title: Chimeric antigen receptors with mutated IgG4 Fc spacer avoid fc receptor binding and improve T cell persistence and antitumor efficacy.
abstract: The success of adoptive therapy using chimeric antigen receptor (CAR)-expressing T cells partly depends on optimal CAR design. CARs frequently incorporate a spacer/linker region based on the constant region of either IgG1 or IgG4 to connect extracellular ligand-binding with intracellular signaling domains. Here, we evaluated the potential for the IgG4-Fc linker to result in off-target interactions with Fc gamma receptors (FcγRs). As proof-of-principle, we focused on a CD19-specific scFv-IgG4-CD28-zeta CAR and found that, in contrast to CAR-negative cells, CAR+ T cells bound soluble FcγRs in vitro and did not engraft in NSG mice. We hypothesized that mutations to avoid FcγR binding would improve CAR+ T cell engraftment and antitumor efficacy. Thus, we generated CD19-specific CARs with IgG4-Fc spacers that had either been mutated at two sites (L235E; N297Q) within the CH2 region (CD19R(EQ)) or incorporated a CH2 deletion (CD19Rch2Δ). These mutations reduced binding to soluble FcγRs without altering the ability of the CAR to mediate antigen-specific lysis. Importantly, CD19R(EQ) and CD19Rch2Δ T cells exhibited improved persistence and more potent CD19-specific antilymphoma efficacy in NSG mice. Together, these studies suggest that optimal CAR function may require the elimination of cellular FcγR interactions to improve T cell persistence and antitumor responses.
authors.affiliation: Department of Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Jonnalagadda
authors.affiliation: Department of Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA.
authors.forename: Armen
authors.initials: A
authors.lastname: Mardiros
authors.affiliation: Department of Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Urak
authors.affiliation: Department of Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA.
authors.forename: Xiuli
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA.
authors.forename: Lauren J
authors.initials: LJ
authors.lastname: Hoffman
authors.affiliation: Department of Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA.
authors.forename: Alyssa
authors.initials: A
authors.lastname: Bernanke
authors.affiliation: Department of Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA.
authors.forename: Wen-Chung
authors.initials: WC
authors.lastname: Chang
authors.affiliation: Department of Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA.
authors.forename: William
authors.initials: W
authors.lastname: Bretzlaff
authors.affiliation: Department of Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA.
authors.forename: Renate
authors.initials: R
authors.lastname: Starr
authors.affiliation: Department of Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA.
authors.forename: Saul
authors.initials: S
authors.lastname: Priceman
authors.affiliation: Department of Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA.
authors.forename: Julie R
authors.initials: JR
authors.lastname: Ostberg
authors.affiliation: Department of Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Forman
authors.affiliation: Department of Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Brown
mesh_terms: D000818:Animals; D007074:Immunoglobulin G / Q000276:immunology*; D007167:Immunotherapy; D051379:Mice; D050506:Mutant Chimeric Proteins / Q000378:metabolism*; D009154:Mutation; D009374:Neoplasms, Experimental / Q000276:immunology / Q000628:therapy*; D011485:Protein Binding; D011946:Receptors, Antigen / Q000235:genetics / Q000378:metabolism*; D011961:Receptors, Fc / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D007074:Immunoglobulin G; D050506:Mutant Chimeric Proteins; D011946:Receptors, Antigen; D011961:Receptors, Fc