Redirecting T cells to Ewing's sarcoma family of tumors by a chimeric NKG2D receptor expressed by lentiviral transduction or mRNA transfection

• Published in: PloS one 2012-02, Vol.7 (2), p.e31210-e31210
• Main Authors: Lehner, Manfred, Götz, Gabriel, Proff, Julia, Schaft, Niels, Dörrie, Jan, Full, Florian, Ensser, Armin, Muller, Yves A, Cerwenka, Adelheid, Abken, Hinrich, Parolini, Ornella, Ambros, Peter F, Kovar, Heinrich, Holter, Wolfgang
• Format: Article
• Language: English
• Subjects: Anchoring; Antigens; Apoptosis; B cells; Biology; Blotting, Western; C-Terminus; Cancer therapies; CD28 antigen; Cell death; Cell Proliferation; Cell recognition; Cell surface; Children & youth; Cytotoxicity; Dermatology; Enzyme-Linked Immunosorbent Assay; Ewing's sarcoma; Ewings sarcoma; Flow Cytometry; Gene expression; Gene transfer; Humans; Immunotherapy; Laboratories; Ligands; Localization; Lymphocyte Activation; Lymphocytes; Lymphocytes T; Major histocompatibility complex; Medical prognosis; Medical research; Medicine; Melanoma; Messenger RNA; Modulation; mRNA; NK Cell Lectin-Like Receptor Subfamily K - genetics; NK Cell Lectin-Like Receptor Subfamily K - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Reengineering; Ribonucleic acid; RNA; RNA, Messenger - administration & dosage; Sarcoma; Sarcoma, Ewing - immunology; Sarcoma, Ewing - therapy; Signal transduction; T cell receptors; T cells; T-Lymphocytes - immunology; T-Lymphocytes - transplantation; Target recognition; Transfection; Tumor Cells, Cultured; Tumors; Virology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0031210

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.