title: Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.
abstract: CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Hannah
authors.initials: H
authors.lastname: Karlsson
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Emma
authors.initials: E
authors.lastname: Svensson
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Camilla
authors.initials: C
authors.lastname: Gigg
authors.affiliation: Department of Medical Sciences, Uppsala University, Uppsala, Sweden.
authors.forename: Malin
authors.initials: M
authors.lastname: Jarvius
authors.affiliation: Department of Medical Sciences, Uppsala University, Uppsala, Sweden.|Section of Hematology, Uppsala University Hospital, Uppsala, Sweden.
authors.forename: Ulla
authors.initials: U
authors.lastname: Olsson-Strömberg
authors.affiliation: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Barbara
authors.initials: B
authors.lastname: Savoldo
authors.affiliation: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Gianpietro
authors.initials: G
authors.lastname: Dotti
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Angelica
authors.initials: A
authors.lastname: Loskog
mesh_terms: D000818:Animals; D018106:CD28 Antigens / Q000276:immunology*; D016022:Case-Control Studies; D005434:Flow Cytometry; D064368:Healthy Volunteers; D006801:Humans; D007167:Immunotherapy; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology* / Q000473:pathology / Q000628:therapy; D008213:Lymphocyte Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D011946:Receptors, Antigen / Q000276:immunology*; D015398:Signal Transduction; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9 / Q000276:immunology*
chemical_list: D018106:CD28 Antigens; D011946:Receptors, Antigen; C505714:TNFRSF9 protein, human; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9