Basic StudiesAn Ig Transmembrane Domain Motif Improves the Function of TCRs Transduced in Human T Cells Implications for ImmunotherapyD’Apice, Luciana*; Cuccaro, Fausta†,‡; Varriale, Sonia†,‡; Cipria, Deborah*; Sartorius, Rossella*; Circosta, Paola§; Cignetti, Alessandro§; Salerno, Massimiliano†,‡; Coscia, Maria R.*; Oreste, Umberto*; Marzullo, Vincenzo M.*; Martini, Giuseppe∥; Acuto, Oreste¶; De Berardinis, Piergiuseppe*Author Information *Institute of Protein Biochemistry, National Research Council †IRBM Science Park, Naples ‡IRBM Science Park, Pomezia ∥Biostella, Via Giovanni da Procida, Rome §Department of Molecular Biotechnology and Health Sciences, Center for Experimental Research and Medical Studies, University of Turin, Turin, Italy ¶Sir William Dunn School of Pathology, Oxford University of Oxford South Parks Road, Oxford, UK L.D. and F.C. contributed equally to this manuscript. Reprints: Piergiuseppe De Berardinis, IBP CNR, Via Pietro Castellino 111, Naples 80131, Italy (e-mail: email@example.com). Received July 30, 2018 Accepted February 6, 2019 Journal of Immunotherapy: May 2019 - Volume 42 - Issue 4 - p 97-109 doi: 10.1097/CJI.0000000000000259 Buy SDC Metrics Abstract Adoptive transfer of T lymphocytes (ACT) engineered with T-cell receptors (TCRs) of known antitumor specificity is an effective therapeutic strategy. However, a major constraint of ACT is the unpredictable interference of the endogenous TCR α and β chains in pairing of the transduced TCR. This effect reduces the efficacy of the genetically modified primary T cells and carries the risk of generating novel TCR reactivities with unintended functional consequences. Here, we show a powerful approach to overcome these limitations. We engineered TCR α and β chains with mutations encompassing a conserved motif (FXXXFXXS) required to stabilize the pairing of immunoglobulin heavy chain transmembrane domains. Molecular modeling supported the preferential pairing of mutated TCR and impaired pairing between mutated and wild-type TCRs. Expression of the mutated TCR was similar to wild type and conferred the expected specificity. Fluorescence resonance energy transfer analysis in mouse splenocytes transduced with mutated or wild-type TCRs showed a higher proximity of the former over the latter. Importantly, we show that mutated TCRs effectively outcompete endogenous TCRs and improve in vitro antitumor cytotoxicity when expressed in ex vivo isolated human T cells. This approach should contribute to improving current protocols of anticancer immunetherapy protocols. Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.