A novel method to quantify IRDye800CW fluorescent antibody probes ex vivo in tissue distribution studies

Abstract

BackgroundWe describe a new method for biodistribution studies with IRDye800CW fluorescent antibody probes. This method allows the quantification of the IRDye800CW fluorescent tracer in percentage of injected dose per gram of tissue (% ID/g), and it is herein compared to the generally used reference method that makes use of radioactivity.MethodsCetuximab was conjugated to both the near-infrared fluorophore IRDye800CW and/or the positron emitter 89-zirconium, which was injected in nude mice bearing A431 human tumor xenografts. Positron emission tomography (PET) and optical imaging were performed 24 h post-injection (p.i.). For the biodistribution study, organs and tumors were collected 24 h p.i., and each of these was halved. One half was used for the determination of probe uptake by radioactivity measurement. The other half was homogenized, and the content of the fluorescent probe was determined by extrapolation from a calibration curve made with the injected probe.ResultsTumors were clearly visualized with both modalities, and the calculated tumor-to-normal tissue ratios were very similar for optical and PET imaging: 3.31 ± 1.09 and 3.15 ± 0.99, respectively. Although some variations were observed in ex vivo analyses, tumor uptake was within the same range for IRDye800CW and gamma ray quantification: 15.07 ± 3.66% ID/g and 13.92 ± 2.59% ID/g, respectively.ConclusionsThe novel method for quantification of the optical tracer IRDye800CW gives similar results as the reference method of gamma ray quantification. This new method is considered very useful in the context of the preclinical development of IRDye800CW fluorescent probes for optical molecular imaging, likely contributing to the selection of lead compounds that are the most promising for clinical translation.

Evaluation of Near Infrared Fluorescent Labeling of Monoclonal Antibodies as a Tool for Tissue Distribution

“…Previous work (Oliveira et al, 2012) highlights the utility of the IR800 dye attached to antibodies as a method for evaluating mAb biodistribution. In that study, the authors employed a dual-labeling approach wherein cetuximab was labeled with desferalchelated positron emitter zirconium ( 89 Zr) and IR800 on the same mAb molecule to yield […”

“…These probe features result in negligible autofluorescence with enhanced signal recovery, and high target-to-background contrast. Last, direct tissue fluorescent measurement has been shown to be quantitative when proper sample dilution is considered (Oliveira et al, 2012). NIR optical probes offer ease of use, are cost effective, and require no specialized equipment or added safety precautions.…”

“…Although characterization of IR800 in tissue has been shown to be quantitative (Oliveira et al, 2012), additional characterization of the pharmacokinetics (PK) of IR800-mAb conjugates is warranted. Specifically, preclinical study of IR800-mAb conjugates that do not contain additional radiolabels has yet to be conducted.…”

Monitoring therapeutic monoclonal antibodies in brain tumor

“…21 The molecular distribution of large molecules (antibodies) has primarily been investigated using widespread approaches of radio-or fluorescence-labeled probes like electron microscopy, 22 intravital fluorescence videomicroscopy, 23 autoradiography, 24 micellarelectrokinetic capillary 25 and positron emission tomography. 26 Imaging mass spectrometry (IMS) provides complementary features that overcome many labeling imaging pitfalls, such as the unspecific reactions, the change of affinity due to labeled probes and the inability to distinguish between the parent compound and metabolites. IMS detects label-free analytes, [27][28][29] measuring the distribution of administered compounds and their metabolites in the same experiment.…”

Improved decision making for prioritizing tumor targeting antibodies in human xenografts: Utility of fluorescence imaging to verify tumor target expression, antibody binding and optimization of dosage and application schedule

“…[25][26][27][28][29] Over the past decades, such optical imaging modalities have undergone rapid development and been used to monitor primary tumor growth and metastases, 30,31 tumor cell and enzyme activity, [32][33][34] tumor cell surface receptors, [35][36][37] apoptosis, 38 anti-tumor treatment effects 39 and biodistribution and accumulation of fluorescencelabeled molecules, such as therapeutic antibodies. [40][41][42][43] The majority of this macroscopic in vivo research was not correlated with microscopic ex vivo fluorescence histology analysis. From our point of view, this is a basic requirement to validate the accuracy of in vivo results on a cellular level and also receive deeper insight and understanding of investigated biological processes.…”