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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

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Abstract

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.

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Abbreviations

6-MP:

6-Mercaptopurine

6-TG:

6-Thioguanine

ARA-C:

Cytarabine

B-NHL:

B-cell non-Hodgkin’s lymphoma

cALL:

Common acute lymphoblastic leukemia

CNS:

Central nervous system

DNR:

Daunorubicin

FMO:

Fluorescence minus one

HIDAC:

High-dose cytarabine

HSA:

Human serum albumin

MOI:

Multiplicity of infection

MTX:

Methotrexate

PB:

Peripheral blood

PEG-ASP:

Pegylated asparaginase

Pre-B ALL:

B-precursor acute lymphoblastic leukemia

Tcm:

Central memory T cells

TCT:

T-cell transduction

Teff:

Effector T cells

Tem:

Effector memory T cells

Tscm:

Stem cell-like memory T cells

VCN:

Vector copy number

VCR:

Vincristine

VP16:

Etoposide

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Acknowledgements

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.

Funding

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.

Author information

Authors and Affiliations

  1. Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Dr. von Hauner University Children’s Hospital, Ludwig Maximilian University Munich, Lindwurmstrasse 4, 80337, Munich, Germany

    Franziska Blaeschke, Dana Stenger, Theresa Kaeuferle, Semjon Willier, Michaela Döring & Tobias Feuchtinger

  2. Institute for Transfusion Medicine, University Hospital Ulm, Ulm, Germany

    Ramin Lotfi

  3. Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Services Baden-Württemberg-Hessen, Ulm, Germany

    Ramin Lotfi

  4. Miltenyi Biotec, Bergisch Gladbach, Germany

    Andrew Didier Kaiser & Mario Assenmacher

  5. Department I - General Pediatrics, Hematology/Oncology, University Hospital Tübingen, Children’s Hospital, Tübingen, Germany

    Michaela Döring & Judith Feucht

  6. Memorial Sloan Kettering Cancer Center, Center for Cell Engineering, New York, USA

    Judith Feucht

Authors
  1. Franziska Blaeschke
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  2. Dana Stenger
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  3. Theresa Kaeuferle
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  4. Semjon Willier
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  5. Ramin Lotfi
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  6. Andrew Didier Kaiser
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  8. Michaela Döring
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  9. Judith Feucht
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  10. Tobias Feuchtinger
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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.

Corresponding author

Correspondence to Tobias Feuchtinger.

Ethics declarations

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).

Additional information

Poster presentation at 58th ASH Annual Meeting and Exposition (American Society of Hematology), December 3–6 2016, San Diego, USA [1].

Poster presentation at Cellular Therapy, International Symposium Erlangen, March 16–17 2017, Erlangen, Germany [2].

Oral presentation at 43rd Annual Meeting of the European Society for Blood and Marrow Transplantation (EBMT), March 26–29 2017, Marseille, France [3].

Oral presentation at Annual Meeting of Paediatrische Arbeitsgemeinschaft für Stammzelltransplantation and Zelltherapie (PAS&ZT), September 14–15 2017, Hamburg, Germany [4].

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Blaeschke, F., Stenger, D., Kaeuferle, T. et al. 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. Cancer Immunol Immunother 67, 1053–1066 (2018). https://doi.org/10.1007/s00262-018-2155-7

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  • DOI: https://doi.org/10.1007/s00262-018-2155-7

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