title: Chip-based platform for dynamic analysis of NK cell cytolysis mediated by a triplebody.
abstract: Cancer therapy via redirected lysis mediated by antibodies and antibody-derived agents relies on the availability of substantial numbers of sufficiently active immune effector cells. To monitor antitumor responses before and during therapy, sensitive methods are needed, capable of quantitating specific lysis of target cells. Here we present a chip-based single-cell cytometric assay, which uses adherent human target cells arrayed in structured micro-fields. Using a fluorescent indicator of cell death and time-lapse microscopy in an automated high-throughput mode, we measured specific target cell lysis by activated human NK cells, mediated by the therapeutic single chain triplebody SPM-2 (33-16-123). This antibody-derived tri-specific fusion protein carries binding sites for the myeloid antigens CD33 and CD123 and recruits NK cells via a binding site for the Fc-receptor CD16. Specific lysis increased with increasing triplebody concentration, and the single-cell assay was validated by direct comparison with a standard calcein-release assay. The chip-based approach allowed measurement of lysis events over 16 hours (compared to 4 hours for the calcein assay) and required far smaller numbers of primary cells. In addition, dynamic properties inaccessible to conventional methods provide new details about the activation of cytolytic effector cells by antibody-derived agents. Thus, the killing rate exhibited a dose-dependent maximum during the reaction interval. In clinical applications ex vivo monitoring of NK activity of patient's endogenous cells will likely help to choose appropriate therapy, to detect impaired or recovered NK function, and possibly to identify rare subsets of cancer cells with particular sensitivity to effector-cell mediated lysis.
authors.affiliation: Faculty of Physics and Graduate School of Quantitative Biosciences (QBM), Ludwig-Maximilians-Universität, Munich, Germany. raedler@lmu.de.
authors.forename: Elisavet I
authors.initials: EI
authors.lastname: Chatzopoulou
authors.affiliation: Division of Hematology and Oncology, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Munich, Germany.
authors.forename: Claudia C
authors.initials: CC
authors.lastname: Roskopf
authors.affiliation: Faculty of Physics and Graduate School of Quantitative Biosciences (QBM), Ludwig-Maximilians-Universität, Munich, Germany. raedler@lmu.de.
authors.forename: Farzad
authors.initials: F
authors.lastname: Sekhavati
authors.affiliation: Division of Hematology and Oncology, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Munich, Germany.
authors.forename: Todd A
authors.initials: TA
authors.lastname: Braciak
authors.affiliation: Department of Biochemistry and Gene Center, Ludwig-Maximilians-Universität, Munich, Germany.
authors.forename: Nadja C
authors.initials: NC
authors.lastname: Fenn
authors.affiliation: Department of Biochemistry and Gene Center, Ludwig-Maximilians-Universität, Munich, Germany.
authors.forename: Karl-Peter
authors.initials: KP
authors.lastname: Hopfner
authors.affiliation: Division of Hematology and Oncology, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Munich, Germany.
authors.forename: Fuat S
authors.initials: FS
authors.lastname: Oduncu
authors.affiliation: Department of Biology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany.
authors.forename: Georg H
authors.initials: GH
authors.lastname: Fey
authors.affiliation: Faculty of Physics and Graduate School of Quantitative Biosciences (QBM), Ludwig-Maximilians-Universität, Munich, Germany. raedler@lmu.de.
authors.forename: Joachim O
authors.initials: JO
authors.lastname: Rädler
mesh_terms: D016923:Cell Death; D045744:Cell Line, Tumor; D057809:HEK293 Cells; D006801:Humans; D007694:Killer Cells, Natural / Q000166:cytology*; D046208:Microchip Analytical Procedures / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*; D057127:Single-Chain Antibodies / Q000378:metabolism*; D013997:Time Factors
chemical_list: D057127:Single-Chain Antibodies