title: Targeting Ewing sarcoma with activated and GD2-specific chimeric antigen receptor-engineered human NK cells induces upregulation of immune-inhibitory HLA-G.
abstract: Activated andin vitroexpanded natural killer (NK) cells have substantial cytotoxicity against many tumor cells, but theirin vivoefficacy to eliminate solid cancers is limited. Here, we used chimeric antigen receptors (CARs) to enhance the activity of NK cells against Ewing sarcomas (EwS) in a tumor antigen-specific manner. Expression of CARs directed against the ganglioside antigen GD2in activated NK cells increased their responses to GD2+ allogeneic EwS cellsin vitroand overcame resistance of individual cell lines to NK cell lysis. Second-generation CARs with 4-1BB and 2B4 co-stimulatory signaling and third-generation CARs combining both co-stimulatory domains were all equally effective. By contrast, adoptive transfer of GD2-specific CAR gene-modified NK cells both by intratumoral and intraperitoneal delivery failed to eliminate GD2-expressing EwS xenografts. Histopathology review revealed upregulation of the immunosuppressive ligand HLA-G in tumor autopsies from mice treated with NK cells compared to untreated control mice. Supporting the relevance of this finding,in vitroco-incubation of NK cells with allogeneic EwS cells induced upregulation of the HLA-G receptor CD85j, and HLA-G1 expressed by EwS cells suppressed the activity of NK cells from three of five allogeneic donors against the tumor cellsin vitro. We conclude that HLA-G is a candidate immune checkpoint in EwS where it can contribute to resistance to NK cell therapy. HLA-G deserves evaluation as a potential target for more effective immunotherapeutic combination regimens in this and other cancers.
authors.affiliation: Department of Pediatric Hematology and Oncology, University Children's Hospital Muenster , Muenster, Germany.
authors.forename: Sareetha
authors.initials: S
authors.lastname: Kailayangiri
authors.affiliation: Department of Pediatric Hematology and Oncology, University Children's Hospital Muenster , Muenster, Germany.
authors.forename: Bianca
authors.initials: B
authors.lastname: Altvater
authors.affiliation: Department of Pediatric Hematology and Oncology, University Children's Hospital Muenster , Muenster, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Spurny
authors.affiliation: Department of Pediatric Hematology and Oncology, University Children's Hospital Muenster , Muenster, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Jamitzky
authors.affiliation: European Institute for Molecular Imaging (EIMI), University of Muenster , Muenster, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Schelhaas
authors.affiliation: European Institute for Molecular Imaging (EIMI), University of Muenster, Muenster, Germany; Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), University of Muenster, Muenster, Germany.
authors.forename: Andreas H
authors.initials: AH
authors.lastname: Jacobs
authors.affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, Children's Hospital, Heinrich Heine University , Duesseldorf, Germany.
authors.forename: Constanze
authors.initials: C
authors.lastname: Wiek
authors.affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, Children's Hospital, Heinrich Heine University , Duesseldorf, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Roellecke
authors.affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, Children's Hospital, Heinrich Heine University, Duesseldorf, Germany; Department of Pediatrics III, University Children's Hospital Essen, University of Duisburg-Essen, Essen, Germany.
authors.forename: Helmut
authors.initials: H
authors.lastname: Hanenberg
authors.affiliation: Gerhard-Domagk Institute for Pathology, University of Muenster , Muenster, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hartmann
authors.affiliation: Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), University of Muenster, Muenster, Germany; Department of Neurology, University Hospital Muenster, Muenster, Germany.
authors.forename: Heinz
authors.initials: H
authors.lastname: Wiendl
authors.affiliation: Department of Neurology, University Hospital Muenster , Muenster, Germany.
authors.forename: Susann
authors.initials: S
authors.lastname: Pankratz
authors.affiliation: Department of Pediatric Hematology and Oncology, University Children's Hospital Muenster , Muenster, Germany.
authors.forename: Jutta
authors.initials: J
authors.lastname: Meltzer
authors.affiliation: Department of Pediatric Hematology and Oncology, University Children's Hospital Muenster , Muenster, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Farwick
authors.affiliation: Department of Pediatric Hematology and Oncology, University Children's Hospital Muenster , Muenster, Germany.
authors.forename: Lea
authors.initials: L
authors.lastname: Greune
authors.affiliation: Department of Pediatric Hematology and Oncology, University Children's Hospital Muenster , Muenster, Germany.
authors.forename: Maike
authors.initials: M
authors.lastname: Fluegge
authors.affiliation: Department of Pediatric Hematology and Oncology, University Children's Hospital Muenster, Muenster, Germany; Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), University of Muenster, Muenster, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Rossig
keywords: Chimeric antigen receptors; Ewing sarcoma; HLA-G; NK cells; inhibitory immune checkpoints