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

Abstract

The pharmacokinetic (PK) behavior of monoclonal antibodies (mAbs) is influenced by target-mediated drug disposition, off-target effects, antidrug antibody-mediated clearance, and interaction with fragmentcrystallizable domain (Fc) receptors such as neonatal Fc receptor. All of these interactions hold the potential to impact mAb biodistribution. Near infrared (NIR) fluorescent probes offer an approach complementary to radionuclides to characterize drug disposition. Notably, the use of IRDye800 (IR800; LI-COR, Lincoln, NE) as a protein-labeling agent in preclinical work holds the potential for quantitative tissue analysis. Here, we tested the utility of the IR800 dye as a quantitative mAb tracer during pharmacokinetic analysis in both plasma and tissues using a model mouse monoclonal IgG1 (8C2) labeled with £1.5 molecules of IR800. The plasma PK parameters derived from a mixture of IR800-8C2 and 8C2 dosed intravenously to C57BL/6 mice at 8 mg/kg exhibited a large discrepancy in exposure depending on the method of quantitation [CL plasma = 8.4 ml/d per kilogram (NIR fluorescence detection) versus 2.5 ml/d per kilogram (enzyme-linked immunosorbent assay)]. The disagreement between measurements suggests that the PK of 8C2 is altered by addition of the IR800 dye. Additionally, direct fluorescence analysis of homogenized tissues revealed several large differences in IR800-8C2 tissue uptake when compared with a previously published study using [125 I]8C2, most notably an over 4-fold increase in liver concentration. Finally, the utility of IR800 in combination with whole body imaging was examined by comparison of IR800-8C2 levels observed in animal sagittal cross-sections to those measured in homogenized tissues. Our results represent the first PK analysis in both mouse plasma and tissues of an IR800-mAb conjugate and suggest that mAb disposition is significantly altered by IR800 conjugation to 8C2.

Tracking Antibody Distribution with Near-Infrared Fluorescent Dyes: Impact of Dye Structure and Degree of Labeling on Plasma Clearance

“…At longer times, the beta phase clearance diverges significantly, indicating 800CW altered the pharmacokinetics of the antibody. These data agree with Conner et al, 6 where the clearance of a different antibody–800CW conjugate showed faster clearance at a similar DoL of 1.5. At a 0.3 DoL, however, both Tras-800CW and Bev-800CW showed similar clearance over the first several days, and only after 4 days does the clearance diverge significantly.…”

Biodistribution of Etanercept to Tissues and Sites of Inflammation in Arthritic Rats

“…Another study concluded that IRDye800 in a low dye/protein labeling ratio does not significantly alter the pharmacokinetics of cetuximab, which has a relatively short half-life at ASPET Journals on May 9, 2018 dmd.aspetjournals.org (2.5 days) (Zinn et al, 2015). In contrast, altered mAb pharmacokinetics in plasma and tissues were observed for bevacizumab, cetuximab, and mouse monoclonal IgG1 (8C2), featuring increased plasma elimination and liver uptake (Cohen et al, 2011;Conner et al, 2014). We also observed the apparent increased plasma elimination as well as enhanced liver and kidney uptake of etanercept measured by fluorescence.…”

Residualization Rates of Near-Infrared Dyes for the Rational Design of Molecular Imaging Agents

“…The degree of labeling (DOL) was kept below 1 for most dyes to minimize the presence of multiple dyes on a single antibody. For in vivo work, this can have a strong impact on distribution [21]. …”