Site-Specifically Labeled Antibody–Drug Conjugate for Simultaneous Therapy and ImmunoPET

The conjugation of antibodies with cytotoxic drugs can alter their <i>in vivo</i> pharmacokinetics. As a result, the careful assessment of the <i>in vivo</i> behavior, and specifically the tumor-targeting properties, of antibody–drug conjugates represents a crucial step in their development. In order to facilitate this process, we have created a methodology that facilitates the dual labeling of an antibody with both a toxin and a radionuclide for positron emission tomography (PET). To minimize the impact of these modifications, this chemoenzymatic approach leverages strain-promoted azide–alkyne click chemistry to graft both cargoes to the heavy chain glycans of the immuoglobulin’s F_c domain. As a proof-of-concept, a HER2-targeting trastuzumab immunoconjugate was created bearing both a monomethyl auristatin E (MMAE) toxin as well as the long-lived positron-emitting radiometal ⁸⁹Zr (<i>t</i>_1/2 ≈ 3.3 days). Both the tumor targeting and therapeutic efficacy of the ⁸⁹Zr-trastuzumab-MMAE immunoconjugate were validated <i>in vivo</i> using a murine model of HER2-expressing breast cancer. The site-specifically dual-labeled construct enabled the clear visualization of tumor tissue via PET imaging, producing tumoral uptake of ∼70%ID/g. Furthermore, a longitudinal therapy study revealed that the immunoconjugate exerts significant antitumor activity, leading to a >90% reduction in tumor volume over the course of 20 days