Targeted Fluorogenic Cyanine Carbamates Enable In Vivo Analysis of Antibody–Drug Conjugate Linker Chemistry

Abstract

Antibody-drug conjugates (ADCs) are a rapidly emerging therapeutic platform. The chemical linker between the antibody and the drug payload plays an essential role in the efficacy and tolerability of these agents. New methods that quantitively assess cleavage efficiency in complex tissue settings could provide valuable insights into the ADC design process. Here we report the development of a near-infrared (NIR) optical imaging approach that measures the site and extent of linker cleavage in mouse models. This approach is enabled by a superior variant of our recently devised cyanine carbamate (CyBam) platform. We identify a novel tertiary amine-containing norcyanine, the product of CyBam cleavage, that exhibits dramatically increased cellular signal due to improved cellular permeability and lysosomal accumulation. The resulting cyanine lysosome-targeting carbamates (CyLBams) are ~50X brighter in cells, and we find this strategy is essential for high-contrast in vivo targeted imaging. Finally, we compare a panel of several common ADC linkers across two antibodies and tumor models. These studies indicate that cathepsin-cleavable linkers provide dramatically higher tumor activation relative to hindered or non-hindered disulfides -an observation that is only apparent with in vivo imaging. This strategy enables quantitative comparisons of cleavable linker chemistries in complex tissue settings with implications across the drug delivery landscape.

Structure–Activity Relationships of Antibody-Drug Conjugates: A Systematic Review of Chemistry on the Trastuzumab Scaffold

“…There remains a significant need for technologies to assess these factors earlier in the ADC design process. A powerful approach is to apply optical imaging to address questions of antibody targeting and linker activation. ,− Such studies have provide significant evidence highlighting the dramatic role of payload properties on antibody targeting. − Thurber and co-workers recently reported efforts to image bystander penetration of microtubule inhibitors, DNA-damaging agents, and topoisomerase inhibitors . They note that MMAE, calicheamicin D, and exatecan showing the greatest bystander killing of the agents measured.…”

“…Our group recently reported fluorogenic (i.e., turn-ON) probes that operate in the near-infrared (NIR) range . We has used the antibody-fluorophore conjugates as a surrogate to compare a panel of several common ADC linkers across two antibodies (anti-EGFR, Cetuximab, and anti-CD-276) . These in vivo imaging studies clearly indicate that cathepsin-cleavable linkers provide higher tumor activation relative to hindered or nonhindered disulfides, at least in this context.…”

Targeted Dual-Modal PET/SPECT-NIR Imaging: From Building Blocks and Construction Strategies to Applications

“…We recently reported a non-FRET-based turn-on strategy in the NIR range. Fluorogenic heptamethine cyanine fluorophores were generated by forming norcyanine carbamates (CyBam) [ 150 , 151 ]. This strategy avoids the chemical complexity of a quencher group.…”

A Hoechst Reporter Enables Visualization of Drug Engagement In Vitro and In Vivo: Toward Safe and Effective Nanodrug Delivery

“…g ., polymers), − or biodistribution of drugs based on mass spectrometry methods ( e . g ., inductively coupled plasma-mass spectrometry). , Recent efforts have also yielded fluorescent “prodrug-like” probes that can detect linker cleavage by a change in fluorescence . Although informative, the above-mentioned methods do not enable in situ detection of drug engagement with its intended cellular target.…”