Stability and activity of MCSP-specific chimeric antigen receptors (CARs) depend on the scFv antigen-binding domain and the protein backbone

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 op...

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Published in:	Cancer Immunology, Immunotherapy Immunotherapy, 2015-12, Vol.64 (12), p.1623-1635
Main Authors:	Krug, Christian, Birkholz, Katrin, Paulus, Alexander, Schwenkert, Michael, Schmidt, Patrick, Hoffmann, Nicole, Hombach, Andreas, Fey, Georg, Abken, Hinrich, Schuler, Gerold, Schuler-Thurner, Beatrice, Dörrie, Jan, Schaft, Niels
Format:	Article
Language:	eng
Subjects:	Animals Cancer Research Carrier Proteins - immunology Cell Line, Tumor Disease Models, Animal Gene Expression Regulation, Neoplastic - immunology Humans Immunology Medicine Medicine & Public Health Melanoma - immunology Melanoma - physiopathology Mice Mitochondrial Proteins - genetics Mitochondrial Proteins - immunology Oncology Original Original Article Protein Stability Protein Structure, Tertiary Receptors, Antigen, T-Cell - genetics Receptors, Antigen, T-Cell - immunology Receptors, Antigen, T-Cell - metabolism Single-Chain Antibodies - metabolism
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Summary:	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.
ISSN:	0340-7004 1432-0851