title: The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity.
abstract: The use of synthetic chimeric antigen receptors (CAR) to redirect T cells to recognize tumor provides a powerful new approach to cancer immunotherapy; however, the attributes of CARs that ensure optimal in vivo tumor recognition remain to be defined. Here, we analyze the influence of length and composition of IgG-derived extracellular spacer domains on the function of CARs. Our studies demonstrate that CD19-CARs with a long spacer from IgG4 hinge-CH2-CH3 are functional in vitro but lack antitumor activity in vivo due to interaction between the Fc domain within the spacer and the Fc receptor-bearing myeloid cells, leading to activation-induced T-cell death. We demonstrate that in vivo persistence and antitumor effects of CAR-T cells with a long spacer can be restored by modifying distinct regions in the CH2 domain that are essential for Fc receptor binding. Our studies demonstrate that modifications that abrogate binding to Fc receptors are crucial for CARs in which a long spacer is obligatory for tumor recognition as shown here for a ROR1-specific CAR. These results demonstrate that the length and composition of the extracellular spacer domain that lacks intrinsic signaling function can be decisive in the design of CARs for optimal in vivo activity.
authors.affiliation: Fred Hutchinson Cancer Research Center, Clinical Research Division, Program in Immunology, Seattle, Washington. University of Würzburg, Department of Medicine II - Hematology and Medical Oncology, Würzburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Hudecek
authors.affiliation: Fred Hutchinson Cancer Research Center, Clinical Research Division, Program in Immunology, Seattle, Washington.
authors.forename: Daniel
authors.initials: D
authors.lastname: Sommermeyer
authors.affiliation: Fred Hutchinson Cancer Research Center, Clinical Research Division, Program in Immunology, Seattle, Washington.
authors.forename: Paula L
authors.initials: PL
authors.lastname: Kosasih
authors.affiliation: Seattle Children's Research Institute, Ben Towne Center for Childhood Cancer Research, Seattle, Washington. Department of Medicine, University of Washington, Seattle, Washington.
authors.forename: Anne
authors.initials: A
authors.lastname: Silva-Benedict
authors.affiliation: Fred Hutchinson Cancer Research Center, Clinical Research Division, Program in Immunology, Seattle, Washington.
authors.forename: Lingfeng
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida. Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, Florida.
authors.forename: Christoph
authors.initials: C
authors.lastname: Rader
authors.affiliation: Fred Hutchinson Cancer Research Center, Clinical Research Division, Program in Immunology, Seattle, Washington. Seattle Children's Research Institute, Ben Towne Center for Childhood Cancer Research, Seattle, Washington. Department of Pediatrics, University of Washington, Seattle, Washington.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jensen
authors.affiliation: Fred Hutchinson Cancer Research Center, Clinical Research Division, Program in Immunology, Seattle, Washington. Department of Medicine, University of Washington, Seattle, Washington. Technical University of Munich, Institute for Advanced Study, Munich, Germany. sriddell@fhcrc.org.
authors.forename: Stanley R
authors.initials: SR
authors.lastname: Riddell
mesh_terms: D000818:Animals; D018941:Antigens, CD19 / Q000276:immunology; D002051:Burkitt Lymphoma / Q000276:immunology / Q000628:therapy*; D016923:Cell Death / Q000276:immunology; D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D016130:Immunophenotyping / Q000379:methods; D016219:Immunotherapy, Adoptive / Q000379:methods*; D016086:Lentivirus / Q000235:genetics; D008168:Lung / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D017434:Protein Structure, Tertiary; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D015398:Signal Transduction / Q000276:immunology; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000276:immunology*; D023041:Xenograft Model Antitumor Assays
chemical_list: D018941:Antigens, CD19; D007074:Immunoglobulin G; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins