title: Influence of chelator and near-infrared dye labeling on biocharacteristics of dual-labeled trastuzumab-based imaging agents.
abstract: OBJECTIVE
  To investigate the effect of fluorescent dye labeling on the targeting capabilities of 111In- (DTPA)n-trastuzumab-(IRDye 800)m.
  
  
  METHODS
  Trastuzumab-based conjugates were synthesized and conjugated with diethylenetriaminepentaacetic acid (DTPA) at molar ratios of 1, 2, 3 and 5 and with a fluorescent dye (IRDye 800CW) at molar ratios of 1, 3 and 5. Immunoreactivity and internalization were assessed on SKBR-3 cells, overexpressing human epidermal growth factor receptor 2. The stability in human serum and phosphate-buffered saline (PBS) was evaluated. The biodistribution of dual-labeled conjugates was compared with that of 111In-(DTPA)2-trastuzumab in a SKBR-3 xenograft model to evaluate the effect of dye-to-protein ratio.
  
  
  RESULTS
  All trastuzumab-based conjugates exhibited a high level of chemical and optical purity. Flow cytometry results showed that increasing dye-to-protein ratios were associated with decreased immunoreactivity. Stability studies revealed that the conjugate was stable in PBS, while in human serum, increased degradation and protein precipitation were observed with increasing dye-to-protein ratios. At 4 h, the percentages of internalization of dual-labeled conjugates normalized by dye-to-protein ratio (m) were 24.88%±2.10%, 19.99%±0.59%, and 17.47%±1.26% for "m" equal to 1, 3, and 5, respectively. A biodistribution study revealed a progressive decrease in tumor uptake with an increase in the dye-to-protein ratios. The liver, spleen and kidney showed a marked uptake with increased dye-to-protein ratios, particularly in the latter.
  
  
  CONCLUSIONS
  With non-specific-site conjugation of the fluorescent dye with a protein based on imaging agent, the increase in dye-to-protein ratios negatively impacted the immunoreactivity and stability, indicating a reduced tumor uptake.
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Nuclear Medicine, Peking University Cancer Hospital & Institute, Beijing 100142, China.
authors.forename: Xuejuan
authors.initials: X
authors.lastname: Wang
authors.affiliation: Center for Molecular Imaging, the Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston TX 77030, USA.
authors.forename: Melissa B
authors.initials: MB
authors.lastname: Aldrich
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Nuclear Medicine, Peking University Cancer Hospital & Institute, Beijing 100142, China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Nuclear Medicine, Peking University Cancer Hospital & Institute, Beijing 100142, China.
authors.forename: Nina
authors.initials: N
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Nuclear Medicine, Peking University Cancer Hospital & Institute, Beijing 100142, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Xie
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Nuclear Medicine, Peking University Cancer Hospital & Institute, Beijing 100142, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Liu
authors.affiliation: Center for Molecular Imaging, the Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston TX 77030, USA.
authors.forename: Eva
authors.initials: E
authors.lastname: Sevick-Muraca
keywords: Influence; duallabeled; human epidermal growth factor receptor 2; near infrared dye; trastuzumab