title: Simplified process for the production of anti-CD19-CAR-engineered T cells.
abstract: OBJECTIVE
  Adoptive immunotherapy with the use of chimeric antigen receptor (CAR)-engineered T cells specific for CD19 has shown promising results for the treatment of B-cell lymphomas and leukemia. This therapy involves the transduction of autologous T cells with a viral vector and the subsequent cell expansion. We describe a new, simplified method to produce anti-CD19-CAR T cells.
  
  
  METHODS
  T cells were isolated from peripheral blood mononuclear cell (PBMC) with anti-CD3/anti-CD28 paramagnetic beads. After 2 days, the T cells were added to culture bags pre-treated with RetroNectin and loaded with the retroviral anti-CD19 CAR vector. The cells, beads and vector were incubated for 24 h, and a second transduction was then performed. No spinoculation was used. Cells were then expanded for an additional 9 days.
  
  
  RESULTS
  The method was validated through the use of two PBMC products from a patient with B-cell chronic lymphoblastic leukemia and one PBMC product from a healthy subject. The two PBMC products from the patient with B-cell chronic lymphoblastic leukemia contained 11.4% and 12.9% T cells. The manufacturing process led to final products highly enriched in T cells with a mean CD3+ cell content of 98%, a mean expansion of 10.6-fold and a mean transduction efficiency of 68%. Similar results were obtained from the PBMCs of the first four patients with acute lymphoblastic leukemia treated at our institution.
  
  
  CONCLUSIONS
  We developed a simplified, semi-closed system for the initial selection, activation, transduction and expansion of T cells with the use of anti-CD3/anti-CD28 beads and bags to produce autologous anti-CD19 CAR-transduced T cells to support an ongoing clinical trial.
authors.affiliation: Cell Processing Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Barbara
authors.initials: B
authors.lastname: Tumaini
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Lee
authors.forename: Tasha
authors.initials: T
authors.lastname: Lin
authors.forename: Luciano
authors.initials: L
authors.lastname: Castiello
authors.forename: David F
authors.initials: DF
authors.lastname: Stroncek
authors.forename: Crystal
authors.initials: C
authors.lastname: Mackall
authors.forename: Alan
authors.initials: A
authors.lastname: Wayne
authors.forename: Marianna
authors.initials: M
authors.lastname: Sabatino
mesh_terms: D018941:Antigens, CD19 / Q000276:immunology*; D018106:CD28 Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D060846:Cell Engineering / Q000379:methods*; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic / Q000235:genetics / Q000276:immunology*; D006801:Humans; D016219:Immunotherapy, Adoptive; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008213:Lymphocyte Activation / Q000276:immunology*; D011946:Receptors, Antigen / Q000235:genetics; D013601:T-Lymphocytes / Q000276:immunology*; D014161:Transduction, Genetic
chemical_list: D018941:Antigens, CD19; D018106:CD28 Antigens; D017252:CD3 Complex; D011946:Receptors, Antigen
keywords: CD19 antigen; adoptive cellular immunotherapy; genetic engineering; genetic transduction; precursor cell lymphoblastic leukemia-lymphoma