In:
Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 4352-4352
Kurzfassung:
Abstract 4352 Background and objectives: We have recently shown that memory B-lymphocytes 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 post-HSCT immunodeficiency (Klenovsek et al., 2007, Blood 110: 3472–9). As adoptive transfer of B-cells has not been used before in a clinical setting, it is necessary to establish a technology for the generation of GMP-grade B-cell products. Methods: Starting from the leukapheresis of healthy donors, B-cells were purified by two different separation strategies using GMP-grade microbeads and the CliniMACS∧TM device. 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. Results: The leukapheresis contained a mean of 9.0×10∧8 CD19-positive B-cells (4.5–12.4 ×10∧8). After the one-step positive purification strategy a mean purity of CD20∧+ B-lymphocytes of 78.1% with a recovery of 32–41% was obtained. With the two-step T-cell depletion/B-cell enrichment protocol we achieved a mean purity of 96.4 % (93.4–97.8%) with a slightly lower recovery of 14–37%. The absolute B-cell numbers obtained in the product were 1.3 to 4.0 ×10∧8 and 1.7 to 2.6 ×10∧8 for the one-step positive enrichment and the two-step protocol, respectively. Importantly, the absolute number of T-cells was lower in cell products after the two-step protocol (0.1 to 0.9 ×10∧6 T-cells) as compared to the one-step positive CD19-enrichment (1.6 to 3.4 ×10∧6 T-cells). Assuming a patient with 70 kg body weight, the B-cell products obtained after the combined CD3-depletion and CD19-enrichment contained less then 4×10∧4 T-lymphocytes/kg bodyweight, which is a critical threshold number of T-cells in haploidentical HSCT. The B-cell products showed antibody production after in vitro stimulation in a limiting dilution assay and showed excellent viability after cryopreservation. Conclusions: 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∧TM technology. (Supported by BayImmuNet) Disclosures: No relevant conflicts of interest to declare.
Materialart:
Online-Ressource
ISSN:
0006-4971
,
1528-0020
DOI:
10.1182/blood.V120.21.4352.4352
Sprache:
Englisch
Verlag:
American Society of Hematology
Publikationsdatum:
2012
ZDB Id:
1468538-3
ZDB Id:
80069-7
