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Molecular Design of the Cβ Interface Favors Specific Pairing of Introduced TCRβ in Human T Cells¹

Ralf-Holger Voss^2,*, Ralph A. Willemsen, Jürgen Kuball, Margarete Grabowski^*, Renate Engel, Ratna S. Intan^*, Philippe Guillaume^¶, Pedro Romero^||, Christoph Huber^* and Matthias Theobald

^* Medical Clinic (III) and Polyclinic, Department of Hematology and Oncology, Johannes Gutenberg-University, Mainz, Germany; Laboratory of Tumor Immunology, Department of Medical Oncology, Erasmus Medical Center-Daniel den Hoed Cancer Center, Rotterdam, The Netherlands; Department of Hematology and Van Creveld Clinic, University Medical Center, Utrecht, The Netherlands; The European Institute for Research and Development of Transplantation Strategies, Idar-Oberstein, Germany; ^¶ Ludwig Institute for Cancer Research, Lausanne Branch, Epalinges, Switzerland; and ^|| Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, Hôpital Orthopédique, Lausanne, Switzerland

A promising approach to adoptive transfer therapy of tumors is to reprogram autologous T lymphocytes by TCR gene transfer of defined Ag specificity. An obstacle, however, is the undesired pairing of introduced TCR- and TCRβ-chains with the endogenous TCR chains. These events vary depending on the individual endogenous TCR and they not only may reduce the levels of cell surface-introduced TCR but also may generate hybrid TCR with unknown Ag specificities. We show that such hybrid heterodimers can be generated even by the pairing of human and mouse TCR- and TCRβ-chains. To overcome this hurdle, we have identified a pair of amino acid residues in the crystal structure of a TCR that lie at the interface of associated TCR C and Cβ domains and are related to each other by both a complementary steric interaction analogous to a "knob-into-hole" configuration and the electrostatic environment. We mutated the two residues so as to invert the sense of this interaction analogous to a charged "hole-into-knob" configuration. We show that this inversion in the CCβ interface promotes selective assembly of the introduced TCR while preserving its specificity and avidity for Ag ligand. Noteworthily, this TCR modification was equally efficient on both a Mu and a Hu TCR. Our data suggest that this approach is generally applicable to TCR independently of their Ag specificity and affinity, subset distribution, and species of origin. Thus, this strategy may optimize TCR gene transfer to efficiently and safely reprogram random T cells into tumor-reactive T cells.

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

¹ This study was supported in part by the Deutsche Forschungsgemeinschaft (SFB 432 A3), the European Commission, and the Wilhelm-Laupitz Foundation. P.R. was supported in part by a grant from the Sixth Framework Program of the European Community (Cancer Immunotherapy).

² Address correspondence and reprint requests to Dr. Ralf-Holger Voss, III. Medical Clinic and Polyclinic, Department of Hematology and Oncology, Johannes Gutenberg-University, Langenbeckstrasse 1, 55101 Mainz, Germany. E-mail address: hvoss{at}uni-mainz.de

³ Abbreviations used in this paper: Hu, human; C/c, constant domain of TCR; Cβ/cβ, constant domain of TCRβ; GMF, geometric mean fluorescence; IMGT, ImMunoGeneTics information system; IRES, internal ribosome entry site; MDM2, mouse double minute 2; MFI, mean fluorescence intensity; Mt, mutant/mutated; RCSB, Research Collaboratory for Structural Bioinformatics; Mu, murine; TAA, tumor-associated Ag; Tg, transgenic; V or Vβ, variable domain of TCR or TCRβ, respectively; Wt, wild type.

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