Page 68 - Tyrosine-Based Bioconjugations - Jorick Bruins
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Chapter 4
4.1. Introduction
Chemical conjugation of functional molecules to monoclonal antibodies is widely applied in fields spanning from fundamental biology research to targeted therapy. These applications include, but are not limited to, cellular imaging,1, 2 antibody-drug conjugates,3, 4 and diagnostics.5 Common conjugation strategies, based on side-chain modification of lysine or cysteine, are straightforward and efficient but provide poor control of regioselectivity and stoichiometry.6, 7 With the expansion of the genetic code to incorporate non-canonical amino acids, chemically orthogonal handles can be introduced at predefined sites in a given protein sequence,8, 9 which allows for chemo-selective oxime ligation10 or click chemistry.11 In addition, a wide arsenal of methods is available for site-specific protein modification with varying degrees of versatility.12
Besides chemical strategies, enzymatic conjugations have emerged recently as they enable highly controlled modification of antibodies through specific peptide tags.13 Examples of enzymatic processes include sortase ligation,14, 15 phosphopantetheinyl transferase,16 and transglutaminase.17 Recently, we demonstrated that selective labelling of monoclonal antibodies can be ensured in a single step based on introduction of a C-terminal tetra-glycyltyrosine tag (G4Y, Figure 1A).18-20 Specifically, oxidation of the phenol moiety of the tyrosine in the G4Y tag to an ortho-quinone by mushroom tyrosinase (mTyr) and in situ Diels-Alder reaction with bicyclo[6.1.0]nonyne (BCN) leads to selective labelling in high yields and under mild conditions, with reaction rates exceeding those of the alkyne–azide cycloaddition (SPAAC) by at least a factor of 500. This strain-promoted oxidation-controlled ortho-quinone cycloaddition (SPOCQ) was subsequently shown to also proceed with cyclopropenes, albeit significantly slower,21 or could be further accelerated by employing cyclopropanated trans-cyclooctene (cpTCO).22
Given the symmetrical nature of an antibody, the site-specific methods described above lead to dual labelling of the protein. However, in some cases forming a [2]:1 antibody format (i.e. two antigen binding sites and one conjugated molecule of interest) may be more desirable, for example in the generation of an antibody-drug conjugate with an extremely potent cytotoxin such as a PBD dimer23 or by radiolabeling with -emitters such as Thorium-227.24 Whilst these formats have been reported, generation of these conjugates relies on approaches with poor control of site and stoichiometry like random lysine conjugation followed by isolation of the mono-functionalized conjugate,24 or required the rearrangement of several disulfide bridges between the light and heavy chains, resulting in a significant loss of binding activity and stability.23 Similarly, various antibody conjugates with a [2]:1 format to enhance the therapeutic window, e.g. with IL-225 and α-CD326, have been reported. The latter conjugates were obtained by fusion of an immunocytokine or T-cell engager to a single chain of an asymmetric antibody format, known as knob-in-hole (KiH) antibodies.27, 28
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