title: CAR-T Cells Inflict Sequential Killing of Multiple Tumor Target Cells.
abstract: Adoptive therapy with chimeric antigen receptor (CAR) T cells shows great promise clinically. However, there are important aspects of CAR-T-cell biology that have not been explored, particularly with respect to the kinetics of activation, immune synapse formation, and tumor cell killing. Moreover, the effects of signaling via the endogenous T-cell receptor (TCR) or CAR on killing kinetics are unclear. To address these issues, we developed a novel transgenic mouse (designated CAR.OT-I), in which CD8(+) T cells coexpressed the clonogenic OT-I TCR, recognizing the H-2K(b)-presented ovalbumin peptide SIINFEKL, and an scFv specific for human HER2. Primed CAR.OT-I T cells were mixed with SIINFEKL-pulsed or HER2-expressing tumor cells and visualized in real-time using time-lapse microscopy. We found that engagement via CAR or TCR did not affect cell death kinetics, except that the time from degranulation to CAR-T-cell detachment was faster when CAR was engaged. We showed, for the first time, that individual CAR.OT-I cells can kill multiple tumor cells ("serial killing"), irrespective of the mode of recognition. At low effector:target ratios, the tumor cell killing rate was similar via TCR or CAR ligation over the first 20 hours of coincubation. However, from 20 to 50 hours, tumor cell death mediated through CAR became attenuated due to CAR downregulation throughout the time course. Our study provides important insights into CAR-T-tumor cell interactions, with implications for single- or dual receptor-focused T-cell therapy.
authors.affiliation: Cancer Immunology Research, Peter MacCallum Cancer Center, East Melbourne, Victoria, Australia. Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia. The ACRF Translational Research Laboratory, Royal Melbourne Hospital, Parkville, Victoria, Australia.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Davenport
authors.affiliation: Cancer Immunology Research, Peter MacCallum Cancer Center, East Melbourne, Victoria, Australia. Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Misty R
authors.initials: MR
authors.lastname: Jenkins
authors.affiliation: Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia. Differentiation and Transcription Laboratory, Peter MacCallum Cancer Center, East Melbourne, Victoria, Australia.
authors.forename: Ryan S
authors.initials: RS
authors.lastname: Cross
authors.affiliation: Cancer Immunology Research, Peter MacCallum Cancer Center, East Melbourne, Victoria, Australia. Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Carmen S
authors.initials: CS
authors.lastname: Yong
authors.affiliation: Department of Cancer Medicine, Peter MacCallum Cancer Center, East Melbourne, Victoria, Australia.
authors.forename: H Miles
authors.initials: HM
authors.lastname: Prince
authors.affiliation: Cancer Immunology Research, Peter MacCallum Cancer Center, East Melbourne, Victoria, Australia. Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia. The ACRF Translational Research Laboratory, Royal Melbourne Hospital, Parkville, Victoria, Australia. Department of Clinical Haematology and Bone Marrow Transplantation, Royal Melbourne Hospital, Parkville, Victoria, Australia.
authors.forename: David S
authors.initials: DS
authors.lastname: Ritchie
authors.affiliation: Cancer Immunology Research, Peter MacCallum Cancer Center, East Melbourne, Victoria, Australia. Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Trapani
authors.affiliation: Cancer Immunology Research, Peter MacCallum Cancer Center, East Melbourne, Victoria, Australia. Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Kershaw
authors.affiliation: Cancer Immunology Research, Peter MacCallum Cancer Center, East Melbourne, Victoria, Australia. Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia. paul.neeson@petermac.org phil.darcy@petermac.org.
authors.forename: Phillip K
authors.initials: PK
authors.lastname: Darcy
authors.affiliation: Cancer Immunology Research, Peter MacCallum Cancer Center, East Melbourne, Victoria, Australia. Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia. paul.neeson@petermac.org phil.darcy@petermac.org.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Neeson
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D045744:Cell Line, Tumor; D052118:Lysosomal-Associated Membrane Protein 1 / Q000276:immunology; D008822:Mice, Transgenic; D009369:Neoplasms / Q000276:immunology*; D054353:Perforin / Q000276:immunology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics; D011946:Receptors, Antigen / Q000276:immunology*; D011948:Receptors, Antigen, T-Cell / Q000276:immunology
chemical_list: D052118:Lysosomal-Associated Membrane Protein 1; D054353:Perforin; D018719:Erb-b2 Receptor Tyrosine Kinases; D011946:Receptors, Antigen; D011948:Receptors, Antigen, T-Cell; C508053:ERBB2 protein, human