title: Target antigen density governs the efficacy of anti-CD20-CD28-CD3 ζ chimeric antigen receptor-modified effector CD8+ T cells.
abstract: The effectiveness of chimeric Ag receptor (CAR)-transduced T (CAR-T) cells has been attributed to supraphysiological signaling through CARs. Second- and later-generation CARs simultaneously transmit costimulatory signals with CD3ζ signals upon ligation, but may lead to severe adverse effects owing to the recognition of minimal Ag expression outside the target tumor. Currently, the threshold target Ag density for CAR-T cell lysis and further activation, including cytokine production, has not yet been investigated in detail. Therefore, we determined the threshold target Ag density required to induce CAR-T cell responses using novel anti-CD20 CAR-T cells with a CD28 intracellular domain and a CD20-transduced CEM cell model. The newly developed CD20CAR-T cells demonstrated Ag-specific lysis and cytokine secretion, which was a reasonable level as a second-generation CAR. For lytic activity, the threshold Ag density was determined to be ∼200 molecules per target cell, whereas the Ag density required for cytokine production of CAR-T cells was ∼10-fold higher, at a few thousand per target cell. CD20CAR-T cells responded efficiently to CD20-downregulated lymphoma and leukemia targets, including rituximab- or ofatumumab-refractory primary chronic lymphocytic leukemia cells. Despite the potential influence of the structure, localization, and binding affinity of the CAR/Ag, the threshold determined may be used for target Ag selection. An Ag density below the threshold may not result in adverse effects, whereas that above the threshold may be sufficient for practical effectiveness. CD20CAR-T cells also demonstrated significant lytic activity against CD20-downregulated tumor cells and may exhibit effectiveness for CD20-positive lymphoid malignancies.
authors.affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan;
authors.forename: Keisuke
authors.initials: K
authors.lastname: Watanabe
authors.affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan; tseit@med.nagoya-u.ac.jp.
authors.forename: Seitaro
authors.initials: S
authors.lastname: Terakura
authors.affiliation: Department of Hematology, VU University Medical Center Amsterdam, 1007 MB Amsterdam, the Netherlands; Department of Immunology, University Medical Center Utrecht, 3508 GA Utrecht, the Netherlands;
authors.forename: Anton C
authors.initials: AC
authors.lastname: Martens
authors.affiliation: Department of Hematology, University Medical Center Groningen, 9700 RB Groningen, the Netherlands;
authors.forename: Tom
authors.initials: T
authors.lastname: van Meerten
authors.affiliation: Division of Advanced Science and Biotechnology, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan;
authors.forename: Susumu
authors.initials: S
authors.lastname: Uchiyama
authors.affiliation: Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan;
authors.forename: Misa
authors.initials: M
authors.lastname: Imai
authors.affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan;
authors.forename: Reona
authors.initials: R
authors.lastname: Sakemura
authors.affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan;
authors.forename: Tatsunori
authors.initials: T
authors.lastname: Goto
authors.affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan;
authors.forename: Ryo
authors.initials: R
authors.lastname: Hanajiri
authors.affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan;
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Imahashi
authors.affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan; Institute for Advanced Research, Nagoya University, Nagoya 464-8601, Japan; and.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Shimada
authors.affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan;
authors.forename: Akihiro
authors.initials: A
authors.lastname: Tomita
authors.affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan;
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kiyoi
authors.affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan;
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Nishida
authors.affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan; National Hospital Organization Nagoya Medical Center, Nagoya 460-0001, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Naoe
authors.affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan;
authors.forename: Makoto
authors.initials: M
authors.lastname: Murata
mesh_terms: D058846:Antibodies, Monoclonal, Murine-Derived / Q000627:therapeutic use; D018951:Antigens, CD20 / Q000276:immunology / Q000378:metabolism; D000954:Antigens, Surface / Q000276:immunology* / Q000378:metabolism; D018106:CD28 Antigens / Q000276:immunology / Q000378:metabolism; D017252:CD3 Complex / Q000276:immunology / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism*; D045744:Cell Line, Tumor; D016207:Cytokines / Q000096:biosynthesis; D003602:Cytotoxicity, Immunologic; D015870:Gene Expression; D023061:Gene Order; D017930:Genes, Reporter; D006801:Humans; D016130:Immunophenotyping; D042541:Intracellular Space; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008213:Lymphocyte Activation; D016393:Lymphoma, B-Cell / Q000188:drug therapy / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000069283:Rituximab; D015398:Signal Transduction; D037182:T-Cell Antigen Receptor Specificity / Q000235:genetics / Q000276:immunology; D016176:T-Lymphocyte Subsets; D014161:Transduction, Genetic
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D000954:Antigens, Surface; D018106:CD28 Antigens; D017252:CD3 Complex; C121704:CD3 antigen, zeta chain; D016207:Cytokines; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins; D000069283:Rituximab