The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR β- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.
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We would like to thank E. Rebar, J. Miller, D. Guschin, E. Leung, X. Meng, S. Hinkley, S. Lam, A. Hassenberg, Z. Zhang, C. Flinders and A. Tam for help in the generation of the TCR-specific ZFNs and the off-target analysis using SELEX, L. Sergi for technical assistance in vector production, M. Bernardi, M.T. Lupo-Stanghellini, A. Forcina, C. Traversari and all of the individuals at the Naldini and Bonini laboratories for fruitful discussion, and C. Tresoldi and the San Raffaele Scientific Institute Leukemia Biobanking for assistance. E.P. and P.G. conducted this study as partial fulfillment of their PhD in molecular medicine at the Program in Immunology, San Raffaele University, Milan, Italy. This work was supported by the Italian Ministry of Health (GR07-5 BO and RO10/07-B-1), the Italian Ministry of Research and University (FIRB-IDEAS, linked to European Research Council (ERC) starting grants), Fondazione Cariplo and the Italian Association for Cancer Research (AIRC) to C. Bonini and by the EU (FP7: GA 222878, PERSIST and ERC advanced grant 249845 TARGETINGGENETHERAPY), AIRC, Italian Ministry of Health (Challenge in Oncology) and Italian Telethon (TELE11/12-D2) to L.N.
P.-Q.L., A.R., V.C., D.E.P., L.Z., M.C.H. and P.D. Gregory are employees of Sangamo BioSciences Inc. C. Bordignon is an employee of Molmed S.p.A.
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Provasi, E., Genovese, P., Lombardo, A. et al. Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer. Nat Med 18, 807–815 (2012). https://doi.org/10.1038/nm.2700
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