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High CAR intensity of expression confers enhanced antitumor effect against lymphoma without functional exhaustion

Cancer Gene Therapy volume 30, pages 51–61 (2023)Cite this article

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A Correction to this article was published on 02 December 2022

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Abstract

Identifying factors that ameliorates clinical outcomes following CART therapy represents an unmet need. We hypothesized that CAR expression level would have a significant impact on CART efficacy and tested this with CAR30+ TSCM-LIKE enriched cells. By sorting T-cells according to CAR mean fluorescence intensity in two markedly different populations (CARHI and CARLO), we showed that a high CAR expression enhances antitumor efficacy in vitro, that is sustained after sequential re-exposures to tumor cells and is not associated with T-cell exhaustion or differentiation. Furthermore, we found a correlation between high surface CAR expression and antitumor effect with CAR19+ T-cells, thus validating our findings with CAR30. Definitive proof of CARHI T-cells improved antitumor efficacy was demonstrated in a human Hodgkin’s lymphoma xenograft mouse model, where CAR30-TSCM-LIKE enriched products with high intensity of CAR expression achieved superior tumor control in vivo and longer survival than those with a low intensity of CAR expression. Our data suggest that modulation of CAR intensity of expression represents an additional strategy to increase CART therapy clinical efficacy.

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Fig. 1: CAR30+ T-cells were sorted according to CAR expression intensity.
Fig. 2: CAR30HI showed a larger proportion of T-cells expressing activation and inhibition markers.
Fig. 3: CAR30HI demonstrated superior specific cytotoxic activity compared to CAR30LO.
Fig. 4: CAR30HI displayed enhanced cytotoxicity after in vitro consecutive re-exposure to tumor cells without functional exhaustion.
Fig. 5: CAR30HI enhanced antitumor effect was accompanied by greater proliferative capacity without further differentiation.
Fig. 6: CAR19HI outperformed CAR19LO in antitumor activity against B-cell lymphoma, confirming that intensity of CAR expression influences the tumour-killing capacity.
Fig. 7: High intensity of CAR expression enhances tumor control in vivo and improves survival.

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The main data supporting the results in this study are available within the paper and its Supplementary Information. All data generated in this study are available from the corresponding authors on reasonable request.

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References

  1. Brentjens RJ, Davila ML, Riviere I, Park J, Wang X, Cowell LG, et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med. 2013;5:177ra38 LP–177ra38.

    Article  Google Scholar 

  2. Grover NS, Savoldo B. Challenges of driving CD30-directed CAR-T cells to the clinic. BMC Cancer. 2019;19:1–8.

    Article  Google Scholar 

  3. Dai H, Wang Y, Lu X, Han W. Chimeric antigen receptors modified T-cells for cancer therapy. J Natl Cancer Inst. 2016;108. https://doi.org/10.1093/jnci/djv439.

  4. Strati P, Neelapu SS. Chimeric antigen receptor–engineered T cell therapy in lymphoma. Curr Oncol Rep. 2019;21. https://doi.org/10.1007/s11912-019-0789-z.

  5. Kochenderfer JN, Dudley ME, Kassim SH, Somerville RPT, Carpenter RO, Maryalice SS, et al. Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol. 2015;33:540–9.

    Article  CAS  Google Scholar 

  6. Neelapu SS, Locke FL, Bartlett NL, Lekakis LJ, Miklos DB, Jacobson CA, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-Cell lymphoma. N. Engl J Med. 2017;377:2531–44.

    Article  CAS  Google Scholar 

  7. Schuster SJ, Bishop MR, Tam CS, Waller EK, Borchmann P, McGuirk JP, et al. Tisagenlecleucel in adult relapsed or refractory diffuse large B-cell lymphoma. N. Engl J Med. 2019;380:45–56.

    Article  CAS  Google Scholar 

  8. Ramos CA, Grover NS, Beaven AW, Lulla PD, Wu MF, Ivanova A, et al. Anti-CD30 CAR-T cell therapy in relapsed and refractory Hodgkin lymphoma. J Clin Oncol. 2020;38:3794–804.

    Article  CAS  Google Scholar 

  9. Wang D, Zeng C, Xu B, Xu JH, Wang J, Jiang LJ, et al. Anti-CD30 chimeric antigen receptor T cell therapy for relapsed/refractory CD30+ lymphoma patients. Blood Cancer J. 2020;10:8–11.

    Article  Google Scholar 

  10. Singh N, Lee YG, Shestova O, Ravikumar P, Hayer KE, Hong SJ, et al. Impaired death receptor signaling in leukemia causes antigen-independent resistance by inducing CAR T-cell dysfunction. Cancer Discov. 2020;10:552–67.

    Article  CAS  Google Scholar 

  11. Fraietta JA, Lacey SF, Orlando EJ, Pruteanu-Malinici I, Gohil M, Lundh S, et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med. 2018;24:563–71.

    Article  CAS  Google Scholar 

  12. Berger C, Jensen MC, Lansdorp PM, Gough M, Elliott C, Riddell SR. Adoptive transfer of effector CD8+ T cells derived from central memory cells establishes persistent T cell memory in primates. J Clin Investig. 2008;118:294–305.

    Article  CAS  Google Scholar 

  13. Xu Y, Zhang M, Ramos CA, Durett A, Liu E, Dakhova O, et al. Closely related T-memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15. Blood. 2014;123:3750–9.

    Article  CAS  Google Scholar 

  14. Deng Q, Han G, Puebla-Osorio N, Ma MCJ, Strati P, Chasen B, et al. Characteristics of anti-CD19 CAR T cell infusion products associated with efficacy and toxicity in patients with large B cell lymphomas. Nat Med. 2020;26:1878–87.

    Article  CAS  Google Scholar 

  15. Gattinoni L, Lugli E, Ji Y, Pos Z, Paulos CM, Quigley MF, et al. A human memory T cell subset with stem cell-like properties. Nat Med. 2011;17:1290–7.

    Article  CAS  Google Scholar 

  16. Biasco L, Izotova N, Rivat C, Ghorashian S, Richardson R, Guvenel A, et al. Clonal expansion of T memory stem cells determines early anti-leukemic responses and long-term CAR T cell persistence in patients. Nat Cancer. 2021;2:629–42.

    Article  CAS  Google Scholar 

  17. Walker AJ, Majzner RG, Zhang L, Wanhainen K, Long AH, Nguyen SM, et al. Tumor antigen and receptor densities regulate efficacy of a chimeric antigen receptor targeting anaplastic lymphoma kinase. Mol Ther. 2017;25:2189–201.

    Article  CAS  Google Scholar 

  18. Majzner RG, Rietberg SP, Sotillo E, Dong R, Vachharajani VT, Labanieh L, et al. Tuning the antigen density requirement for car T-cell activity. Cancer Discov. 2020;10:702–23.

    Article  CAS  Google Scholar 

  19. Brudno JN, Maric I, Hartman SD, Rose JJ, Wang M, Lam N, et al. T cells genetically modified to express an anti–B-Cell maturation antigen chimeric antigen receptor cause remissions of poor-prognosis relapsed multiple myeloma. J Clin Oncol. 2018;36:2267–80.

    Article  CAS  Google Scholar 

  20. Fry TJ, Shah NN, Orentas RJ, Stetler-Stevenson M, Yuan CM, Ramakrishna S, et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nat Med. 2018;24:20–28.

    Article  CAS  Google Scholar 

  21. Alvarez-Fernández C, Escribà-Garcia L, Caballero AC, Escudero-López E, Ujaldón-Miró C, Montserrat-Torres R, et al. Memory stem T cells modified with a redesigned CD30-chimeric antigen receptor show an enhanced antitumor effect in Hodgkin lymphoma. Clin Transl Immunol. 2021;10:1–15.

    Article  Google Scholar 

  22. Nagata S, Ise T, Onda M, Nakamura K, Ho M, Raubitschek A, et al. Cell membrane-specific epitopes on CD30: Potentially superior targets for immunotherapy. 2005 https://www.pnas.org.

  23. Jena B, Maiti S, Huls H, Singh H, Lee DA, Champlin RE, et al. Chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials. PLoS ONE 2013;8. https://doi.org/10.1371/journal.pone.0057838.

  24. Yam P, Jensen M, Akkina R, Anderson J, Villacres MC, Wu J, et al. Ex vivo selection and expansion of cells based on expression of a mutated inosine monophosphate dehydrogenase 2 after HIV vector transduction: effects on lymphocytes, monocytes, and CD34+ stem cells. Mol Ther. 2006;14:236–44.

    Article  CAS  Google Scholar 

  25. Wang X, Chang WC, Wong CLW, Colcher D, Sherman M, Ostberg JR, et al. A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood. 2011;118:1255–63.

    Article  CAS  Google Scholar 

  26. Zola H, MacArdle PJ, Bradford T, Weedon H, Yasui HKY. Preparation and characterization of a chimeric CD19 monoclonal antibody. Immunol Cell Biol. 1991;69: 411–22.

  27. Alvarez-Fernández C, Escribà-Garcia L, Vidal S, Sierra J, Briones J. A short CD3/CD28 costimulation combined with IL-21 enhance the generation of human memory stem T cells for adoptive immunotherapy. J Transl Med. 2016. https://doi.org/10.1186/s12967-016-0973-y.

  28. Cieri N, Camisa B, Cocchiarella F, Forcato M, Oliveira G, Provasi E, et al. IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors. Blood. 2013;121:573–84.

    Article  CAS  Google Scholar 

  29. Lugli E, Gattinoni L, Roberto A, Mavilio D, Price DA, Restifo NP, et al. Identification, isolation and in vitro expansion of human and nonhuman primate T stem cell memory cells. Nat Protoc. 2013;8:33–42.

    Article  CAS  Google Scholar 

  30. Mamonkin M, Rouce RH, Tashiro H, Brenner MK. A T-cell-directed chimeric antigen receptor for the selective treatment of T-cell malignancies. Blood. 2015;126:983–92.

    Article  CAS  Google Scholar 

  31. Pasquini MC, Hu ZH, Curran K, Laetsch T, Locke F, Rouce R, et al. Real-world evidence of tisagenlecleucel for pediatric acute lymphoblastic leukemia and non-Hodgkin lymphoma. Blood Adv. 2020;4. https://doi.org/10.1182/bloodadvances.2020003092.

  32. Roberts ZJ, Better M, Bot A, Roberts MR, Ribas A. Axicabtagene ciloleucel, a first-in-class CAR T cell therapy for aggressive NHL. Leuk Lymphoma. 2018;59:1785–96.

    Article  CAS  Google Scholar 

  33. Locke FL, Ghobadi A, Jacobson CA, Miklos DB, Lekakis LJ, Oluwole OO, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. Lancet Oncol. 2019;20:31–42.

    Article  CAS  Google Scholar 

  34. Ruella M, Maus MV. Catch me if you can: leukemia escape after CD19-directed T cell immunotherapies. Comput. Struct. Biotechnol. J. 2016;14:357–62.

    Article  CAS  Google Scholar 

  35. Watanabe K, Terakura S, Martens AC, van Meerten T, Uchiyama S, Imai M, et al. Target antigen density governs the efficacy of anti–CD20-CD28-CD3 ζ chimeric antigen receptor–modified effector CD8 + T Cells. J Immunol. 2015;194:911–20.

    Article  CAS  Google Scholar 

  36. Caruso HG, Hurton LV, Najjar A, Rushworth D, Ang S, Olivares S, et al. Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue while maintaining potent antitumor activity. Cancer Res. 2015;75:3505–18.

    Article  CAS  Google Scholar 

  37. Xiong W, Chen Y, Kang X, Chen Z, Zheng P, Hsu YH, et al. Immunological synapse predicts effectiveness of chimeric antigen receptor cells. Mol Ther. 2018;26:963–75.

    Article  CAS  Google Scholar 

  38. Davenport AJ, Cross RS, Watson KA, Liao Y, Shi W, Prince HM, et al. Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity. Proc Natl Acad Sci USA. 2018;115:E2068–E2076.

    Article  CAS  Google Scholar 

  39. Gudipati V, Rydzek J, Doel-Perez I, Gonçalves VDR, Scharf L, Königsberger S, et al. Inefficient CAR-proximal signaling blunts antigen sensitivity. Nat Immunol. 2020;21:848–56.

    Article  CAS  Google Scholar 

  40. Caballero AC, Escribà-Garcia L, Montserrat-Torres R, Escudero-López E, Pujol-Fernández P, Ujaldón-Miró C, et al. S257 A phase 1, first-in-human, dose-escalation clinical trial of memory-enriched Cd30-Car T-cell therapy for the treatment of relapsed or refractory hodgkin lymphoma and Cd30+ T-cell lymphoma. 2022 https://journals.lww.com/hemasphere/pages/default.aspx.

  41. Sabins NC, Harman BC, Barone LR, Shen S, Santulli-Marotto S. Differential expression of immune checkpoint modulators on in vitro primed CD4+ and CD8+ T cells. Front Immunol. 2016;7:1–11.

    Article  Google Scholar 

  42. Chen L, Flies DB. Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol. 2013;13:227–42.

    Article  Google Scholar 

  43. Agata Y, Kawasaki A, Nishimura H, Ishida Y, Tsubata T, Yagita H, et al. Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. Int Immunol. 1996;8:765–72.

    Article  CAS  Google Scholar 

  44. Chang ZNL, Silver PA, Chen YY. Identification and selective expansion of functionally superior T cells expressing chimeric antigen receptors. J Transl Med. 2015;13:1–16.

    Article  CAS  Google Scholar 

  45. Nakazawa Y, Huye LE, Salsman VS, Leen AM, Ahmed N, Rollins L, et al. PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor. Mol Ther. 2011;19:2133–43.

    Article  CAS  Google Scholar 

  46. Song DG, Ye Q, Poussin M, Harms GM, Figini M, Powell DJ. CD27 costimulation augments the survival and antitumor activity of redirected human T cells in vivo. Blood. 2012;119:696–706.

    Article  CAS  Google Scholar 

  47. Alcover A, Alarcón B, di Bartolo V. Cell biology of T cell receptor expression and regulation. Annu Rev Immunol. 2018;36:103–25.

    Article  CAS  Google Scholar 

  48. D’Oro U, Munitic I, Chacko G, Karpova T, McNally J, Ashwell JD. Regulation of constitutive TCR internalization by the ζ-chain. J Immunol. 2002;169:6269–78.

    Article  Google Scholar 

  49. Shum T, Omer B, Tashiro H, Kruse RL, Wagner DL, Parikh K, et al. Constitutive signaling from an engineered IL7 receptor promotes durable tumor elimination by tumor-redirected T cells. Cancer Discov. 2017;7:1238–47.

    Article  CAS  Google Scholar 

  50. Abramson JS, Palomba ML, Gordon LI, Lunning MA, Wang M, Arnason J, et al. Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study. Lancet. 2020;396:839–52.

    Article  Google Scholar 

  51. Schuster SJ, Maziarz RT, Rusch ES, Li J, Signorovitch JE, Romanov VV, et al. Grading and management of cytokine release syndrome in patients treated with tisagenlecleucel in the JULIET trial. Blood Adv. 2020;4:1432–9.

    Article  CAS  Google Scholar 

  52. Alabanza L, Pegues M, Geldres C, Shi V, Wiltzius JJW, Sievers SA, et al. Function of novel anti-CD19 chimeric antigen receptors with human variable regions is affected by hinge and transmembrane domains. Mol Ther. 2017;25:2452–65.

    Article  CAS  Google Scholar 

  53. Brudno JN, Lam N, Vanasse D, Shen YW, Rose JJ, Rossi J, et al. Safety and feasibility of anti-CD19 CAR T cells with fully human binding domains in patients with B-cell lymphoma. Nat Med. 2020;26:270–80.

    Article  CAS  Google Scholar 

  54. Wang CM, Wu ZQ, Wang Y, Guo YL, Dai HR, Wang XH, et al. Autologous T cells expressing CD30 chimeric antigen receptors for relapsed or refractory hodgkin lymphoma: an open-label phase i trial. Clin Cancer Res. 2017;23:1156–66.

    Article  CAS  Google Scholar 

  55. Arcangeli S, Bove C, Mezzanotte C, Camisa B, Falcone L, Manfredi F, et al. CAR T cell manufacturing from naive/stem memory T lymphocytes enhances antitumor responses while curtailing cytokine release syndrome. J Clin Investig 2022;132. https://doi.org/10.1172/jci150807.

  56. Li W, Qiu S, Chen J, Jiang S, Chen W, Jiang J, et al. Chimeric antigen receptor designed to prevent ubiquitination and downregulation showed durable antitumor efficacy. Immunity. 2020;53:456–470.e6.

    Article  CAS  Google Scholar 

  57. Frigault MJ, Lee J, Basil MC, Carpenito C, Motohashi S, Scholler J, et al. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells. Cancer Immunol Res. 2015;3:356–67.

    Article  CAS  Google Scholar 

  58. Long AH, Haso WM, Shern JF, Wanhainen KM, Murgai M, Ingaramo M, et al. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med. 2015;21:581–90.

    Article  CAS  Google Scholar 

  59. Kong W, Dimitri A, Wang W, Jung IY, Ott CJ, Fasolino M, et al. BET bromodomain protein inhibition reverses chimeric antigen receptor extinction and reinvigorates exhausted T cells in chronic lymphocytic leukemia. J Clin Investig. 2021;131:1–16.

    Article  CAS  Google Scholar 

  60. Davenport AJ, Jenkins MR, Cross RS, Yong CS, Prince HM, Ritchie DS, et al. CAR-T cells inflict sequential killing of multiple tumor target cells. Cancer Immunol Res. 2015;3:483–94.

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported in part by grants from La Marató TV3 (Exp. 20130710), Deutsche José Carreras Leukämie Stiftung (DJCSL 10R/2016), Beca Carlos Antonio López (Gobierno paraguayo), Instituto de Salud Carlos III (PI15/1383y PI18/01023; Fondos FEDER), Fundación Bancaria ‘La Caixa’, TerCel (SG/11/2008), Ministerio de Economía y Competitividad (RETOS; RTC 2015-3393-1), AGAUR (2017SGR1395) and Red de Terapias Avanzadas (RICORS, ISCIII; RD21/0017/0011; Fondos Next Generation, UE). Graphical abstract was created with BioRender.com support.

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Author notes

  1. These authors contributed equally: Alvarez-Fernández, Carmen, Briones, Javier.

Authors and Affiliations

  1. Hematology Service, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain

    Ana Carolina Caballero, Laura Escribà-Garcia, Paula Pujol-Fernández, Eva Escudero-López, Cristina Ujaldón-Miró, Rosanna Montserrat-Torres, Jorge Sierra, Carmen Alvarez-Fernández & Javier Briones

  2. Laboratory of Experimental Hematology-IIB, Institut Recerca Hospital de la Santa Creu i Sant Pau, Barcelona, Spain

    Ana Carolina Caballero, Laura Escribà-Garcia, Paula Pujol-Fernández, Eva Escudero-López, Cristina Ujaldón-Miró, Rosanna Montserrat-Torres, Carmen Alvarez-Fernández & Javier Briones

  3. Josep Carreras Leukemia Research Institute, Barcelona, Spain

    Ana Carolina Caballero, Laura Escribà-Garcia, Paula Pujol-Fernández, Eva Escudero-López, Cristina Ujaldón-Miró, Rosanna Montserrat-Torres, Jorge Sierra, Carmen Alvarez-Fernández & Javier Briones

  4. Department of Medicine, Autonomous University of Barcelona, Barcelona, Spain

    Ana Carolina Caballero

  5. Autonomous University of Barcelona, Barcelona, Spain

    Eva Escudero-López, Cristina Ujaldón-Miró, Jorge Sierra & Javier Briones

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Contributions

A.C.C., C.A.F., L.E.G., P.P.F., E.E.L., C.U.M., and R.M.T. conducted the experiments. A.C.C, C.A.F., and J.B. conceived study. A.C.C, C.A.F., and J.B. designed the experiments. A.C.C., L.E.G., C.A.F., and J.B. wrote the paper. J.S. and J.B. supplied essential samples or reagents. All authors reviewed and approved the final version of the manuscript.

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Correspondence to Carmen Alvarez-Fernández or Javier Briones.

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All experiments were performed in accordance with a protocol approved by Ethics Committee of Hospital de la Santa Creu i Sant Pau.

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Caballero, A.C., Escribà-Garcia, L., Pujol-Fernández, P. et al. High CAR intensity of expression confers enhanced antitumor effect against lymphoma without functional exhaustion. Cancer Gene Ther 30, 51–61 (2023). https://doi.org/10.1038/s41417-022-00518-6

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