Details

Autor(en) / Beteiligte

• Darowski, Diana
• Jost, Christian
• Stubenrauch, Kay
• Wessels, Uwe
• Benz, Jörg
• Ehler, Andreas
• Freimoser-Grundschober, Anne
• Brünker, Peter
• Mössner, Ekkehard
• Umaña, Pablo
• Kobold, Sebastian
• Klein, Christian
• Batty, Sharon

Titel

P329G-CAR-J: a novel Jurkat-NFAT-based CAR-T reporter system recognizing the P329G Fc mutation

Ist Teil von

• Protein engineering, design and selection, 2019-12, Vol.32 (5), p.207

Ort / Verlag

England

Erscheinungsjahr

2019

Quelle

Oxford Journals 2020 Medicine

Beschreibungen/Notizen

• Monoclonal antibody-based therapeutics are an integral part of treatment of different human diseases, and the selection of suitable antibody candidates during the discovery phase is essential. Here, we describe a novel, cellular screening approach for the identification and characterization of therapeutic antibodies suitable for conversion into T cell bispecific antibodies using chimeric antigen receptor (CAR) transduced Jurkat-NFAT-luciferase reporter cells (CAR-J). For that purpose, we equipped a Jurkat-NFAT reporter cell line with a universal CAR, based on a monoclonal antibody recognizing the P329G mutation in the Fc-part of effector-silenced human IgG1-antibodies. In addition to scFv-based second generation CARs, Fab-based CARs employing the P329G-binder were generated. Using these anti-P329G-CAR-J cells together with the respective P329G-mutated IgG1-antibodies, we established a system, which facilitates the rapid testing of therapeutic antibody candidates in a flexible, high throughput setting during early stage discovery. We show that both, scFv- and Fab-based anti-P329G-CAR-J cells elicit a robust and dose-dependent luciferase signal if the respective antibody acts as an adaptor between tumor target and P329G-CAR-J cells. Importantly, we could demonstrate that functional characteristics of the antibody candidates, derived from the anti-P329G-CAR-J screening assay, are predictive for the functionality of these antibodies in the T cell bispecific antibody format.