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
Abstract 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 st...
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| Format: | Article |
| Language: | English |
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BMC
2022-12-01
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| Series: | Journal of Translational Medicine |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12967-022-03833-6 |
| _version_ | 1797977275515273216 |
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| author | Haiying Wang Shih-Ting Tsao Mingyuan Gu Chengbing Fu Feng He Xiu Li Mian Zhang Na Li Hong-Ming Hu |
| author_facet | Haiying Wang Shih-Ting Tsao Mingyuan Gu Chengbing Fu Feng He Xiu Li Mian Zhang Na Li Hong-Ming Hu |
| author_sort | Haiying Wang |
| collection | DOAJ |
| description | Abstract 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 |
| first_indexed | 2024-04-11T05:04:19Z |
| format | Article |
| id | doaj.art-86fbe28d249b4aa9811640eeca5bb880 |
| institution | Directory Open Access Journal |
| issn | 1479-5876 |
| language | English |
| last_indexed | 2024-04-11T05:04:19Z |
| publishDate | 2022-12-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Translational Medicine |
| spelling | doaj.art-86fbe28d249b4aa9811640eeca5bb8802022-12-25T12:27:28ZengBMCJournal of Translational Medicine1479-58762022-12-0120111510.1186/s12967-022-03833-6A 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 countHaiying Wang0Shih-Ting Tsao1Mingyuan Gu2Chengbing Fu3Feng He4Xiu Li5Mian Zhang6Na Li7Hong-Ming Hu8Department of Research and Development, Hrain Biotechnology Co., Ltd.Department of Research and Development, Hrain Biotechnology Co., Ltd.Department of Research and Development, Hrain Biotechnology Co., Ltd.Department of Research and Development, Hrain Biotechnology Co., Ltd.Department of Manufacturing, Hrain Biotechnology Co., Ltd.Department of Research and Development, Hrain Biotechnology Co., Ltd.Department of Research and Development, Hrain Biotechnology Co., Ltd.Department of Research and Development, Hrain Biotechnology Co., Ltd.Department of Research and Development, Hrain Biotechnology Co., Ltd.Abstract 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, 2019https://doi.org/10.1186/s12967-022-03833-6Chimeric antigen receptor T (CAR-T) cellsT-cell purificationT-cell activationMonocyteDesign of experiments (DoE) |
| spellingShingle | Haiying Wang Shih-Ting Tsao Mingyuan Gu Chengbing Fu Feng He Xiu Li Mian Zhang Na Li Hong-Ming Hu 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 Journal of Translational Medicine Chimeric antigen receptor T (CAR-T) cells T-cell purification T-cell activation Monocyte Design of experiments (DoE) |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | Chimeric antigen receptor T (CAR-T) cells T-cell purification T-cell activation Monocyte Design of experiments (DoE) |
| url | https://doi.org/10.1186/s12967-022-03833-6 |
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