title: Tetraspecific scFv construct provides NK cell mediated ADCC and self-sustaining stimuli via insertion of IL-15 as a cross-linker.
abstract: BACKGROUND
  The design of a highly effective anti-cancer immune-engager would include targeting of highly drug refractory cancer stem cells (CSC). The design would promote effective antibody-dependent cell-mediated cytotoxicity (ADCC) and simultaneously promote costimulation to expand and self-sustain the effector NK cell population. Based on our bispecific NK cell engager platform we constructed a tetraspecific killer engager (TetraKE) comprising single-chain variable fragments (scFvs) binding FcγRIII (CD16) on NK cells, EpCAM on carcinoma cells and CD133 on cancer stem cells in order to promote ADCC. Furthermore, an Interleukin (IL)-15-crosslinker enhanced NK cell related proliferation resulting in a highly active drug termed 1615EpCAM133.
  
  
  RESULTS
  Proliferation assays showed TetraKE promoted proliferation and enhanced NK cell survival. Drug-target binding, NK cell related degranulation, and IFN-γ production was specific for both tumor related antigens in EpCAM and CD133 bearing cancer cell lines. The TetraKE showed higher killing activity and superior dose dependent degranulation. Cytokine profiling showed a moderately enhanced IFN-γ production, enhanced GM-CSF production, but no evidence of induction of excessive cytokine release.
  
  
  METHODS
  Assembly and synthesis of hybrid genes encoding the TetraKE were performed using DNA shuffling and ligation. The TetraKE was tested for efficacy, specificity, proliferation, survival, and cytokine production using carcinoma cell lines and functional assays measuring NK cell activity.
  
  
  CONCLUSIONS
  1615EpCAM133 combines improved induction of ADCC with enhanced proliferation, limited cytokine response, and prolonged survival and proliferation of NK cells. By linking scFv-related targeting of carcinoma and CSCs with a sustaining IL-15 signal, our new construct shows great promise to target cancer and CSCs.
authors.affiliation: University of Minnesota, Masonic Cancer Center, Section of Molecular Cancer Therapeutics, Therapeutic Radiology-Radiation Oncology, University of Minnesota, Minneapolis, MN, USA.|University of Tuebingen, Department for Hematology and Oncology, Medicine Department 2, University Hospital of Tuebingen, Tuebingen, Germany.
authors.forename: Joerg U
authors.initials: JU
authors.lastname: Schmohl
authors.affiliation: University of Minnesota, Department of Medicine, Division of Hematology, Oncology, and Transplantation, Minneapolis, MN, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Felices
authors.affiliation: University of Minnesota, Masonic Cancer Center, Section of Molecular Cancer Therapeutics, Therapeutic Radiology-Radiation Oncology, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Deborah
authors.initials: D
authors.lastname: Todhunter
authors.affiliation: University of Minnesota, Masonic Cancer Center, Section of Molecular Cancer Therapeutics, Therapeutic Radiology-Radiation Oncology, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Taras
authors.affiliation: University of Minnesota, Department of Medicine, Division of Hematology, Oncology, and Transplantation, Minneapolis, MN, USA.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Miller
authors.affiliation: University of Minnesota, Masonic Cancer Center, Section of Molecular Cancer Therapeutics, Therapeutic Radiology-Radiation Oncology, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Vallera
mesh_terms: D000071916:AC133 Antigen / Q000235:genetics / Q000378:metabolism; D000920:Antibody-Dependent Cell Cytotoxicity; D015415:Biomarkers; D015550:Cell Degranulation / Q000276:immunology; D045744:Cell Line, Tumor; D016207:Cytokines / Q000096:biosynthesis; D000071858:Epithelial Cell Adhesion Molecule / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D019409:Interleukin-15 / Q000235:genetics / Q000378:metabolism*; D007694:Killer Cells, Natural / Q000276:immunology* / Q000378:metabolism*; D014411:Neoplastic Stem Cells / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology*
chemical_list: D000071916:AC133 Antigen; D015415:Biomarkers; D016207:Cytokines; D000071858:Epithelial Cell Adhesion Molecule; D019409:Interleukin-15; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D007371:Interferon-gamma
keywords: CD133; EpCAM; TetraKE; cancer stem cells; carcinoma