Site-Specific ⁸⁹Zr- and ¹¹¹In-Radiolabeling and In Vivo Evaluation of Glycan-free Antibodies by Azide–Alkyne Cycloaddition with a Non-natural Amino Acid

Antibody–drug conjugates (ADCs) are a class of targeted therapeutics consisting of a monoclonal antibody coupled to a cytotoxic payload. Various bioconjugation methods for producing site-specific ADCs have been reported recently, in efforts to improve immunoreactivity and pharmacokinetics and minimize batch variancepotential issues associated with first-generation ADCs prepared via stochastic peptide coupling of lysines or reduced cysteines. Recently, cell-free protein synthesis of antibodies incorporating para-azidomethyl phenylalanine (pAMF) at specific locations within the protein sequence has emerged as a means to generate antibody–drug conjugates with strictly defined drug–antibody-ratio, leading to ADCs with markedly improved stability, activity, and specificity. The incorporation of pAMF enables the conjugation of payloads functionalized for strain-promoted azide–alkyne cycloaddition. Here, we introduce two dibenzylcyclooctyne-functionalized bifunctional chelators that enable the incorporation of radioisotopes for positron emission tomography with ⁸⁹Zr (t_1/2 = 78.4 h, β⁺ = 395 keV (22%), γ = 897 keV) or single photon emission computed tomography with ¹¹¹In (t_1/2 = 67.3 h, γ = 171 keV (91%), 245 keV (94%)) under physiologically compatible conditions. We show that the corresponding radiolabeled conjugates with site-specifically functionalized antibodies targeting HER2 are amenable to targeted molecular imaging of HER2+ expressing tumor xenografts in mice and exhibit a favorable biodistribution profile in comparison with conventional, glycosylated antibody conjugates generated by stochastic bioconjugation.