title: Stability and activity of MCSP-specific chimeric antigen receptors (CARs) depend on the scFv antigen-binding domain and the protein backbone.
abstract: Chimeric antigen receptor (CAR)-modified T cells emerged as effective tools in the immunotherapy of cancer but can produce severe on-target off-tissue toxicities. This risk can conceivably be overcome, at least partially, by transient transfection. The design of CARs, however, has so far not been optimized for use in non-permanent T cell modification. Here we compared the performance of T cells modified with three different first- and second-generation CARs, each specific for MCSP (HMW-MAA) which is commonly expressed by melanoma cells. Upon RNA transfer, the expression of all receptors was limited in time. The second-generation CARs, which combined CD28-CD3ζ signaling, were expressed at higher levels and more prolonged than first-generation CARs with CD3ζ only. The CD28 domain increased the cytokine production, but had only an indirect effect on the lytic capacity, by prolonging the CAR expression. Especially for the second-generation CARs, the scFv clearly impacted the level and duration of CAR expression and the T cell performance. Thus, we identified a CAR high in both expression and anti-tumor cell reactivity. T cells transfected with this CAR increased the mean survival time of mice after challenge with melanoma cells. To facilitate clinical application, this CAR was used to redirect T cells from late-stage melanoma patients by RNA transfection. These T cells mediated effective antigen-specific tumor cell lysis and release of pro-inflammatory cytokines, even after cryoconservation of the transfected T cells. Taken together, the analysis identified a CAR with superior anti-melanoma performance after RNA transfer which is a promising candidate for clinical exploration.
authors.affiliation: Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany.|Department of Biology, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Krug
authors.affiliation: Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Birkholz
authors.affiliation: Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Paulus
authors.affiliation: Department of Biology, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schwenkert
authors.affiliation: Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.|Department I Internal Medicine, University Hospital Cologne, Cologne, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Schmidt
authors.affiliation: Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.|Department I Internal Medicine, University Hospital Cologne, Cologne, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Hoffmann
authors.affiliation: Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.|Department I Internal Medicine, University Hospital Cologne, Cologne, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Hombach
authors.affiliation: Department of Biology, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Fey
authors.affiliation: Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.|Department I Internal Medicine, University Hospital Cologne, Cologne, Germany.
authors.forename: Hinrich
authors.initials: H
authors.lastname: Abken
authors.affiliation: Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany.
authors.forename: Gerold
authors.initials: G
authors.lastname: Schuler
authors.affiliation: Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Schuler-Thurner
authors.affiliation: Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Dörrie
authors.affiliation: Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany. niels.schaft@uk-erlangen.de.
authors.forename: Niels
authors.initials: N
authors.lastname: Schaft
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000276:immunology; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D015972:Gene Expression Regulation, Neoplastic / Q000276:immunology; D006801:Humans; D008545:Melanoma / Q000276:immunology / Q000503:physiopathology; D051379:Mice; D024101:Mitochondrial Proteins / Q000235:genetics / Q000276:immunology*; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D024101:Mitochondrial Proteins; D011948:Receptors, Antigen, T-Cell; C484385:SMCP protein, human; D057127:Single-Chain Antibodies
keywords: Adoptive T cell therapy; Chimeric antigen receptor; Melanoma; Melanoma-associated chondroitin sulfate proteoglycan (MCSP); Single-chain variable fragment (scFv); mRNA transfection