title: A clinically adaptable method to enhance the cytotoxicity of natural killer cells against B-cell malignancies.
abstract: OBJECTIVE
  Retroviral transduction of anti-CD19 chimeric antigen receptors significantly enhances the cytotoxicity of natural killer (NK) cells against B-cell malignancies. We aimed to validate a more practical, affordable and safe method for this purpose.
  
  
  METHODS
  We tested the expression of a receptor containing CD3ζ and 4-1BB signaling molecules (anti-CD19-BB-ζ) in human NK cells after electroporation with the corresponding mRNA using a clinical-grade electroporator. The cytotoxic capacity of the transfected NK cells was tested in vitro and in a mouse model of leukemia.
  
  
  RESULTS
  Median anti-CD19-BB-ζ expression 24 h after electroporation was 40.3% in freshly purified (n =18) and 61.3% in expanded (n = 31) NK cells; median cell viability was 90%. NK cells expressing anti-CD19-BB-ζ secreted interferon (IFN)-γ in response to CD19-positive target cells and had increased cytotoxicity. Receptor expression was detectable 6 h after electroporation, reaching maximum levels at 24-48 h; specific anti-CD19 cytotoxicity was observed at 96 h. Levels of expression and cytotoxicities were comparable with those achieved by retroviral transduction. A large-scale protocol was developed and applied to expanded NK cells (median NK cell number 2.5 × 10(8), n = 12). Median receptor expression after 24 h was 82.0%; NK cells transfected under these conditions exerted considerable cytotoxicity in xenograft models of B-cell leukemia.
  
  
  CONCLUSIONS
  The method described here represents a practical way to augment the cytotoxicity of NK cells against B-cell malignancies. It has the potential to be extended to other targets beyond CD19 and should facilitate the clinical use of redirected NK cells for cancer therapy.
authors.affiliation: Department of Oncology, St Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Noriko
authors.initials: N
authors.lastname: Shimasaki
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Fujisaki
authors.forename: Duck
authors.initials: D
authors.lastname: Cho
authors.forename: Marika
authors.initials: M
authors.lastname: Masselli
authors.forename: Timothy
authors.initials: T
authors.lastname: Lockey
authors.forename: Paul
authors.initials: P
authors.lastname: Eldridge
authors.forename: Wing
authors.initials: W
authors.lastname: Leung
authors.forename: Dario
authors.initials: D
authors.lastname: Campana
mesh_terms: D000818:Animals; D018941:Antigens, CD19 / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use; D017252:CD3 Complex / Q000235:genetics / Q000627:therapeutic use; D045744:Cell Line, Tumor; D064987:Cell- and Tissue-Based Therapy; D003602:Cytotoxicity, Immunologic / Q000235:genetics; D015973:Gene Expression Regulation, Leukemic; D006801:Humans; D007694:Killer Cells, Natural / Q000166:cytology* / Q000378:metabolism; D008213:Lymphocyte Activation; D008228:Lymphoma, Non-Hodgkin / Q000628:therapy; D051379:Mice; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000628:therapy; D012333:RNA, Messenger / Q000235:genetics / Q000627:therapeutic use; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9 / Q000235:genetics / Q000627:therapeutic use
chemical_list: D018941:Antigens, CD19; D017252:CD3 Complex; C121704:CD3 antigen, zeta chain; D012333:RNA, Messenger; C505714:TNFRSF9 protein, human; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9