Site-specific antibody conjugation of metal chelators for nuclear imaging and therapy.

66 Introduction: Classical antibody bioconjugation techniques rely on non-site specific conjugation to amino acids, typically accessible lysines or reduced cysteines; however, stochastic conjugation can alter targeting affinity and pharmacokinetics of the antibody. ImmunoPET requires favorable in vivo pharmacokinetics and high binding affinity to the target for clinical applicability. Here, we describe the preparation and in vivo application of site-specifically functionalized immunoconjugates with chelators designed for diagnostic and therapeutic radiometals. The on-target uptake of site-specific immunoconjugates is compared to the uptake of classical stochastic conjugates via nuclear imaging and biodistribution in a murine xenograft model. Methods: Dibenzocyclooctyne (DBCO) functionalized radiometal chelators desferrioxamine (DFO) and 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were synthesized and conjugated to site-specifically incorporated para-azido phenylalanine of glycan-free variants of the antibody trastuzumab.1 Clinical trastuzumab was conjugated to isothiocyanate-functionalized chelators via lysine residues in a non-site-specific fashion. ESI-TOF MS/MS was employed to characterize the corresponding immunoconjugates and radiolabeling was carried out with 89Zr and 111In. The radiolabeled immunoconjugates were injected via tail-vein catheter into athymic nude mice with HER2-expressing SKOV-3 xenografts, and PET images were obtained 24, 48, 72 and 96 h post injection (p.i.). Biodistribution was carried out at the 96 h time point. Results: ESI-TOF MS/MS analyses show that site-specific, copper-free click chemistry results in predominantly one immunoconjugate product with 4 chelators appended, whereas stochastically labeled conjugate ranges from 0 to 6 chelators per antibody. Both DFO-functionalized immunoconjugates were efficiently radiolabeled with yields of 82±5% for stochastic 89Zr-DFO-trastuzumab and 78±6% for site-specific 89Zr-DFO-DBCO-trastuzumab, with specific activity of 0.85±0.15 mCi/mg and 0.79±0.05 mCi/mg respectively. SDS PAGE analysis confirms introduction of DBCO conjugates to be site-specific, occurring only at the heavy chains of the antibody. The presence of DBCO linker molecule does not alter the targeting affinity of the immunoconjugate, as evidenced by ROI analyses of PET images and biodistribution. Imaging indicates gradual accumulation of the conjugate at the target and rapid clearance from background organs. Biodistribution at 96 h p.i. reveal statistically equivalent target uptake, 19.5±9.0% ID/g for 89Zr-DFO-trastuzumab and 39.9±29.1% ID/g for 89Zr-DFO-DBCO-trastuzumab. We note a significantly lower liver uptake for 89Zr-DFO-DBCO-trastuzumab (1.73±0.90%) than 89Zr-DFO-trastuzumab (14.0±6.4%), underscoring the improved clearance properties of the site-specifically functionalized construct. The evaluation of 111In-DOTA-DBCO-trastuzumab and 177Lu-DOTA-DBCO-trastuzumab conjugates is ongoing. Conclusions: We have successfully prepared site-specifically radiolabeled immunoconjugates and assessed their performance in vivo. Site-specific functionalization using copper-free click chemistry produces immunoPET and immunoSPECT probes with retained high targeting affinity and favorable in vivo performance. The efficient click reaction between azide-functionalized antibody and DBCO linker provides a modular platform for preparing site-guaranteeing minimal batch variation and consistent in vivo performance.Reference: 1. Zimmerman, E.S., et al. Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system. Bioconjugate Chem. 2017, 25, 351-361.