title: Ex vivo expansion of natural killer cells from human peripheral blood mononuclear cells co-stimulated with anti-CD3 and anti-CD52 monoclonal antibodies.
abstract: OBJECTIVE
  This study developed a new method to expand CD3(-)CD56(+) natural killer (NK) cells from human peripheral blood mononuclear cells (PBMCs) without feeder cells for clinical trials.
  
  
  METHODS
  PBMCs from healthy subjects were co-stimulated with anti-CD3 and anti-CD52 monoclonal antibodies and cultured for 14 days in newly developed NKGM-1 medium containing autologous plasma and interleukin-2. Expanded NK cells were examined for cell number, phenotype, in vitro and in vivo cytotoxicity and interferon (IFN)-γ secretion. We also evaluated the proliferative ability of NK cells after cryopreservation. A patient with advanced pancreatic cancer was treated with autologous-expanded NK cells through the use of this method in combination with chemotherapy.
  
  
  RESULTS
  Expanded NK cells expressed higher levels of activating molecules compared with resting NK cells and exhibited potent cytotoxicity against K562 cells and IFN-γ secretion by cytokine stimulation. Significant anti-tumor activity was observed in immunodeficient mice injected with the human pancreatic cancer cell line BxPC-3. Large-scale cultures generated a median 5.7 × 10(9) NK cells from 20 mL of peripheral blood (n = 38) after 14 days of culture and 8.4 × 10(9) NK cells after 18 days of culture through the use of a cryopreservation procedure. The number of NK cells and cytotoxic activity in the peripheral blood of the patient with pancreatic cancer greatly increased, and successful clinical responses were observed after multiple infusions of expanded NK cells.
  
  
  CONCLUSIONS
  These data demonstrate that this simple and safe methodology for the ex vivo expansion of NK cells can be used for cancer immunotherapy.
authors.affiliation: New City Osaki Clinic, Tokyo, Japan; Division of Inflammation Research, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan. Electronic address: jmas@nco-clinic.jp.
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Masuyama
authors.affiliation: Laboratory of Tumor Biology, Takasaki University of Health and Welfare, Takasaki, Gunma, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Murakami
authors.affiliation: Department of Biochemistry, School of Medicine, Showa University, Tokyo, Japan.
authors.forename: Sanju
authors.initials: S
authors.lastname: Iwamoto
authors.affiliation: New City Osaki Clinic, Tokyo, Japan.
authors.forename: Sanehiko
authors.initials: S
authors.lastname: Fujita
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D015703:Antigens, CD / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D017252:CD3 Complex / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D000074301:CD52 Antigen; D049109:Cell Proliferation; D002469:Cell Separation; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000166:cytology* / Q000276:immunology*; D008213:Lymphocyte Activation / Q000187:drug effects*; D008297:Male; D051379:Mice; D008875:Middle Aged; D010641:Phenotype; D011956:Receptors, Cell Surface / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D017252:CD3 Complex; D000074301:CD52 Antigen; C000613862:CD52 protein, human; D006023:Glycoproteins; D011956:Receptors, Cell Surface; D007371:Interferon-gamma
keywords: CD52; NK cell expansion; co-stimulation; immunotherapy