A simple and effective method to purify and activate T cells for successful generation of chimeric antigen receptor T (CAR-T) cells from patients with high monocyte count

J Transl Med. 2022 Dec 19;20(1):608. doi: 10.1186/s12967-022-03833-6.


Background: Chimeric antigen receptor T (CAR-T) cells are genetically modified T cells with redirected specificity and potent T-cell-mediated cytotoxicity toward malignant cells. Despite several CAR-T products being approved and commercialized in the USA, Europe, and China, CAR-T products still require additional optimization to ensure reproducible and cost-effective manufacture. Here, we investigated the critical parameters in the CD3+ T-cell isolation process that significantly impacted CAR-T manufacturing's success.

Methods: CAR-T cells were prepared from cryopreserved peripheral blood mononuclear cells (PBMC). The thawed PBMC was rested overnight before the CD3+ T cell isolation process using CTS Dynabeads CD3/CD28. Different isolation media, cell-bead co-incubation time, and cell density were examined in this study. Activated CD3+ T cells were transduced with a gamma retroviral vector carrying the CD19 or BCMA CAR sequence. The CAR-T cells proliferated in a culture medium supplemented with interleukin 2 (IL-2).

Results: CD14+ monocytes hindered T-cell isolation when X-VIVO 15 basic medium was used as the selection buffer. The activation of T cells was blocked because monocytes actively engulfed CD3/28 beads. In contrast, when DPBS was the selection medium, the T-cell isolation and activation were no longer blocked, even in patients whose PBMC contained abnormally high CD14+ monocytes and a low level of CD3+ T cells.

Conclusions: In this study, we discovered that selecting CD3+ T-cell isolation media is critical for improving T-cell activation, transduction, and CAR-T proliferation. Using DPBS as a CD3+ T cell isolation buffer significantly improved the success rate and shortened the duration of CAR-T production. The optimized process has been successfully applied in our ongoing clinical trials. Trial registration NCT03798509: Human CD19 Targeted T Cells Injection Therapy for Relapsed and Refractory CD19-positive Leukemia. Date of registration: January 10, 2019. NCT03720457: Human CD19 Targeted T Cells Injection (CD19 CAR-T) Therapy for Relapsed and Refractory CD19-positive Lymphoma. Date of registration: October 25, 2018. NCT04003168: Human BCMA Targeted T Cells Injection Therapy for BCMA-positive Relapsed/Refractory Multiple Myeloma. Date of registration: July 1, 2019.

Keywords: Chimeric antigen receptor T (CAR-T) cells; Design of experiments (DoE); Monocyte; T-cell activation; T-cell purification.

Publication types

  • Clinical Trial
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD19
  • B-Cell Maturation Antigen
  • Humans
  • Immunotherapy, Adoptive / methods
  • Leukocytes, Mononuclear
  • Lymphoma*
  • Monocytes
  • Receptors, Antigen, T-Cell
  • Receptors, Chimeric Antigen*
  • T-Lymphocytes


  • Antigens, CD19
  • B-Cell Maturation Antigen
  • Receptors, Antigen, T-Cell
  • Receptors, Chimeric Antigen

Associated data

  • ClinicalTrials.gov/NCT03798509
  • ClinicalTrials.gov/NCT03720457
  • ClinicalTrials.gov/NCT04003168