Biomedical Microdevices

, Volume 12, Issue 5, pp 855–863 | Cite as

A high throughput microelectroporation device to introduce a chimeric antigen receptor to redirect the specificity of human T cells

  • Yoonsu Choi
  • Carrie Yuen
  • Sourindra N. Maiti
  • Simon Olivares
  • Hillary Gibbons
  • Helen Huls
  • Robert Raphael
  • Thomas C. Killian
  • Daniel J. Stark
  • Dean A. Lee
  • Hiroki Torikai
  • Daniel Monticello
  • Susan S. Kelly
  • Partow Kebriaei
  • Richard E. Champlin
  • Sibani L. Biswal
  • Laurence J. N. Cooper
Article

Abstract

It has been demonstrated that a chimeric antigen receptor (CAR) can directly recognize the CD19 molecule expressed on the cell surface of B-cell malignancies independent of major histocompatibility complex (MHC). Although T-cell therapy of tumors using CD19-specific CAR is promising, this approach relies on using expression vectors that stably integrate the CAR into T-cell chromosomes. To circumvent the potential genotoxicity that may occur from expressing integrating transgenes, we have expressed the CD19-specific CAR transgene from mRNA using a high throughput microelectroporation device. This research was accomplished using a microelectroporator to achieve efficient and high throughput non-viral gene transfer of in vitro transcribed CAR mRNA into human T cells that had been numerically expanded ex vivo. Electro-transfer of mRNA avoids the potential genotoxicity associated with vector and transgene integration and the high throughput capacity overcomes the expected transient CAR expression, as repeated rounds of electroporation can replace T cells that have lost transgene expression. We fabricated and tested a high throughput microelectroporator that can electroporate a stream of 2 × 108 primary T cells within 10 min. After electroporation, up to 80% of the passaged T cells expressed the CD19-specific CAR. Video time-lapse microscopy (VTLM) demonstrated the redirected effector function of the genetically manipulated T cells to specifically lyse CD19+ tumor cells. Our biomedical microdevice, in which T cells are transiently and safely modified to be tumor-specific and then can be re-infused, offers a method for redirecting T-cell specificity, that has implications for the development of adoptive immunotherapy.

Keywords

Electroporation Cancer High throughput mRNA Chimeric antigen receptor T cells 

Supplementary material

Supplementary material 1

Video of the electro-transfer process of CAR transgene mRNA using HiTMeD. Extended cables deliver the electrical signal to HiTMeD that operates in a fume hood to maintain sterility. Sampling of electroporated cells is performed using multi-well plates. (MPG 4323 kb)

Supplementary material 2

Time lapse video microscopy redirected killing of adherent tumor targets by CAR+ T cells after electro-transfer. (MPG 4035 kb)

References

  1. C. Baum, J. Dullmann, Z. Li, B. Fehse, J. Meyer, D.A. Williams, Blood 101, 2099 (2003)CrossRefGoogle Scholar
  2. C.J. Cohen, Y.F. Li, M. El-Gamil, P.F. Robbins, S.A. Rosenberg, R.A. Morgan, Cancer Res. 67, 3898 (2007)CrossRefGoogle Scholar
  3. L.J.N. Cooper, Blood 112, 2172 (2008)CrossRefGoogle Scholar
  4. L.J.N. Cooper, M.S. Topp, L.M. Serrano, S. Gonzalez, W. Chang, A. Naranjo, Blood 101, 1637 (2003)CrossRefGoogle Scholar
  5. L.J.N. Cooper, Z. Al-Kadhimi, L.M. Serrano, T. Pfeiffer, S. Olivares, A. Castro, Blood 105, 1622 (2005)CrossRefGoogle Scholar
  6. A. Craiu, D. Scadden, Methods Mol. Biol. 423, 301 (2008)CrossRefGoogle Scholar
  7. K.A. DeBruin, W. Krassowska, Biophys. J. 77, 1213 (1999)CrossRefGoogle Scholar
  8. M.L. Edelstein, M.R. Abedi, J. Wixon, R.M. Edelstein, J. Gene. Med. 6, 597 (2004)CrossRefGoogle Scholar
  9. N. Elango, S. Elango, P. Shivshankar, M.S. Katz, Biochem. Biophys. Res. Commun. 330, 958 (2005)CrossRefGoogle Scholar
  10. J.C. Fratantoni, S. Dzekunov, V. Singh, L.N. Liu, Cytotherapy 5, 208 (2003)CrossRefGoogle Scholar
  11. S. Hacein-Bey-Abina, C. Von Kalle, M. Schmidt, F. Le Deist, N. Wulffraat, E. McIntyre, N. Engl. J. Med. 348, 255 (2003)CrossRefGoogle Scholar
  12. S. Holtkamp, S. Kreiter, A. Selmi, P. Simon, M. Koslowski, C. Huber, Blood 108, 4009 (2006)CrossRefGoogle Scholar
  13. C.M. Kowolik, M.S. Topp, S. Gonzalez, T. Pfeiffer, S. Olivares, N. Gonzalez, Cancer Res. 66, 10995 (2006)CrossRefGoogle Scholar
  14. W. Krassowska, P.D. Filev, Biophys. J. 92, 404 (2007)CrossRefGoogle Scholar
  15. S. Li, Curr. Gene Ther. 4, 309 (2004)Google Scholar
  16. P.C.H. Li, D.J. Harrison, Anal. Chem. 69, 1564 (1997)CrossRefGoogle Scholar
  17. C.U. Louis, M.K. Brenner, Curr. Pharm. Des. 15, 424 (2009)CrossRefGoogle Scholar
  18. M.S. Mahmoud, R. Fujii, H. Ishikawa, M.M. Kawano, Blood 94, 3551 (1999)Google Scholar
  19. J.F. Miller, W.J. Dower, L.S. Tompkins, Proc. Natl. Acad. Sci. USA 85, 856 (1988)CrossRefGoogle Scholar
  20. D.A. Mitchell, I. Karikari, X. Cui, W. Xie, R. Schmittling, J.H. Sampson, Hum. Gene Ther. 19, 511 (2008)CrossRefGoogle Scholar
  21. M. Mockey, C. Goncalves, F.P. Dupuy, F.M. Lemoine, C. Pichon, P. Midoux, Biochem. Biophys. Res. Commun. 340, 1062 (2006)CrossRefGoogle Scholar
  22. A.C. Nathwani, K.M. Gale, K.D. Pemberton, D.C. Crossman, E.D.G. Tuddenham, J.H. McVey, Br. J. Haematol. 88, 122 (1994)CrossRefGoogle Scholar
  23. R.J. O’Reilly, Nat. Med. 14, 1148 (2008)CrossRefGoogle Scholar
  24. M.A. Pule, B. Savoldo, G.D. Myers, C. Rossig, H.V. Russell, G. Dotti, Nat. Med. 14, 1264 (2008)CrossRefGoogle Scholar
  25. L.M. Serrano, T. Pfeiffer, S. Olivares, T. Numbenjapon, J. Bennitt, D. Kim, Blood 107, 2643 (2006)CrossRefGoogle Scholar
  26. H. Singh, L.M. Serrano, T. Pfeiffer, S. Olivares, G. McNamara, D.D. Smith, Cancer Res. 67, 2872 (2007)CrossRefGoogle Scholar
  27. H. Singh, P.R. Manuri, S. Olivares, N. Dara, M.J. Dawson, H. Huls, Cancer Res. 68, 2961 (2008)CrossRefGoogle Scholar
  28. J. Stepinski, C. Waddell, R. Stolarski, E. Darzynkiewicz, R.E. Rhoads, RNA 7, 1486 (2001)Google Scholar
  29. B.G. Till, M.C. Jensen, J. Wang, E.Y. Chen, B.L. Wood, H.A. Greisman, Blood 112, 2261 (2008)CrossRefGoogle Scholar
  30. H. Wang, A.K. Bhunia, C. Lu, Biosens. Bioelectron. 22, 582 (2006)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Yoonsu Choi
    • 1
  • Carrie Yuen
    • 1
  • Sourindra N. Maiti
    • 1
  • Simon Olivares
    • 1
  • Hillary Gibbons
    • 1
  • Helen Huls
    • 1
  • Robert Raphael
    • 4
  • Thomas C. Killian
    • 5
  • Daniel J. Stark
    • 5
  • Dean A. Lee
    • 1
  • Hiroki Torikai
    • 1
  • Daniel Monticello
    • 6
  • Susan S. Kelly
    • 1
  • Partow Kebriaei
    • 2
  • Richard E. Champlin
    • 2
  • Sibani L. Biswal
    • 3
  • Laurence J. N. Cooper
    • 1
  1. 1.Division of Pediatrics, Children’s Cancer Hospital, The University of Texas Graduate School of Biomedical Sciences at HoustonThe University of Texas M. D. Anderson Cancer CenterHoustonUSA
  2. 2.Department of Stem Cell Transplantation and Cellular TherapyThe University of Texas M. D. Anderson Cancer CenterHoustonUSA
  3. 3.Department of Chemical and Biomolecular EngineeringRice UniversityHoustonUSA
  4. 4.Department of BioengineeringRice UniversityHoustonUSA
  5. 5.Department of Physics and AstronomyRice UniversityHoustonUSA
  6. 6.InCellerate Inc.HoustonUSA

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