Human TCR That Incorporate CD3{zeta} Induce Highly Preferred Pairing between TCR{alpha} and {beta} Chains following Gene Transfer

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Human TCR That Incorporate CD3 ${zeta}$ Induce Highly Preferred Pairing between TCR ${alpha}$ and β Chains following Gene Transfer¹

Zsolt Sebestyén^*, Erik Schooten^*, Tamara Sals^*, Irene Zaldivar, Esther San José, Balbino Alarcón, Sara Bobisse, Antonio Rosato, János Szöll $o$ si, Jan Willem Gratama^*, Ralph A. Willemsen^* and Reno Debets²^,*

^* Unit Clinical and Tumor Immunology, Department of Medical Oncology, Erasmus Medical Center–Daniel den Hoed Cancer Center, Rotterdam, The Netherlands; Consejo Superior de Investigaciones Cientificas–Universidad Autónoma de Madrid, Madrid, Spain; Department of Oncology and Surgical Sciences, University of Padova and Instituto Oncologico Veneto, Padova, Italy; and Department of Biophysics and Cell Biology, Research Center for Molecular Biology, University of Debrecen, Debrecen, Hungary

TCR gene therapy is adversely affected by newly formed TCR ${alpha}$ βheterodimers comprising exogenous and endogenous TCR chainsthat dilute expression of transgenic TCR ${alpha}$ β dimers and arepotentially self-reactive. We have addressed TCR mispairingby using a modified two-chain TCR that encompasses total humanCD3 ${zeta}$ with specificities for three different Ags. Transfer ofeither TCR ${alpha}$ :CD3 ${zeta}$ or β:CD3 ${zeta}$ genes alone does not result insurface expression, whereas transfer of both modified TCR chainsresults in high surface expression, binding of peptide-MHC complexesand Ag-specific T cell functions. Genetic introduction of TCR ${alpha}$ β: ${zeta}$ does not compromise surface expression and functions of an endogenousTCR ${alpha}$ β. Flow cytometry fluorescence resonance energy transferand biochemical analyses demonstrate that TCR ${alpha}$ β:CD3 ${zeta}$ is thefirst strategy that results in highly preferred pairing betweenCD3 ${zeta}$ -modified TCR ${alpha}$ and β chains as well as absence of TCRmispairing between TCR:CD3 ${zeta}$ and nonmodified TCR chains. Intracellularassembly and surface expression of TCR:CD3 ${zeta}$ chains is independentof endogenous CD3 ${gamma}$ , ${delta}$ , and ${epsilon}$ . Taken together, our data supportthe use of TCR ${alpha}$ β:CD3 ${zeta}$ to prevent TCR mispairing, which mayprovide an adequate strategy to enhance efficacy and safetyof TCR gene transfer.

The costs of publication of this article were defrayed in partby the payment of page charges. This article must thereforebe hereby marked advertisement in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

¹ This work was supported by a European Community 6th FrameworkGrant 018914, entitled "Adoptive Engineered T-Cell Targetingto Activate Cancer Killing (ATTACK)."

² Address correspondence and reprint requests to Dr. Reno Debets,Department of Medical Oncology, Erasmus Medical Center–Danielden Hoed Cancer Center, Groene Hilledijk 301, Rotterdam 3075EA,The Netherlands. E-mail address: j.debets{at}erasmusmc.nl

³ Abbreviations used in this paper: FRET, fluorescence resonanceenergy transfer; IP, immune precipitation; pMHC, peptide-MHC;RLU, relative light unit; MFI, mean fluorescence intensity.

Human TCR That Incorporate CD3 Induce Highly Preferred Pairing between TCR and β Chains following Gene Transfer1

Human TCR That Incorporate CD3 ${zeta}$ Induce Highly Preferred Pairing between TCR ${alpha}$ and β Chains following Gene Transfer¹