The role of the near-infrared fluorescent dye on the pharmacokinetic behavior of fluorescent nanobody-based tracers for intra-operative cancer imaging

Introduction

Near-infrared fluorescence molecular imaging is a propitious tool aimed at supporting intra-operative

decision-making during cancer resection. Most targeted contrast agents currently under development are labelled with IRDye800CW®, however this dye is known to cause non-specific background signals. [1-5] Here, tumor-specific Nanobodies (Nbs) were labelled with alternative dyes, namely ZW800-1 [4,5], FNIR-Tag [6] and s775z [7], and a side-by-side comparison of their in vivo biodistribution and tumor targeting was performed to determine whether these dyes possess improved characteristics for the development of fluorescent Nb-based tracers. Furthermore, the impact of a higher dye density on the tracer’s pharmacokinetics was investigated.

 

Methods

An anti-EGFR Nb was randomly labeled with NHS-activated IRDye800CW, ZW800-1, FNIR-tag or s775z.

Excess of the dyes was adapted to yield a degree of labeling of 1 or 2 on average. Following the in vitro assessment of purity, composition, spectral properties, functionality, and serum protein binding, the in vivo behavior of all fluorescent tracers was evaluated in EGFR⁺ tumor-bearing mice (n=4/group, N=32). At 1, 3, 6, 12, and 24 hours after i.v. injection of 2nmol of the anti-EGFR tracer or a control tracer, 2D images were acquired using the Fluobeam. In addition, major organs and tissues excised at 1h and 24h post-injection were imaged ex vivo. For all images, mean fluorescent signals and tumor-to-background ratios (TBRs) were determined. An analogous protocol was applied to prepare and evaluate fluorescent anti-HER2 and anti-CEA Nbs.

 

Results/Discussion

The Nbs were successfully labeled with IRDye800CW, ZW800-1, FNIR-tag or s775z. The in vivo evaluation of the tracers with a DOL1 showed that FNIR-Tag- and s775z-Nbs clearly accumulate in the tumor at 1h post-injection with little background signal, except in the kidneys due to renal clearance of the tracer. In comparison IRDye800CW- and ZW800-1-tracers demonstrate considerable aspecific accumulation leading to low TBRs within the rapid imaging timeframe Nb-based tracers aim at. The ex vivo analysis confirmed the in vivo results, showing tumor-to-liver of 1.7 ± 0.1 and 9.0 ± 0.9, respectively for FNIR-Tag and s775z-labeled Nbs. Similar findings were observed for the anti-HER2 and -CEA Nbs. As compared to Nbs labeled with a DOL1, Nbs labeled with on average 2 FNIR-Tag dyes showed higher mean fluorescent tumor signals, but no increased TBRs, because of a higher background, mainly in the liver. Nbs labeled with on average 2 s775z show similar results, albeit the increase in aspecific liver accumulation was more limited.

 

 

Conclusions

FNIR-Tag- and s775z-labeled Nbs possess a clinically relevant biodistribution profile with rapid tumor

visualization and low background signals. Thus, these dyes can be used as a platform technique for the development of translatable Nb-based tracers. We could not show a clear benefit for conjugating more than 1 dye per Nb. However, our data further underlines the importance of the chosen dye on the pharmacokinetic profile of a molecular tracer.

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