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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from $8.99

All prices are NET prices.

References

  1. 1.

    Haematopoietic cell transplantation as immunotherapy. Nature 411, 385–389 (2001).

  2. 2.

    et al. Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens. J. Clin. Oncol. 26, 5233–5239 (2008).

  3. 3.

    et al. Treatment of metastatic melanoma with autologous CD4+ T cells against NY-ESO-1. N. Engl. J. Med. 358, 2698–2703 (2008).

  4. 4.

    , , , & Immunotherapy through TCR gene transfer. Nat. Immunol. 2, 957–961 (2001).

  5. 5.

    et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314, 126–129 (2006).

  6. 6.

    et al. Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood 114, 535–546 (2009).

  7. 7.

    et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J. Clin. Oncol. 29, 917–924 (2011).

  8. 8.

    et al. Efficiency of T-cell receptor expression in dual-specific T cells is controlled by the intrinsic qualities of the TCR chains within the TCR-CD3 complex. Blood 109, 235–243 (2007).

  9. 9.

    et al. Mixed T cell receptor dimers harbor potentially harmful neoreactivity. Proc. Natl. Acad. Sci. USA 107, 10972–10977 (2010).

  10. 10.

    et al. Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy. Nat. Med. 16, 565–570 (2010).

  11. 11.

    , , , & Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability. Cancer Res. 66, 8878–8886 (2006).

  12. 12.

    et al. Improved expression and reactivity of transduced tumor-specific TCRs in human lymphocytes by specific silencing of endogenous TCR. Cancer Res. 69, 9003–9011 (2009).

  13. 13.

    et al. Development of human anti-murine T-cell receptor antibodies in both responding and nonresponding patients enrolled in TCR gene therapy trials. Clin. Cancer Res. 16, 5852–5861 (2010).

  14. 14.

    et al. Codon modification of T cell receptors allows enhanced functional expression in transgenic human T cells. Clin. Immunol. 119, 135–145 (2006).

  15. 15.

    et al. Targeting the Wilms tumor antigen 1 by TCR gene transfer: TCR variants improve tetramer binding but not the function of gene modified human T cells. J. Immunol. 179, 5803–5810 (2007).

  16. 16.

    et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005).

  17. 17.

    , , , & Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).

  18. 18.

    et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat. Biotechnol. 25, 1298–1306 (2007).

  19. 19.

    et al. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat. Methods 8, 861–869 (2011).

  20. 20.

    et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26, 808–816 (2008).

  21. 21.

    et al. Central memory self/tumor-reactive CD8+ T cells confer superior antitumor immunity compared with effector memory T cells. Proc. Natl. Acad. Sci. USA 102, 9571–9576 (2005).

  22. 22.

    et al. Adoptive transfer of effector CD8+ T cells derived from central memory cells establishes persistent T cell memory in primates. J. Clin. Invest. 118, 294–305 (2008).

  23. 23.

    et al. IL-7 and IL-15 allow the generation of suicide gene-modified alloreactive self-renewing central memory human T lymphocytes. Blood 113, 1006–1015 (2009).

  24. 24.

    et al. IL-7 receptor expression identifies suicide gene-modified allospecific CD8+ T cells capable of self-renewal and differentiation into antileukemia effectors. Blood 117, 6469–6478 (2011).

  25. 25.

    , , , & IL-15 mimics T cell receptor crosslinking in the induction of cellular proliferation, gene expression, and cytotoxicity in CD8+ memory T cells. Proc. Natl. Acad. Sci. USA 99, 6192–6197 (2002).

  26. 26.

    et al. Facilitating matched pairing and expression of TCR chains introduced into human T cells. Blood 109, 2331–2338 (2007).

  27. 27.

    , , , & Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters. Nat. Biotechnol. 23, 108–116 (2005).

  28. 28.

    et al. Enhanced antitumor activity of T cells engineered to express T-cell receptors with a second disulfide bond. Cancer Res. 67, 3898–3903 (2007).

  29. 29.

    et al. Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion. Nucleic Acids Res. 38, 8269–8276 (2010).

  30. 30.

    et al. Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients. Blood 115, 925–935 (2010).

  31. 31.

    & Generation of HIV-1 derived lentiviral vectors. Methods Enzymol. 346, 454–465 (2002).

  32. 32.

    et al. Development of an adenoviral vector system with adenovirus serotype 35 tropism; efficient transient gene transfer into primary malignant hematopoietic cells. J. Gene Med. 6, 631–641 (2004).

  33. 33.

    & The use of anti-CD3 and anti-CD28 monoclonal antibodies to clone and expand human antigen-specific T cells. J. Immunol. Methods 128, 189–201 (1990).

  34. 34.

    et al. Transient cold shock enhances zinc-finger nuclease-mediated gene disruption. Nat. Methods 7, 459–460 (2010).

  35. 35.

    et al. Repertoire, diversity, and differentiation of specific CD8 T cells are associated with immune protection against human cytomegalovirus disease. J. Exp. Med. 201, 1999–2010 (2005).

  36. 36.

    et al. LPS antagonism reduces graft-versus-host disease and preserves graft-versus-leukemia activity after experimental bone marrow transplantation. J. Clin. Invest. 107, 1581–1589 (2001).

  37. 37.

    et al. Suicide gene therapy of graft-versus-host disease induced by central memory human T lymphocytes. Blood 107, 1828–1836 (2006).

Download references

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

    • Jurgen Kuball

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

    • Elena Provasi
    •  & Pietro Genovese

    These authors equally contributed to this work.

Affiliations

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

    • Fabio Ciceri
    •  & Chiara Bonini
  9. MolMed S.p.A., Milan, Italy.

    • Claudio Bordignon

Authors

  1. Search for Elena Provasi in:

  2. Search for Pietro Genovese in:

  3. Search for Angelo Lombardo in:

  4. Search for Zulma Magnani in:

  5. Search for Pei-Qi Liu in:

  6. Search for Andreas Reik in:

  7. Search for Victoria Chu in:

  8. Search for David E Paschon in:

  9. Search for Lei Zhang in:

  10. Search for Jurgen Kuball in:

  11. Search for Barbara Camisa in:

  12. Search for Attilio Bondanza in:

  13. Search for Giulia Casorati in:

  14. Search for Maurilio Ponzoni in:

  15. Search for Fabio Ciceri in:

  16. Search for Claudio Bordignon in:

  17. Search for Philip D Greenberg in:

  18. Search for Michael C Holmes in:

  19. Search for Philip D Gregory in:

  20. Search for Luigi Naldini in:

  21. Search for Chiara Bonini in:

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.

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.

Corresponding authors

Correspondence to Luigi Naldini or Chiara Bonini.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Tables 1 and 2 and Supplementary Figures 1–8

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nm.2700