Near-infrared Fluorescence Labeled Anti-TAG-72 Monoclonal Antibodies for Tumor Imaging in Colorectal Cancer Xenograft Mice

Anti-TAG-72 monoclonal antibodies target the tumor-associated glycoprotein (TAG)72 in various solid tumors. This study evaluated the use of anti-TAG-72 monoclonal antibodies, both murine CC49 and humanized CC49 (HuCC49 Delta C(H)2), for near-infrared fluorescent (NIR) tumor imaging in colorectal cancer xenograft models. The murine CC49 and HuCC49 Delta C(H)2 were conjugated with Cy7 monofunctional N-hydroxysuccinimide ester (Cy7-NHS). Both in vitro and in vivo anti-TAG-72 antibody binding studies were performed. The in vitro study utilized the human colon adenocarcinoma cell line LS174T that was incubated with Cy7, antibody-Cy7 conjugates, or excessive murine CC49 followed by the antibody-Cy7 conjugates and was imaged by fluorescence microscopy. The in vivo study utilized xenograft mice, bearing LS174T subcutaneous tumor implants, that received tail vein injections of Cy7, murine CC49-Cy7, HuCC49 Delta C(H)2-Cy7, or nonspecific IgG-Cy7 and were imaged by the Xenogen IVIS 100 system from 15 min to 288 h. The biodistribution of the fluorescence labeled antibodies was determined by imaging the dissected tissues. The in vitro study revealed that the antibody-Cy7 conjugates bound to LS174T cells and were blocked by excessive murine CC49. The in vivo study demonstrated that murine CC49 achieved a tumor/blood ratio of 15 at 96 h postinjection. In comparison, HuCC49 Delta C(H)2-Cy7 cleared much faster than murine CC49-Cy7 from the xenograft mice, and HuCC49 Delta C(H)2-Cy7 achieved a tumor/blood ratio of 12 at 18 h postinjection. In contrast, Cy7 and Cy7 labeled nonspecific IgG resulted in no demonstrable tumor accumulation. When mice were injected with excessive unlabeled murine CC49 at 6 h before the injection of murine CC49-Cy7 or HuCC49 Delta C(H)2-Cy7, both the intensity and retention time of the fluorescence from the tumor were reduced. In summary, the Cy7 labeled murine CC49 and HuCC49 Delta C(H)2 demonstrate tumor-targeting capabilities in living colorectal cancer xenograft mice and provide an alternative modality for tumor imaging.