title: Redirecting T cells to Ewing's sarcoma family of tumors by a chimeric NKG2D receptor expressed by lentiviral transduction or mRNA transfection.
abstract: We explored the possibility to target Ewing's sarcoma family of tumors (ESFT) by redirecting T cells. To this aim, we considered NKG2D-ligands (NKG2D-Ls) as possible target antigens. Detailed analysis of the expression of MICA, MICB, ULBP-1, -2, and -3 in fourteen ESFT cell lines revealed consistent expression of at least one NKG2D-L. Thus, for redirecting T cells, we fused a CD3ζ/CD28-derived signaling domain to the ectodomain of NKG2D, however, opposite transmembrane orientation of this signaling domain and NKG2D required inverse orientation fusion of either of them. We hypothesized that the particularly located C-terminus of the NKG2D ectodomain should allow reengineering of the membrane anchoring from a native N-terminal to an artificial C-terminal linkage. Indeed, the resulting chimeric NKG2D receptor (chNKG2D) was functional and efficiently mediated ESFT cell death triggered by activated T cells. Notably, ESFT cells with even low NKG2D-L expression were killed by CD8(pos) and also CD4(pos) cells. Both, mRNA transfection and lentiviral transduction resulted in high level surface expression of chNKG2D. However, upon target-cell recognition receptor surface levels were maintained by tranfected RNA only during the first couple of hours after transfection. Later, target-cell contact resulted in strong and irreversible receptor down-modulation, whereas lentivirally mediated expression of chNKG2D remained constant under these conditions. Together, our study defines NKG2D-Ls as targets for a CAR-mediated T cell based immunotherapy of ESFT. A comparison of two different methods of gene transfer reveals strong differences in the susceptibility to ligand-induced receptor down-modulation with possible implications for the applicability of RNA transfection.
authors.affiliation: Laboratory of Cellular Therapy, Children's University Hospital, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. manfred.lehner@uk-erlangen.de
authors.forename: Manfred
authors.initials: M
authors.lastname: Lehner
authors.forename: Gabriel
authors.initials: G
authors.lastname: Götz
authors.forename: Julia
authors.initials: J
authors.lastname: Proff
authors.forename: Niels
authors.initials: N
authors.lastname: Schaft
authors.forename: Jan
authors.initials: J
authors.lastname: Dörrie
authors.forename: Florian
authors.initials: F
authors.lastname: Full
authors.forename: Armin
authors.initials: A
authors.lastname: Ensser
authors.forename: Yves A
authors.initials: YA
authors.lastname: Muller
authors.forename: Adelheid
authors.initials: A
authors.lastname: Cerwenka
authors.forename: Hinrich
authors.initials: H
authors.lastname: Abken
authors.forename: Ornella
authors.initials: O
authors.lastname: Parolini
authors.forename: Peter F
authors.initials: PF
authors.lastname: Ambros
authors.forename: Heinrich
authors.initials: H
authors.lastname: Kovar
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Holter
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D049109:Cell Proliferation; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007167:Immunotherapy; D008213:Lymphocyte Activation; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000008:administration & dosage*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012512:Sarcoma, Ewing / Q000276:immunology* / Q000628:therapy*; D013601:T-Lymphocytes / Q000276:immunology* / Q000637:transplantation; D014407:Tumor Cells, Cultured
chemical_list: D055655:NK Cell Lectin-Like Receptor Subfamily K; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins