title: A novel assay for monitoring internalization of nanocarrier coupled antibodies.
abstract: BACKGROUND
  Discovery of tumor-selective antibodies or antibody fragments is a promising approach for delivering therapeutic agents to antigen over-expressing cancers. Therefore it is important to develop methods for the identification of target- and function specific antibodies for effective drug delivery. Here we describe a highly selective and sensitive method for characterizing the internalizing potential of multivalently displayed antibodies or ligands conjugated to liposomes into tumor cells. The assay requires minute amounts of histidine-tagged ligand and relies on the non-covalent coupling of these antibodies to fluorescent liposomes containing a metal ion-chelating lipid. Following incubation of cells with antibody-conjugated liposomes, surface bound liposomes are gently removed and the remaining internalized liposomes are quantitated based on fluorescence in a high throughput manner. We have termed this methodology "Chelated Ligand Internalization Assay", or CLIA.
  
  
  RESULTS
  The specificity of the assay was demonstrated with different antibodies to the ErbB-2 and EGF receptors. Antibody-uptake correlated with receptor expression levels in tumor cell lines with a range of receptor expression. Furthermore, Ni-NTA liposomes containing doxorubicin were used to screen for the ability of antibodies to confer target-specific cytotoxicity. Using an anti-ErbB2 single chain Fv (scFv) (F5) antibody, cytotoxicity could be conferred to ErbB2-overexpressing cells; however, a poly(ethylene glycol)-linked lipid (DSPE-PEG-NTA-Ni) was necessary to allow for efficient loading of the drug and to reduce nonspecific drug leakage during the course of the assay.
  
  
  CONCLUSIONS
  The CLIA method we describe here represents a rapid, sensitive and robust assay for the identification and characterization of tumor-specific antibodies capable of high drug-delivery efficiency when conjugated to liposomal nanocarriers.
authors.affiliation: Department of Anesthesia, University of California San Francisco, San Francisco, CA 94110, USA. unielsen@merrimackpharma.com
authors.forename: Ulrik B
authors.initials: UB
authors.lastname: Nielsen
authors.forename: Dmitri B
authors.initials: DB
authors.lastname: Kirpotin
authors.forename: Edward M
authors.initials: EM
authors.lastname: Pickering
authors.forename: Daryl C
authors.initials: DC
authors.lastname: Drummond
authors.forename: James D
authors.initials: JD
authors.lastname: Marks
mesh_terms: D000912:Antibodies, Neoplasm / Q000008:administration & dosage* / Q000032:analysis*; D000970:Antineoplastic Agents / Q000378:metabolism; D016923:Cell Death; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000737:chemistry; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology; D007158:Immunologic Techniques; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry; D050356:Lipid Metabolism; D008081:Liposomes / Q000008:administration & dosage* / Q000378:metabolism*; D008727:Methotrexate / Q000737:chemistry; D009532:Nickel / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology; D012680:Sensitivity and Specificity
chemical_list: D000912:Antibodies, Neoplasm; D000970:Antineoplastic Agents; D004317:Doxorubicin; D006639:Histidine; D007135:Immunoglobulin Variable Region; D008024:Ligands; D008051:Lipid Bilayers; D008081:Liposomes; D008727:Methotrexate; D009532:Nickel; D009842:Oligopeptides; D018719:Erb-b2 Receptor Tyrosine Kinases; C471213:His-His-His-His-His-His