Bioorthogonal Fluorophore Linked DFO—Technology Enabling Facile Chelator Quantification and Multimodal Imaging of Antibodies

Abstract

Herein we describe the development and application of a bioorthogonal fluorogenic chelate linker that can be used for facile creation of labeled imaging agents. The chelate linker is based on the trans-cyclooctene(TCO)-tetrazine(Tz) chemistry platform and incorporates deferoxamine (DFO) as a 89Zr PET tracer and a BODIPY fluorophore for multimodal imaging. The rapid (<3 min) ligation between mAb-TCO and Tz-BODIPY-DFO chelator is monitored using fluorescence and allows for determination of labeling completion. Utilizing BODIPY as the linker between mAb and DFO facilitates in chelator quantification using spectrophotometry, allowing for an alternative to traditional methods (mass and isotope dilution assay). Radiolabeling with 89Zr to form 89Zr-DFO-BODIPY-trastuzumab was found to be quantitative after incubation at room temperature for 1 h (1.5 mCi/mg specific activity). The cell binding assay using HER2+ (BT474) and HER2- (BT20) cell lines showed significant binding to 89Zr-DFO-BODIPY-trastuzumab (6.45 ± 1.87% in BT474 versus 1.47 ± 0.39% in BT20). In vivo PET imaging of mice bearing BT20 or BT474 xenografts with 89Zr-DFO-BODIPY-trastuzumab showed high tumor conspicuity, and biodistribution confirmed excellent, specific probe uptake of 237.3 ± 14.5% ID/g in BT474 xenografts compared to low, nonspecific probe uptake in BT20 xenografts (16.4 ± 5.6% ID/g) 96 h p.i. . Ex vivo fluorescence (465ex/520em) of selected tissues confirmed superb target localization and persistence of the fluorescence of 89Zr-DFO-BODIPY-trastuzumab. The described platform is universally adaptable for simple antibody labeling.

Multifunctional Desferrichrome Analogues as Versatile 89Zr(IV) Chelators for ImmunoPET Probe Development

“…We have used a similar approach to quantify conjugation efficiency of fluorogenic BODIPY-DFO derivatives to antibodies. 44 The trifunctional trastuzumab conjugate 8 –trastuzumab was also utilized for the study of probe uptake in cells using fluorescence microscopy. We incubated BT474 cells (5 × 10 4 ) with 15 μ g of conjugate for 24 h and subsequently assessed cell uptake by fluorescence microscopy.…”

Emerging Applications of Fluorogenic and Non‐fluorogenic Bifunctional Linkers

“… Hetero‐bifunctional SCF linkers for the fabrication of tumor‐targeting dual‐modality imaging probes, antibody–probe conjugates, through fluorogenic conjugation between an antibody and nuclear imaging moiety: a) conjugation of DFO, a 89 Zr PET tracer, with tz‐TCO by utilizing SCF4 as the bridging unit; b) conjugation of 2,2′‐[7‐(3‐amino‐1‐carboxypropyl)‐1,4,7‐triazonane‐1,4‐diyl]diacetic acid (NH 2 ‐NOTA), a 64Cu PET tracer, with acrylamide‐functionalized cetuximab (ct‐Acm) by utilizing SCF5 as the bridging unit …”

“…In this respect, Weissleder et al reported fluorogenic conjugation between deferoxamine (DFO), a 89 Zr PET tracer,a nd transcyclooctene-functionalized trastuzumab (tz-TCO) by utilizing SCF4 as af luorogenic linker. [60] Notably,t he fluorogenic nature allows for the quantification of coupling efficiency. An onfluo-rescenti ntermediate, SCF4-DFO, was synthesized first through an amidation reaction.…”

Cherenkov Radiation‐Mediated In Situ Excitation of Discrete Luminescent Lanthanide Complexes

“…[14–15] Subsequently, we designed and synthesized a Tb(III) complex that allows for covalent attachment of the CR emitting isotope 89 Zr, generating a Tb(III) complex with an intramolecular CR source (Scheme 2). …”