title: Transiently redirected T cells for adoptive transfer.
abstract: OBJECTIVE
  T cells can be redirected to reject cancer by retroviral transduction with a chimeric antigen receptor (CAR) or by administration of a bispecific T cell engager (BiTE). We demonstrate that transfection of T cells with messenger (m) RNA coding for CAR is an alternative strategy.
  
  
  METHODS
  We describe the pre-clinical evaluation of a method based on transient modification of expanded T cells with a CD19 CAR directed against B-cell malignancies. CAR mRNA was generated under cell-free conditions in a scalable process using recombinant RNA polymerase. Efficient and non-toxic square-wave electroporation was used to load the mRNA into the cytoplasm of T cells with no risk of insertional mutagenesis.
  
  
  RESULTS
  After transfection >80% of T cells were viable, with 94% CAR expression. Transfected T cells were cytolytic to CD19(+) targets and produced interferon (IFN)-γ in response. Killing of CD19(+) target cells was demonstrated even at day 8 with undetectable CAR expression. Increasing the concentration of mRNA resulted in higher surface CAR expression, better killing and more IFN-γ release but at the expense of increased activation-induced cell death. Finally, we demonstrated that a second transgene could be introduced by co-electroporation of CXCR4 or CCR7 with CAR to also modify chemotactic responses.
  
  
  CONCLUSIONS
  We advocate the transient redirection approach as well suited to meet safety aspects for early phase studies, prior to trials using stably transduced cells once CAR has been proven safe. The simplicity of this methodology also facilitates rapid screening of candidate targets and novel receptors in pre-clinical studies.
authors.affiliation: Section for Immunology, Radiumhospitalet, Oslo University Hospital, Oslo, Norway. hilde.almasbak@rr-research.no
authors.forename: Hilde
authors.initials: H
authors.lastname: Almåsbak
authors.forename: Edith
authors.initials: E
authors.lastname: Rian
authors.forename: Hanna Julie
authors.initials: HJ
authors.lastname: Hoel
authors.forename: Martin
authors.initials: M
authors.lastname: Pulè
authors.forename: Sébastien
authors.initials: S
authors.lastname: Wälchli
authors.forename: Gunnar
authors.initials: G
authors.lastname: Kvalheim
authors.forename: Gustav
authors.initials: G
authors.lastname: Gaudernack
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Rasmussen
mesh_terms: D019264:Adoptive Transfer / Q000379:methods*; D018941:Antigens, CD19 / Q000032:analysis; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D006801:Humans; D007938:Leukemia / Q000628:therapy*; D008223:Lymphoma / Q000628:therapy*; D011946:Receptors, Antigen / Q000235:genetics*; D054400:Receptors, CCR7 / Q000235:genetics*; D019718:Receptors, CXCR4 / Q000235:genetics*; D012190:Retroviridae; D013601:T-Lymphocytes / Q000276:immunology* / Q000637:transplantation*; D014161:Transduction, Genetic
chemical_list: D018941:Antigens, CD19; C515370:CCR7 protein, human; C527276:CXCR4 protein, human; D011946:Receptors, Antigen; D054400:Receptors, CCR7; D019718:Receptors, CXCR4