title: 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 DsRed-expressing 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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Center for Molecular Imaging, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, Texas 77030, USA.
authors.forename: Mary A
authors.initials: MA
authors.lastname: Hall
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Pinkston
authors.forename: Nathaniel
authors.initials: N
authors.lastname: Wilganowski
authors.forename: Holly
authors.initials: H
authors.lastname: Robinson
authors.forename: Pradip
authors.initials: P
authors.lastname: Ghosh
authors.forename: Ali
authors.initials: A
authors.lastname: Azhdarinia
authors.forename: Karina
authors.initials: K
authors.lastname: Vazquez-Arreguin
authors.forename: Arseniy M
authors.initials: AM
authors.lastname: Kolonin
authors.forename: Barrett R
authors.initials: BR
authors.lastname: Harvey
authors.forename: Eva M
authors.initials: EM
authors.lastname: Sevick-Muraca
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000276:immunology*; D001667:Binding, Competitive; D015815:Cell Adhesion Molecules / Q000276:immunology*; D045744:Cell Line, Tumor; D003287:Contrast Media; D003301:Copper Radioisotopes; D003429:Cross Reactions; D000071858:Epithelial Cell Adhesion Molecule; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007259:Infrared Rays; D008207:Lymphatic Metastasis; D008297:Male; D051379:Mice; D064847:Multimodal Imaging; D049268:Positron-Emission Tomography; D011471:Prostatic Neoplasms / Q000000981:diagnostic imaging* / Q000473:pathology*; D012372:ROC Curve; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D014057:Tomography, X-Ray Computed
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D003287:Contrast Media; D003301:Copper Radioisotopes; D000071858:Epithelial Cell Adhesion Molecule; D006573:Heterocyclic Compounds, 1-Ring; D018796:Immunoconjugates; D007140:Immunoglobulin Fab Fragments; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid