Title |
GMP-production of purified human B lymphocytes for the adoptive transfer in patients after allogeneic hematopoietic stem cell transplantation
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Published in |
Journal of Translational Medicine, November 2017
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DOI | 10.1186/s12967-017-1330-5 |
Pubmed ID | |
Authors |
Hannes Tittlbach, Andrea Schneider, Julian Strobel, Robert Zimmermann, Stefanie Maas, Bernd Gebhardt, Georg Rauser, Michael Mach, Andreas Mackensen, Thomas H. Winkler, Julia Winkler |
Abstract |
We have recently shown that memory B cells from murine CMV immune donor animals adoptively transferred into immunodeficient mice were highly effective in protecting from a viral infection indicating a therapeutic potential of virus specific memory B cells. These preclinical data provided evidence that a cell-based strategy supporting the humoral immune response might be effective in a clinical setting of immunodeficiency after allogeneic hematopoietic stem cell transplantation. As adoptive transfer of B cells has not been used before in a clinical setting it was necessary to establish a technology for the generation of good manufacturing practice (GMP)-grade B cell products. Starting from the leukapheresis product of healthy blood donors, B cells were purified by two different separation strategies using GMP-grade microbeads and the CliniMACS system. A one-step protocol was used for positive enrichment of B lymphocytes with anti-CD19 microbeads. In a two-step enrichment protocol, first T lymphocytes were depleted by anti-CD3 microbeads and the remaining fraction was positively selected by anti-CD19 microbeads. The purity and recovery after enrichment of B lymphocytes from the leukapheresis material in both separations strategies was not statistically different. However, contamination of the B-cell product with T cells was significantly lower after the two-step protocol (0.16%, range 0.01-0.43% after two-step separation and 0.55%, range 0.28-0.85% after one-step separation, p < 0.05). Therefore, a combined CD3 depletion and CD19 enrichment was used for the production of GMP-conform B-cell products from the leukapheresis material of 17 healthy stem cell donors. The absolute B-cell numbers obtained in the final product was 4.70 ± 3.64 × 10(8) with a purity of 95.98 ± 3.31% B lymphocytes and a recovery of 18.9 ± 10.6%. Importantly, the contamination with CD3(+) T cells was extremely low in the final B- cell products (0.10 ± 0.20%). Purified B cells exhibited normal antibody production after in vitro stimulation and showed excellent viability after cryopreservation. A GMP-grade B-cell product can be obtained with high purity and very low T-cell contamination using the two-step enrichment protocol based on CliniMACS® technology. |
X Demographics
Geographical breakdown
Country | Count | As % |
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United States | 1 | 50% |
Unknown | 1 | 50% |
Demographic breakdown
Type | Count | As % |
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Scientists | 1 | 50% |
Members of the public | 1 | 50% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
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Unknown | 31 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
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Researcher | 7 | 23% |
Student > Ph. D. Student | 4 | 13% |
Student > Master | 2 | 6% |
Student > Doctoral Student | 1 | 3% |
Other | 1 | 3% |
Other | 3 | 10% |
Unknown | 13 | 42% |
Readers by discipline | Count | As % |
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Medicine and Dentistry | 4 | 13% |
Biochemistry, Genetics and Molecular Biology | 3 | 10% |
Agricultural and Biological Sciences | 3 | 10% |
Engineering | 3 | 10% |
Pharmacology, Toxicology and Pharmaceutical Science | 2 | 6% |
Other | 3 | 10% |
Unknown | 13 | 42% |