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

Background: Photoimmunodetection, 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 Zr-89-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. Methods: Zr-89-cetuximab and Zr-89-bevacizumab, containing 0.5 Zr-89-desferal group per mAb molecule, were incubated with 1 to 10 eq IRDye800CW at pH 8.5 for 2 h at 35 degrees C, and Zr-89-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 degrees C and 37 degrees C and human serum at 37 degrees C. 89Zr-mAb-IRDye800CW conjugates and Zr-89-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. Results: Conjugation efficiency of IRDye800CW to Zr-89-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 degrees C and 37 degrees C and human serum at 37 degrees C for at least 96 h. In biodistribution studies with Zr-89-cetuximabIRDye800CW, 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. (89)Zrbevacizumab-IRDye800CW showed a similar tendency, with an impaired biodistribution when 2 eq of dye had been coupled to mAb. Conclusion: Usage of Zr-89-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.