Cancer Immunology, Immunotherapy

, Volume 67, Issue 7, pp 1053–1066 | Cite as

Induction of a central memory and stem cell memory phenotype in functionally active CD4+ and CD8+ CAR T cells produced in an automated good manufacturing practice system for the treatment of CD19+ acute lymphoblastic leukemia

  • Franziska Blaeschke
  • Dana Stenger
  • Theresa Kaeuferle
  • Semjon Willier
  • Ramin Lotfi
  • Andrew Didier Kaiser
  • Mario Assenmacher
  • Michaela Döring
  • Judith Feucht
  • Tobias Feuchtinger
Original Article


Relapsed/refractory B-precursor acute lymphoblastic leukemia (pre-B ALL) remains a major therapeutic challenge. Chimeric antigen receptor (CAR) T cells are promising treatment options. Central memory T cells (Tcm) and stem cell-like memory T cells (Tscm) are known to promote sustained proliferation and persistence after T-cell therapy, constituting essential preconditions for treatment efficacy. Therefore, we set up a protocol for anti-CD19 CAR T-cell generation aiming at high Tcm/Tscm numbers. 100 ml peripheral blood from pediatric pre-B ALL patients was processed including CD4+/CD8+-separation, T-cell activation with modified anti-CD3/-CD28 reagents and transduction with a 4-1BB-based second generation CAR lentiviral vector. The process was performed on a closed, automated device requiring additional manual/open steps under clean room conditions. The clinical situation of these critically ill and refractory patients with leukemia leads to inconsistent cellular compositions at start of the procedure including high blast counts and low T-cell numbers with exhausted phenotype. Nevertheless, a robust T-cell product was achieved (mean CD4+ = 50%, CD8+ = 39%, transduction = 27%, Tcm = 50%, Tscm = 46%). Strong proliferative potential (up to > 100-fold), specific cytotoxicity and low expression of co-inhibitory molecules were documented. CAR T cells significantly released TH1 cytokines IFN-γ, TNF-α and IL-2 upon target-recognition. In conclusion, partly automated GMP-generation of CAR T cells from critically small blood samples was feasible with a new stimulation protocol that leads to high functionality and expansion potential, balanced CD4/CD8 ratios and a conversion to a Tcm/Tscm phenotype.


CAR T cells GMP production Tscm/cm Pediatric ALL 









B-cell non-Hodgkin’s lymphoma


Common acute lymphoblastic leukemia


Central nervous system




Fluorescence minus one


High-dose cytarabine


Human serum albumin


Multiplicity of infection




Peripheral blood


Pegylated asparaginase


B-precursor acute lymphoblastic leukemia


Central memory T cells


T-cell transduction


Effector T cells


Effector memory T cells


Stem cell-like memory T cells


Vector copy number







The authors thank all patients and their parents for participating in the study. Nadine Stoll, Tanja Weisser, Nicola Habjan, Florian Jurgeleit, Carola Barth and Daniela Mauer are acknowledged for excellent technical assistance. The authors thank Katharina Drechsel and Nadine Mockel-Tenbrinck for helpful advice.

Author contributions

Experiments were designed by TF, FB and ADK; the automated process was developed by ADK and MA; patient samples were provided by TF, SW and MD; experiments were performed by FB, DS and TK; JF set up experiments and provided protocols; RL provided healthy donor starting fractions and human serum. Data analysis was done by FB, DS and TF; the manuscript was written by FB and TF and was reviewed by all co-authors.


This work was supported by Elterninitiative Ebersberg, Elterninitative Intern3 and Bettina Braeu Stiftung, Adler Stiftung and the Care for Rare Foundation. Miltenyi Biotec provided reagents free of charge.

Compliance with ethical standards

Conflict of interest

Andrew Didier Kaiser and Mario Assenmacher are employees of Miltenyi Biotec. This work has been performed as a collaboration between Tobias Feuchtinger, Franziska Blaeschke and Miltenyi Biotec. Miltenyi Biotec provided reagent free of charge. All other authors declare that they have no conflict of interest.

Ethical approval and ethical standards

This study was approved by the Institutional Ethical Review Board (“Ethikkommission bei der LMU München”), approval number 435 − 15, and was performed in accordance with the Declaration of Helsinki.

Informed consent

Patients/their representatives gave written informed consent according to the guidelines and approval of the Institutional Ethical Review Board.

Cell line authentication

Cell lines Raji, Jeko, Molm-13, U-266 were routinely tested for identity by short-tandem repeat analyses (DSMZ, Braunschweig, Germany).

Supplementary material

262_2018_2155_MOESM1_ESM.pdf (492 kb)
Supplementary material 1 (PDF 491 KB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell TransplantationDr. von Hauner University Children’s Hospital, Ludwig Maximilian University MunichMunichGermany
  2. 2.Institute for Transfusion MedicineUniversity Hospital UlmUlmGermany
  3. 3.Institute for Clinical Transfusion Medicine and Immunogenetics UlmGerman Red Cross Blood Services Baden-Württemberg-HessenUlmGermany
  4. 4.Miltenyi BiotecBergisch GladbachGermany
  5. 5.Department I - General Pediatrics, Hematology/OncologyUniversity Hospital Tübingen, Children’s HospitalTübingenGermany
  6. 6.Memorial Sloan Kettering Cancer Center, Center for Cell EngineeringNew YorkUSA

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