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

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.

Keywords

CAR T cells GMP production Tscm/cm Pediatric ALL 

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

Notes

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.

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.

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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