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

Abstract

Monoclonal antibodies labeled with near-infrared (NIR) fluorophores have potential use in disease detection, intraoperative imaging, and pharmacokinetic characterization of therapeutic antibodies in both the preclinical and clinical setting. Recent work has shown conjugation of NIR fluorophores to antibodies can potentially alter antibody disposition at a sufficiently high degree of labeling (DoL); however, other reports show minimal impact after labeling with NIR fluorophores. In this work, we label two clinically approved antibodies, Herceptin (trastuzumab) and Avastin (bevacizumab), with NIR dyes IRDye 800CW (800CW) or Alexa Fluor 680 (AF680), at 1.2 and 0.3 dyes/antibody and examine the impact of fluorophore conjugation on antibody plasma clearance and tissue distribution. At 0.3 DoL, AF680 conjugates exhibited similar clearance to unlabeled antibody over 17 days while 800CW conjugates diverged after 4 days, suggesting AF680 is a more suitable choice for long-term pharmacokinetic studies. At the 1.2 DoL, 800CW conjugates cleared faster than unlabeled antibodies after several hours, in agreement with other published reports. The tissue biodistribution for bevacizumab–800CW and −AF680 conjugates agreed well with literature reported biodistributions using radiolabels. However, the greater tissue autofluorescence at 680 nm resulted in limited detection above background at low (∼2 mg/kg) doses and 0.3 DoL for AF680, indicating that 800CW is more appropriate for short-term biodistribution measurements and intraoperative imaging. Overall, our work shows a DoL of 0.3 or less for non-site-specifically labeled antibodies (with a Poisson distribution) is ideal for limiting the impact of NIR fluorophores on antibody pharmacokinetics.

Low‐Fouling Fluoropolymers for Bioconjugation and In Vivo Tracking

“…[4] However,t he additional chemical steps increase synthetic complexity;q uestions over the stability of the conjugation chemistry and the effect of the conjugated moiety on biological fate have been raised. [5] It therefore follows that there is an evident need and interest in developing selftrackable polymers for use in bioconjugation. Such selftrackable polymers remove the need for reaction with additional imaging moieties and inherently provide am easurable signal in the commonly used imaging modalities to allow quantitative tracking of the polymer-conjugated therapeutics.…”

Repurposing Cyanine NIR‐I Dyes Accelerates Clinical Translation of Near‐Infrared‐II (NIR‐II) Bioimaging

“…Due to the interaction between these dyes and proteins, the brightness of NIR‐I dyes was higher in serum compared with that in phosphate buffered saline (PBS) or water . In order to investigate the maximized brightness of NIR‐I dyes in the form of protein complex, we tested the NIR‐I brightness in bovine serum albumin (BSA), fetal bovine serum (FBS), and PBS as a function of temperature.…”

In Vitro Characterization and Stability Profiles of Antibody–Fluorophore Conjugates Derived from Interchain Cysteine Cross-Linking or Lysine Bioconjugation

“…We chose the anti-HER2 trastuzumab (TTZ) and the anti-CD20 rituximab (RTX) as antibody carriers (IgG1κ) to analyze potential antibody-dependent discrepancies during the bioconjugation process with two different well-known bioconjugation method. FAR value should be limited to avoid AFC aggregation and the loss of mAb biological properties [21]. Therefore, we produced all labelled mAbs (Table 1) with an average FAR of 1.5 verified through mass spectroscopy analysis.…”

“…Cross-linked mAbs were compared to their labelled counterparts resulting from NHS conjugation in order to evaluate the discrepancies between their respective fluorescence emissions, stability upon storage, or pH shift. To avoid aggregation, [21] labelled mAbs were produced with an average FAR of 1.5.…”