Comparison of mAbs Targeting Epithelial Cell Adhesion Molecule for the Detection of Prostate Cancer Lymph Node Metastases with Multimodal Contrast Agents: Quantitative Small-Animal PET/CT and NIRF

Abstract

The proliferation of most carcinomas is associated with an overexpression of epithelial cell adhesion molecule (EpCAM), a 40-kDa type I transmembrane protein found on epithelial cells yet absent from other cell types. The absence of EpCAM in normal lymphatics makes it an attractive marker for studying lymph node (LN) metastases of carcinomas to improve LN staging accuracy. Herein, we developed and quantitatively compared dual-labeled monoclonal antibodies (mAbs) of varying affinities against EpCAM for both noninvasive and intraoperative detection of metastatic LNs in prostate cancer. Methods: A panel of hybridoma-derived anti-EpCAM mAbs was generated and screened. Two high-affinity candidate mAbs with specificity for nonoverlapping epitopes on the EpCAM extracellular domain were chosen for further evaluation. After conjugation with DOTA for 64 Cu radiolabeling and IRDye 800CW as a fluorophore, dual-labeled specific or isotype control mAb was administered intravenously to male nu/nu mice at 10-12 wk after orthotopic implantation of DsRedexpressing PC3 cells. Within 18-24 h, noninvasive small-animal PET/CT and in vivo, in situ, and ex vivo DsRed reporter gene and near-infrared fluorescence (NIRF) imaging were performed to detect primary tumors and metastatic LNs. Using DsRed fluorescence as the true indicator of cancer-positive tissue, we performed receiver operating characteristic curve analyses of percentage injected dose per gram measured from quantitative small-animal PET/CT and fluorescence intensity measured from semiquantitative NIRF imaging for each LN examined to compare mAb sensitivity and specificity. Results: mAbs 7 and 153 generated in-house were found to have higher affinity than commercial mAb 9601. Accuracy, as a function of sensitivity and specificity, for the detection of cancer-positive LNs during in vivo small-animal PET/CT was highest for mAbs 7 (87.0%) and 153 (78.0%) and significantly greater (P , 0.001) than random chance (50.0%). Rates for mAb 9601 (60.7%) and control mAb 69 (27.0%) were not significantly different from chance. Similarly, mAb 7 had significant detection accuracy by NIRF imaging (96.0%, P , 0.001). Conclusion: mAbs 7 and 153 are attractive, high-affinity candidates for further multimodal imaging agent optimization aimed at enhancing sensitivity and specificity for detection of metastatic LNs in prostate cancer. Fully quantitative NIRF imaging is needed for comprehensive analyses of NIRF-labeled agent accuracy for intraoperative guidance. Dual -labeled contrast agents promise the advantage of noninvasive imaging via nuclear techniques and molecularly guided surgical resection via near-infrared fluorescence (NIRF) imaging (1). Currently, nodal staging of most cancers is performed after lymph node (LN) biopsy and dissection for subsequent pathologic examination. As in most cancers, imaging of LN involvement in prostate cancer (PCa) lacks sensitivity and specificity (2) and as a result, extended pelvic LN dissection, which provides higher staging accuracy (3), is rapidly becomi...

In vivoimaging of orthotopic prostate cancer with far-red gene reporter fluorescence tomography andin vivoandex vivovalidation

“…We found that PC3-IFP1.4 cells exhibited fast tumor growth compared to wild type (WT) or prior orthotopic studies employing PC3-DsRed. 16 The potential reason may be the effect of the virus transfection method and DNA combination. Although in vivo PET imaging can provide validation information, it is not always possible to discriminate bladder uptake from prostate uptake in the PET/CT images.…”

“…16 A previously described flow cytometry method 17 was used to assess the biological activity of the antibody conjugates and showed that conjugation of a DOTA chelating agent did not affect binding compared to unlabeled antibody.…”

Emerging lymphatic imaging technologies for mouse and man

“…The ability to non-invasively image cancer-positive LNs could obviate the need for LN dissection for the purposes of cancer staging. Molecularly specific imaging for detection of cancer-positive LNs has been proposed, employing intravenous administration of radiolabeled and fluorescently labeled antibodies for targeting cancer cells (106,107). These antibody-based approaches possess high specificity to the cancer-specific antigen, but also typically contain the Fc domain to which immune cells resident within LNs can bind (108).…”

Targeting Pili in Enterococcal Pathogenesis

“…ELISA procedures utilizing rEbpC and recombinant human cell adhesion molecule/Fc (rHuEpCAM/Fc) have been previously described (25). Briefly, rEbpC or rHuEpCAM/Fc was coated onto Microlon 600 ELISA plates (Greiner BioOne).…”