Inert coupling of IRDye800CW to monoclonal antibodies for clinical optical imaging of tumor targets

Abstract

BackgroundPhotoimmunodetection, in which monoclonal antibodies [mAbs] are labeled with fluorescent dyes, might have clinical potential for early detection and characterization of cancer. For this purpose, the dye should be coupled in an inert way to mAb. In this study, different equivalents of IRDye800CW, a near-infrared fluorescent dye, were coupled to 89Zr-labeled cetuximab and bevacizumab, and conjugates were evaluated in biodistribution studies. Radiolabeled mAbs were used to allow accurate quantification for assessment of the number of dye groups that can be coupled to mAbs without affecting their biological properties.Methods89Zr-cetuximab and 89Zr-bevacizumab, containing 0.5 89Zr-desferal group per mAb molecule, were incubated with 1 to 10 eq IRDye800CW at pH 8.5 for 2 h at 35°C, and 89Zr-mAb-IRDye800CW conjugates were purified by a PD10 column using 0.9% NaCl as eluent. HPLC analysis at 780 nm was used to assess conjugation efficiency. In vitro stability measurements were performed in storage buffer (0.9% NaCl or PBS) at 4°C and 37°C and human serum at 37°C. 89Zr-mAb-IRDye800CW conjugates and 89Zr-mAb conjugates (as reference) were administered to nude mice bearing A431 (cetuximab) or FaDu (bevacizumab) xenografts, and biodistribution was assessed at 24 to 72 h after injection.ResultsConjugation efficiency of IRDye800CW to 89Zr-mAbs was approximately 50%; on an average, 0.5 to 5 eq IRDye800CW was conjugated. All conjugates showed optimal immunoreactivity and were > 95% stable in storage buffer at 4°C and 37°C and human serum at 37°C for at least 96 h. In biodistribution studies with 89Zr-cetuximab-IRDye800CW, enhanced blood clearance with concomitant decreased tumor uptake and increased liver uptake was observed at 24 to 72 h post-injection when 2 or more eq of dye had been coupled to mAb. No significant alteration of biodistribution was observed 24 to 48 h after injection when 1 eq of dye had been coupled. 89Zr-bevacizumab-IRDye800CW showed a similar tendency, with an impaired biodistribution when 2 eq of dye had been coupled to mAb.ConclusionUsage of 89Zr-mAbs allows accurate quantification of the biodistribution of mAbs labeled with different equivalents of IRDye800CW. Alteration of biodistribution was observed when more than 1 eq of IRDye800CW was coupled to mAbs.

Dual-Modality Image-Guided Surgery of Prostate Cancer with a Radiolabeled Fluorescent Anti-PSMA Monoclonal Antibody

“…Enhanced hepatic uptake of IRDye800CW-conjugated antibodies has been reported in previous studies. Cohen et al (23) reported enhanced liver uptake of the dual-labeled antibodies 89 Zr-cetuximab-IRDye800CW and 89 Zr-bevacizumab-IRDye800CW in vivo, which was attributed to the lipophilicity and charge of the antibody conjugate. In this study, a substitution ratio of less than 2 was found to be optimal to prevent enhanced blood clearance and hepatic liver uptake.…”

Tissue Distribution Studies of Protein Therapeutics Using Molecular Probes: Molecular Imaging

“…This has been a major barrier to the adoption of many otherwise excellent fluorescent labels. Controlling the labeling sites and limiting the stoichiometry (19,20) should minimize the risk of immunogenicity and the problems of batch-to-batch heterogeneity, helping the validation of fluorescent labels in clinical and other critical settings (4). With optical and radioactive labels alike, incorporating more labels per protein [up to 10 of each in Sampath et al (21)] gives rise to a temptingly "brighter" protein with higher specific activity or fluorescent yield, but this is a siren call risking compromised behavior in vivo (4) and would require even more cautious validation.…”

“…Figure 1 schematically illustrates an antibody labeled with lysine-linked moieties that facilitate detection by PET (Zr-89 chelated in desferrioxamine) and near-infrared fluorescence (IRDye800CW). This reagent was used in elegant dual-modality studies of antibody imaging reagents by Cohen et al (4).…”

“…NIRF labels make whole-body studies feasible in small animals (3) and may be suitable for applications such as illuminating positive margins in real time during tumor resection surgeries (4,5). They should also allow microscopic examination of antibody distribution and the cellular origins of the signal (tumor cell versus macrophage, for example).…”

Optical Imaging of Renal Cell Carcinoma with Anti–Carbonic Anhydrase IX Monoclonal Antibody Girentuximab

“…IRDye800CW can be stably coupled to targeting molecules, such as peptides and monoclonal antibodies (mAbs), and can be detected with an NIR imaging camera (7,8).…”