title: A GMP-compliant protocol to expand and transfect cancer patient T cells with mRNA encoding a tumor-specific chimeric antigen receptor.
abstract: Chimeric antigen receptors (CARs), which combine an antibody-derived binding domain (single chain fragment variable) with T-cell-activating signaling domains, have become a promising tool in the adoptive cellular therapy of cancer. Retro- and lenti-viral transductions are currently the standard methods to equip T cells with a CAR; permanent CAR expression, however, harbors several risks like uncontrolled auto-reactivity. Modification of T cells by electroporation with CAR-encoding RNA to achieve transient expression likely circumvents these difficulties. We here present a GMP-compliant protocol to activate and expand T cells for clinical application. The protocol is optimized in particular to produce CAR-modified T cells in clinically sufficient numbers under full GMP-compliance from late-stage cancer patients. This protocol allows the generation of 6.7 × 10(8) CAR-expressing T cells from one patient leukapheresis. The CAR-engineered T cells produced pro-inflammatory cytokines after stimulation with antigen-bearing tumor cells and lysed tumor cells in an antigen-specific manner. This functional capacity was maintained after cryopreservation. Taken together, we provide a clinically applicable protocol to transiently engineer sufficient numbers of antigen-specific patient T cells for use in adoptive cell therapy of cancer.
authors.affiliation: Department of Dermatology, Universtitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Krug
authors.forename: Manuel
authors.initials: M
authors.lastname: Wiesinger
authors.forename: Hinrich
authors.initials: H
authors.lastname: Abken
authors.forename: Beatrice
authors.initials: B
authors.lastname: Schuler-Thurner
authors.forename: Gerold
authors.initials: G
authors.lastname: Schuler
authors.forename: Jan
authors.initials: J
authors.lastname: Dörrie
authors.forename: Niels
authors.initials: N
authors.lastname: Schaft
mesh_terms: D045744:Cell Line, Tumor; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D008545:Melanoma / Q000276:immunology* / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D014162:Transfection / Q000592:standards
chemical_list: D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins