conjugation leads to a highly heterogeneous mixture of conjugates with non-optimal therapeutic index due to decreased binding affinity, accelerated clearance and increased toxicity.35 Therefore, site-specific conjugation techniques have been widely investigated.36-39
1.2. Bioconjugate chemistry
Bioconjugate chemistry is the discipline of using chemistry to form covalent bonds between two molecules, of which at least one is a protein, a glycan, a nucleic acid or a lipid.36 The resulting modified biomolecules can fulfil a plethora of purposes such as targeted drug delivery,40, 41 cell imaging,38, 42 improved pharmacokinetics (e.g. lower clearance rate, increased thermostability),43 and diagnostic tools.44, 45 In this thesis, the focus is entirely on the modification of proteins.
Protein conjugation is unique in the sense that several of the 20 canonical amino acids have specific reactivities,45 combined with the vast variety of protein structures and functions this leads to endless possibilities for protein conjugates. Employing the innate reactivity of the various amino acids side-chains, in particular lysine and cysteine, is the most straightforward and abundant way to generate protein conjugates.45 The full extent of these strategies are meticulously described in several review papers,45-47 therefore only a few examples will be discussed here.
1.2.1. Labeling based on natural amino acids
Lysine residues (1) are widely used in protein modification, as the e-amino function on the side- chains readily reacts with electrophilic reagents such as activated esters (Figure 4A, 2), yielding stable adducts (3).45 A downside of this innate reactivity is the general lack of selectivity for a specific residue in a given protein, as all or the majority of solvent-accessible residues will undergo acylation, leading to heterogeneous mixtures. Amongst various efforts, one recent
i The therapeutic index (TI), or safety window, refers to the ratio between toxic and effective dose. While an indicative TI may be quickly derived based on various animal models, in humans, the TI is defined as the dose where 50% of humans experience adverse side-effects (TD50), divided by the dose where 50% of humans experience desired pharmacological effects (ED50), resulting in: TI = (TD50) / (ED50).
General Introduction
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