title: Manufacturing of gene-modified cytotoxic T lymphocytes for autologous cellular therapy for lymphoma.
abstract: BACKGROUND
  The production of therapeutic T-cell populations for adoptive immunotherapy of cancer requires extensive ex vivo cell processing, including the isolation or creation of Ag-specific T cells and their subsequent propagation to clinically relevant numbers. These procedures must be performed according to the principles of current good manufacturing practices (cGMP) for phase I clinical trials to ensure the identity, purity potency and safety of the cellular product. In this report we describe our approach to manufacturing and characterizing bulk populations of gene-modified autologous T cells for use in treating follicular lymphoma.
  
  
  METHODS
  PBMC from healthy donors, obtained after informed consent, were stimulated in vitro with Ab to CD3epsilon (OKT3) and recombinant human IL-2 and then electroporated with plasmid DNA containing a human CD19-specific chimeric Ag receptor (CAR) gene and HSV-1 thymidine kinase (TK) gene. Stably transfected cells were selected in cytocidal concentrations of hygromycin B over multiple 14-day stimulation culture cycles and then cryopreserved. Vials of cryopreserved/selected T cells were used to initiate T-cell expansion cultures to produce cell products for clinical infusion. These cultures were characterized for phenotype, function and suitability for use in adoptive immunotherapy studies.
  
  
  RESULTS
  Our results demonstrate that bulk populations of gene-modified T cells derived from peripheral blood of healthy donors express CD19+ chimeric Ag receptor at low levels and can specifically lyse CD19+ target cells in vitro. These cells display a differentiated T-effector phenotype, are sensitive to ganciclovir-mediated killing and display a non-transformed phenotype. TCR Vbeta usage indicated that all populations tested were polyclonal. Ex vivo cell expansion from cryopreserved cell banks is sufficient to produce doses of between 5 x 10(9) and 1 x 10(10) cells/run. One of three transductions resulted in a population of cells that was not suitable for infusion but was identified during release testing. No populations displayed any evidence of bacterial, fungal or mycoplasma contamination.
  
  
  CONCLUSIONS
  We have established a manufacturing strategy that is being used to produce T cells for a phase I clinical trial for follicular lymphoma. Genetically modified T cells have been characterized by cell-surface marker phenotype, functional activity against CD19+ targets and requisite safety testing. These pre-clinical data confirm the feasibility of this approach to manufacturing T-cell products.
authors.affiliation: Division of Pediatric Hematology-Oncology, City of Hope National Medical Center, California 91010, USA.
authors.forename: L J N
authors.initials: LJ
authors.lastname: Cooper
authors.forename: L
authors.initials: L
authors.lastname: Ausubel
authors.forename: M
authors.initials: M
authors.lastname: Gutierrez
authors.forename: S
authors.initials: S
authors.lastname: Stephan
authors.forename: R
authors.initials: R
authors.lastname: Shakeley
authors.forename: S
authors.initials: S
authors.lastname: Olivares
authors.forename: L M
authors.initials: LM
authors.lastname: Serrano
authors.forename: L
authors.initials: L
authors.lastname: Burton
authors.forename: M C V
authors.initials: MC
authors.lastname: Jensen
authors.forename: S J
authors.initials: SJ
authors.lastname: Forman
authors.forename: D L
authors.initials: DL
authors.lastname: DiGiusto
mesh_terms: D015703:Antigens, CD / Q000276:immunology; D018941:Antigens, CD19 / Q000276:immunology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic; D015774:Ganciclovir / Q000494:pharmacology; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D007376:Interleukin-2 / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D016014:Linear Models; D008224:Lymphoma, Follicular / Q000235:genetics / Q000276:immunology / Q000628:therapy*; D016853:Muromonab-CD3 / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection
chemical_list: D015703:Antigens, CD; D018941:Antigens, CD19; D007376:Interleukin-2; D016853:Muromonab-CD3; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D013937:Thymidine Kinase; D015774:Ganciclovir