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Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer

Nature Medicine volume 18, pages 807–815 (2012)Cite this article

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Abstract

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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Figure 1: ZFN-mediated disruption of TRBC genes in human T lymphocytes.
Figure 2: Expansion and purification of CD3− primary T lymphocytes.
Figure 3: TCR-β gene editing results in high-avidity tumor-specific T lymphocytes.
Figure 4: Full gene editing of TCR-α and TCR-β results in high-avidity tumor-specific lymphocytes with sharply reduced alloreactivity.
Figure 5: TCR-α/β–edited lymphocytes inhibit leukemia development without inducing GvHD in immunodeficient mice.

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Acknowledgements

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.

Author information

Author notes

  1. Jurgen Kuball

    Present address: Present address: Department of Hematology and Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.,

  2. Elena Provasi and Pietro Genovese: These authors equally contributed to this work.

Authors and Affiliations

  1. Division of Regenerative Medicine, Experimental Hematology Unit, Gene Therapy and Stem Cells, Program of Immunology, Gene Therapy and Bio-Immunotherapy of Cancer, Leukemia Unit, San Raffaele Scientific Institute, Milan, Italy

    Elena Provasi, Zulma Magnani, Barbara Camisa, Attilio Bondanza, Fabio Ciceri & Chiara Bonini

  2. Vita Salute San Raffaele University, Milan, Italy

    Elena Provasi, Pietro Genovese, Claudio Bordignon, Luigi Naldini & Chiara Bonini

  3. San Raffaele Telethon Institute for Gene Therapy and Division of Regenerative Medicine, Gene Therapy and Stem Cells, San Raffaele Scientific Institute, Milan, Italy

    Pietro Genovese, Angelo Lombardo & Luigi Naldini

  4. Sangamo BioSciences Inc., Richmond, California, USA

    Pei-Qi Liu, Andreas Reik, Victoria Chu, David E Paschon, Lei Zhang, Michael C Holmes & Philip D Gregory

  5. Fred Hutchinson Cancer Research Center and University of Washington, Seattle, Washington, USA

    Jurgen Kuball & Philip D Greenberg

  6. Division of Immunology, Experimental Immunology Unit, Transplantation and Infectious Diseases, Program of Immunology, Gene Therapy and Bio-Immunotherapy of Cancer, San Raffaele Scientific Institute, Milan, Italy

    Giulia Casorati

  7. Department of Pathology, Leukemia Unit, San Raffaele Scientific Institute, Milan, Italy

    Maurilio Ponzoni

  8. Division of Regenerative Medicine, Hematology Clinical Unit, Gene Therapy and Stem Cells, San Raffaele Scientific Institute, Milan, Italy

    Fabio Ciceri & Chiara Bonini

  9. MolMed S.p.A., Milan, Italy

    Claudio Bordignon

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  1. Elena Provasi
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Contributions

E.P. and P.G. designed experiments, performed research, analyzed data and wrote the manuscript. A.L. designed research and analyzed data. Z.M. performed research and analyzed data. P.-Q.L., A.R. and V.C. designed and were responsible for assembly of the TRBC-ZFN. D.E.P. and L.Z. designed and were responsible for assembly of the TRAC-ZFN. J.K. optimized the WT1-TCR genes. B.C. performed in vivo experiments. A.B. set up the protocols of the T cell culture and assisted with experimental design. M.P. supervised the histological analyses. G.C., F.C., C. Bordignon, P.D. Greenberg, M.C.H. and P.D. Gregory assisted with experimental design and revised the paper. L.N. designed the research, analyzed the data and wrote the manuscript. C. Bonini designed the research, analyzed the data, wrote the manuscript and acted as senior author of the study.

Corresponding authors

Correspondence to Luigi Naldini or Chiara Bonini.

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Competing interests

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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