title: In vivo target-specific activatable near-infrared optical labeling of humanized monoclonal antibodies.
abstract: Imaging with labeled monoclonal antibodies may be useful in detecting, staging, and monitoring tumors. Despite their high affinity and specificity, a critical limitation of antibody imaging is the high background signal due to prolonged clearance from the blood, which reduces the tumor-to-background ratio. To address this problem, we developed a molecular imaging probe consisting of multiple self-quenching fluorophores [Cy5.5 or Alexa Fluor 680 (Alexa680)] conjugated to a monoclonal antibody (trastuzumab) to synthesize Tra-Cy5.5(SQ) or Tra-Alexa680(SQ), respectively. This agent only becomes fluorescently "active" after cellular internalization but is quenched in the unbound state leading to high tumor-to-background ratios. The in vitro quenching capacity for both conjugates was approximately 9-fold. In vivo imaging experiments were done in mice bearing both 3T3/HER-2+ and BALB/3T3/ZsGreen/HER-2- xenografts. Tra-Alexa680(SQ) produced specific enhancement in the 3T3/HER-2+ tumors but not in the HER-2- control tumors. However, Tra-Cy5.5(SQ) produced nonspecific enhancement in both 3T3/HER-2+ and control tumors. In conclusion, whereas Cy5.5-conjugates produced nonspecific results as well as rapid liver accumulation, conjugating multiple Alexa680 molecules to a single monoclonal antibody resulted in a near-infrared optical agent that activated within specific target tumors with high tumor-to-background ratio with considerable potential for clinical translation.
authors.affiliation: Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, NIH, Building 10, Room 1B40, MSC1088, Bethesda, MD 20892-1088, USA.
authors.forename: Mikako
authors.initials: M
authors.lastname: Ogawa
authors.forename: Celeste A S
authors.initials: CA
authors.lastname: Regino
authors.forename: Peter L
authors.initials: PL
authors.lastname: Choyke
authors.forename: Hisataka
authors.initials: H
authors.lastname: Kobayashi
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D061067:Antibodies, Monoclonal, Humanized; D000918:Antibody Specificity / Q000276:immunology*; D002232:Carbocyanines; D002460:Cell Line; D004195:Disease Models, Animal; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity; D000068878:Trastuzumab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D011994:Recombinant Proteins; D000068878:Trastuzumab