Platforms for Clinical-Grade CAR-T Cell Expansion

  • Amanda MizukamiEmail author
  • Kamilla Swiech
Part of the Methods in Molecular Biology book series (MIMB, volume 2086)


Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the immunotherapy field with high rate complete responses especially for hematological diseases. Despite the diversity of tumor specific-antigens, the manufacturing process is consistent and involves multiple steps, including selection of T cells, activation, genetic modification, and in vitro expansion. Among these complex manufacturing phases, the choice of culture system to generate a high number of functional cells needs to be evaluated and optimized. Flasks, bags, and rocking motion bioreactor are the most used platforms for CAR-T cell expansion in the current clinical trials but are far from being standardized. New processing options are available and a systematic effort seeking automation, standardization and the increase of production scale, would certainly help to bring the costs down and ultimately democratize this personalized therapy. In this review, we describe different cell expansion platforms available as well as the quality control requirements for clinical-grade production.

Key words

Chimeric antigen receptor CAR-T cells Manufacturing Culture systems Bioreactors Quality control 



This work was financially supported by FAPESP (2016/19741-9), CTC Center for Cell-based Therapies (FAPESP 2013/08135-2) and National Institute of Science and Technology in Stem Cell and Cell Therapy (CNPq 573754-2008-0 and FAPESP 2008/578773). The authors also acknowledge financial support from Secretaria Executiva do Ministério da Saúde (SE/MS), Departamento de Economia da Saúde, Investimentos e Desenvolvimento (DESID/SE), Programa Nacional de Apoio à Atenção Oncológica (PRONON) Process 25000.189625/2016-16.


  1. 1.
    Geyer MB, Brentjens RJ (2016) Current clinical applications of chimeric antigen receptor (CAR) modified T cells. Cytotherapy 18:1393–1409CrossRefGoogle Scholar
  2. 2.
    Jürgens B, Clarke NS (2019) Evolution of CAR T-cell immunotherapy in terms of patenting activity. Nat Biotechnol 37(4):370–375. Scholar
  3. 3.
    Levine BL, Miskin J, Wonnacott K, Keir C (2017) Global manufacturing of CAR T cell therapy. Mol Ther 4:92–101Google Scholar
  4. 4.
    Calmels B, Mfarrej B, Chabannon C (2018) From clinical proof-of-concept to commercialization of CAR T cells. Drug Discov Today 23:758–762CrossRefGoogle Scholar
  5. 5.
    Piscopo NJ, Mueller KP, Das A et al (2018) Bioengineering solutions for manufacturing challenges in CAR T cells. Biotechnol J 13(2):1–21CrossRefGoogle Scholar
  6. 6.
    Vormittag P, Gunn R, Ghorashian S et al (2018) A guide to manufacturing CAR T cell therapies. Curr Opin Biotechnol 53:164–181CrossRefGoogle Scholar
  7. 7.
    Iyer RK, Bowles PA, Kim H et al (2018) Industrializing autologous adoptive immunotherapies: manufacturing advances and challenges. Front Med 5:150CrossRefGoogle Scholar
  8. 8.
    Jenkins MJ, Farid SS (2018) Cost-effective bioprocess design for the manufacture of allogeneic CAR-T cell therapies using a decisional tool with multi-attribute decision-making analysis. Biochem Eng J 137:192–204CrossRefGoogle Scholar
  9. 9.
    Fekete N, Béland AV, Campbell K et al (2018) Bags versus flasks: a comparison of cell culture systems for the production of dendritic cell–based immunotherapies. Transfusion 58:1800–1813CrossRefGoogle Scholar
  10. 10.
    Lamers CHJ, van Elzakker P, van Steenbergen SCL et al (2008) Retronectin®-assisted retroviral transduction of primary human T lymphocytes under good manufacturing practice conditions: tissue culture bag critically determines cell yield. Cytotherapy 10(4):406–416CrossRefGoogle Scholar
  11. 11.
    Till BG, Jensen MC, Wang J et al (2008) Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells. Blood 112(6):2261–2271CrossRefGoogle Scholar
  12. 12.
    Zuliani T, David J, Bercegeay S et al (2011) Value of large scale expansion of tumor infiltrating lymphocytes in a compartmentalised gas-permeable bag: interests for adoptive immunotherapy. J Transl Med 9:63CrossRefGoogle Scholar
  13. 13.
    Tumaini B, Lee DW, Lin T et al (2013) Simplified process for the production of anti-CD19-CAR-engineered T cells. Cytotherapy 15(11):1406–1415CrossRefGoogle Scholar
  14. 14.
    Wang X, Rivière I (2016) Clinical manufacturing of CAR T cells: foundation of a promising therapy. Mol Ther Oncolytics 3:16015CrossRefGoogle Scholar
  15. 15.
    Bajgain P, Mucharla R, Wilson J et al (2014) Optimizing the production of suspension cells using the G-Rex M series. Mol Ther Methods Clin Dev 1:14015CrossRefGoogle Scholar
  16. 16.
    Vera JF, Brenner LJ, Gerdemann U et al (2010) Accelerated production of antigen-specific T cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (G-Rex). J Immunother 33(3):305–315CrossRefGoogle Scholar
  17. 17.
    Lapteva N, Parihar R, Rollins LA et al (2016) Large-scale culture and genetic modification of human natural killer cells for cellular therapy. Methods Mol Biol 1441:195–202CrossRefGoogle Scholar
  18. 18.
    Forget MA, Haymaker C, Dennison JB et al (2016) The beneficial effects of a gas-permeable flask for expansion of tumor-infiltrating lymphocytes as reflected in their mitochondrial function and respiration capacity. Oncoimmunology 5(2):e1057386CrossRefGoogle Scholar
  19. 19.
    Chakraborty R, Mahendravada A, Perna SK et al (2013) Robust and cost-effective expansion of human regulatory T cells highly functional in a xenograft model of graft-versus-host disease. Haematologica 98(4):533–537CrossRefGoogle Scholar
  20. 20.
    Nakazawa Y, Huye LE, Salsman VS et al (2011) PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor. Mol Ther 19(12):2133–2143CrossRefGoogle Scholar
  21. 21.
    Davis BM, Loghin ER, Conway KR et al (2018) Automated closed-system expansion of pluripotent stem cell aggregates in a rocking-motion bioreactor. SLAS Technol 23(4):364–373PubMedPubMedCentralGoogle Scholar
  22. 22.
    Sadeghi A, Pauler L, Annerén C et al (2011) Large-scale bioreactor expansion of tumor-infiltrating lymphocytes. J Immunol Methods 364(1–2):94–100CrossRefGoogle Scholar
  23. 23.
    Hollyman D, Stefanski J, Przybylowski M et al (2009) Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy. J Immunother 32(2):169–180CrossRefGoogle Scholar
  24. 24.
    Brentjens RJ, Rivière I, Park JH et al (2011) Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood 118(18):4817–4828CrossRefGoogle Scholar
  25. 25.
    Davila ML, Riviere I, Wang X et al (2014) Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med 6(224):224ra25CrossRefGoogle Scholar
  26. 26.
    Brentjens RJ, Davila ML, Riviere I et al (2013) CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med 5(177):177ra38CrossRefGoogle Scholar
  27. 27.
    Mock U, Nickolay L, Philip B et al (2016) Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS Prodigy. Cytotherapy 18(8):1002–1011CrossRefGoogle Scholar
  28. 28.
    Fesnak AD, Hanley PJ, Levine BL (2017) Considerations in T cell therapy product development for B cell Leukemia and lymphoma immunotherapy. Curr Hematol Malig Rep 12:335–343CrossRefGoogle Scholar
  29. 29.
    Lock D, Mockel-Tenbrinck N, Drechsel K et al (2017) Automated manufacturing of potent CD20-directed CAR T cells for clinical use. Hum Gene Ther 28(10):914–925CrossRefGoogle Scholar
  30. 30.
    Zhang W, Jordan KR, Schulte B et al (2018) Characterization of clinical grade CD19 chimeric antigen receptor T cells produced using automated CliniMACS Prodigy system. Drug Des Devel Ther 12:3343–3356CrossRefGoogle Scholar
  31. 31.
    Kaiser A (2015) Method for automated generation of genetically modified T cells. CA 2946222, 29 Oct 2015Google Scholar
  32. 32.
  33. 33.
    Köhl U, Arsenieva S, Holzinger A et al (2018) CAR T cells in trials: recent achievements and challenges that remain in the production of modified T cells for clinical applications. Hum Gene Ther 29(5):559–568CrossRefGoogle Scholar
  34. 34.
    Shi Y (2019) End-to-end cell therapy automation. US patent WO/2019/046766, 7 Mar 2019Google Scholar
  35. 35.
    Kaiser AD, Assenmacher M, Schröder B et al (2015) Towards a commercial process for the manufacture of genetically modified T cells for therapy. Cancer Gene Ther 22:72–78CrossRefGoogle Scholar
  36. 36.
    Xu Y, Zhang M, Ramos CA et al (2014) 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 123(24):3750–3759CrossRefGoogle Scholar
  37. 37.
    Jensen MC, Riddell SR (2014) Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells. Immunol Rev 257(1):127–144CrossRefGoogle Scholar
  38. 38.
    Ramanayake S, Bilmon I, Bishop D et al (2015) Low-cost generation of good manufacturing practice-grade CD19-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials. Cytotherapy 17(9):1251–1267CrossRefGoogle Scholar
  39. 39.
    Siegler EL, Wang P (2018) Preclinical models in chimeric antigen receptor–engineered T-cell therapy. Hum Gene Ther 29(5):534–546CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Center for Cell-Based Therapy CTC, Regional Blood Center of Ribeirão PretoUniversity of São PauloSão PauloBrazil
  2. 2.School of Pharmaceutical Sciences of Ribeirão PretoUniversity of São PauloRibeirão Preto, São PauloBrazil

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