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Less expensive CARs?
Cytotherapy, 2012; 14: 773–774
COMMENTARY
Less expensive CARs?
JOHN C. RICHES & JOHN G. GRIBBEN Barts Cancer Institute, Queen Mary University of London, London, UK Key Words: Immunotherapy, gene therepay, natural killer cells
Immunotherapy has the potential to have a major impact on cancer treatment, especially by targeting drug-resistant tumor subclones. The B-cell malignancies are good models for testing novel immunotherapeutic approaches, and there has been recent excitement about the apparent efficacy of chimeric antigen receptor (CAR) T cells in the treatment of chronic lymphocytic leukemia (CLL) (1). To be applicable clinically, cell-based therapies need to be translated from the laboratory into practical good manufacturing practice (GMP)-compliant methods. The excitement surrounding these approaches is dampened somewhat by their high expense, and the need for very specialized facilities to produce cells that have been retrovirally transduced to GMP requirements. In this issue, Shimasaki et al. (2) demonstrate that they are able to electroporate human natural killer (NK) cells with a receptor encoding anti-CD19 with CD3ζ and CD137 (4 - 1BB) using a GMP-compliant electroporator. They were able to achieve anti-CD19BB-ζ receptor expression of 40% in freshly purified and more than 60% in expanded NK cells, levels of expression comparable to those achieved with retroviral transduction, even after scale up to clinicalgrade material. The NK cells expressing the chimeric receptor had increased cytotoxicity, as demonstrated in a xenograft model of B-cell leukemia. Most studies to date on the use of CAR have been with T cells (3). A major advantage of this approach is that it eliminates the need for major histocompatibility complex (MHC) restriction, thus enabling the same CAR to be used for different patients. The use of an antibody receptor means that potential targets can be increased to include a wide range of surface proteins, sugars and lipids (4). A number of phase I/II clinical trials are currently underway using anti-CD19
CAR T cells for the treatment of B-cell malignancies (5). A key finding in pre-clinical studies has been that the addition of a co-stimulatory domain, such as CD28 or CD137, significantly improves the efficacy of these CAR T cells (6,7). A clinical trial with anti-CD19 CAR T cells in a patient with advanced follicular lymphoma resulted in regression of lymphadenopathy, associated with B lymphopenia and hypogammaglobulinemia. However, the CAR T cells did not persist long-term, with the anti-CD19 CAR becoming undetectable at 27 weeks, followed by the development of progressive disease 5 weeks later (8). Subsequent studies of anti-CD19 CAR T cells in CLL following conditioning with fludarabine, cyclophosphamide and high-dose interleukin (IL)-2 have demonstrated impressive clinical results (9). The importance of conditioning has also been shown by other groups, with no objective responses seen in patients who did not receive conditioning, in contrast to disease stabilization or lymph node responses in three out of four of the patients who had had cyclophosphamide as part of the trial design. However, a further patient in this study rapidly developed complications and died within 48 h of an infusion of a higher dose of CAR T cells, highlighting the risks associated with this therapy (10,11). Although this work is still in the early stages, it has underscored the importance of the conditioning regimen in promoting T-cell engraftment and activation, analogous to the impact of conditioning in allogeneic transplantation. In particular, it may be vital to eliminate regulatory T cells, which are known to be expanded in CLL and can be suppressed with fludarabine treatment (12–14). It may also be important to eliminate other subpopulations, such as immature dendritic cells, as well as cell populations that act as ‘cytokine sinks’ by competing for the same survival and stimulatory factors (5).
Correspondence: Professor John G. Gribben MD DSc FMedSci, Chair of Medical Oncology, Barts Cancer Institute, Queen Mary University of London, 3rd Flr John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK. E-mail: [email protected] ISSN 1465-3249 print/ISSN 1477-2566 online © 2012 Informa Healthcare DOI: 10.3109/14653249.2012.698119
774
J. C. Riches & J. G. Gribben
NK cells can also exert impressive anti-tumor effects, and large-scale NK cell expansion protocols are already in clinical use. It has been demonstrated previously that NK cell cytotoxicity against acute lymphoblastic leukemia (ALL) cells can be markedly enhanced by retroviral transduction of an anti-CD19 chimeric signaling receptor, and that this could be further augmented by the addition of the co-stimulatory molecule CD137 (15). The results presented demonstrate that this technique is feasible, leads to increased NK activity, and has demonstrable anti-leukemic activity in xenograft models. However, receptor expression was transient, and in the murine studies persisted for only 4 days, and therefore the results may not be sufficient to support the authors’ statement that ‘this should have durable effects in vivo’. However, therapeutic approaches using CAR NK cells present a viable alternative to CAR T cells, with the results of clinical studies using this approach being awaited with interest. In particular, T-cell depleted allogeneic CAR NK cells may have a role in lymphopenic patients, where the number of functional autologous T cells is compromised by previous chemotherapy and/or resistant disease. Further studies are required to determine the role of conditioning prior to infusion of these cells, and in what clinical scenarios this should occur. This is all part of a fascinating area of active ongoing clinical translational research, which has the potential to offer massive benefits to patients, so long as the price is right! Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References 1. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011;365:725–33. 2. Shimasaki N, Fujisaki H, Cho D, Masselli M, Lockey T, Eldridge P, et al. A clinically adaptable method to enhance the cytotoxicity of natural killer cells against B-cell malignancies. Cytotherapy. 2012;14:830–840.
3. June CH, Blazar BR, Riley JL. Engineering lymphocyte subsets: tools, trials and tribulations. Nat Rev Immunol. 2009;9:704–16. 4. Cartellieri M, Bachmann M, Feldmann A, Bippes C, Stamova S, Wehner R, et al. Chimeric antigen receptor-engineered T cells for immunotherapy of cancer. J Biomed Biotechnol. 2010:956304. 5. Koehler P, Schmidt P, Hombach AA, Hallek M, Abken H. Engineered T cells for the adoptive therapy of B-cell chronic lymphocytic leukaemia. Adv Hematol. 2012:595060. 6. Dazzi F, D’Andrea E, Biasi G, De Silvestro G, Gaidano G, Schena M, et al. Failure of B cells of chronic lymphocytic leukemia in presenting soluble and alloantigens. Clin Immunol Immunopathol. 1995;75:26–32. 7. Maher J, Brentjens RJ, Gunset G, Riviere I, Sadelain M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta/CD28 receptor. Nat Biotechnol. 2002;20:70–5. 8. Kochenderfer JN, Wilson WH, Janik JE, Dudley ME, StetlerStevenson M, Feldman SA, et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically-engineered to recognize CD19. Blood. 2010;116:4099–102. 9. Kochenderfer JN, Dudley ME, Stetler-Stevenson M, Wilson WH, Janik JE, Nathan DA, et al. A phase I clinical trial of treatment of B-cell malignancies with autologous anti-CD19CAR-transduced T cells. Blood. 2010;116:Abstract 2865. 10. Brentjens R, Yeh R, Bernal Y, Riviere I, Sadelain M. Treatment of chronic lymphocytic leukemia with genetically targeted autologous T cells: case report of an unforeseen adverse event in a phase I clinical trial. Mol Ther. 2010;18:666–8. 11. Brentjens RJ, Riviere I, Park JH, Davila ML, Wang X, Stefanski J, et al. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood. 2011;118: 4817–28. 12. Beyer M, Kochanek M, Darabi K, Popov A, Jensen M, Endl E, et al. Reduced frequencies and suppressive function of CD4 ⫹ CD25hi regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine. Blood. 2005;106:2018–25. 13. Giannopoulos K, Schmitt M, Kowal M, Wlasiuk P, BojarskaJunak A, Chen J, et al. Characterization of regulatory T cells in patients with B-cell chronic lymphocytic leukemia. Oncol Rep. 2008;20:677–82. 14. Jak M, Mous R, Remmerswaal EBM, Spijker R, Jaspers A, Yague A, et al. Enhanced formation and survival of CD4 ⫹ CD25hi Foxp3 ⫹ T-cells in chronic lymphocytic leukemia. Leuk Lymph. 2009;50:788–801. 15. Imai C, Iwamoto S, Campana D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood. 2005;106: 376–83.
COMMENTARY
Less expensive CARs?
JOHN C. RICHES & JOHN G. GRIBBEN Barts Cancer Institute, Queen Mary University of London, London, UK Key Words: Immunotherapy, gene therepay, natural killer cells
Immunotherapy has the potential to have a major impact on cancer treatment, especially by targeting drug-resistant tumor subclones. The B-cell malignancies are good models for testing novel immunotherapeutic approaches, and there has been recent excitement about the apparent efficacy of chimeric antigen receptor (CAR) T cells in the treatment of chronic lymphocytic leukemia (CLL) (1). To be applicable clinically, cell-based therapies need to be translated from the laboratory into practical good manufacturing practice (GMP)-compliant methods. The excitement surrounding these approaches is dampened somewhat by their high expense, and the need for very specialized facilities to produce cells that have been retrovirally transduced to GMP requirements. In this issue, Shimasaki et al. (2) demonstrate that they are able to electroporate human natural killer (NK) cells with a receptor encoding anti-CD19 with CD3ζ and CD137 (4 - 1BB) using a GMP-compliant electroporator. They were able to achieve anti-CD19BB-ζ receptor expression of 40% in freshly purified and more than 60% in expanded NK cells, levels of expression comparable to those achieved with retroviral transduction, even after scale up to clinicalgrade material. The NK cells expressing the chimeric receptor had increased cytotoxicity, as demonstrated in a xenograft model of B-cell leukemia. Most studies to date on the use of CAR have been with T cells (3). A major advantage of this approach is that it eliminates the need for major histocompatibility complex (MHC) restriction, thus enabling the same CAR to be used for different patients. The use of an antibody receptor means that potential targets can be increased to include a wide range of surface proteins, sugars and lipids (4). A number of phase I/II clinical trials are currently underway using anti-CD19
CAR T cells for the treatment of B-cell malignancies (5). A key finding in pre-clinical studies has been that the addition of a co-stimulatory domain, such as CD28 or CD137, significantly improves the efficacy of these CAR T cells (6,7). A clinical trial with anti-CD19 CAR T cells in a patient with advanced follicular lymphoma resulted in regression of lymphadenopathy, associated with B lymphopenia and hypogammaglobulinemia. However, the CAR T cells did not persist long-term, with the anti-CD19 CAR becoming undetectable at 27 weeks, followed by the development of progressive disease 5 weeks later (8). Subsequent studies of anti-CD19 CAR T cells in CLL following conditioning with fludarabine, cyclophosphamide and high-dose interleukin (IL)-2 have demonstrated impressive clinical results (9). The importance of conditioning has also been shown by other groups, with no objective responses seen in patients who did not receive conditioning, in contrast to disease stabilization or lymph node responses in three out of four of the patients who had had cyclophosphamide as part of the trial design. However, a further patient in this study rapidly developed complications and died within 48 h of an infusion of a higher dose of CAR T cells, highlighting the risks associated with this therapy (10,11). Although this work is still in the early stages, it has underscored the importance of the conditioning regimen in promoting T-cell engraftment and activation, analogous to the impact of conditioning in allogeneic transplantation. In particular, it may be vital to eliminate regulatory T cells, which are known to be expanded in CLL and can be suppressed with fludarabine treatment (12–14). It may also be important to eliminate other subpopulations, such as immature dendritic cells, as well as cell populations that act as ‘cytokine sinks’ by competing for the same survival and stimulatory factors (5).
Correspondence: Professor John G. Gribben MD DSc FMedSci, Chair of Medical Oncology, Barts Cancer Institute, Queen Mary University of London, 3rd Flr John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK. E-mail: [email protected] ISSN 1465-3249 print/ISSN 1477-2566 online © 2012 Informa Healthcare DOI: 10.3109/14653249.2012.698119
774
J. C. Riches & J. G. Gribben
NK cells can also exert impressive anti-tumor effects, and large-scale NK cell expansion protocols are already in clinical use. It has been demonstrated previously that NK cell cytotoxicity against acute lymphoblastic leukemia (ALL) cells can be markedly enhanced by retroviral transduction of an anti-CD19 chimeric signaling receptor, and that this could be further augmented by the addition of the co-stimulatory molecule CD137 (15). The results presented demonstrate that this technique is feasible, leads to increased NK activity, and has demonstrable anti-leukemic activity in xenograft models. However, receptor expression was transient, and in the murine studies persisted for only 4 days, and therefore the results may not be sufficient to support the authors’ statement that ‘this should have durable effects in vivo’. However, therapeutic approaches using CAR NK cells present a viable alternative to CAR T cells, with the results of clinical studies using this approach being awaited with interest. In particular, T-cell depleted allogeneic CAR NK cells may have a role in lymphopenic patients, where the number of functional autologous T cells is compromised by previous chemotherapy and/or resistant disease. Further studies are required to determine the role of conditioning prior to infusion of these cells, and in what clinical scenarios this should occur. This is all part of a fascinating area of active ongoing clinical translational research, which has the potential to offer massive benefits to patients, so long as the price is right! Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References 1. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011;365:725–33. 2. Shimasaki N, Fujisaki H, Cho D, Masselli M, Lockey T, Eldridge P, et al. A clinically adaptable method to enhance the cytotoxicity of natural killer cells against B-cell malignancies. Cytotherapy. 2012;14:830–840.
3. June CH, Blazar BR, Riley JL. Engineering lymphocyte subsets: tools, trials and tribulations. Nat Rev Immunol. 2009;9:704–16. 4. Cartellieri M, Bachmann M, Feldmann A, Bippes C, Stamova S, Wehner R, et al. Chimeric antigen receptor-engineered T cells for immunotherapy of cancer. J Biomed Biotechnol. 2010:956304. 5. Koehler P, Schmidt P, Hombach AA, Hallek M, Abken H. Engineered T cells for the adoptive therapy of B-cell chronic lymphocytic leukaemia. Adv Hematol. 2012:595060. 6. Dazzi F, D’Andrea E, Biasi G, De Silvestro G, Gaidano G, Schena M, et al. Failure of B cells of chronic lymphocytic leukemia in presenting soluble and alloantigens. Clin Immunol Immunopathol. 1995;75:26–32. 7. Maher J, Brentjens RJ, Gunset G, Riviere I, Sadelain M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta/CD28 receptor. Nat Biotechnol. 2002;20:70–5. 8. Kochenderfer JN, Wilson WH, Janik JE, Dudley ME, StetlerStevenson M, Feldman SA, et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically-engineered to recognize CD19. Blood. 2010;116:4099–102. 9. Kochenderfer JN, Dudley ME, Stetler-Stevenson M, Wilson WH, Janik JE, Nathan DA, et al. A phase I clinical trial of treatment of B-cell malignancies with autologous anti-CD19CAR-transduced T cells. Blood. 2010;116:Abstract 2865. 10. Brentjens R, Yeh R, Bernal Y, Riviere I, Sadelain M. Treatment of chronic lymphocytic leukemia with genetically targeted autologous T cells: case report of an unforeseen adverse event in a phase I clinical trial. Mol Ther. 2010;18:666–8. 11. Brentjens RJ, Riviere I, Park JH, Davila ML, Wang X, Stefanski J, et al. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood. 2011;118: 4817–28. 12. Beyer M, Kochanek M, Darabi K, Popov A, Jensen M, Endl E, et al. Reduced frequencies and suppressive function of CD4 ⫹ CD25hi regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine. Blood. 2005;106:2018–25. 13. Giannopoulos K, Schmitt M, Kowal M, Wlasiuk P, BojarskaJunak A, Chen J, et al. Characterization of regulatory T cells in patients with B-cell chronic lymphocytic leukemia. Oncol Rep. 2008;20:677–82. 14. Jak M, Mous R, Remmerswaal EBM, Spijker R, Jaspers A, Yague A, et al. Enhanced formation and survival of CD4 ⫹ CD25hi Foxp3 ⫹ T-cells in chronic lymphocytic leukemia. Leuk Lymph. 2009;50:788–801. 15. Imai C, Iwamoto S, Campana D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood. 2005;106: 376–83.