title: Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity.
abstract: Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard (51)Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the (51)Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies.
authors.affiliation: Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Freiligrathstrasse 12, 55131 Mainz, Germany; BioNTech Cell & Gene Therapies GmbH, An der Goldgrube 12, 55131 Mainz, Germany.
authors.forename: Tana A
authors.initials: TA
authors.lastname: Omokoko
authors.affiliation: Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Freiligrathstrasse 12, 55131 Mainz, Germany; BioNTech AG, An der Goldgrube 12, 55131 Mainz, Germany.
authors.forename: Uli
authors.initials: U
authors.lastname: Luxemburger
authors.affiliation: BioNTech Cell & Gene Therapies GmbH, An der Goldgrube 12, 55131 Mainz, Germany.
authors.forename: Shaheer
authors.initials: S
authors.lastname: Bardissi
authors.affiliation: Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Freiligrathstrasse 12, 55131 Mainz, Germany; BioNTech Cell & Gene Therapies GmbH, An der Goldgrube 12, 55131 Mainz, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Simon
authors.affiliation: Ganymed Pharmaceuticals AG, An der Goldgrube 12, 55131 Mainz, Germany.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Utsch
authors.affiliation: Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Freiligrathstrasse 12, 55131 Mainz, Germany; BioNTech Cell & Gene Therapies GmbH, An der Goldgrube 12, 55131 Mainz, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Breitkreuz
authors.affiliation: Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Freiligrathstrasse 12, 55131 Mainz, Germany; Ganymed Pharmaceuticals AG, An der Goldgrube 12, 55131 Mainz, Germany.
authors.forename: Özlem
authors.initials: Ö
authors.lastname: Türeci
authors.affiliation: Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Freiligrathstrasse 12, 55131 Mainz, Germany; BioNTech AG, An der Goldgrube 12, 55131 Mainz, Germany.
authors.forename: Ugur
authors.initials: U
authors.lastname: Sahin
mesh_terms: D000938:Antigen-Presenting Cells / Q000166:cytology / Q000276:immunology*; D000941:Antigens / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D002470:Cell Survival; D018920:Coculture Techniques; D003602:Cytotoxicity, Immunologic; D017930:Genes, Reporter; D006801:Humans; D007111:Immunity, Cellular; D056724:Immunity, Humoral; D007118:Immunoassay; D008156:Luciferases / Q000235:genetics / Q000276:immunology; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology*; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D000941:Antigens; D012333:RNA, Messenger; D008156:Luciferases